Fast hydrazone reactants: Electronic and acid/base effects strongly influence rate at biological pH

Article abstract of DOI:10.1021/ja407407h

Kinetics studies with structurally varied aldehydes and ketones in aqueous buffer at pH 7.4 reveal that carbonyl compounds with neighboring acid/base groups form hydrazones at accelerated rates. Similarly, tests of a hydrazine with a neighboring carboxylic acid group show that it also reacts at an accelerated rate. Rate constants for the fastest carbonyl/hydrazine combinations are 2-20 M^-1 s^-1, which is faster than recent strain-promoted cycloaddition reactions.

Full text of DOI:10.1021/ja407407h

Communication
pubs.acs.org/JACS
Fast Hydrazone Reactants: Electronic and Acid/Base Effects Strongly
Influence Rate at Biological pH
Eric T. Kool,* Do-Hyoung Park, and Pete Crisalli
Department of Chemistry, Stanford University, Stanford, California 94305-5080, United States
S
* Supporting Information
However, there is a significant limitation of hydrazone and
ABSTRACT: Kinetics studies with structurally varied
aldehydes and ketones in aqueous buffer at pH 7.4 reveal
that carbonyl compounds with neighboring acid/base
groups form hydrazones at accelerated rates. Similarly,
tests of a hydrazine with a neighboring carboxylic acid
group show that it also reacts at an accelerated rate. Rate
constants for the fastest carbonyl/hydrazine combinations
are 2−20 M−1 s−1, which is faster than recent strain-
promoted cycloaddition reactions.
oxime formation that hinders its broader use: the slow rate of
reaction of most substrates at neutral pH. This can be
inconvenient for reactions in vitro (sometimes requiring hours
to days8b,15) and can be strongly limiting in vivo, where
concentrations of reactants are low. Although aniline can be
used as a nucleophilic catalyst,7c high concentrations are
required, and the rate acceleration is moderate.8b,14a New-
generation catalysts such as anthranilic acids and phosphonates
have improved upon aniline catalysis,9b,c but in some
applications the use of a catalyst is undesirable, adding
complexity and, in some cases, toxicity.16
Thus the identification of structural features that might speed
hydrazone/oxime formation without the addition of a catalyst
could significantly enhance the utility of this reaction.
Surprisingly, there exist few general studies of what structural
features in carbonyl substrates affect reaction rates, particularly
at pH 7.4.15 Early mechanistic studies were typically carried out
at acidic pH, where rates are much more rapid.17 However, the
modern expansion of biological chemistry has greatly increased
the relevance of reactivity at pH values near neutral.
Extensive efforts1 to develop new bond-forming reactions
having improved selectivity, lower interference and cross-
reactivity with biological molecules, and enhanced rates have
resulted in the development of important classes of reactions
such as Cu-catalyzed azide−alkyne cycloadditions,2 strain-
driven cycloadditions,3,4 photoclick reactions,5 and Staudinger
ligations.6 Relative to earlier, less-selective reactions, these new
bond-forming strategies greatly enhance the ability to construct
conjugates of biomolecules, particularly under challenging
aqueous conditions at pH 7.4, at low concentrations, and in
cellular settings.
Here we have performed kinetics studies on a range of
aldehyde and ketone substrates, to examine how structure
affects reactivity at pH 7.4 in aqueous buffer. We find that
substrate structures can have marked effects on reaction rate,
and we have identified specific structural features that yield
reactions with rates that rival many modern cycloaddition
reactions.
One of the earliest reactions used for bioconjugations is that
of hydrazone/oxime formation (Figure 1), involving the stable
Reaction rates were measured for a range of commercially
available carbonyl reactants (Tables 1, 2) dissolved in
phosphate-buffered saline (137 mM Na+, 2.7 mM K+, 12 mM
phosphate, pH 7.4, 25 °C). A small amount of organic
cosolvent (10% DMF) was added to ensure the solubility of
both reactants. To evaluate structural effects, we tested a range
of aldehydes and ketones, both aryl- and alkyl-substituted.
Electron-rich and -poor cases were included, and steric
substitution near the carbonyl was varied. Finally, carbonyl
compounds with proximal acid/base groups were examined as
well. A standard hydrazine (phenylhydrazine) was chosen to
react with these; in limited cases we tested other hydrazines as
well. Rates were measured under pseudo-first-order conditions
by following changes in absorption, with aldehydes at 125 μM−
1 mM and hydrazines at 2.5−20 μM. In general, fits were very
good (see Supporting Information), although a few examples
Figure 1. Mechanism of hydrazone formation. Breakdown of the
tetrahedral intermediate is typically rate limiting at neutral pH.7a,b
imine formation of aldehydes and ketones with α-nucleophiles
such as hydrazines and aminooxy groups. This venerable
reaction7 has been widely useful in bioconjugation,8 due to its
biomolecular orthogonality and because carbonyl and hydrazine
functional groups are readily installed into small molecules.
Early mechanistic studies of the reaction were performed by
Jencks in the 1960s,7a and work by Dawson8d and Tam8a has
highlighted the utility of the reaction in peptide labeling. Very
recent studies by our laboratory9 and by Raines,10 Distefano,11
and Canary12 are also contributing to the utility of the reaction,
which is employed not only in bioconjugations but also in other
fields, such as polymer chemistry13 and dynamic combinatorial
chemistry.8d,14
Received: July 18, 2013
Published: November 12, 2013
© 2013 American Chemical Society
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Table 1. Rates of Reaction of Varied Carbonyl Compounds
with Phenylhydrazine in Aqueous Buffer at pH 7.4
Table 2. Rates of Reaction of Aldehydes and Ketones with
a
Phenylhydrazine and Other Hydrazines in Aqueous Buffer at
a
pH 7.4
a
Conditions: 137 mM NaCl, 2.7 mM KCl, 12 mM phosphate, 25 °C.
Values measured 3−6 times and averaged (std. dev. in parentheses).
Pseudo-first-order k(obs) normalized to standard 1 mM aldehyde
concentration.
a
Conditions are the same as those in Table 1. Values measured 3−6
showed some curvature at later time points, indicating a
deviation from strictly second-order behavior.
times and averaged (standared deviations in parentheses). Pseudo-
first-order k(obs) normalized to the standard 1 mM aldehyde
concentration.
Structures and rate constants are given in Tables 1 and 2.
Examination of the whole set shows some broad trends and
identifies specific features of interest that affect reaction rates
very significantly. First, among all substrates, simple alkyl
aldehydes are the fastest reactants. Indeed, the three fastest-
reacting carbonyl compounds in the study are butyraldehyde, 2-
methylbutyraldehyde, and pivaldehyde (entries 10−12, Table
1). Butyraldehyde, the fastest carbonyl substrate overall, formed
a hydrazone 65-fold more rapidly than did benzaldehyde (entry
2). It seems likely that the faster reaction of alkyl carbonyls
compared to aryl substrates is due to the conjugation in the aryl
cases, which is disrupted upon formation of the tetrahedral
intermediate. This is also consistent with the observation that
cinnamaldehyde reacts 6-fold more slowly than phenyl-
acetaldehyde. A second general observation is that aldehydes
are in most cases faster than comparable ketones (e.g.,
butyraldehyde is faster than 2-butanone by 44-fold and
pivaldehyde is 2.3-fold faster than di-tert-butylketone (entries
17, 19). One exception to this general rule is acetophenone
(entry 20), which reacted 2.4-fold more rapidly than
benzaldehyde. Overall, the two fastest-reacting ketones in the
study were 2-acetylpyridine and 1,1,1-trifluoroacetone (entries
18, 31), which may be due in part to their more electron-
deficient character.
general trend favoring electron-deficient aldehydes and ketones
over electron-rich ones. For example, the slowest-reacting
compound in the study was 4-methoxybenzaldehyde (entry 1),
while 4-nitrobenzaldehyde reacted 4.5-fold more rapidly.
Similarly, trifluoroacetone formed a hydrazone 3.4-fold more
rapidly than 2-butanone, which could also be attributed to the
electron deficiency of the former. However, this effect did not
always hold; for example, dimethoxyacetaldehyde reacted 8-fold
more slowly than 2-methylbutyraldehyde (compare entries
11,13).
Carbohydrates are of special interest for hydrazone formation
because they naturally contain reactive ketone or aldehyde
functional groups, which can be useful in labeling and
bioconjugation.18 Interestingly, the three aldoses tested
hereribose, deoxyribose, and glucosereacted at reasonably
good rates despite the fact that they exist largely in the
hemiacetal state in solution. Ribose and deoxyribose displayed
rates similar to that of benzaldehyde, while glucose reacted 3.5-
fold more rapidly and was similar in reactivity to an electron-
deficient alkyl aldehyde (entry 13). Reactions of anomeric
carbons in deoxyribose are of interest in the development of
reagents for detecting abasic damage in DNA,18a and reactions
of hexoses are useful in making bioconjugates;18b the current
results show that such reactions can proceed with moderate to
good rates.
On this topic, it has long been known that electron-
withdrawing groups can increase the reactivity of aldehydes in
hydrazone formation.7d,17 However, the overall effect has been
found to be small (Jencks observed a Hammett ρ value of 0.91
at pH 1.75).17 In our experiments at pH 7.4 we observed a
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Communication
Steric effects could potentially have significant effects on the
rate of hydrazone formation, by blocking the approach of the
hydrazine nucleophile to the carbonyl carbon. However, the
rate of breakdown of the tetrahedral intermediate might well be
enhanced by the relief of steric crowding. We are unaware of
any prior literature studies of steric effects on hydrazone or
oxime formation. In the current study, steric effects with the
phenylhydrazine standard reactant are found to be small and
inconsistent. For example, we observed a 7.5-fold decrease in
rate for the reaction of pivaldehyde relative to butyraldehyde
(Table 1). Interestingly, however, in the ketone series, di-tert-
butylketone reacts faster than diisopropyl ketone, which in turn
reacts faster than 2-butanone (entries 15−17), showing a
significant enhancement by alkyl substitution. Use of the more
bulky diphenylhydrazine (entries 32−36) did not result in
magnified effects on rate. Overall we conclude that steric effects
are generally moderate for this reaction and that increased
substitution is not always detrimental to the rate, especially for
ketones.
Figure 2. Proposed mechanism for rate enhancement by acid/base
groups near the carbonyl center. Intramolecular protonation of the
leaving group speeds breakdown of the tetrahedral intermediate.
observed in a hydrazine reactant. 2-Carboxyphenylhydrazine
was reacted with selected aldehydes and ketones (Table 2
entries 37−43), and rates were compared to those with
phenylhydrazine. In every case the reaction was significantly
faster with the 2-carboxy-substituted hydrazine (Figure S5).
The rate enhancement by the carboxy group varied from a
factor of 1.6-fold (in the reaction with 2-butanone) to 8.7-fold
(benzaldehyde). With the quinoline substrate, already
enhanced by the nearby imino group, we observed a further
2.3-fold rate acceleration in reaction with the 2-carboxyphe-
nylhydrazine derivative (Figure 3). Importantly, the rate
Finally, we explored the effects of substituting acid/base
groups near the reactive center on the rate of reaction. It was
observed several decades ago that ortho-substitution in
benzaldehydes could speed hydrazone formation, although a
satisfactory explanation for the effect was elusive.19 Pyridoxal
(which has an ortho hydroxy group) has long been known as an
efficient reactant for imine and hydrazone formation,20 and a
recent report employed rapidly reacting pyridoxal phosphor-
amide derivatives in bioconjugations.12 In our recent study, we
observed that 2-carboxybenzaldehyde reacted more rapidly
than the 4-isomer;9c we proposed that the acid group might
donate a proton at the transition state, acting as an
intramolecular catalyst in the reaction.
To address this hypothesized self-catalytic effect, we tested
several compounds with imino, hydroxy, or carboxy groups
substituted near the reactive carbonyl (Table 2, entries 23−31
and Figure S4). Significantly, all cases were found to react more
rapidly at pH 7.4 than do control compounds that either lack
the group or have it substituted remotely from the carbonyl.
Imino groups (as pyridine derivatives) and ortho carboxy
groups had similar effects. Ortho hydroxy groups accelerate the
reaction by 2−4-fold (see entries 26, 27 relative to
benzaldehyde). Finally, the greatest effect occurred with the
imino group of quinoline-8-carboxaldehyde (entry 30), which
accelerates the rate by a sizable factor of 8.3 relative to 1-
naphthaldehyde (entry 9). Notably, while pyridoxal is a
moderately fast reactant in the aromatic carbonyl series, both
this new quinoline aldehyde substrate and 2-acetylpyridine are
considerably faster, and thus these latter two are prime
candidates for further development in bioconjugation reactions.
The rate-limiting step for hydrazone formation at neutral pH
is the breakdown of the tetrahedral intermediate to eliminate
water.7b Thus, catalysis of the reaction could occur by
intramolecular transfer of a proton to the leaving hydroxy
group.9c We tentatively propose that an arylhydroxy group (pKa
∼9−10 in the substrates tested here), a pyridine-type imino
group (pKa ∼5.2), or a carboxy group (pKa ∼4.2) could achieve
proton transfer via 5-, 6-, or 7-membered ring transition states
(Figure 2). More studies will be needed to test this mechanistic
hypothesis, but the results suggest that careful tuning of pKa
and geometry in the future could result in new and yet faster
carbonyl substrates for this reaction.
Figure 3. Time course plots for four related hydrazone-forming
reactions, showing favorable effects of the imino group in the quinoline
electrophile and of a carboxy group in the arylhydrazine nucleophile.
Conditions are same as those in Table 1, with 500 μM aldehyde and
10 μM hydrazine.
constant for reaction of this pair of enhanced substrates was
1.9 M−1 s−1, which is faster than many recently developed
bioorthogonal bond-forming reactions. Similarly high rates and
enhancements were also seen for ketone substrates (entries 38,
41, 43). Remarkably, the rate constant for the fastest substrate
in this study, butyraldehyde, with this enhanced hydrazine was
24 M−1 s−1, which is an order of magnitude more rapid than the
rates of strain-promoted azide−alkyne cycloadditions and a
tetrazine-norbornene addition/rearrangement.3,4b Thus
although hydrazone formation has been generally characterized
as slow at neutral pH, it is clear that specialized hydrazine and
carbonyl substrates can undergo very rapid reactions.
We conclude that with the careful choice of carbonyl and
hydrazine substrate structure, hydrazone formation can be rapid
at biological pH even in the absence of a catalyst. Future studies
will test whether additional structural tuning can yield yet faster
hydrazone-forming reactants. With the advent of such rapid
substrates, there are multiple reasons why this reaction may
become more broadly useful, including ease of synthesis, water
solubility of reactants, and facile introduction into biomole-
cules.
Spurred by this finding, we carried out preliminary studies to
investigate whether a similar self-catalytic effect might be
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details, kinetic traces and plots. This information
is available free of charge via the Internet at http://pubs.acs.org.
Broaders, K. E.; Cohen, J. L.; Engleman, E. G.; Frec
Chem. Soc. 2009, 131, 10360.
(19) Wolfenden, R.; Jencks, W. P. J. Am. Chem. Soc. 1961, 83, 2763.
(20) French, T. C.; Auld, D. S.; Bruice, T. C. Biochemistry 1965, 4,
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AUTHOR INFORMATION
■
Corresponding Author
kool@stanford.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the National Institutes of Health (GM068122) for
support.
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