Rapid and reversible hydrazone bioconjugation in cells without the use of extraneous catalysts

Article abstract of DOI:10.1039/c8ob00946e

The amenability of hydrazone linkages to disassemble via either hydrolysis in mildly acidic aqueous solutions or transimination upon treatment with amine nucleophiles renders them extremely attractive for applications in chemical biology, drug delivery and materials science. Unfortunately, however, the use of hydrazones is hampered by the extremely slow intrinsic rates of their formation from their hydrazine and carbonyl precursors. Consequently, hydrazone formation is typically performed in the presence of a large excess of cytotoxic aniline-based nucleophilic catalysts, rendering hydrazones unsuitable for biological applications that entail their formation in cells. Herein, we report a hydrazine scaffold - o-amino benzyl hydrazine - that rapidly forms hydrazones via intramolecular nucleophilic catalysis, thereby obviating the use of extraneous catalysts. We demonstrate the use of this scaffold for rapid and reversible peptide and protein hydrazone bioconjugation and also for reversible fluorescent labeling of sialylated glycoproteins and choline lipids in mammalian cells.

Full text of DOI:10.1039/c8ob00946e

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Rapid and reversible hydrazone bioconjugation in cells without the use of extraneous catalysts
Rahul Nisal, Gregor P. Jose, Chitra Shanbhag and Jeet Kalia*
Indian Institute of Science Education and Research (IISER) Pune
Dr. Homi Bhabha Road, Pashan, Pune–411008, Maharashtra, India.
Current address: Department of Biological Sciences,
Indian Institute of Science Education and Research (IISER) Bhopal
Bhopal Bypass Road, Bhauri, Bhopal–462066, Madhya Pradesh, India
*Correspondence may be addressed to:
Dr. Jeet Kalia,
Assistant Professor and Wellcome TrustꢀDBT India Alliance Intermediate Fellow
Tel.: +91ꢀ755ꢀ6691437; Email: jeet@iiserb.ac.in
1

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Abstract:
The amenability of hydrazone linkages to disassemble via either hydrolysis in mildly acidic aqueous solutions
or transimination upon treatment with amine nucleophiles renders them extremely attractive for applications in
chemical biology, drug delivery and material science. Unfortunately, however, the use of hydrazones is
hampered by the extremely slow intrinsic rates of their formation from their hydrazine and carbonyl precursors.
Consequently, hydrazone formation is typically performed in presence of a large excess of cytotoxic anilineꢀ
based nucleophilic catalysts rendering hydrazones unsuitable for biological applications that entail their
formation in cells. Herein, we report a hydrazine scaffold—oꢀamino benzyl hydrazine—that rapidly forms
hydrazones via intramolecular nucleophilic catalysis, thereby obviating the use of extraneous catalysts. We
demonstrate the use of this scaffold for rapid and reversible peptide and protein hydrazone bioconjugation and
also for reversible fluorescent labeling of sialylated glycoproteins and choline lipids in mammalian cells.
Introduction:
Covalent linkages that disassemble when subjected to specific chemical stimuli are extremely attractive for
applications in a wide variety of research areas including materials sciences1 and both clinical and basic
biological research.2ꢀ5 Among the handful of such linkages known,6ꢀ15 hydrazones16 are unique in possessing the
ability to undergo breakdown in two ways—hydrolysis to form their hydrazine and aldehyde/ketone precursors
under mildly acidic conditions,17 and transimination to form imines, oximes or other hydrazones when treated
with amines, alkoxyamines or hydrazines respectively. The hydrolytic instability of hydrazones has been
exploited for a diverse range of applications including drug delivery,18ꢀ23 and generating smart polymeric
scaffolds.13, 24ꢀ25 Transimination of hydrazones, on the other hand, has facilitated development of dynamic
combinatorial libraries26ꢀ27 and has also been employed for chemical biologyꢀbased applications.28ꢀ29
Additionally, hydrazones have also been utilized for applications that do not rely on their ability to disassemble,
2

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including protein immobilization30ꢀ31 and labeling of proteins with fluorophores,32ꢀ35 affinity tags29, 33, 36ꢀ37, mass
spectrometry labels37ꢀ39 and polyethylene glycol moieties.40ꢀ42
Among the previously reported biological applications of hydrazone linkages, very few32, 43ꢀ44 involve
their formation within cells. Indeed, perhaps the most widely used application of hydrazones is in drug
delivery18ꢀ23 that entails conjugate formation in vitro followed by administration into cells. A major reason for
the lack of use of hydrazones for performing bioconjugation within cells is the slow rate of hydrazone formation
in aqueous solutions,45ꢀ47 a drawback that can be partly overcome by the use of large amounts (usually ~50 fold
molar excess or more) of aniline or its derivatives that function as nucleophilic catalysts for hydrazone
formation (Figure 1a).45, 48ꢀ55 Use of such large amounts of aniline derivatives is undesirable due to their
cytotoxicity,47, 56ꢀ57 as well as the likelihood of these compounds triggering undesirable changes in cellular
physiology due to their wellꢀdocumented ability to modulate the function of cellular proteins including
enzymes58 and ion channel proteins.59 These drawbacks of aniline catalysis have motivated several studies
aimed at developing new methods for rapidly forming hydrazones and oximes that have resulted in the
discovery of hyperreactive aldehydes such as butyraldehyde47, oꢀboronic acid aldehydes6, 60ꢀ63 and dialdehydes64
that may be amenable to biomolecular incorporation by employing modern chemical biologyꢀbased
approaches65ꢀ73 leading to their use for bioconjugation. Despite this recent success, new efficacious approaches
for hydrazone bioconjugation are urgently
needed, especially with respect to their
formation within cells.
Results and Discussion:
We envisaged a strategy for rapidly making
Figure 1. Hydrazone bioconjugation via a) the traditional anilineꢀcatalyzed
hydrazones by designing bifunctional
approach, and b) our approach involving intramolecular nucleophilic catalysis.
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compounds containing both the hydrazine nucleophile and
the catalytic aniline moiety that upon treatment with
aldehydes/ketones could form anilineꢀiminium intermediates,
which could then potentially undergo an intramolecular
attack by the adjacent hydrazine group to rapidly form the
desired hydrazones (Figure 1b). The oꢀamino phenyl
hydrazine scaffold can potentially undergo such a reaction
but due to its lack of stability,74ꢀ75 we instead focused on
studying the effects of appending the aniline moiety
adjacent to the hydrazide, alkyl hydrazine and the benzyl
hydrazine scaffolds.
Figure 2. Kinetic studies on hydrazone formation. a) The
reaction employed for the assay. b) Kinetic traces for
hydrazine compounds appended with aniline are depicted as
solid symbols, whereas those for the corresponding
scaffolds lacking aniline substituents are depicted as open
symbols of the same shape. The hydrazine compounds were
used at a concentration of 250 µM whereas the aldehyde (2ꢀ
formylpyridine) was used at a concentration of 50 µM in the
assays.
In agreement with our hypothesis, UV
spectroscopyꢀbased kinetic assays revealed that hydrazine
compounds appended with a suitablyꢀplaced adjacent
aniline moiety (compounds 2, 4 and 7) formed hydrazones
faster than their respective counterparts not appended with aniline— benzhydrazide 1, propyl hydrazine 3, and
benzyl hydrazine 6 (Figure 2b and Table 1; all kinetic traces are depicted in Figure S1, supporting information
section). These studies identified oꢀamino benzyl hydrazine (7) as our most rapidly reacting hydrazine scaffold.
With this scaffold as a benchmark, we attempted to further enhance its reactivity by synthesizing compounds 8
and 9 (Figure 2b). The design of 8 was based on the expectation that it would demonstrate faster hydrazone
formation rates as compared to 7 due to the presence of two electron donating –OMe substituents that could
potentially enhance the electron density on the nitrogen atom of aniline making it a better nucleophilic catalyst.
On the other hand, 9 was expected to form hydrazones faster than 7 as it would form a reactive positively
4

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k2
charged iminium intermediate upon reacting
S. No.
1
Compound
Catalyst
None
Relative k2
(Mꢀ1Sꢀ1)
with aldehydes which would be more
susceptible to a
0.08 ± 0.02
0.89
1
2
3
4
None
None
None
0.14 ± 0.01
1.05 ± 0.02
4.23 ± 0.04
1.56
11.67
47.00
2
3
intramolecular hydrazine attack as
opposed to the imine intermediate formed by
7 that requires protonation to form an
iminium prior to hydrazine attack (Figure S2,
supporting information section). Consistent
with these hypotheses, both 8 and 9 formed
hydrazones faster than all compounds tested
(Figure 2b and Table 1).
4
5
None
4.56 ± 0.11
50.67
5
6
6
7
8
None
None
None
2.49 ± 0.03
5.50 ± 0.22
7.90 ± 0.27
27.67
61.11
87.78
7
8
9
The rate constant of hydrazone
formation obtained for 8 and 9 were ~90ꢀ100
fold higher than those for benzhydrazide 1
and the alkyl hydrazide 10 (Table 1). These
observation have important implications in
hydrazone conjugation technology as an
overwhelming majority of the reported
examples of the use of hydrazones for
biomedical and materials science applications
employ these slowꢀreacting hydrazide
scaffolds.13, 18ꢀ19, 25ꢀ26, 31, 33ꢀ35, 37ꢀ42 We also
9
None
None
8.56 ± 0.12
0.09 ± 0.01
95.11
1.00
10
10
10
11
12
1.69 ± 0.01
8.60 ± 0.13
18.78
95.56
10
13
14
15
None
None
None
1.61 ± 0.01
2.83 ± 0.18
1.59 ± 0.04
17.89
31.44
17.67
11
12
13
Table 1. Rate constants obtained from the kinetic assays. For entries 11
and 12, 50ꢀfold molar excess of anilineꢀbased catalysts were used.
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compared the rates of hydrazone formation demonstrated by 8 and 9 with those observed for the hydrazide 10 in
presence of 50ꢀfold excess of aniline (entry 11 in Table 1) and its derivative, pꢀmethoxy anthranilic acid (entry
12 in Table 1), which is one of the most proficient catalysts known for hydrazone formation.16, 48 These
experiments demonstrated that our compounds, 8 and 9, form hydrazones much faster than those formed by the
hydrazide in presence of aniline and as fast as that formed in presence of pꢀmethoxy anthranilic acid.
We next proceeded to test our hypothesis that hydrazone formation by these compounds is preceded by
transient imine formation via their oꢀamino groups. Towards this goal, we employed a previously reported
strategy of trapping imine intermediates by utilizing NaCNBH3 to reduce them to form stable secondary amines
that can then be characterized via HPLCꢀMS analysis.76 We employed this approach (Figure S3, supporting
information section) on our slowest anilineꢀappended hydrazine substrate, compound 2, to maximize the
chances of reductively trapping the imine to form S13. HPLC analysis of the hydrazone formation reaction prior
to NaCNBH3 addition gave, as expected, peaks for the hydrazone (S31) and the reactants (fourth panel, Figure
S3). Treatment of the hydrazone formation reaction mixture with NaCNBH3, on the other hand, yielded the
reduced imine, S13, as demonstrated by comparison of the retention times with that of the pure reduced imine
standard (third and fifth panels, Figure S3) and also by mass spectrometric characterization (Table S1), thereby
demonstrating the formation of our hypothesized imine intermediate.
In addition to intramolecular nucleophilic catalysis (Figure 1b), the enhanced rates of hydrazone
formation demonstrated by oꢀamino benzyl hydrazines can also be explained by the electronꢀdonating effect of
their oꢀamino groups resulting in an increase in the nucleophilicity of the hydrazine moiety. To evaluate the
contribution of this effect on hydrazone formation rates, we synthesized two benzyl hydrazine derivatives
appended with electronꢀdonating groups at the ortho position that are incapable of forming imines (compound
11 containing an –OMe group and compound 12 containing the –NMe2 group depicted as entries 13 and 14
respectively in Table 1). These compound was observed to form hydrazones much slower as compared to the oꢀ
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amino benzyl hydrazines 7, 8 and 9 (Table 1), demonstrating that the oꢀamino group enables rapid hydrazone
formation due to its ability to orchestrate nucleophilic catalysis rather than by increasing the electron density on
the hydrazine group.
Although these mechanistic and kinetics studies establish that hydrazone formation by oꢀamino benzyl
hydrazines is preceded by an imine intermediate, they do not rule out the possibility that the imine undergoes an
intermolecular attack by the hydrazine group of another molecule of oꢀamino benzyl hydrazine, rather than an
intramolecular hydrazine attack to form the hydrazone (Figure S4, Supporting Information section). To
distinguish between these two possibilities, we synthesized a benzyl hydrazine derivative containing a pꢀamino
substituent (compound 13, entry 15, Table 1)—a compound that can participate in imineꢀmediated
intermolecular catalysis but cannot orchestrate intramolecular catalysis. This compound was observed to form
hydrazones at much slower rates as compared to the oꢀamino benzyl hydrazines 7, 8 and 9 (Table 1), suggesting
that consistent with our proposed hypothesis, the enhanced rates of hydrazone formation by these compounds
were due to intramolecular hydrazine attack onto the transient imine. Furthermore, to directly compare intra and
intermolecular catalysis, we performed kinetic assays on benzyl hydrazine in presence of equimolar aniline as a
catalyst (Figure S1, S. No. 16) and obtained a rate constant that was lower (k2 = 3.37 ± 0.08 Mꢀ1sꢀ1) than that of
oꢀamino benzyl hydrazine derivatives, 7, 8 and 9 (Table 1). This result also supports our proposed
intramolecular catalysis mechanism.
To compare the efficaciousness of oꢀamino benzyl hydrazines with that of benzyl hydrazine for
bioconjugation, we performed peptide bioconjugation with these two scaffolds. Specifically, we treated 6 and 9
with angiotensinꢀketone which we generated via pyridoxal phosphate (PLP) chemistry (Figure S5).73 Consistent
with the trend obtained with our small molecule studies (Figure 2b and Table 1), HPLC analyses revealed that
the oꢀamino benzyl hydrazine 9 formed its hydrazone more rapidly, giving a yield of 76% yield of the
bioconjugate (angiotensin hydrazone) in 15 h as compared to benzyl hydrazine 6 that took 36 h to give a similar
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yield (Figure S6). We also compared the hydrolytic stability of the angiotensin hydrazones formed with
compounds 6 and 9 and observed that interestingly, the hydrazone formed by 9 hydrolyzed much more rapidly
at pH 5 (81 % in 2 h) as compared to the hydrazone of 6 that did not demonstrate any observable hydrolysis
within 2 h (Figure S7).
To explore the potential of oꢀamino benzyl hydrazines for labeling peptides and proteins with
fluorophores, we synthesized a fluorescent version of this scaffold by appending it with the dansyl fluorophore
(compound 14, Figure 3a). Treatment of angiotensinꢀketone with 10ꢀfold excess of 14 followed by HPLCꢀMS
analysis yielded a peak corresponding to the desired fluorescently labeled peptide, and was accompanied by the
concomitant disappearance of the reactant angiotensinꢀketone (Figure 3a, top panel; Table S1 for mass
characterization), thereby demonstrating the suitability of this scaffold for peptide bioconjugation. Incubation of
this fluorescent peptide conjugate at pH 5 and 37 °C for 18 h resulted in complete hydrazone hydrolysis to form
the precursor hydrazine and angiotensinꢀketone (Figure 3b), demonstrating that fluorescent hydrazone
bioconjugates formed by this scaffold retain the desirable property of undergoing hydrolysis in mildly acidic
solutions. To evaluate the scope of oꢀamino benzyl hydrazines for protein bioconjugation, we generated an
Figure 3. Peptide and protein bioconjugation with the oꢀamino benzyl hydrazine scaffold. a) HPLC traces for labeling the angiotensin peptide with a
fluorescent oꢀamino benzyl hydrazine derivative (14). b) Evaluation of the ability of the angiotensinꢀ14 conjugate to undergo hydrolysis in acidic
aqueous solutions. c) Labeling of aldehydeꢀderivatized myoglobin protein with hydrazines 8 and 9. d) SDSꢀPAGE analysis of bioconjugation of the
myoglobin protein (Mb) with 14. In both c) and d), the aldehyde was generated on the Nꢀterminus of the protein via PLP chemistry.73
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aldehydeꢀcontaining protein (myoglobinꢀCHO) via PLP chemistry73 and treated it with 20 foldꢀexcess of oꢀ
amino benzyl hydrazines 8 and 9 (detailed procedures described in the supporting information section). Mass
spectrometry analysis of these reaction mixtures gave single peaks corresponding to the expected masses of the
desired hydrazone bioconjugates (Figure 3c). Next, we proceeded to fluorescently label myoglobin with this
hydrazine scaffold by treating our dansyl analog 14 with myoglobinꢀCHO. SDSꢀPAGE analysis of this reaction
yielded a bright fluorescent band, whereas no observable fluorescent band was obtained in a control experiment
where wildꢀtype myoglobin lacking the aldehyde functional group was incubated with 14 (Figure 3d). MALDI
analysis yielded a mass corresponding to that of the expected myoglobin dansyl hydrazone conjugate (observed
m/z = 17350.1; expected m/z = 17348.5).
After demonstrating efficacious reversible in vitro bioconjugation with the oꢀamino benzyl hydrazine
scaffold, we proceeded to evaluate its suitability for bioconjugation in cells. Towards this goal, we attempted
hydrazone bioconjugation on two different cellular biomolecules—choline lipids and sialylated glycoproteins.
Interestingly, both these biomolecules have been subjected to hydrazoneꢀmediated bioconjugation previously.
Whereas a recent attempt at imaging choline lipids in cells by employing anilineꢀcatalyzed hydrazone
bioconjugation was unsuccessful,77 imaging of glycoproteins by hydrazone bioconjugation proceeded
Figure 4. Imaging choline lipids in mammalian cells using our oꢀamino benzyl hydrazine scaffold. a) Overview of the imaging approach involving
metabolic labeling of choline lipids with the ketoꢀcholine derivative 15 followed by hydrazoneꢀmediated labeling. b) Mass spectrometryꢀbased
targeted lipidomics for testing metabolic labeling of choline lipids with 15 (5 mM). The m/z values obtained for the three major peaks are labeled.
c) Fluorescent labeling of choline lipids via hydrazone bioconjugation performed in cells treated with 15 (5 mM) by employing a fluorescein
derivative of the oꢀamino benzyl hydrazine scaffold (16, 100 µM). The white line at the bottom right portion of both images depicts a scale bar
denoting a length of 20 µm. The Hoechst stain (blue) was used for nuclear staining.
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efficiently only upon the of use of millimolar concentrations of a hydrazide.78
To image choline lipids, we followed the previously attempted procedure77 that involved metabolic
labeling of choline lipids with the ketoꢀcholine molecule, 15 (Figure 4a). Whether the reported lack of success
of this approach was due to the inefficient metabolic labeling of choline lipids by 15 or due to poor
bioconjugation yields was not reported.77 Therefore, to determine if choline lipids of mammalian cells were
amenable to be metabolically labeled with 15, we synthesized it and administered it to HEK293 cells in culture.
After a 24 hꢀlong incubation, the cells were lysed and the lipids were extracted and subjected to mass
spectrometry analysis via direct infusion into a triple quadrupole ion trap mass spectrometer in the precursor ion
mode (see supporting information section for details). This analysis convincingly established the robust
metabolic labeling of choline lipids by 15 as demonstrated by the appearance of masses of parent ions
corresponding to various phosphatidylcholine and sphingomyelin lipids containing the phosphocholine
derivative of 15 (Figure 4b, left panel; see supporting information section Figure S8 for all the metabolically
labeled phosphatidylcholine lipids identified via this mass spectrometric characterization), whereas, control
experiments wherein cells were not treated with 15 did not yield any peaks for the labeled choline lipids (Figure
4b, right panel). After this demonstration that mammalian cells are amenable to metabolic labeling with 15, we
proceeded to fluorescently label these ketoneꢀappended cellular lipids via our hydrazone bioconjugation
chemistry. Towards this goal, we first synthesized a fluoresceinꢀappended oꢀamino benzyl hydrazine derivative,
16, and treated HEK293 cells metabolically labeled with 15 with 100 µM of 16. This treatment endowed the
cells with green fluorescence (Figure 4c, left panel), whereas cells that were not metabolically labeled with 15
were not rendered green upon treatment with 16 (Figure 4c, right panel). These experiments suggest that the
previously reported failure77 of fluorescent labeling of cells treated with 15 was possibly due to inefficient
hydrazone bioconjugation of cellular choline lipids appended with the ketone functional group via the
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conventional hydrazine dyes that were employed in those experiments, thereby demonstrating the advancement
that our new and improved hydrazine moiety has achieved.
Although the above results on cellular choline lipid imaging were extremely encouraging, this
experimental system precluded testing of hydrolysis and transiminationꢀmediated hydrazone disassembly in
cells as choline lipids are found in both the surfaceꢀexposed outer leaflet of the plasma membrane as well as in
the inaccessible intracellular membranes that cannot be easily subjected to low pH and transimination
conditions. Consequently, to test whether the hydrazone linkages formed by our hydrazine derivatives were
amenable to hydrolysis and transimination in cells, we decided to generate these linkages on sialylated
glycoproteins that are expressed in the outer leaflet of the plasma membrane such that their sialic acid moieties
are exposed extracellularly where after being labeled with hydrazones, they can be subjected to low pH
treatment for hydrolysis studies and amine nucleophiles for performing transimination. To fluorescently label
sialylated glycoproteins via hydrazone bioconjugation in cells, we first converted their sialic acid moieties to
their aldehyde derivatives by treating cells with NaIO4 according to previously reported protocols79 and then
treated the resulting aldehydeꢀdecorated cells with 100 µM of 16 (Figure 5a). Upon imaging, we observed
Figure 5. Glycoprotein imaging followed by hydrolysis and transiminationꢀbased disassembly of hydrazone bioconjugates formed with oꢀamino
benzyl hydrazines on mammalian cells. a) Overview of our strategy of fluorescently labeling sialylated glycoproteins expressed in the plasma
membranes of mammalian cells. b) Oxidation of sialic acid moieties of sialylated glycoproteins followed by fluorescent hydrazone formation (first
panel) upon treatment with 16 (100 µM), followed by bioconjugate hydrolysis upon a 1h incubation in a buffer at pH 4 (second panel) and
transimination by a 1h treatment with 10 mM NH2OH (third panel). The last panel is that for the control experiment where cells not exposed to
NaIO4 were treated with 16 (100 µM). The white line at the bottom right portion of both images depicts a scale bar denoting a length of 20 µm.
The Hoechst stain (blue) was used for nuclear staining.
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robust green fluorescence on the cellular plasma membranes (Figure 5b, first panel) whereas the plasma
membranes of cells not subjected to NaIO4 treatment were not rendered fluorescent upon treatment with 16
(fourth panel), demonstrating efficacious catalystꢀfree hydrazone bioconjugation with our chemistry. To trigger
hydrazone breakdown via hydrolysis, we incubated these fluorescently labeled cells in a buffer at pH 4 for 1 h.
Subsequent imaging showed that the fluorescence was significantly diminished (Figure 5b, second panel)
demonstrating reversible bioconjugation in cells by using our chemistry. To test hydrazone breakdown via
transimination, we treated fluorescentlyꢀlabeled cells with hydroxylamine (10 mM) for 1 h. This treatment
resulted in complete disappearance of green fluorescence (Figure 5b, third panel) demonstrating efficient
transimination on cells.
Finally, we performed MTTꢀbased cellular viability studies on HEK293 mammalian cells in culture to
test whether our oꢀamino benzylhydrazine derivatives, 7, 8, 9 and 16 were cytotoxic. These experiments (Figure
S9) revealed that there was no observable cytotoxicity demonstrated by any of these compounds at
concentrations upto 0.5 mM which is 5ꢀfold higher than that required for cellular imaging applications depicted
in Figures 4 and 5 above, establishing their suitability for cellꢀbased experiments. Interestingly, cytotoxicity
experiments on one of the best anilineꢀbased catalysts, pꢀmethoxy anthranilic acid, revealed that it is cytotoxic
at 10 mM and 50 mM—concentrations at which aniline derivatives are typically utilized to catalyze hydrazone
formation (Figure S9).
To summarize, we have developed a novel hydrazine scaffold, oꢀamino benzyl hydrazine, which forms
hydrazones ~100ꢀfold faster than routinelyꢀused hydrazides under physiological conditions. We demonstrated
the applicability of this scaffold for bioconjugation by using it to perform catalystꢀfree and reversible
fluorescent labeling of a peptide and a protein. Furthermore, we demonstrate that this scaffold can be used for
fluorescently labeling cellular choline lipids and glycoproteins, and that furthermore, the resulting linkages can
be diassembled on cells via hydrolysis and also by transimination. Based on our results, we conclude that oꢀ
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amino benzyl hydrazine is an optimal scaffold for hydrazone bioconjugation and will find applications in a
variety of in vitro and in vivo applications.
Experimental Section. Experimental protocols for kinetic assays, mechanistic studies, in vitro and cellꢀbased
bioconjugation and the synthesis of compounds 4, 5, 7, 8, 9, 11, 12, 13, 14, 15, 16 and S13 are provided in the
supporting information section along with all the kinetic traces and the detailed characterization of all
synthesized compounds. The HEK293 cell line was purchased from the repository of National Centre for Cell
Sciences (NCCS), Pune, India.
Acknowledgements. This work was supported by the Wellcome Trust/DBT India Alliance Fellowship [grant
number IA/I/14/2/501551] awarded to J. K. and funds from IISER Pune. R. N. thanks UGC for a fellowship and
G. P. J. thanks SERB for National Post Doctoral Fellowship (NPDF) research grant [File No:
PDF/2016/003750]. Aditi Dixit is duly acknowledged for providing 5ꢀazidofluorescein which was used for the
synthesis of compound 16.
References:
1. E. Aznar, M. Oroval, L. Pascual, J. R. Murguia, R. MartinezꢀManez and F. Sancenon, Chem. Rev., 2016,
116, 561ꢀ718.
2. A. Bandyopadhyay and J. Gao, Curr .Opin. Chem .Biol., 2016, 34, 110ꢀ116.
3. N. Krall, F. P. da Cruz, O. Boutureira and G. J. L. Bernardes, Nat. Chem., 2016, 8, 103ꢀ113.
4. D. A. Fulton, Nat. Chem., 2016, 8, 899ꢀ900.
5. N. Stephanopoulos and M. B. Francis, Nat. chem.biol., 2011, 7, 876ꢀ884.
6. P. M. Cal, J. B. Vicente, E. Pires, A. V. Coelho, L. F. Veiros, C. Cordeiro and P. M. Gois, J. Am. Chem.
Soc., 2012, 134, 10299ꢀ10305.
7. Y. Zhang, X. Zhou, Y. Xie, M. M. Greenberg, Z. Xi and C. Zhou, J. Am. Chem. Soc., 2017.
8. C. J. Choy, J. J. Geruntho, A. L. Davis and C. E. Berkman, Bioconjugate Chem., 2016, 27, 824ꢀ830.
9. I. M. Serafimova, M. A. Pufall, S. Krishnan, K. Duda, M. S. Cohen, R. L. Maglathlin, J. M. McFarland, R.
M. Miller, M. Frodin and J. Taunton, Nat. Chem. Biol., 2012, 8, 471ꢀ476.
10. K. L. Diehl, I. V. Kolesnichenko, S. A. Robotham, J. L. Bachman, Y. Zhong, J. S. Brodbelt and E. V.
Anslyn, Nat. Chem., 2016, 8, 968ꢀ973.
11. M. E. Smith, F. F. Schumacher, C. P. Ryan, L. M. Tedaldi, D. Papaioannou, G. Waksman, S. Caddick and J.
R. Baker, J. Am. Chem. Soc., 2010, 132, 1960ꢀ1965.
12. G. J. Bernardes, M. Steiner, I. Hartmann, D. Neri and G. Casi, Nat. Protoc., 2013, 8, 2079ꢀ2089.
13. W. G. Skene and J. M. Lehn, Proc. Natl. Acad. Sci. U. S. A., 101, 8270ꢀ8275.
13

Organic & Biomolecular Chemistry
Page 14 of 16
View Article Online
DOI: 10.1039/C8OB00946E
14. G. W. Ashley, J. Henise, R. Reid and D. V. Santi, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 2318ꢀ2323.
15. E. R. Gillies, A. P. Goodwin and J. M. Frechet, Bioconjugate Chem., 2004, 15, 1254ꢀ1263.
16. D. K. Kolmel and E. T. Kool, Chem. Rev., 2017, 117, 10358ꢀ10376.
17. J. Kalia and R. T. Raines, Angew. Chem. Int. Ed., 2008, 47, 7523ꢀ7526.
18. X. Jia, X. B. Zhao, K. Tian, T. T. Zhou, J. G. Li, R. N. Zhang and P. Liu, Biomacromolecules, 2015, 16,
3624ꢀ3631.
19. K. Zuberbuhler, G. Casi, G. J. Bernardes and D. Neri, Chem. Commun., 2012, 48, 7100ꢀ7102.
20. A. Beck, L. Goetsch, C. Dumontet and N. Corvaia, Nat. Rev. Drug. Discov., 2017, 16, 315ꢀ337.
21. V. Chudasama, A. Maruani and S. Caddick, Nat. Chem., 2016, 8, 114ꢀ119.
22. P. F. Bross, J. Beitz, G. Chen, X. H. Chen, E. Duffy, L. Kieffer, S. Roy, R. Sridhara, A. Rahman and G.
Williams, Clin. Cancer Res., 2001, 7, 1490ꢀ1496.
23. A. D. Ricart, Clin. Cancer Res., 2011, 17, 6417ꢀ6427.
24. C. S. McKay and M. Finn, Angew. Chem., Int. Ed., 2016, 55, 12643−12649
25. G. H. Deng, C. M. Tang, F. Y. Li, H. F. Jiang and Y. M. Chen, Macromolecules, 2010, 43, 1191ꢀ1194.
26. R. T. Lam, A. Belenguer, S. L. Roberts, C. Naumann, T. Jarrosson, S. Otto and J. K. Sanders, Science,
2005, 308, 667ꢀ669.
27. A. J. Clipson, V. T. Bhat, I. McNae, A. M. Caniard, D. J. Campopiano and M. F. Greaney, Chem.–Eur. J.,
2012, 18, 10562ꢀ10570.
28. J. Furukawa, Y. Shinohara, H. Kuramoto, Y. Miura, H. Shimaoka, M. Kurogochi, M. Nakano and S.
Nishimura, Anal. Chem., 2008, 80, 1094ꢀ1101.
29. A. Dirksen, S. Yegneswaran and P. E. Dawson, Angew. Chem. Int. Ed., 2010, 49, 2023ꢀ2027.
30. J. Y. Byeon, F. T. Limpoco and R. C. Bailey, Langmuir, 2010, 26, 15430ꢀ15435.
31. M. Rashidian, J. M. Song, R. E. Pricer and M. D. Distefano, J. Am. Chem. Soc., 2012, 134, 8455ꢀ8467.
32. L. H. Yuen, N. S. Saxena, H. S. Park, K. Weinberg and E. T. Kool, ACS Chem. Biol., 2016, 11, 2312ꢀ2319.
33. G. M. Eldridge and G. A. Weiss, Bioconjugate chem., 2011, 22, 2143ꢀ2153.
34. X. H. Shi, Y. Jung, L. J. Lin, C. Liu, C. Wu, I. K. O. Cann and T. Ha, Nat. Methods, 2012, 9, 499ꢀU128.
35. I. Chen, M. Howarth, W. Y. Lin and A. Y. Ting, Nat. Methods, 2005, 2, 99ꢀ104.
36. D. Blanot, J. Lee and S. E. Girardin, Chem. Biol. Drug. Des., 2012, 79, 2ꢀ8.
37. B. N. Han, J. F. Stevens and C. S. Maier, Anal. Chem., 2007, 79, 3342ꢀ3354.
38. A. Palmese, C. De Rosa, G. Chiappetta, G. Marino and A. Amoresano, Anal. Bioanal. Chem., 2012, 404,
1631ꢀ1635.
39. H. Hahne, P. Neubert, K. Kuhn, C. Etienne, R. Bomgarden, J. C. Rogers and B. Kuster, Anal. Chem., 2012,
84, 3716ꢀ3724.
40. J. Thom, D. Anderson, J. McGregor and G. Cotton, Bioconjugate chem., 2011, 22, 1017ꢀ1020.
41. S. Salmaso, A. Semenzato, S. Bersani, F. Mastrotto, A. Scomparin and P. Caliceti, Eur. Polym. J., 2008, 44,
1378ꢀ1389.
42. A. A. Kale and V. P. Torchilin, J. Drug Target, 2007, 15, 538ꢀ545.
43. I. Chen, M. Howarth, W. Lin and A. Y. Ting, Nat. Methods, 2005, 2, 99ꢀ104.
44. P. Wu, W. Shui, B. L. Carlson, N. Hu, D. Rabuka, J. Lee and C. R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A.,
2009, 106, 3000ꢀ3005.
45. M. Wendeler, L. Grinberg, X. Wang, P. E. Dawson and M. Baca, Bioconjugate chem., 2013, 25, 93ꢀ101.
46. E. T. Kool, P. Crisalli and K. M. Chan, Org. lett., 2014, 16, 1454ꢀ1457.
47. E. T. Kool, D.ꢀH. Park and P. Crisalli, J. Am. Chem. Soc., 2013, 135, 17663ꢀ17666.
48. P. Crisalli and E. T. Kool, J. Org. Chem., 2013, 78, 1184ꢀ1189.
49. P. Crisalli and E. T. Kool, Org. lett., 2013, 15, 1646ꢀ1649.
50. D. Larsen, M. Pittelkow, S. Karmakar and E. T. Kool, Org. lett., 2014, 17, 274ꢀ277.
14

Page 15 of 16
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB00946E
51. A. Dirksen, T. M. Hackeng and P. E. Dawson, Angew. Chem., Int. Ed., 2006, 45, 7581ꢀ7584.
52. A. R. Blanden, K. Mukherjee, O. Dilek, M. Loew and S. L. Bane, Bioconjugate chem., 2011, 22, 1954ꢀ
1961.
53. M. M. Mahmoodi, M. Rashidian, Y. Zhang and M. D. Distefano, Curr. Protoc. Protein Sci., 2015, 15.14.
11ꢀ15.14. 28.
54. M. Rashidian, M. M. Mahmoodi, R. Shah, J. K. Dozier, C. R. Wagner and M. D. Distefano, Bioconjugate
chem., 2013, 24, 333ꢀ342.
55. E. Cordes and W. Jencks, J. Am. Chem. Soc., 1962, 84, 832ꢀ837.
56. M. Curvall, C. R. Enzell and B. Pettersson, Cell biol. toxicol., 1984, 1, 173ꢀ193.
57. U. G. Sauer, S. Vogel, A. Hess, S. N. Kolle, L. MaꢀHock, B. van Ravenzwaay and R. Landsiedel, Toxicol.
In Vitro, 2013, 27, 174ꢀ190.
58. J. O. Westerik, B. Sarkar, R. Deslauriers and I. C. Smith, J. Biol. Chem., 1978, 253, 8499ꢀ8506.
59. U.S.A. Pat., US 6,251,919 B1, 2001.
60. O. Dilek, Z. Lei, K. Mukherjee and S. Bane, Chem. Commun., 2015, 51, 16992ꢀ16995.
61. A. Bandyopadhyay and J. Gao, Chem.–Eur. J., 2015, 21, 14748ꢀ14752.
62. P. Schmidt and D. Gillingham, Chem. Sci., 2015, 6, 3329ꢀ3333.
63. C. J. Stress, P. J. Schmidt and D. G. Gillingham, Org. Biomol.Chem., 2016, 14, 5529ꢀ5533.
64. P. Schmidt, L. Zhou, K. Tishinov, K. Zimmermann and D. Gillingham, Angew. Chem. Int. Ed., 2014, 53,
10928ꢀ10931.
65. R. J. Spears and M. A. Fascione, Org. Biomol.Chem., 2016, 14, 7622ꢀ7638.
66. B. Sklarz, Q. Rev. Chem. Soc., 1967, 21, 3ꢀ28.
67. R. A. Scheck, M. T. Dedeo, A. T. Iavarone and M. B. Francis, J. Am. Chem. Soc., 2008, 130, 11762ꢀ11770.
68. G. Reuter, R. Schauer, C. Szeiki, J. P. Kamerling and J. F. Vliegenthart, Glycoconj. J., 1989, 6, 35ꢀ44.
69. D. S. Hage, C. A. Wolfe and M. R. Oates, Bioconjugate chem., 1997, 8, 914ꢀ920.
70. C. G. Gahmberg and S.ꢀi. Hakomori, J. Biol. Chem., 1973, 248, 4311ꢀ4317.
71. M. Rashidian, J. K. Dozier, S. Lenevich and M. D. Distefano, Chem. Commun., 2010, 46, 8998ꢀ9000.
72. I. S. Carrico, B. L. Carlson and C. R. Bertozzi, Nat. chem. biol., 2007, 3, 321ꢀ322.
73. J. M. Gilmore, R. A. Scheck, A. P. Esser‐Kahn, N. S. Joshi and M. B. Francis, Angew. Chem. Int. Ed., 2006,
45, 5307ꢀ5311.
74. A. Bandyopadhyay, S. Cambray and J. Gao, J. Am. Chem. Soc., 2017, 139, 871ꢀ878.
75. M. Falsig and P. E. Iversen, Acta Chem. Scand. B., 1977, 31, 15ꢀ20.
76. A. Dirksen, S. Dirksen, T. M. Hackeng and P. E. Dawson, J. Am. Chem. Soc., 2006, 128, 15602ꢀ15603.
77. C. Li, J. A. Key, F. Jia, A. Dandapat, S. Hur and C. W. Cairo, Photochem. Photobiol., 2014, 90, 686ꢀ695.
78. D. A. Nauman and C. R. Bertozzi, Biochim. Biophys. Act., 2001, 1568, 147ꢀ154.
79. Y. Zeng, T. N. Ramya, A. Dirksen, P. E. Dawson and J. C. Paulson, Nat. Methods, 2009, 6, 207ꢀ209.
15

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