Exceptionally rapid oxime and hydrazone formation promoted by catalytic amine buffers with low toxicity

Article abstract of DOI:10.1039/c8sc01082j

Hydrazone and oxime bond formation between α-nucleophiles (e.g. hydrazines, alkoxy-amines) and carbonyl compounds (aldehydes and ketones) is convenient and is widely applied in multiple fields of research. While the reactants are simple, a substantial drawback is the relatively slow reaction at neutral pH. Here we describe a novel molecular strategy for accelerating these reactions, using bifunctional buffer compounds that not only control pH but also catalyze the reaction. The buffers can be employed at pH 5-9 (5-50 mM) and accelerate reactions by several orders of magnitude, yielding second-order rate constants of >10 M^-1 s^-1. Effective bifunctional amines include 2-(aminomethyl)imidazoles and N,N-dimethylethylenediamine. Unlike previous diaminobenzene catalysts, the new buffer amines are found to have low toxicity to human cells, and can be used to promote reactions in cellular applications.

Full text of DOI:10.1039/c8sc01082j

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Exceptionally Rapid Oxime and Hydrazone Formation Promoted
by Catalytic Amine Buffers with Low Toxicity
Received 00th January 20xx,
Accepted 00th January 20xx
Dennis Larsen,† Anna M. Kietrys, Spencer A. Clark, Hyun Shin Park, Andreas Ekebergh,‡ and Eric T.
Kool*
DOI: 10.1039/x0xx00000x
Hydrazone and oxime bond formation between α-nucleophiles (e.g. hydrazines, alkoxy-amines) and carbonyl compounds
(aldehydes and ketones) is convenient and is widely applied in multiple fields of research. While the reactants are simple, a
substantial drawback is the relatively slow reaction at neutral pH. Here we describe a novel molecular strategy for
accelerating these reactions, using bifunctional buffer compounds that not only control pH but also catalyze the reaction.
The buffers can be employed at pH 5-9 (5-50 mM) and accelerate reactions by several orders of magnitude, yielding
second-order rate constants of >10 M-1s-1. Effective bifunctional amines include 2-(aminomethyl)imidazoles and N,N-
dimethylethylenediamine. Unlike previous dia-minobenzene catalysts, the new buffer amines are found to have low
toxicity to human cells, and can be used to promote reactions in cellular applications.
www.rsc.org/
speed up the hydrazone and oxime formation reactions.
Gillingham and others have developed specialized aromatic
aldehydes or ketones with a boronic acid group in the o-
Introduction
Bioconjugation techniques are a cornerstone of the field of
chemical biology, and much scientific interest has focused on
developing new bioorthogonal chemical transformations that
can be performed in water at neutral pH.1 Particular attention
has been given to developing reactions with very high second-
order rate constants in order to promote rapid bond formation
even when the reactants are present at low concentrations.2
In addition to development of new conjugation reactions,
substantial research efforts have gone toward accelerating
existing reactions that have broad application but are limited
by slow rates. In this light, we have recently focused on
developing catalysts for the well-established hydrazone and
oxime conjugation reactions.3 Hydrazones and oximes, once
formed, can be hydrolytically stable species4 that have been
employed extensively for bioconjugations and polymer
functionalization.5 The reactions remain appealing because of
the simplicity of the reacting groups and the ease of
introducing the reactants into biomolecules and probes.
However, the relatively slow rate of product formation under
neutral conditions (ca. 0.01 – 0.1 M-1s-1)1d has limited the
utility of these reactions.
position that result in extremely rapid bond formation with
aminooxy groups.6 Another approach to speed up this reaction
without requirement of specialized reactant structures is the
development of catalysts for hydrazone and oxime formations.
Building on seminal work by Cordes and Jencks,7 Dawson
introduced the use of aniline as a nucleophilic amine catalyst
for hydrazone and oxime bioconjugations,4d, 8 and subsequent
work in several groups has identified simple aniline derivatives
that exceed aniline’s catalytic ability.9
We have focused on developing bifunctional catalysts that
employ intramolecular proton transfer from acid/base groups
in proximity to a nucleophilic amino-group to facilitate rapid
hydrazone and oxime formation,3 building on the early work of
Hine.10 A substantial advantage of the use of catalysis in this
reaction (rather than specialized substrates) is that the
reaction can be reversed or equilibrated under the influence of
a catalyst, while the products remain relatively stable in its
absence. This enables diverse applications such as exchange
reactions for cellular imaging,11 dynamic combinatorial
12
selections,8a,
and reversal of formalin crosslinks in fixed
tissue specimens.13
To overcome this, researchers have developed strategies to
In a recent study, we identified 2-(aminomethyl)imidazoles
as a new, highly active bifunctional catalyst scaffold for these
transformations.3c Further work in our laboratory led us to
realize that these and related compounds have pKa values near
the neutral range, and that the best way to increase the
catalyst concentration, and thus speed up the desired reaction
even further, without exceeding the buffer capacity of the
system, is to use the catalysts themselves as buffering agents.
Note that these agents are used here in super-stoichiometric
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amounts, but still follow a catalytic mechanism (see below). This is a reasonable assumption at a fifty-fold excess of one
Here we study this new strategy of using catalyzing amine reagent, and was also confirmed by chromatographic analysis
buffers (Amine Buffer/Catalysts, ABCs) to dramatically enhance of reactions in ABC
4 (Fig. S9), eliminating the concern that
the rate of oxime and hydrazone formation at neutral pH (Fig. these buffers can form stable Schiff-bases or (hemi)aminals
1).
that could preclude full conversion.
Scheme 1 Model reaction between benzaldehyde and phenylhydrazine
Table 1. Second-Order Rate Constants for the Model Reaction in a Range of Buffers at
pH 7.4a
k2
k2
no.
1
Buffer
krel no.
1.00 11
Buffer
krel
(
M
ꢀ1sꢀ1)
(
M
ꢀ1sꢀ1)
0.466
± 0.007
0.34
± 0.01
Phosphate
0.72
Fig. 1 In previous studies, nucleophilic amines were used to catalyze hydrazone (X=NH)
and oxime (X=O) formations in a phosphate buffer at pH 7.4. In the present study,
nucleophilic amines with proximal acid/base groups with pKa values in the 6-9 range
are used both as catalysts for the reactions as well as buffering agents for the aqueous
reaction medium.
0.481
± 0.002
2.15
± 0.02
2
3
Tris
1.0 12
0.60 13
4.6
5.9
0.28
± 0.02
2.77
± 0.06
HEPES
10.6
± 0.6
2.26
± 0.02
Results and discussion
4
5
23
18
14
15
4.9
ABC screening
8.6
± 0.9
0.34
± 0.02
0.74
A range of ABCs were chosen for the initial screening (Table 1).
To emulate the conditions found in buffered saline solutions
often used in biological experiments, all the catalyzing buffers
were screened in 150 mM NaCl (saline). Although recent
studies have highlighted that high salt concentrations (>1 M)
influence formation rates of oximes,14 at the NaCl
concentrations applied here we did not see remarkable
changes when compared to control experiments with no NaCl
(see details in Table S1 in the ESI). For the initial testing of
buffers, we used a buffer concentration of 50 mM, but the
best catalyzing buffers were also found to be effective at 10
mM (Table S1). All the ABCs have an acid/base group with a
pKa in the biologically relevant range, defined here as 6-9,
ensuring that they can in fact function as buffering agents at
biologically relevant pH levels. No pKa values have been
reported for 2-(aminomethyl)pyrimidine 20, nor have they
been determined for the two phosphonic acids 17 and 18, but
they are estimated to fall within the 6-9 pKa range (see full list
of known pKa values in Table S2). A potentiometric titration
was used to determine the pKa values of 2-
10.7
± 0.5
(Not soluble
at 50 mM)
Me2N NH2
6
7
8
23
17
16
17
O
P
OH
OH
8.0
± 0.5
(Not soluble
at 50 mM)
Me
NH2
O
P
H
OH
OH
0.222
± 0.004
2.9
6.3
N
Me2N
0.48 18
NH2
N
± 0.4
Me
NH2
0.304
± 0.004
6.2
13
9
0.65 19
0.46 20
± 0.2
0.22
± 0.01
1.46
3.1
10
± 0.04
aSecond-order rate constants, k2, listed as mean values ± standard deviations
based on triplicate measurements. Relative reaction rates, krel, in comparison to
reaction in phosphate buffer (entry 1). Reactions were performed in saline
solution (150 mM NaCl), and buffer pH was adjusted (using either 1 M NaOH or 1
M HCl) to pH 7.40 ± 0.04 prior to use.
(aminomethyl)imidazole
ESI).
4 to be 4.38 and 8.13 in water (see
The reaction rate determined in phosphate buffer was used as
the point of reference (Table 1, entry 1). Two other common
buffers (Tris and HEPES, entries 2 and 3) were also employed
for comparison, but although Tris is a primary amine buffer, it
did not afford any significant improvement compared to the
phosphate buffer. HEPES buffered saline was found to give an
even slower reaction rate.
To initially evaluate the catalyzing buffers, we followed the
reaction between benzaldehyde and phenylhydrazine (Scheme
1). The reaction is conveniently monitored by absorption at or
near 340 nm (approx. λmax of the hydrazone product), and the
reaction rates were determined by non-linear regression
assuming full conversion of the limiting reagent into product.3c
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It is worth mentioning that the reaction rate in phosphate (aminomethyl)(benz)imidazoles
ARTICLE
4
and 5 catalyze hydrazone
buffer reported here (ca. 0.47 M-1s-1) is more than two-fold and oxime formations when used at only 1 mM concentration
faster than the known rate in less concentrated phosphate in phosphate buffer,3c and when employed as catalyzing
buffered saline (0.21 M-1s-1 in ca. 12 mM phosphate),3c buffers here, we find that they achieve remarkably high
illustrating that the reaction is dependent on buffer second-order reaction rates of greater than 10 M-1s-1. This
concentration. This is indicative of a reaction following a represents a roughly 20-fold enhancement compared to
general acid/base mechanism, and supports Jencks’ well- phosphate at the same buffer concentration, and the rates are
established conclusion that the reaction proceeds via a rate- higher than could be achieved with the best previously
determining acid-catalyzed dehydration of the intermediate identified catalyst, phosphonic acid 17, on the model reaction
hemiaminal.15
Comparative evaluation of twenty buffers revealed (Table 1) aqueous media using our previous conditions.3a Thus, using 17
that aminopyridines and aminoimidazoles (compounds 11 as a catalyst at 1.0 mM in a 12 mM phosphate buffered saline
were the least effective catalysts, giving rates equal to or at pH 7.4, the rate constant was found to be 6.1 ± 0.3 M-1s-1.
below that of phosphate buffer. Histidine 12 and its simple Hine showed that N,N-dimethylethylenediamine forms
between benzaldehyde and phenylhydrazine in purely
8-
)
6
derivatives 13 and 14 were moderately effective as catalytic imines much faster than other primary amines, due to
buffers, showing improved reaction rates compared to both intramolecular acid-catalyzed dehydration of the intermediate
phosphate or the more structurally similar and well-known hemi-aminal.10b Here, we find that it is highly effective as a
biological buffer imidazole (15), but still falling well below the catalytic buffer for hydrazone formation at 50 mM. Due to the
best buffers in efficiency.
proximal location of the two amino groups in 6, the primary
Anthranilic and phosphanilic acids are among the most amine has a pKa of 6.93, while the tertiary amine has a pKa of
effective catalysts known for this reaction.3a, 3b We found that 9.53,18 leaving the primary amine mostly in its nucleophilic
the poor solubility of anthranilic acids prevent their use at 50 state at biological pH levels. Although
6 was found to be a
mM, and addition of a ring nitrogen (16) still did not confer relatively poor catalyst under previous conditions,3c in this
solubility at 50 mM. In tests at lower buffer concentrations of catalyzing buffer context it achieves a second-order rate
10 mM, 16 was found to give a rate constant almost 10-fold constant in excess of 10 M-1s-1, a >20-fold rate enhancement
slower than with
4
(see ESI). Catalyst 17 is the most efficient of compared to phosphate. As was later discovered (see below),
in the presence of phenylhydrazine is limited,
previously identified catalysts in hydrazone formation when the stability of
6
employed at lower concentrations in phosphate buffered especially at pH levels of 5.5-6.5, somewhat limiting its
saline.3a It is hampered in this catalyzing buffer context by applicability in the lower part of the biological pH range.
relatively low solubility at neutral pH (estimated to be in the
Both meta- and para-diaminobenzene have been identified
15-20 mM range at room temperature, pH 7).13 In fact, when as catalysts that are slightly superior to aniline for hydrazone
employed as a catalyzing buffer at 10 mM, 17 fared quite well, and oxime ligation, and due in part to a much higher solubility
giving a relatively high second-order rate constant of 3.3 ± 0.2 in water, the meta derivative has been praised as a ‘highly
M-1s-1, though it was not as high as the 4.6 ± 0.2 M-1s-1 efficient’ catalyst when employed at concentrations as high as
achieved with ABC
4 7 is the only isomer of the
at 10 mM concentration (see further 0.9 M.9b para-Diaminobenzene
examples in the ESI). Therefore, we synthesized the more diaminobenzenes with a pKa value in the desired range (pKa
water-soluble 18, hoping it would retain the catalytic effect of 6.46),19 and thus the only derivative to be potentially useful as
17. While 18 was soluble enough to be used at 50 mM, it was a catalyzing buffer at a biologically relevant pH. As is evident
also found that the rate enhancements achieved (ca. 6-fold from Table 1, ABC
compared to phosphate) were not as high as for the best ABCs of ca. 8 M-1s-1, meaning it belongs in the ranks of the four
. We attribute the poorer effectiveness of 18 relative to 17 standout catalyzing buffers discovered in this screening, ABCs
7 achieves a reasonably high rate constant
4-7
to the undesired inductive effect of the dimethylammonium
group on the aromatic ring.
4
-
7
. The oxidative instability of
7, however, leads to buffered
solutions turning colored within minutes. This severely limits
(Aminomethyl)imidazoles (4, 5) proved to be stable under the applicability of the buffer for most UV/vis based assays,
all conditions tested here, and provided among the highest except where those assays are performed in less than ca. 30
rate enhancements (18- to 23-fold over phosphate buffer) for min. Due to the large absorbance by the breakdown products
the test reaction. For comparison, we examined other of
aminomethyl-substituted heterocycles 19 20). 500 nm appears over the course of an hour), it was not
2-(Aminomethyl)pyridine 19 gives a high rate enhancement of possible to evaluate whether use of a more than one-hour old
13-fold. Structurally similar to imidazoles and , the relatively buffer solution significantly altered the rate of hydrazone
high pKa value of 8.6 for 19 may explain why it does not formation.
7 (a broad, strongly absorbing band with a λmax at ca. 450-
(
,
4
5
perform as well as
17
4
and
5
, whose pKa values (8.13 and 7.83
All four ABCs 4-7 exhibited a linear relationship between
respectively)16,
are closer to the pH level used in these reaction rate and buffer concentration in experiments from
screening reactions. Pyrimidine compound 20 is less effective, 10-50 mM in saline at pH 7.4 (see ESI for full details), although
likely due to its significantly lower pKa. for ABC the linearity was not very good, likely due to optical
Next, we turned our attention to the most effective interference from oxidation byproducts. Using the highly
7
catalytic buffers,
4
-7
. As we found in a recent study, 2- soluble
4
as a buffer at 500 mM in water, a second-order rate
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constant of 33.6 ± 1.0 M-1s-1 was achieved for the model phosphate buffer), now makes the use of oxime ligations with
reaction between benzaldehyde and phenylhydrazine.
standard, non-activated substrates a viable alternative without
requiring the synthesis of specialized reactants.
Applicability of ABCs at pH 4.5 – 9.0
1000
100
10
Building on these results, we proceeded to evaluate the
applicability of ABCs 4-7 at a range of pH levels from pH 4.5 to
pH 9.0. All the catalyzing buffers improved the rate of reaction
compared to phosphate buffer throughout the range, except
for ABC 6, which proved to be unstable at pH 5.5 and pH 6.5 in
the presence of phenylhydrazine (Fig. 2). The reaction rates
were found to be higher below neutral pH, with a maximum
near pH 5.5 in most cases, including with the use of phosphate
buffer. This is in accord with the extensive research on imine
formation reaction mechanisms by Cordes and Jencks.20
1
When comparing the reaction rates with ABCs 4-7 to that in
Phosphate
4
5
6
phosphate buffer at different pH levels, an interesting pattern
emerges. The rate enhancements relative to phosphate buffer
0.1
go from below 5-fold (with exception of
than 20- or even 40-fold rate enhancements at pH 8.2 (Fig. 3).
This strongly suggests that the catalysts function in
7) at pH 4.5 to more
7
a
0.01
bifunctional manner by way of an intramolecular general
acid/base mechanism, and it also demonstrates that these
catalyzing buffers are highly effective in improving the reaction
rate specifically in the biologically relevant part of the pH scale.
4
5
6
7
8
9
pH
Fig. 2 Second-order rate constants for the test reaction (Scheme 1) as a function of
solution pH in selection of ABCs and in phosphate buffer (50 mM buffer
a
concentration). All reactions performed in saline (150 mM NaCl). Error bars show
standard deviations based on triplicate measurements (see full data in Table S3); where
no error bars are visible, they are smaller than or equal to the size of the data point.
The dashed part of the yellow line illustrates the pH levels where ABC 6 was found to
be unstable in presence of phenylhydrazine.
Scope investigations
We next employed ABCs 4-7 in a range of experiments to
investigate the scope of their abilities to catalyze hydrazone,
oxime and acylhydrazone formations. First, phenylhydrazine
was reacted with three different carbonyl compounds in both
60
phosphate buffer and using ABCs 4-7 (Table 2). The results
4
show that ABCs give higher rate enhancements with otherwise
disfavored electron-rich carbonyl electrophiles like 4-
5
50
methoxybenzaldehyde
than
with
electron-poor
3-
6
formylpyridine or neutral benzaldehyde (compare to values in
Table 1). The data also show high rate enhancements for
otherwise challenging ketone substrates such as
7
40
acetophenone. Using ABC
6, a heretofore unseen rate of
reaction between phenylhydrazine and acetophenone of
almost 10 M-1s-1 is achieved, representing a more than 60-fold
rate enhancement in comparison to the phosphate buffer. This
is a potentially useful finding for protein functionalization, as
robust methods to incorporate 4-acetylphenylalanine into
proteins have been developed.1a, 1b, 21
30
20
10
0
Next,
we
reacted
O-benzylhydroxylamine
and
benzoylhydrazide with benzaldehyde to evaluate the
catalyzing buffer method in oxime and acylhydrazone
formations, respectively (Table 3). As is widely observed in the
literature, oxime bond formations are roughly an order of
magnitude slower than hydrazone formations, which is also
reflected in the quite low rate constant seen here in
phosphate buffer for this reaction (0.0213 M-1s-1). However,
4
5
6
7
8
9
pH
Fig. 3 Comparison of rate enhancements of the model reaction (Scheme 1) for a
selection of ABCs relative to phosphate buffer as a function of solution pH (buffer
concentration 50 mM). All reactions performed in saline (150 mm NaCl). Error bars
show compounded standard deviations based on triplicate measurements (see full data
in Table S3); where no error bar is visible, the standard deviation is smaller than or
equal to the size of the data point. The greatest rate enhancements in comparison to
phosphate buffer are achieved in the biologically relevant region of pH 6-9. The dashed
using ABC buffers we were delighted to see that using either
or
we could achieve reaction rates in excess of 1.0 M-1s-1.
Although even higher oxime formation rates have been
4
6
reported using specially activated aldehydes,6a,
the rate
22
enhancements reported here (75 – 140-fold in comparison to
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part of the yellow line illustrates the pH levels where ABC 6 was found to be unstable in
Table 3. Reaction rates of benzaldehyde with different α-nucleophiles in a selection of
catalyzing buffers at pH 7.4a
presence of phenylhydrazine.
Table 2. Reaction rates of phenylhydrazine with a range of carbonyl compounds in a
selection of catalyzing buffers at pH 7.4a
αꢀnucleophile:
_
_
__
__
krel
Carbonyl compound
Buffer
k2
krel
k2
0.0213
± 0.0013
0.00141
± 0.00004
Phosphate
1.00
140
1.00
420
__
k2
__
krel
_
_
__
k2
__
krel
Buffer
k2
krel
2.9
± 0.2
0.59
± 0.01
4
5
0.407
± 0.007
0.093
0.118
± 0.015
Phosphate
1.00
14
1.00
1.00
13
± 0.007
0.32
± 0.08
5.7
± 0.6
2.8
1.5
± 0.4
n.d.
220
220
4
5
30
27
± 0.2
1.6
± 0.2
0.31
± 0.02
6
7
75
5.4
± 0.3
2.47
0.55
± 0.03
13
4.6
67
± 0.12
n.d.
n.d.
2.13
± 0.04
3.77
8.0
± 1.2
6
7
5.2
29
41
28
± 0.06
aSecond-order rate constants (M-1s-1), k2, listed as mean values ± standard
deviations based on triplicate measurements. Conditions as per Table 1. Relative
reaction rates, krel, is in comparison to reaction in phosphate buffer for each
substrate respectively. n.d., no data could be obtained due to UV spectral overlap
from the buffer.
11.9
± 0.8
2.60
n.d.
± 0.16
aSecond-order rate constants (M-1s-1), k2, listed as mean values ± standard
deviations based on triplicate measurements. Conditions as per Table 1. Relative
reaction rates, krel, is in comparison to reaction in phosphate buffer for each
substrate respectively. n.d., no data could be obtained due to UV spectral overlap
from the buffer.
Protein labelling studies
ABCs
4 and 6 were found to be useful for catalyzing hydrazone
formation between phenylhydrazine and the aldehyde
produced in-situ by periodate oxidation of threonine (i.e.
glyoxalic aldehyde), reaching second-order rate constants of
0.459 ± 0.005 and 0.318 ± 0.009 M-1s-1, respectively (see ESI for
Due to the electron-withdrawing effect of the carbonyl,
acylhydrazines (i.e. carbohydrazides) are also known to be
significantly less nucleophilic than hydrazines, and therefore
acylhydrazone formation is much slower than hydrazone
formation. However, because of the ease of preparation and
handling of hydrazides, many hydrazone conjugation
procedures are performed with hydrazides as the
nucleophile.23 In our tests, using benzoylhydrazide as the test
nucleophile instead of phenylhydrazine leads in phosphate
buffer to a dramatic loss of reactivity of more than two orders
details). ABCs
5 and 7, on the other hand, were found to be
unstable in solutions containing periodate, which we
tentatively ascribe to oxidative polymerization processes.24
To test a labelling application of the catalysis, we applied a
periodate-based oxidation method to illustrate improved
labelling of protein fragments using buffer
4 (compared to
PBS) with a commercially available fluorescent hydrazide for
visualisation of the peptides in a gel (Figure S8). This shows
that the catalyzing buffer method is also applicable to systems
relying on in-situ formation of aldehydes by periodate
oxidation of naturally occurring 1,2-diols, such as sialylated
glycoproteins and nucleic acids that display 3’-
ribonucleotides,11b, 25 or 1,2-amino alcohols such as N-terminal
serine or threonine residues in peptides and proteins.26
of magnitude (Table 3). Importantly, use of ABCs
for this lost reactivity, with rate enhancements of more than
200-fold, and even more than 400-fold for ABC . The high rate
4-6 makes up
4
enhancements achieved with benzoylhydrazide underline the
broad applicability of the catalyzing buffer method, and makes
the use of more easily-attained hydrazides as conjugation
partners much more feasible.
Toxicity studies
To evaluate whether ABCs 4-7 might be useful in biological
studies, we carried out toxicity studies of the buffers with the
HeLa cell line. Living cells were subjected to ABCs (0.2 – 20
mM) in the DMEM growth medium (supplemented with HEPES
buffer to maintain pH) for 6 hours. The results show (Figs. 4
and S6) that ABCs
4,
5, and
6
show generally low toxicities,
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allowing for use of these ABCs in cellular applications.
Diaminobenzene 7, on the other hand, shows significant
toxicity at concentrations of 5 mM and higher, suggesting that
ABC 7 is not well suited for use in live cell applications.
4
5
6
7
120
100
80
60
40
20
0
20
10
5
1
0.5
0.2
Ctrl.
ABC concentration (mM)
Fig.
5
Epifluorescence images of cells treated with fluorescent coumarin 21 and
Fig. 4 The viability of HeLa cells in presence of ABCs 4-7 were evaluated by subjecting
the living cells to 0.2 20 mM concentration of the ABCs in DMEM medium
incubated for 1 h. (A) In PBS, (B) in PBS with oxyamine quencher 22, (C) in buffer 4 (10
mM), (D) in buffer 4 (10 mM) with quencher 22. Excitation 400-440 nm; [21] = 10 µM
and [22] = 50 µM; pH 7.4, 37°C. The scale bar represents 10ꢀμm.
–
supplemented with HEPES buffer for 6 h at 37 °C and determining the metabolic
activity by measuring resorufin fluorescence (see details in SI); error bars show
standard deviations based on measurements performed in multiple wells (n>4). These
results were supported by similar results using the MTT assay (Fig. S6).
Conclusions
Live cell experiments
Our data show that catalytic amine buffers can provide a
simple and low-toxicity strategy for speeding hydrazone and
oxime conjugations. We show that reaction rate constants of
ca. 20-30 M-1s-1 can be achieved even at pH 7 and above, with
rate enhancements of up to 400-fold possible. The buffer
compounds are commercially available and serve a dual
purpose, buffering solution pH and greatly accelerating these
reactions both in vitro and in living cells.
Given the low toxicity of ABCs such as
4, we tested the
application of this catalytic amine buffer to promote oxime
and hydrazone formation in living cells. To provide optical
evidence of oxime formation, we incubated the aldehyde-
functionalized coumarin 21 with cells, visible by the coumarin’s
bright fluorescence, and then incubated the cells with the
oxyamine-substituted dabcyl quencher 22 in phosphate-
buffered saline (PBS) or in buffer
The results show that 21 was quenched relatively slowly in
PBS, while in catalyzing buffer the quenching was
4 (10 mM) at pH 7.4 (Fig. 5).
Conflicts of interest
4,
There are no conflicts to declare.
considerably more rapid, and was almost complete within one
hour (compare images B and D in Fig. 5).
To further test the scope of hydrazone formation promoted
Acknowledgements
by ABC
4 in living cells, we employed the DarkZone reagent
developed recently for imaging of cellular aldehydic loads.11a In
HeLa cells incubated in a medium supplied with formaldehyde
(500 µM), a dramatic improvement in light-up of the DarkZone
We thank the U.S. National Institutes of Health (GM110050
and CA217809) for support. D.L. acknowledges a postdoctoral
fellowship from the Carlsberg Foundation, and A.E.
acknowledges a postdoctoral stipend from the Foundation
Blanceflor. We thank the Vincent Coates Foundation Mass
Spectrometry Laboratory, Stanford University, for assistance
with MS measurements.
dye was seen in the presence of ABC
4 in comparison to
medium in cell visualization studies (Figure S7). Taken
together, these results provide evidence that an ABC can
promote oxime and hydrazone formations, ordinarily slow
reactions at physiological pH, at convenient time scales in
living cells.
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