Intramolecular Catalysis of Hydrazone Formation of Aryl-Aldehydes via ortho -Phosphate Proton Exchange

Article abstract of DOI:10.1055/s-0035-1561387

Bioorthogonal site-specific chemical reaction to label biomolecules in vitro and in living cells is one of the most powerful and convenient tools in chemical biology. Reactive pairs frequently used for chemical conjugation are aldehydes/ketones with hydrazines/hydrazides/hydroxylamines. Although the reaction is generally specific for the two components, even in a cellular environment, the reaction is very slow under physiological conditions. Addition of a phosphate group at the ortho position of an aromatic aldehyde increases the reaction rate by an order of magnitude and enhances the aqueous solubility of the reagent and the product. We have synthesized phosphate-substituted aldehyde synthetic models to study kinetics of their reactions with hydrazines and hydrazides that contain a fluorophore. This rapid bioorthogonal reaction should therefore be potentially a very useful reaction for routine site-specific chemical ligations to study and image complex cellular processes in biological systems.

Full text of DOI:10.1055/s-0035-1561387

SYNLETT
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
©
Georg Thieme Verlag Stuttgart · New York
2016, 27, A–D
A
letter
Synlett
O. Dilek et al.
Letter
Intramolecular Catalysis of Hydrazone Formation of Aryl-Alde-
hydes via ortho-Phosphate Proton Exchange
Ozlem Dilek*a
Anthony M. Sorrentinob
Susan Baneb
OH
OH
R
NHNH2
NHR
O
H
N
H
pH 7
k2 = 52 M–1min–1
a
Istanbul Kemerburgaz University, School of Medicine,
Bagcilar, Istanbul 34217, Turkey
ozlem.dilek@kemerburgaz.edu.tr
State University of New York at Binghamton,
Department of Chemistry, Binghamton, NY 13901,
USA
O
O
b
HO
P
O
HO
P
O
R
NHNH2
pH 7
O
O
NHR
O
N
H
H
k2 = 1034 M–1min–1
Received: 27.09.2015
Accepted after revision: 19.01.2016
Published online: 17.02.2016
rate normally peaks around pH 4, which is well below phys-
iological pH. The reaction rate can be increased by addition
of a nucleophilic catalyst such as aniline and derivatives of
aniline.5 Although impressive rate enhancement can be
DOI: 10.1055/s-0035-1561387; Art ID: st-2015-b0769-l
Abstract Bioorthogonal site-specific chemical reaction to label bio-
molecules in vitro and in living cells is one of the most powerful and
convenient tools in chemical biology. Reactive pairs frequently used for
chemical conjugation are aldehydes/ketones with hydrazines/hydra-
zides/hydroxylamines. Although the reaction is generally specific for
the two components, even in a cellular environment, the reaction is
very slow under physiological conditions. Addition of a phosphate
group at the ortho position of an aromatic aldehyde increases the reac-
tion rate by an order of magnitude and enhances the aqueous solubility
of the reagent and the product. We have synthesized phosphate-substi-
tuted aldehyde synthetic models to study kinetics of their reactions
with hydrazines and hydrazides that contain a fluorophore. This rapid
bioorthogonal reaction should therefore be potentially a very useful re-
action for routine site-specific chemical ligations to study and image
complex cellular processes in biological systems.
6
achieved by substituted anilines, large excess of the cata-
lyst is required to overcome the generally low equilibrium
constants for imine formation in neutral aqueous solution.
Another approach to increasing the rate of hydrazone or
oxime formation is to modify the structure of the reagent.
Nucleophilicity of the hydrazine amine is dependent of the
7
substitutents on the hydrazine. In very general terms, aro-
matic hydrazines are more reactive toward carbonyls than
aliphatic hydrazines. Attaching electron-withdrawing
groups to the α nitrogen, such as in a hydrazide, decreases
the reactivity of the functional group, while other groups,
such as a β-dialkylamine, can enhance the rate.
Less attention has been focused on enhancing the reac-
tion by changing the structure of the electrophile. This ap-
proach is more limiting in the sense that naturally occur-
ring carbonyls, such as those generated in proteins, lipids,
and nucleic acids by reactive oxygen species, by definition
have a fixed structure of the electrophile. Site-specific cou-
pling is often performed with exogenous aldehydes or ke-
tones and carbonyl-containing partner in the conjugation
reaction, however, modification of the carbonyl-containing
partner can also be considered.
Key words site-specific conjugation, kinetics, bioorthogonal reaction
Site-specific conjugation of molecules in biological sys-
1
tems is a general method with many applications. Ideally,
two molecules are joined in a reaction that occurs rapidly
under physiologically compatible conditions without cross-
reaction with other functional groups in the molecules or
milieu. Multiple reactive pairs are in use, such as
azide/phosphine, alkyne/azide, tetrazine/alkyne, and new
2
bioorthogonal reactions continue to be developed. It has
The reactivity of an aromatic aldehyde varies sharply
with the structure of the molecule. This property has been
recently exploited by Wang and Canary, who take advan-
tage of the naturally occurring pyridoxal phosphate (PLP) as
the source of the aldehyde for hydrazone ligation reac-
long been known that amino-substituted heteroatoms,
amines bonded to oxygen or nitrogen, react with aldehydes
and ketones in aqueous solutions to form reversible but sta-
3
ble covalent bonds. Generally, hydrazines or hydroxyl-
8
amines are used as nucleophiles, forming hydrazones or ox-
imes, respectively, the carbonyl-containing molecule. The
mechanism of this class of reactions has been very well
tions. Both the phosphate and the phenol are known to
participate in imine formation in PLP reactions in solution.
When PLP is used as a ligation ligand, though, the phos-
phate is converted into a phosphoamidate, which is less
likely to participate in the hydrazone-forming reaction.
4
studied. In particular, it is known that the rate of product
formation as a function of pH is a bell-shaped curve. The
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–D

B
Synlett
O. Dilek et al.
Letter
Recently, Cristalli and Kool showed that aniline deriva-
agent while still providing a visible signal. In addition, we
have shown previously that hydrazone formation affects
the absorption and emission properties of the fluorophore
tives with certain proton-donating substituents ortho to the
9
amine display enhanced catalytic activity. The best catalyst
10
possessed a phosphonate substituent, which has a pK close
and were thus assured of a signal to monitor kinetics.
a
to the pH of the reaction solution. They also examined a
small series of substituted aromatic aldehydes with the
new catalysts and noted that certain ortho substituents on
the aldehyde can produce a synergistic effect on the reac-
tion rate.
Kinetics of hydrazone formation was assessed at 25 °C
using absorption difference spectroscopy as described in
Supplementary Material. Figure 2 shows a kinetic trace at a
single wavelength for the reaction of fY or SA-P with the
same concentration of CH, forming hydrazones 5 or 6. Data
were fit to a single pseudo-first order reaction to obtain
peudo-first order rate constant. The kinetics for SA-P hy-
drazone formation at pH 4 and pH 5 was too fast to be mea-
sured on a standard spectrophotometer. Therefore these
data were collected under pseudo-first order conditions us-
ing twofold lower concentrations of reagents. In order to
compare all of the reactions, apparent second-order rate
constants were calculated (Table 1). A plot of these data is
shown in Figure 3.
We have been interested in developing hydrazine-con-
taining fluorophores that undergo a spectroscopic change
upon hydrazone formation for use as probes in biological
10
systems. Hydrazine-containing fluorophores were first
11
synthesized and characterized later. These fluorophores
do react with aromatic aldehydes at neutral pH, but the re-
action is sluggish. In this work we show that the reaction
can be accelerated by an order of magnitude by a phosphate
substituent.
Model compounds (Figure 1) were used to assess the ef-
fect of an ortho phosphate on the kinetics of hydrazone for-
mation between an aromatic aldehyde and an aromatic hy-
drazine. The aldehyde components were salicylaldehyde (1,
SA) or 3-formyltyrosine (2, fY), and 2-formylphenyldihydro-
gen phosphate (3, SA-P); fY is of interest because it can be
incorporated into proteins. Furthermore, it is much more
water soluble than SA. Since formation of hydrazone makes
a more hydrophobic product enhanced water solubility of
the components of the model reaction is desirable.
OR1
O
H
Figure 2 Formation of hydrazone 5 (black circles) or 6 (red circles) at
25 °C. The concentration of CH was 325 μM and the concentration of fY
or SA-P was 32.5 μM.
R2
H2NHN
O
O
1
2
: R1 = R2 = H
4
1
: R = H,
R2 = CH2CH(NH2)CO2H
: R = P(O)(OH)2
1
3
Table 1 Apparent Second-Order Rate Constants for Hydrazone For-
mation at 25 °C
R2 = H
1
5
: R = H,
2
R
= CH2CH(NH2)CO2H
R2
OR1
Apparent k
2
, M–1 min–1
Relative rate
1
6
: R = P(O)(OH)2
R2 = H
CH + fY
CH + SA-P
NHN
O
O
pH 4
pH 5
pH 6
pH 7
pH 8
pH 9
pH 10
804 ± 14
548 ± 2.5
196 ± 1.0
52 ± 0.4
50 ± 0.9
41 ± 1.4
40 ± 0.4
3950 ± 166
3270 ± 260
2790 ± 56
1034 ± 73
186 ± 1.2
86 ± 0.5
1.8
5
H
Figure 1 Structures of model aldehydes, hydrazine, and hydrazones
14
20
The hydrazine, 7-hydrazino-4-methyl coumarin (4, CH),
was selected as a model aromatic hydrazine because its ab-
sorption maximum at longer wavelength is more than the
commonly used 2-hydrazinopyridine. It also has a larger
extinction coefficient so it can be used as the limiting re-
3.8
2.1
1.6
64 ± 0.2
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–D

C
Synlett
O. Dilek et al.
Letter
Figure 3 Apparent second-order rate constants for hydrazone forma-
tion as a function of pH. Open circles: fY plus CH. Filled circles: SA-P
plus CH.
Figure 5 Formation of hydrazone 8 (black circles) or 9 (red circles) at
25 °C. The concentration of coumarin hydrazide (7) was 325 μM, and
the concentration of fY or SA-P was 32.5 μM. Note the unit of the X-axis
is hours.
As expected, the rate of both the fY-CH and SA-P-CH re-
12
actions increases with decreasing pH. The rate of the fY-CH
reaction increases gradually with decreasing pH, but the
rate if the SA-P-CH reaction increases suddenly at pH 7 and
The hydrazone forming reactions are much slower. The
reaction between SA-P and coumarin hydrazide is essen-
tially complete in about 12 hours. The reaction between fY
and coumarin hydrazide, however, is not complete after 36
hours. Therefore, an ortho-phosphate increases the speed of
the reaction with hydrazides as well as hydrazines at neu-
tral pH. These data also serve to illustrate the large differ-
ence in reactivity between an aromatic hydrazine and a hy-
drazide. Under the same conditions, the hydrazine reaction
is complete in less than 30 minutes, while the hydrazide re-
action requires at least 12 hours to finish.
In conclusion, we show that directly appending a phos-
phate to an aromatic ring and positioning it adjacent to the
aldehyde gives advantages in a hydrazone ligation reaction.
First, the phosphate serves as an intramolecular general
acid catalyst, which increases the rate of reaction over an
order of magnitude compared to a phenol. Second, the
phosphate group increases the aqueous solubility of the al-
dehyde and the hydrazone product. This should be particu-
larly useful when relatively hydrophobic probes such as fluo-
rophores are part of the ligation product.
then more gradually as the pH decreases. Since the pK of
a
12
the phosphate is near 7, the 20-fold faster rate can be at-
tributed to intramolecular acid catalysis by the phosphate.
The difference in the apparent second order rate constants
decreases with decreasing pH, which reflects the increasing
influence of the protons in the buffer.
Hydrazides are much less reactive with aldehydes and
ketones than aromatic hydrazines. Biological probes with
hydrazides are frequently commercially available, so it was
of interests to assess whether an ortho-phosphate would af-
fect the rate of hydrazone formation. A commercially avail-
able coumarin hydrazide (7-diethylaminocoumarin-3-car-
bohydrazide 7, Figure 4) was allowed to react with fY or SA-P
at pH 7 using the same concentrations employed for the CH
reactions. A typical kinetic trace is shown in Figure 5.
O
NHNH2
Et2N
O
O
O
7
1
R2
R O
Acknowledgment
NHN
We gratefully acknowledge NIH Grant R15 GM-102867 for financial
support. Mr. Sorrentino thanks the Harpur College Dean’s office and
the Starzak Award for additional support. The Regional NMR Facility
(600 MHz instrument) at Binghamton University is supported by NSF
(CHE-0922815).
H
Et2N
O
O
1
8
9
: R = H,
2
R
= CH2CH(NH2)CO2H
1
: R = P(O)(OH)2
R2 = H
Supporting Information
Figure 4 Structures of coumarin hydrazide (7) and hydrazones formed
with fY (8) and SA-P (9).
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1561387.
S
u
p
p
ortioIgnfmr oaitn
S
u
p
p
o
nrtogI
i
f
rm oaitn
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–D

D
Synlett
O. Dilek et al.
Letter
References and Notes
(11) Synthesis of Compound 5
2
Coumarin hydrazine (100 mg, 0.44 mmol) was added to diben-
zyl-SA-P (0.162 g, 0.44 mmol) in MeOH. The reaction was
stirred at r.t. for 2 h. Completion of the reaction was monitored
by TLC. The precipitate was filtered and washed with excess
(1) Chattopadhaya, S.; Abu Bakar, F. B.; Yao, S. Q. Curr. Med. Chem.
2009, 16, 4527.
(
(
(
2) King, M.; Wagner, A. Bioconjugate Chem. 2014, 25, 825.
3) Jencks, W. P. J. Am. Chem. Soc. 1959, 81, 475.
4) (a) Jencks, W. P. Catalysis in Chemistry and Enzymology
MeOH and dried. Yellow product was obtained (130 mg, yield
1
5
5%). H NMR (DMSO-d ): δ = 2.36 (s, 3 H), 5.17 (s, 2 H), 7.26 (s,
6
(McGraw-Hill Series in Advanced Chemistry); McGraw-Hill: New
2
H), 5.20 (s, 2 H), 6.07 (s, 1 H), 6.99–7.05 (m, 2 H), 7.25–7.39
York, 1969, 644. (b) Schmidt, P.; Zhou, L.; Tishinov, K.;
Zimmermann, K.; Gillingham, D. Angew Chem. Int. Ed. 2014, 53,
13
(m, 14 H), 7.62 (d, 1 H), 8.23 (s, 1 H), 11.09 (s, 1 H) ppm.
C
NMR (DMSO-d ): δ = 18.0, 69.6, 97.9, 109.3, 109.5, 111.8, 120.6,
6
1
6
1
0928. (c) Schmidt, P.; Stress, C.; Gillingham, D. Chem. Sci. 2015,
, 329. (d) Bandyopadhyay, A.; Gao, J. Chem. Eur. J. 2015, 21,
4748. (e) Cal, P. M. S. D.; Vicente, J. B.; Pires, E.; Coelho, A. V.;
1
1
25.6, 125.9, 126.5, 126.6, 127.9, 128.5, 128.6, 129.7, 133.7,
35.5, 135.6, 147.7, 147.8, 148.3, 158.4, 155.1, 160.4 ppm.
Synthesis of Compound 6 (Deprotection)
Compound 5 was added to TFA–CH Cl (1:1) and stirred at r.t.
for overnight. Solvent was removed to yield actual product (60
mg, yield 100%). H NMR (DMSO-d ): δ = 2.35 (s, 3 H), 6.05 (s, 1
Veiros, L. F.; Cordeiro, C.; Gois, P. M. P. J. Am. Chem. Soc. 2012,
34, 10299.
2
2
1
(
(
5) Dirksen, A.; Dawson, P. E. Bioconjugate Chem. 2008, 19, 2543.
6) Wendeler, M.; Grinberg, L.; Wang, X.; Dawson, P. E.; Baca, M.
Bioconjugate Chem. 2014, 25, 93.
7) Kool, E. T.; Crisalli, P.; Chan, K. M. Org. Lett. 2014, 16, 1454.
8) Wang, X.; Canary, J. W. Bioconjugate Chem. 2012, 23, 2329.
9) Crisalli, P.; Kool, E. T. Org. Lett. 2013, 15, 1646.
1
6
H), 6.95–7.06 (m, 2 H), 7.14–7.39 (m, 3 H), 7.60 (d, 1 H), 8.01 (d,
13
1
1
1
H), 8.26 (s, 1 H), 11.09 (s, 1 H) ppm. C NMR (DMSO-d ): δ =
6
(
(
(
8.0, 97.8, 109.3, 111.6, 120.9, 124.4, 125.4, 126.5, 126.7, 129.6,
35.1, 148.6, 149.4, 149.5, 153.6, 155.2, 160.5 ppm. ESI-MS
+
[
M ]: 373.08; found: 374.
(
10) (a) Dilek, Ö.; Bane, S. L. Tetrahedon Lett. 2008, 49, 1413.
b) Banerjee, A.; Panosian, T. D.; Mukherjee, K.; Ravindra, R.;
Gal, S.; Sackett, D. L.; Bane, S. ACS Chem. Biol. 2010, 5, 777.
(
(
12) Bender, M. L.; Lawlor, J. M. J. Am. Chem. Soc. 1963, 85, 3010.
13) Silverberg, J. L.; Dillon, L. J.; Vemishetti, P. Tetrahedron Lett.
(
1996, 27, 771.
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–D