Fast alpha nucleophiles: Structures that undergo rapid hydrazone/oxime formation at neutral pH

Article abstract of DOI:10.1021/ol500262y

Hydrazones and oximes are widely useful structures for conjugate formation in chemistry and biology, but their formation can be slow at neutral pH. Kinetics studies were performed for a range of structurally varied hydrazines, and a surprisingly large variation in reaction rate was observed. Structures that undergo especially rapid reactions were identified, enabling reaction rates that rival orthogonal cycloaddition-based conjugation chemistries.

Full text of DOI:10.1021/ol500262y

Letter
pubs.acs.org/OrgLett
Fast Alpha Nucleophiles: Structures that Undergo Rapid Hydrazone/
Oxime Formation at Neutral pH
Eric T. Kool,* Pete Crisalli,† and Ke Min Chan
Department of Chemistry, Stanford University, Stanford, California 94305-5080, United States
S
* Supporting Information
ABSTRACT: Hydrazones and oximes are widely useful structures for conjugate
formation in chemistry and biology, but their formation can be slow at neutral pH.
Kinetics studies were performed for a range of structurally varied hydrazines, and a
surprisingly large variation in reaction rate was observed. Structures that undergo
especially rapid reactions were identified, enabling reaction rates that rival orthogonal
cycloaddition-based conjugation chemistries.
he formation of imines by hydrazines and aminooxy
compounds has been an exceedingly useful strategy for
Nucleophilic catalysis can speed hydrazone and oxime
formation. Aniline has been traditionally used for this
purpose;1d,9 however, it exhibits relatively low efficiency and
significant toxicity.10 As a result, we and others were motivated
to find water-soluble organocatalysts that are considerably more
effective and less toxic than aniline,7,9b,11,12 and we
subsequently described the development of further improved
third-generation catalysts as well.13 However, catalysts add
complexity to the reaction and may not be compatible with
some reactant structures, or with cellular experiments.
T
formation of conjugates in chemistry and biology (Figure 1).1−3
To address these issues, we recently undertook more general
studies of aldehyde and ketone structure and their effects on
reaction rate in the absence of catalysts. We found a large range
of reactivates, depending on structure, and identified specially
reactive carbonyl compounds with acid/base groups near the
reactive center.14 These latter compounds formed products
rapidly even without an added catalyst at biological pH.
Figure 1. Formation of hydrazones and oximes by reaction of alpha-
nucleophiles with carbonyl compounds.
Although carbonyl reactivity in hydrazone and oxime
formation is now becoming better understood, the reactivity
of the other partner is less well-defined. Indeed, we are aware of
no general studies of the effects of structure on alpha
nucleophile reaction rates in imine formation. One might
expect that, since the nucleophilic amino group is generally not
the site of most structural variation in hydrazines, reaction rates
might be relatively insensitive to structural differences. Here we
report that, on the contrary, these alpha nucleophiles vary
considerably in their rates of hydrazone formation. We survey a
range of structurally varied hydrazines and find a ≥100-fold
range of rate constants for reaction. The studies have allowed
us to identify structural features that yield surprisingly rapid
Alpha nucleophiles such as hydrazines and aminooxy groups act
as stronger nucleophiles than standard amines,4 and their low
basicity allows them to form more stable imine products as
well.5 Given these favorable attributes, hydrazone and oxime
formation is of general interest and utility not only in biological
chemistry1 but also in polymer chemistry,2 dynamic combina-
torial chemistry,3 and reaction development.6
Despite this widespread interest, one issue that limits the
practical utility of these imine-forming reactions is their
relatively slow rate, particularly at neutral pH.1d,7 For one
example, the reaction of aminooxy Peg with glyoxyl modified
peptides has been reported to proceed with an observed
second-order rate constant of 6 × 10−3 M−1 s−1 at pH 7.0.7 This
is much slower than ideal for reactions in, for example,
biological settings where reactants occur at micromolar
concentrations.8
Received: January 23, 2014
Published: February 21, 2014
© 2014 American Chemical Society
1454
dx.doi.org/10.1021/ol500262y | Org. Lett. 2014, 16, 1454−1457

Organic Letters
Letter
rates in hydrazone and oxime formation comparable to other
existing rapid bioorthogonal reactions.
We began by performing kinetics studies of hydrazone
formation with 20 structurally varied commercial hydrazines.
The reactions were carried out at pH 7.4 in phosphate-buffered
saline at room temperature. 10% DMF was added to ensure
solubility; the cosolvent was not needed for many substrates
but was included in all cases for consistency, and does not
greatly affect rates (see Supporting Information (SI), Table S2).
As a reaction partner for this first survey we chose 2-
formylpyridine, which provides a useful chromophore during
hydrazone formation for measuring reaction progress by UV−
vis spectroscopy (Table S1). All reactions were performed in
triplicate under standard pseudo-first-order conditions, with
hydrazine in excess ([RNHNH2] = 500 μM; [RCHO] = 10
μM). Linear first-order fits were quite good; UV−vis scans,
reaction progress curves, and line fits are provided in the SI
(Figures S1−S3).
Table 1 displays observed first-order rate constants and
apparent second-order rate constants as a function of hydrazine
Table 1. Reactivity of Varied Hydrazines with 2-
a
Formylpyridine
Figure 2. Examples of strongly varied reaction rates with changes in
hydrazine structure, as shown by curves of reaction progress. (A)
Alkylhydrazines with and without a basic amino group; (B) electron-
rich vs electron-poor arylhydrazines. Conditions same as those in
Table 1.
trimethylphenylhydrazine, entry 12 and Figure 2). A fit of
sigma values in the aryl cases afforded a roughly linear
correlation with ρ = −1.3 (Figure S5), consistent with a
nonconjugated inductive effect lowering the nucleophilicity of
the reacting amino group. Similarly, acylhydrazides and
sulfonylhydrazides were also sluggish reactants, consistent
with this explanation. Second, simple alkylhydrazines react at
similar rates as phenylhydrazine and show little variation in rate.
Finally, two hydrazines containing acid/base groups escape
these trends by reacting significantly more rapidly: ortho-
carboxyphenylhydrazine (OCPH; 13-fold more reactive than
the slowest hydrazine) and 2-(dimethylamino)ethylhydrazine
(DMAEH; 23-fold more reactive; see Figure 2). A similar
survey (excluding alkylhydrazines due to the lack of a
chromophore) was carried out with 2-butanone, and again
the electron-poor hydrazines reacted more slowly than the
electron-rich ones (Table S3). A correlation plot of reaction
rates for these two carbonyl substrates shows a general
correlation of reactivity of most aryl hydrazines, although the
o-carboxy compound and (to a lesser extent) trimethylphenyl-
hydrazine fall well off the line due to their substantially higher
reactivity with the aldehyde (Figure S6).
a
Conditions: 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate, 10%
DMF, 25 °C. Values measured 3 times and averaged (std. dev. in
parentheses). Pseudo-first-order k(abs) normalized to standard 500 μM
[hydrazine].
structure. Note that overall second-order behavior is expected
for hydrazone formation at the low concentrations employed.15
In the current study, second-order behavior was documented
for two cases (Figure S4). Analyzing the data, we find that the
hydrazines vary by over 20-fold in their rate of reaction with 2-
formylpyridine (see Figure 2 for two examples). The slowest
reactions were observed with the electron-deficient penta-
fluorophenylhydrazine and diphenylhydrazine, while methoxy-
and methyl-substituted arylhydrazines were substantially faster.
Some general trends were noted: first, electron-poor arylhy-
drazines react more slowly than electron-rich ones (see
Having identified two exceptionally reactive hydrazines (fast
alpha nucleophiles, FANs) for the aldehyde substrate, we then
explored the scope of their reactivity by reacting them with a
range of aldehydes and ketones. The data are presented in
Table 2. The o-carboxy compound OCPH reacts more rapidly
with all new aldehyde and ketone substrates than it does with 2-
1455
dx.doi.org/10.1021/ol500262y | Org. Lett. 2014, 16, 1454−1457

Organic Letters
Letter
Table 2. Scope of Reactivity of Two Fast Hydrazines
a
(Boxed)
Figure 3. Favorable effect of a dimethylamino group on oxime
formation rate, as shown by reaction progress curves for aminooxy
compounds reacting with 2-formylpyridine at pH 7.4.
a
Conditions: 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate, 10%
DMF, 25 °C. Values measured 3 times and averaged (std. dev. in
parentheses). Pseudo-first-order k(abs) normalized to standard 500 μM
[carbonyl].
formed when the alpha nucleophile attacks the carbonyl
carbon.9a,15 We speculate that the origin of the high reactivity
of the two fast-reacting hydrazines in this study (OCPH and
DMAEH) is their ability to donate a proton intramolecularly at
the transition state of the reaction. Although detailed
mechanistic studies will be needed to test this, such proton
donation could assist in the elimination of water from this
tetrahedral intermediate, accelerating formation of the imine
product.15 The current FAN substrates have nonideal pKa’s for
such a proton transfer, so we speculate that analogs with pKa
values closer to solution pH would be yet faster in hydrazone or
oxime formation. This has indeed been found to be the case
with nucleophilic catalysts for the reaction,13 which adopt a
closely analogous transition state. Future studies of this
possibility for new, designed alpha nucleophiles are planned.
Significantly, the apparent second-order rate constants for
reaction of the current two FAN hydrazines with a range of
aldehydes are greater than 1 M−1 s−1 and, with the fastest
substrate, over 10 M−1 s−1 (Table 2). This is more rapid than
many bioorthogonal conjugation reactions, including most
azide/strained-alkyne cycloadditions,16 and is competitive even
with recently described strain-driven Diels−Alder reactions.17
Although we have recently reported structural features in
aldehydes and ketones that yield rapid hydrazone formation,14
the identification of rapid-reacting hydrazines and aminooxy
compounds has special significance, because aldehyde reactive
groups can be generated on a wide range of native
biomolecules. For example, sugars and oligosaccharides can
react directly with hydrazines18 (see Table 2 for an example
with a sugar). RNAs can be readily modified to generate
reactive aldehydes in one step at the 3′ end via periodate
oxidation,19 and peptides and proteins can similarly be oxidized
at N-terminal serine residues to generate a reactive aldehyde.20
Thus one can envision the design of FAN hydrazine and
aminooxy reagents carrying useful labels for rapid conjugation
to these biomolecules. Future studies will be directed toward
this possibility.
butanone. It forms hydrazones rapidly with aryl and alkyl
aldhehydes and with aryl and alkyl ketones as well. Among aryl
substrates, the fastest reactions occur with carbonyl compounds
having proximal imino groups14 (e.g., quinoline-8-carboxalde-
hyde, entry 8; 2-acetylpyridine, entry 10). The latter case
proceeds with a rate 24-fold faster than the slowest reaction in
the study. Alkyl aldehydes are yet faster, yielding rates up to
100-fold greater (butyraldehyde, entry 9) than the reference
slow reaction. Intriguingly, dimethylaminoethylhydrazine
(DMAEH, entries 11−14) is found to react even more rapidly
than OCPH. Although it could be measured with only a few
aryl aldehydes due to the lack of a chromophore in the
hydrazone product, in each case the reaction rate was 2−4
times that of OCPH with the same carbonyl compound. We
speculate that reactions of DMAEH hydrazine with alkyl
aldehydes would yield the highest rates of all, but this
possibility could not be measured with current methods.
Oxime bonds are in some applications more useful than
hydrazones because of their greater hydrolytic stability.5,9b
Inspired by this fastest FAN reactant, we synthesized the
aminooxy analogue of DMAEH in order to test its reactivity in
oxime formation. The new compound, dimethylaminoethyl-
oxyamine (see SI for synthetic details), was reacted with 2-
formylpyridine, and its rate was compared to that of the
simplest alkyl aminooxy compound, methoxyamine. The results
show (Figure 3) that the dimethylamino-substituted compound
reacts with a rate 3-fold higher than that of the control
compound. Thus, the data suggest that the favorable effect of
the dimethylaminoethyl group on reaction rates may be
general. More detailed studies and synthesis of analogs will
be needed to test this possibility, but strategies for accelerating
reaction rates may be especially useful for oxime ligations,
which are slower than corresponding hydrazone formation
reactions at biological pH.5,9b
The rate-limiting step for hydrazone formation at neutral pH
is generally the breakdown of the tetrahedral intermediate
1456
dx.doi.org/10.1021/ol500262y | Org. Lett. 2014, 16, 1454−1457

Organic Letters
Letter
(c) Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc. 2010,
132, 3688.
(17) Devaraj, N. K.; Upadhyay, R.; Haun, J. B.; Hilderbrand, S. A.;
Weissleder, R. Angew. Chem., Int. Ed. 2009, 48, 7013.
(18) (a) Kubo, K.; Ide, H.; Wallace, S. S.; Kow, Y. W. Biochemistry
1992, 31, 3703. (b) Beaudette, T. T.; Cohen, J. A.; Bachelder, E. M.;
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis and kinetics procedures, kinetic fit data, and
supporting figures are provided. This material is available free
of charge via the Internet at http://pubs.acs.org.
Broaders, K. E.; Cohen, J. L.; Engleman, E. G.; Frec
́
het, J. M. J. Am.
Chem. Soc. 2009, 131, 10360.
AUTHOR INFORMATION
■
(19) Proudnikov, D.; Mirzabekov, A. Nucleic Acids Res. 1996, 24,
4535.
(20) (a) Melnyk, O.; Fehrentz, J. A.; Martinez, J.; Gras-Masse, H.
Biopolymers 2000, 55, 165. (b) Geoghegan, K. F.; Stroh, J. G.
Bioconjug. Chem. 1992, 3, 138.
Corresponding Author
*E-mail: kool@stanford.edu.
Present Address
†Department of Mechanical Engineering, University of
California, Santa Barbara
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
(GM068122, GM067201, and GM072705).
REFERENCES
■
(1) (a) Tam, J. P.; Xu, J.; Eom, K. D. Biopolymers 2001, 60, 194.
(b) Cornish, V. W.; Hahn, K. M.; Schultz, P. G. J. Am. Chem. Soc.
1996, 118, 8150. (c) Zeng, Y.; Ramya, T. N. C.; Dirksen, A.; Dawson,
P. E.; Paulson, J. C. Nat. Methods 2009, 6, 207. (d) Dirksen, A.;
Dirksen, S.; Hackeng, T. M.; Dawson, P. E. J. Am. Chem. Soc. 2006,
128, 15602.
(2) (a) Gauthier, M. A.; Klok, H. A. Chem. Commun. 2008, 2591.
(b) Bandyopadhyay, S.; Xia, X.; Maiseiyeu, A.; Mihai, G.; Rajagopalan,
S.; Bong, D. Macromolecules 2012, 45, 6766. (c) Alconcel, S. N. S.;
Kim, S. H.; Tao, L.; Maynard, H. D. Macro. Rapid Commun. 2013, 34,
983.
(3) (a) Otto, S.; Furlan, R. L. E.; Sanders, J. K. M. Curr. Opin. Chem.
Biol. 2002, 6, 321. (b) Nguyen, R.; Huc, I. Chem. Commun. 2003, 942.
(c) Simpson, M. G.; Pittelkow, M.; Watson, S. P.; Sanders, J. K. M.
Org. Biomol. Chem. 2010, 8, 1181. (d) Algar, W. R.; Prasuhn, D. E.;
Stewart, M. H.; Jennings, T. L.; Blanco-Canosa, J. B.; Dawson, P. E.;
Medintz, I. L. Bioconj. Chem. 2011, 22, 825. (e) Rodriguez-Docampo,
Z.; Otto, S. Chem. Commun. 2008, 5301. (f) Roberts, S. L.; Furlan, R.
L. E.; Otto, S.; Sanders, K. M. Org. Biomol. Chem. 2003, 1, 1625.
(4) Fina, N. J.; Edwards, J. O. Int. J. Chem. Kinetics 1978, 5, 1.
(5) Kalia, J.; Raines, R. T. Angew. Chem., Int. Ed. 2008, 47, 7523.
(6) Loskot, S. A.; Zhang, J.; Langenhan, J. M. J. Org. Chem. 2013, 78,
12189.
(7) Wendeler, M.; Grinberg, L.; Wang, X.; Dawson, P. E.; Baca, M.
Bioconjug. Chem. 2014, 25, 93.
(8) Kalia, J.; Raines, R. T. Curr. Org. Chem. 2010, 14, 138.
(9) (a) Cordes, E. H.; Jencks, W. P. J. Am. Chem. Soc. 1962, 84, 826.
(b) Dirksen, A.; Dawson, P. E. Bioconjug. Chem. 2008, 19, 2543.
(10) Kennedy, D. C.; McKay, C. S.; Legault, M. C.; Danielson, D. C.;
Blake, J. A.; Pegoraro, A. F.; Stolow, A.; Mester, Z.; Pezacki, J. P. J. Am.
Chem. Soc. 2011, 133, 17993.
(11) Crisalli, P.; Kool, E. T. J. Org. Chem. 2013, 78, 1184.
(12) (a) Rashidian, M.; Mahmoodi, M. M.; Shah, R.; Dozier, J. K.;
Wagner, C. R.; Distefano, M. D. Bioconjug. Chem. 2013, 24, 333.
(b) Blanden, A. R.; Mukherjee, K.; Dilek, O.; Loew, M.; Bane, S. L.
Bioconjug. Chem. 2011, 22, 1954.
(13) Crisalli, P.; Kool, E. T. Org. Lett. 2013, 15, 1646.
(14) Kool, E. T.; Park, D.; Crisalli, P. J. Am. Chem. Soc. 2013, 135,
17663.
(15) Jencks, W. P. Prog. Phys. Org. Chem. 1964, 2, 63.
(16) (a) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.;
Chang, P. V.; Miller, I. A; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 16793. (b) Ning, X. H.; Guo, J.;
Wolfert, M. A.; Boons, G.-J. Angew. Chem., Int. Ed. 2008, 47, 2253.
1457
dx.doi.org/10.1021/ol500262y | Org. Lett. 2014, 16, 1454−1457