Importance of ortho proton donors in catalysis of hydrazone formation

Article abstract of DOI:10.1021/ol400427x

Anthranilic acids were recently reported as superior catalysts for hydrazone and oxime formation compared to aniline, the classic catalyst for these reactions. Here, alternative proton donors were examined with varied pK_a in an effort to enhanc

Full text of DOI:10.1021/ol400427x

ORGANIC
LETTERS
2013
Vol. 15, No. 7
1646–1649
Importance of ortho Proton Donors
in Catalysis of Hydrazone Formation
Pete Crisalli and Eric T. Kool*
Department of Chemistry, Stanford University, Stanford, California 94305-5080,
United States
kool@stanford.edu
Received February 13, 2013
ABSTRACT
Anthranilic acids were recently reported as superior catalysts for hydrazone and oxime formation compared to aniline, the classic catalyst for
these reactions. Here, alternative proton donors were examined with varied pKa in an effort to enhance activity at biological pH. The experiments
show that 2-aminobenzenephosphonic acids are superior to anthranilic acids in catalyzing hydrazone formation with common aldehyde
substrates.
The rapid development of bioorthogonal reactions has
been broadly important for applications in bioconjuga-
tion chemistry.1,2 Common goals of this research include
increased specificity while avoiding cross-reactivity with
biological components, and increasing reaction rates.
Recent useful developments in the field such as strain-
promoted click chemistry and DielsÀAlder reactions of
tetrazines and strained alkenes have provided relatively
rapid reactions that can be performed at moderately low
reactant concentrations.3À5 However, the adoption and
application of these reactions has required specialized
substrates that are not always readily synthetically acces-
sible and can have limited aqueous solubility. Additional
bioorthogonal reactions are still sought in order to carry
out multiple distinct modifications of biomolecules and to
provide synthetic flexibility.
readily synthetically accessed.1,6 However, the slow rate
of product formation, particularly at biological pH, has
placed limits on the utility of this reaction in the past.7 An
important improvement was Jencks’ establishment of ani-
line as a nucleophilic catalyst for the reaction, and Dawson
has recently demonstrated useful applications of this
compound.7À10 The syntheticaccessibilityofthe substrates
and the development of this catalysis has led to consider-
able growth in the applications of hydrazone and oxime
formation for bioorthogonal labeling as well as in other
fields, such as dynamic covalent chemistry.1,2,8À14
However, the use of aniline as a nucleophilic catalyst is
still less than ideal, as large concentrations of the com-
pound (10À100 mM) are required to yield useful rates.7À9,14
Alternatively, more acidic buffers can improve aniline
A considerably older bioorthogonal reaction, hydra-
zone/oxime formation, has proven quite generally useful
in bioconjugations. Many aldehydes, hydrazines, and
aminooxy compounds are widely available and can be
(6) Cornish, V. W.; Hahn, K. M.; Schultz, P. G. J. Am. Chem. Soc.
1996, 118, 8150.
(7) Dirksen, A.; Dawson, P. E. Bioconjugate Chem. 2008, 19, 2543.
(8) Dirksen, A.; Dirksen, S.; Hackeng, T. M.; Dawson, P. E. J. Am.
Chem. Soc. 2006, 128, 15602.
(9) Dirksen, A.; Hackeng, T. M.; Dawson, P. E. Angew. Chem., Int.
Ed. 2006, 45, 7581.
(10) Cordes, E. H.; Jencks, W. P. J. Am. Chem. Soc. 1962, 84, 826.
(11) Carrico, I. S.; Carlson, B. L.; Bertozzi, C. R. Nat. Chem. Biol.
2007, 3, 321.
(12) Byeon, J.-Y.; Limpoco, F. T.; Bailey, R. C. Langmuir 2010, 26,
15430.
(13) Miller, B. L. Nat. Chem. 2010, 2, 433.
(14) Zeng, Y.; Ramya, T. N. C.; Dirksen, A.; Dawson, P. E.; Paulson,
(1) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48,
6974.
(2) Lim, R. K. V.; Lin, Q. Chem. Commun. 2010, 46, 1589.
(3) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc.
2004, 126, 15046.
(4) Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc.
2010, 132, 3688.
(5) Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A. Bioconjugate
Chem. 2008, 19, 2297.
J. C. Nat. Methods 2009, 6, 207.
r
10.1021/ol400427x
Published on Web 03/11/2013
2013 American Chemical Society

Figure 1. Structures of nucleophilic catalysts for hydrazone formation having ortho proton donors of varied pKa.
catalysis, although 10 mM catalyst is still recommended.14,15
Unfortunately, acidic pH can be detrimental to some
biomolecules, not to mention unachievable in cells. As a
result, the development of improved nucleophilic catalysts
for hydrazone- and oxime-forming reactions would be of
considerable utility in bioorthogonal conjugations.7
Scheme 1. Colorimetric Reaction for Monitoring Hydrazone
Formation
Recently we reported that anthranilic acid and its
derivatives can serve as catalysts superior to aniline for
hydrazone formation at biological pH.16 At 1 mM cat-
alyst concentration, 5-methoxyanthranilic acid (5MA)
displayed a greater than 5-fold advantage over aniline in
the second-order rate constant for hydrazone formation,
while higher concentrations afforded an advantage of over
100-fold.16 We hypothesized that the ortho carboxylic acid
of these compounds was the primary source of the en-
hanced catalysis, serving as an intramolecular proton
donor during imine and hydrazone formation.16 Indeed,
modification of the carboxylic acid to nonacidic deriva-
tives (e.g., anthranilamide or anthranilonitrile) eliminated
catalytic activity.16 However, the low pKa of the carboxy-
late group in anthranilic acid (2.18)17 implies that verylittle
of the catalyst exists in the presumed active protonated
state at neutral pH. Thus we reasoned that developing new
derivatives with a pKa closer to that of biological buffers
could provide further rate enhancements.
To examine the role of the pKa of the proton donor, we
explored moieties that could serve as carboxylic acid
surrogates in the anthranilic acid scaffold (Figure 1). We
started with simple carboxylic acid replacements, such
as sulfonic acid, tetrazole, phosphonic acid, hydroxamic
acid, and sulfonamide. Similarly, we tested conjugate
acids of basic moieties, choosing amine or imidazole
functionalities. We tested the new catalyst analogs with
the chromogenic reaction between NBD hydrazine and
4-nitrobenzaldehyde (Scheme 1), which yields an increase
in absorbance at 504 nm with hydrazone formation.16
Plots of reactions are shown in Figure S1 (Supporting
Information (SI)), and apparent second-order rate con-
stants are given in Table 1. 2-Aminobenzenesulfonic acid
2, with a pKa far below the buffer pH, performed poorly as
expected (Table 1, Figure S1) and was inferior to aniline
itself, likely because the strongly electron-withdrawing
sulfonate lowers the nucleophilicity of the amine. Indeed,
the sulfonate group in compound 2 reduces the pKa of
the anilinic nitrogen from the 4.6 of aniline to 2.5.18 As
the pKa of the acidic moieties increased, catalytic activity
was observed to increase as well. Most notably, 5-(2-
aminophenyl)tetrazole 3 was superior to aniline and only
slightly less active than anthranilic acid. This is reasonable,
considering the similar pKa values of benzoic acid and
5-phenyltetrazole (4.19 and 4.30 respectively).19 2-Amino-
benzenephosphonic acid 4, with a pKa of 7.29,20 was
almost 4-fold more active than aniline and displayed
a 2-fold improvement in rate constant over anthranilic
acid. As the pKa of the acid moiety increased further,
to 2-aminobenzenehydroxamic acid 6 (pKa = 9.2921) or
2-aminobenzenesulfonamide 5 (pKa = 10.022), catalytic
activity at pH 7.4 fell dramatically. These results indicate
that the pKa of the proton donor has great importance
and suggest that a pKa near that of the solution pH may
provide optimal catalysis (see Figure S2).
(18) Perrin, D. D. Aust. J. Chem. 1964, 17, 484.
(19) Scherrer, R. A. Biolipid pKa Values and the Lipophilicity of
Ampholytes and Ion Pairs. In Pharmacokinetic Optimization in Drug
Research; Testa, B., van de Waterbeemd, H., Folkers, G., Guy, R., Eds.;
Wiley-VCH: Z€urich, 2001; pp 351À382.
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(15) Jencks, W. P. J. Am. Chem. Soc. 1959, 81, 475.
(16) Crisalli, P.; Kool, E. T. J. Org. Chem. 2013, 78, 1184.
(17) Zapaza, L.; Kalembkiewicz; Sitarz-Palczak, E. Biophys. Chem.
2009, 140, 91.
(20) Jaffe, H. H.; Freedman, L. D.; Doak, G. O. J. Am. Chem. Soc.
1954, 76, 1548.
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~
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Org. Lett., Vol. 15, No. 7, 2013
1647

hydrazine concentration gave no significant change in reac-
tion rates under the conditions tested (Table S1, Figure S5).
Next we sought to optimize the 2-aminobenzene-
phosphonic acid framework to further improve cata-
lytic efficiency. We hypothesized that modification of the
5-position of the aromatic ring with electron-donating
groups might enhance the basicity and nucleophilicity
of the amine.16 To this end, we prepared 5-methoxy-2-
aminobenzenephosphonic acid 11 and compared its ac-
tivity to 5-methoxyanthranilic acid (5MA), which was
previously determined to be an optimal anthranilic
acid derivative (Figure 2).16 Despite displaying a greatly
Table 1. Apparent Second-Order Rate Constants (MÀ1 minÀ1
for Different ortho Proton Donor Nucleophilic Catalystsa
)
second-order
rate constant
rel. to
b
catalyst
pKa
uncat. rxn.
(none)
À
0.10 ( 0.01
1.1 ( 0.1
2.3 ( 0.3
0.14 ( 0.03
1.7 ( 0.2
4.1 ( 0.2
0.09 ( 0.03
0.25 ( 0.06
0.04 ( 0.02
0.03 ( 0.01
0.05 ( 0.06
0.12 ( 0.03
1
aniline
À
11
1
2
2.18
22
À1.41c
1.4
16
3
À
4
7.29
10.0
9.29
9.60c
9.78c
6.51c
5.63c
40
5
0.8
2.5
0.4
0.3
0.5
1.2
6
7
8
9
10
a Reaction conditions: 18 μM NBD hydrazine, 1 mM 4-nitrobenzal-
dehyde, 1 mM catalyst in 10:1 PBS (pH 7.4)/DMF, 23 °C. Reaction
monitored at 504 nm. b pKa values of acidic moiety from literature (see
text for references). c pKa calculated by ACD/Laboratories (V11.02
1994À2013); no literature value available.
Figure 2. Improved benzenephosphonic acid and 5-phenyltetra-
zole derivatives for catalysis of hydrazone formation.
Interestingly, the cationic conjugate acids of basic func-
tional groups displayed strongly contrasting behavior,
with the imidazoles and amines showing a near-complete
lack of reactivity in catalyzing hydrazone formation de-
spite relatively favorable pKa values of the proton donors
(Table 1, Figure S3). We hypothesize that positively
charged groups conjugated to the ortho amine lower its
nucleophilicity more than neutral or negatively charged
groups do.
Since the mechanism of catalysis by anilines and anthranilic
acids involves nucleophilic catalysis via an imine inter-
mediate, it is clearly important that a catalyst maintain
nucleophilicity in the anilinic nitrogen. This was indicated
by the activity of anthranilic acid, which has amine
basicity similar to that of aniline.17 2-Aminobenzenesul-
fonic acid 2 demonstrates the adverse effect of a strongly
electron-withdrawing ortho substituent. The superior new
catalyst 2-aminobenzenephosphonic acid 4, however,
maintains a reasonably basic aniline pKa of 4.1.20
We sought to test the mechanism of catalysis by amino-
benzenephosphonic acid derivatives to observe similarities
and differences with anthranilic acid and aniline. Previous
studies of catalyst imine formation showed that anthranilic
acid forms a relatively unstable imine intermediate, with
imine formation acting as the rate-limiting step (consistent
with the aniline-catalyzed reaction).10,16,23 Similar studies
performed with 2-aminobenzenephosphonic acid yielded
no spectroscopically observable imine formation at 1 mM
catalyst, suggesting the imine-forming equilibrium lies
considerably to the side of free aldehyde and catalyst
(Figure S4). In further studies, we confirmed that the
rate-determining step in hydrazone formation catalyzed
by phosphonic acid 4 is imine formation: varying NBD
improved initial reaction rate, compound 11 lost much
of its catalytic ability after approximately 30 min in buffer.
Use of deoxygenated buffer did not prevent the loss of
activity (data not shown), indicating a degradation path-
way other than oxidation. As an alternative, we prepared
5-methyl-2-aminobenzenephosphonic acid 12. We were
gratified to observe that this derivative not only was stable
in buffer but also afforded further enhanced catalysis, with
a second-order rate constant almost 8-fold higher than
that for aniline catalysis in hydrazone formation at 1 mM
catalyst concentration (Table 2, Figure S6). Control reac-
tions showed that the catalyst, hydrazine, and aldehyde were
all required for efficient reaction as expected (Figure S7).
In an analogous experiment, we also prepared a 5-methoxy
derivative of the tetrazole catalyst (13) and this also
showed improved catalysis, with activity similar to that
of 5MA, the previous best catalyst for this reaction.
We proceeded to explore the substrate scope of the new
phosphonic acid and tetrazole catalysts relative to aniline
and anthranilates. We were particularly interested in
whether the increased size and added negative charge of
the phosphonic acid of 4 and 12 relative to a carboxylic
acid might be problematic on certain substrates. Data are
shown in Table 3 (also see Figures S8ÀS18). In general, the
new compounds displayed enhanced catalysis with a range
of electron-rich and -poor benzaldehyde derivatives, with
12 proving superior in most cases to the previous best
catalyst 5MA. 2-Formylpyridine also served as a good
substrate for 12 and 13 and was the fastest aromatic
substrate of all. Tests with an aliphatic aldehyde, butyr-
aldehyde, showed a particularly rapid reaction, with 5MA,
12, and 13 proving most efficient. We also tested the
reactivity with 2-butanone to assay for catalysis with
aliphatic ketones, but observed only negligible product
ꢀ
(23) Thygesen, M. B.; Munch, H.; Sauer, J.; Clo, E.; Jørgensen,
M. R.; Hindsgaul, O.; Jensen, K. L. J. Org. Chem. 2010, 75, 1752.
1648
Org. Lett., Vol. 15, No. 7, 2013

Table 2. Apparent Second-Order Rate Constants (MÀ1 minÀ1
for Different ortho Proton Donor Nucleophilic Catalystsa
)
Table 3. Substrate Scope of Catalysts As Measured by
Hydrazone Yields (%) with Aldehydes and Ketones after
4 h Reactiona
no
substrate
catalyst
aniline
5MA
4
12
13
benzaldehyde
p-anisaldehyde
4-carboxy-
1.1
0.3
3.6
4.1
1.4
26
9.4
2.3
44
10
2.3
28
17
4.8
40
7.7
1.9
39
benzaldehyde
2-carboxy-
7.9
2.8
11
68
9.4
82
99
26
99
55
36
35
94
42
65
46
72
59
98
17
92
56
benzaldehyde
4-chloro-
benzaldehyde
2-formyl-
pyridine
4-formyl-
3.3
29
pyridine
cinnamaldehyde
salicylaldehyde
butyraldehydeb
acetophenone
1.1
4.1
13
3.5
13
5.8
34
9.2
18
14
27
96
0.0
5.4
23
83
94
75
94
0.5
2.9
0.4
0.4
0.0
a Conditions: 18 μM NBD hydrazine, 1 mM 4-nitrobenzaldehyde,
1 mM catalyst in 10:1 PBS (pH 7.4)/DMF, 23 °C.
a Conditions: 18 μM NBD Hydrazine, 1 mM catalyst and 1 mM
substrate in 10:1 PBS (pH 7.4)/DMF. Reactions monitored by UV/vis
spectroscopy (see Supporting Information). b Yields after 15 min of
reaction.
formation even after extended reaction, consistent with
reports that NBD hydrazine reacts poorly with ketones
at pH 7.4.24 Like the anthranilic acids, the new analogs
were not suitable for more hindered substrates such as
acetophenone.16 Overall, the substrate studies show that
sterically unhindered aldehydes are the best suited for
catalysis by aminobenzenephosphonic acids such as 12
and that 5MA orthe new tetrazole 13are preferable if there
is steric or electronic interference.
Interestingly, we observed that 2-carboxybenzaldehyde
reacts faster than 4-carboxybenzaldehyde in both the
uncatalyzed and catalyzed reactions. Similar results were
observed with 2-formylpyridine relative to 4-formylpyridine.
These results indicate that it may not necessarily need to be
the catalyst that contains an ortho proton donor for proton
transfer in imine and hydrazone formation, but that such
a donor may also function in the substrate itself. This has
been hinted at by a recent literature report that pyridoxal
phosphate can serve as a rapidly reacting substrate for
hydrazone formation.25
In conclusion, we have shown that ortho proton donor
groups strongly enhance the activity of nucleophilic cata-
lysts in hydrazone formation and that tuning the pKa
of the proton donor toward the biological buffer pH
further enhances catalysis. 2-Aminobenzenephosphonic
acid derivatives in particular provide useful enhancements
over known compounds and act as the best existing
new catalysts for hydrazone formation on many carbonyl
substrates.
Acknowledgment. This work was supported by the
National Institutes of Health (GM068122, GM067201,
and GM072705).
Supporting Information Available. Synthesis procedures,
screening conditions, kinetic fit information characteriza-
tion of prepared compounds, and supporting figures are
provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
(24) Key, J. A.; Li, C.; Cairo, C. W. Bioconjugate Chem. 2012, 23, 363.
(25) Wang, X.; Canary, J. W. Bioconjugate Chem. 2012, 23, 2329.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 7, 2013
1649