Comparison of boron-assisted oxime and hydrazone formations leads to the discovery of a fluorogenic variant

Article abstract of DOI:10.1039/c6ob00168h

We use kinetic data, photophysical properties, and mechanistic analyses to compare recently developed high-rate constant oxime and hydrazone formations. We show that when Schiff base formation between aldehydes and arylhydrazines is carried out with an appropriately positioned boron atom, then aromatic B-N heterocycles form irreversibly. These consist of an extended aromatic structure amenable to the tailoring of specific properties such as reaction rate and fluorescence. The reactions work best in neutral aqueous buffer and can be designed to be fluorogenic-properties which are particularly interesting in bioconjugation.

Full text of DOI:10.1039/c6ob00168h

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Comparison of boron-assisted oxime and
hydrazone formations leads to the discovery
Cite this: DOI: 10.1039/c6ob00168h
of a fluorogenic variant†
Received 20th January 2016,
Accepted 2nd February 2016
Cedric J. Stress, Pascal J. Schmidt and Dennis. G. Gillingham*
DOI: 10.1039/c6ob00168h
www.rsc.org/obc
We use kinetic data, photophysical properties, and mechanistic nucleophiles. The inspiration for this choice came from two
analyses to compare recently developed high-rate constant oxime reports, published more than fifty years ago, demonstrating
and hydrazone formations. We show that when Schiff base for- that with hydrazines the initial Schiff base adducts (structure 3
mation between aldehydes and arylhydrazines is carried out with in Fig. 1) undergo a secondary intramolecular cyclization to
an appropriately positioned boron atom, then aromatic B–N deliver aromatic, boron-substituted isoquinoline derivatives,
heterocycles form irreversibly. These consist of an extended aromatic which were stable to prolonged boiling in acid or base.13,14
structure amenable to the tailoring of specific properties such as These reports on 4,3-borazaroisoquinolines (BIQs) were largely
reaction rate and fluorescence. The reactions work best in neutral forgotten until the 1990s when reports from Groziak15 struc-
aqueous buffer and can be designed to be fluorogenic – properties turally confirmed and extended16 the earlier findings. The
which are particularly interesting in bioconjugation.
Groziak group also proposed these materials as the basis of
unique pharmacophores.17,18 Indeed several BIQs have shown
growth inhibitory activity against different types of
bacteria.18–20 In a very recent development, the Bane lab has
shown that BIQ formation works well under aqueous con-
ditions and could be used for bioconjugation.21 In this work
we present a mechanistic analysis and comparison of oxime,
hydrazone, and BIQ formation (i.e. structures 1, 2, 3, and 4 in
Fig. 1). The data should serve as a guidebook to potential
users for determining which conjugation is best suited to their
specific application. Furthermore, we demonstrate that when
the aromatic system of the BIQ is extended and appropriately
substituted to deliver a push–pull system, then a fluorogenic
reaction is possible. Turn-on fluorescence is important in
Introduction
Condensation of aldehydes or ketones with α-effect nucleophiles
such as hydroxylamines and hydrazines is an important ligation
reaction in chemical biology.1–3 Reports from us4 and others5,6
have shown that proximal functional groups enhance the rate of
oxime/hydrazone condensation at neutral pH. Recent work from
our group has demonstrated that proximal boronic acids
increase the rate of oxime condensation up to more than
104 M−1 s−1 under neutral aqueous conditions.7 Nevertheless,
oximes and hydrazones are unstable to hydrolysis under
aqueous conditions.8 We have quantified the reversibility of
aldoximes that bear proximal boronic acids and found a stability
on the order of several hours in neutral aqueous buffer; others
have found that the reversibility problem is particularly acute
with ketoximes,9 which equilibrate in minutes. While reversibil-
ity may be beneficial for certain applications,10 a durable link is
often required for biological studies11 or in practical applications
such as for antibody–drug conjugates.12
In the search for an irreversible variant of an oxime or
hydrazone condensation we turned to hydrazines as α-effect
Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056 Basel,
Switzerland. E-mail: dennis.gillingham@unibas.ch
†Electronic supplementary information (ESI) available. CCDC 1448404. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c6ob00168h
Fig. 1 Boron accelerates the formation of Schiff bases of type 1, 2, and
3 from carbonyl compounds and α-effect amines; 3, however, engages
in a secondary reaction that leads to the stable BIQ 4.
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cellular assays since it reduces background during imaging we turned to substrate variation to probe this mechanistic
and can remove the need for washing steps.22,23
hypothesis. We had already shown7 that aldehyde positioning
relative to the boronic acid is important for high reaction
rates, with only ortho-positioning leading to any acceleration.
para- or meta-Formylphenylboronic acids led to oxime conden-
sations with rates comparable to benzaldehyde. The new sub-
strates added in Fig. 2 (see c & d) are also consistent with the
mechanistic hypothesis. The condensation of 10 and 11 (see c
in Fig. 2) would require the formation of an intermediate
8-membered ring if the boronic acid were involved. The rate of
this transformation is comparable to substrates that bear no
boronic acid and hence a small ring size seems crucial. Our
attempts to look at other ring sizes were thwarted by synthetic
difficulties. An important aspect of the proposed mechanism
is the direct involvement of boron. 2-Formylphenyltrifluoro-
borate 13 maintains the boron, but does not have an open
coordination site. Treatment of 13 with 6 (see d in Fig. 2)
delivers product far slower than 2-FPBA but still faster than
benzaldehyde (t1/2 = 48 h at 1 mM; Note: extrapolated from
four data points). In addition the hydrolyzed molecule 7 is the
only observed product (i.e. no fluoroborates); hence in this
case hydrolysis of the trifluoroborate must occur before con-
densation. Indeed the rate of oxime formation is consistent
with rates of hydrolysis for related aryltrifluoroborates (see the
ESI page 18† for an additional example with a coordinatively
saturated arylboronate ester).28
Results and discussion
We have shown recently that boronic acid groups in ortho posi-
tion to an aldehyde greatly enhance the rate of oxime for-
mation (see a in Fig. 2).7 We hypothesize that this is due to the
formation of a five-membered ring intermediate prior to the
normally rate-limiting dehydration step (see b in Fig. 2). This
intermediate must eliminate a boronate rather than a water
molecule to deliver the oxime – a process that should be far
more facile since protonation of the cyclic boronate would
occur right around neutral pH.24–26 This mechanistic pathway
is supported by a previous study which examined the effect of
boron on labelling the ε-amino group of lysines.27 The overall
oxime formation is too rapid for us to detect intermediates so
In our previous report we found that aldoximes are revers-
ible but took several hours until the equilibrium was reached.7
To further investigate the influence of substituents on the
stability and reversibility of oximes we treated ketoxime 14
with a five-fold excess of 2-formylphenylboronic acid (2-FPBA).
NMR analysis indicated rapid equilibration and a strong ener-
getic preference for the aldoxime: after the first measurement
(15 min) about 60% of the ketoxime was converted to the
corresponding aldoxime, and after 75 minutes only the aldo-
xime was observed (compare time points in Fig. 3). A substan-
tial reduction in transition state energy for formyl versus acetyl
Schiff bases has also been seen by others9 and is likely a result
of the preference for the ketoximine nitrogen to bind to the
boron in an iminoboronate interaction, thus activating it for
hydrolysis; X-Ray crystallography indicates no such interaction
in the ground-state for the aldoxime (see B in Fig. 3). We
speculate that the energetic reason for this difference is
related to Raine’s hypothesis that the basicity of the nitrogen
of the Schiff base is decisive in determining stability of oximes
and hydrazones. The additional σ-donation of the methyl sub-
stituent should render its nitrogen more basic (compare X =
Me to X = H in panel c, Fig. 3) and therefore more amenable to
protonation or interaction with a Lewis acid.
In the search for an irreversible bioconjugation we turned
to arylhydrazines. Although hydrazones (such as 16 in Fig. 4)
are known to be more labile than oximes,8 reports from the
1960s showed that hydrazones with a boronic acid group in
ortho position undergo an intramolecular cyclization reaction
to 4,3-borazaroisoquinolines (BIQ).13,14 The Bane lab has very
recently described that this reaction could offer a powerful tool
Fig. 2 The prototype boron assisted oxime condensation (a), our
working mechanistic hypothesis (b) and substrate variations that provide
some insight on the mechanism (c & d).
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slower, with a rate constant of 2.5 × 10−3 M−1 s−1 (see Fig. 4).
The rates were measured at 10 μM concentration by following
hydrazone formation and disappearance at 340 nm. As
expected, acidification increased the reaction rates, whereas
under more basic conditions the transformations proceeded
more slowly (see ESI†). We also found that polar aprotic sol-
vents like DMF and MeCN drastically slowed down the BIQ for-
mation and led to undesired oxidation of the boronic acid
leaving a hydroxy substituted hydrazone. As a general rule we
found that higher percentages of water increased the reaction
rates even when the starting materials and products were
poorly soluble. Complex media like human serum or milli-
molar concentrations of glutathione (see also ref. 21) only
slightly affected the reaction rates (see ESI page 26†).
The relatively slow BIQ formation allowed us to employ
NMR to follow the cyclization and to examine whether it was
reversible. As shown in Fig. 5 arylhydrazone 16 cyclizes to BIQ
17 in aqueous phosphate buffer over the course of several
hours – a rate that is consistent with the UV measurements. If
purified product 17 is then treated with 2-FPBA (see panel B in
Fig. 5), no change in the amount of 17 or 5 is observed over
24 h. If 2-FPBA is added to 16 before cyclization is complete
then small amounts of the oxime product are observed, indi-
cating that arylhydrazone formation is reversible.
BIQ formation leads to an unusual aromatic scaffold whose
optical properties might be amenable to tuning. Particularly
interesting is the possibility that the de novo construction of
Fig. 3 A. NMR indicates rapid equilibration and a strong preference for
7 over 14. B. X-Ray analysis of compound 7 suggests no iminoboronate
in the ground state. C. The increased σ-donation of methyl versus
hydrogen likely increases basicity and hence reduces stability of oximes.
Fig. 4 BIQ formation proceeds at low concentration under mild
conditions.
in aqueous conjugations.21 We present here our own explora-
tion of the reaction, which is fully consistent with the reports
of the Bane lab. We also include additional mechanistic data,
new substrates, and the development of a fluorogenic process.
We began by examining the conjugation of compound 15
with phenylhydrazine in pH 7.2 phosphate buffer. UV analysis
could not provide the time resolution to determine the rate of
hydrazone (16) formation but a lower bound of >103 M−1 s−1
can be established. The cyclization step to deliver BIQ 17 was Fig. 5 NMR analysis of BIQ formation (A) and reversibility (B).
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rescent with a maximum emission at 502 nm. BIQ formation
led to a more than five-fold increase in fluorescence and an
improvement in fluorescent quantum yield (ϕ = 0.39 for 27
versus 0.28 for 28). Although improvements still need to be
made for this fluorophore to service areas of unmet need in
chemical biology, the fact that a fluorogenic reaction could be
arrived at from a simple two-step optimization with less than
fifteen compounds bodes well for future optimization.
Conclusions
Fig. 6 Representative examples of BIQs synthesized (see the ESI† for
We have presented mechanistic data and a comparison of
strengths and weaknesses of various Schiff base reactions with
α-effect amines. Arylhydrazines are unique in that they
undergo a secondary reaction to deliver aromatic BIQs that are
highly stable. Optimization of substituents led to the develop-
ment of a BIQ which exhibits a large fluorescence enhance-
ment in comparison to its constituent components. Our
studies should provide guidance for practitioners in chemical
biology who would like to employ the process: acyl hydrazone
and ketoxime formation is rapidly reversible and would likely
be ideal in dynamic covalent chemistry; aldoximes are slowly
reversible and might be ideally suited for applications where
targeted slow release or exchange is desired; arylhydrazones
lead to BIQ formation and would be ideal when the linkage is
meant to be permanent.
additional examples and information).
the ring might allow turn-on fluorescence. The modularity of
the reaction allowed us to quickly access a variety of substituted
BIQs and study their absorbance and fluorescence. In general
all tested BIQs showed some fluorescence at excitation wave-
lengths <300 nm, but most with an efficiency that would not be
useful in practice (see panel A in Fig. 6 for structures and the
ESI page 30† for absorbance and fluorescence spectra).
Two thoughts guided the design of our next series of BIQs:
We considered the B-OH group as a potential fluorescence
quencher and we also thought that additional annulation
might red-shift the absorption wavelength. Accordingly, we
synthesized another series of substituted BIQs (see panel b in
Fig. 6) with an additional O-substituted five-membered ring
(see as well the ESI† for an example of a six-membered ring).
In general the alkoxy or hydroxyl substitutions did not lead to
substantial shifts in absorbance maximum or improvements
in fluorescence efficiency. Substitution with the electron rich
dimethylamino group in the boronic acid component,
however, delivered a good blue fluorophore (27 in Fig. 6). As
shown in Fig. 7, the starting boronic acid, 28, was poorly fluo-
Acknowledgements
Professor Oliver Wenger and Mr Angelo Lanzilotto are grate-
fully acknowledged for discussions on photochemistry and
help in optical measurements. Mr Thomas Müntener is grate-
fully acknowledged for help with NMR measurements.
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