Nucleophilic catalysis of oxime ligation

Article abstract of DOI:10.1002/anie.200602877

(Chemical Equation Presented) Aniline acts as a nucleophilic catalyst of oxime ligation in aqueous solution through formation of the orange-colored intermediate. The method is demonstrated by acceleration of the oxime ligation of peptides at pH 4.5 and pH

Full text of DOI:10.1002/anie.200602877

Angewandte
Chemie
Oxime Ligation
DOI: 10.1002/anie.200602877
Nucleophilic Catalysis of Oxime Ligation**
Anouk Dirksen, Tilman M. Hackeng, and
Philip E. Dawson*
Imine-based reactions have found wide application in the
conjugation of biomolecules as a result of their high chemo-
[
1–8]
selectivity.
Aldehydes and ketones can be readily intro-
duced into biomolecules and are virtually inert towards
reaction with other functional groups in these molecules.
Under acidic conditions the carbonyl group reacts with
primary amines to form a reversible imine (Scheme 1a),
and the equilibrium favors the free carbonyl. However, when
[
9]
a-effect nitrogens such as aminooxy groups and hydrazides
[
1–8]
are used, the equilibrium favors the imine.
Scheme 1. a) Imine formation and b) transimination under acidic con-
ditions.
Oxime ligations are often called upon to linkcomplex and
[
7,8]
precious macromolecules.
The oxime bond is stable under
physiological conditions, whereas more dynamic imines, such
as hydrazones, are often reduced to obtain a stable linkage.
Oxime ligations proceed with modest reaction rates in acidic
solution but are poorly reactive at pH 7. which limits their use
in many biological applications. To improve the reaction rate,
oxime ligations typically require millimolar concentrations of
[
*] Dr. A. Dirksen, Dr. P. E. Dawson
Departments of Cell Biology and Chemistry
Skaggs Institute for Chemical Biology
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA92037 (USA)
Fax: (+1)858-784-7319
E-mail: dawson@scripps.edu
Dr. A. Dirksen, Dr. T. M. Hackeng
Cardiovascular Research Institute Maastricht
University Maastricht
P.O. Box 616, 6200 MD Maastricht (The Netherlands)
[
**] This work was supported by the Netherlands Organization for
Scientific Research (NWO; to A.D.) and by the National Institutes of
Health (GM059380 to P.E.D.).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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each reactant or a large excess of one component to establish
pseudo-first-order kinetics.
that is, the time required to reach 50% conversion, is 310 min
À1 À1
(kobs = 0.057 Æ 0.010m s ; Figure 1). However, in the pres-
Here we show that oxime ligations can be significantly
accelerated by using aniline as a nucleophilic catalyst. Rate
enhancements are achieved by changing the electrophile from
a weakly populated protonated carbonyl (Scheme 1a) to a
ence of 10mm aniline t
1
is reduced to 25 min (kobs = 0.68 Æ
=
2
À1 À1
0.05 m s ). A further increase in the reaction rate is
established by using a 100mm anilinium acetate buffer
(pH 4.6; Figure 1). Consistent with the proposed mechanism
of nucleophilic catalysis, aniline does not change the equilib-
rium for the oxime ligation, and all reactions eventually reach
over 99% conversion to 3.
[
10]
more highly populated protonated aniline Schiff base
(
[
11]
[11]
Scheme 1b). The transimination (“trans-Schiffization”)
of the protonated aniline Schiff base to the oxime proceeds
rapidly under acidic aqueous conditions. The resulting
ligation rates (kobs) are increased up to 400-fold in aqueous
solution at pH 4.5 and up to 40-fold at pH 7. Such rate
enhancements enable the use of equimolar reactants at
submillimolar concentrations, meeting the stringent require-
ments of challenging macromolecular ligation.
To illustrate the method, two unprotected peptides,
glyoxylyl-LYRAG 1 and aminooxyacetyl-GRGDSGG 2
(
Scheme 2) were ligated in the presence and absence of
aniline at pH 4.5. The ligation reactions were performed at
ambient temperature with each peptide at a concentration of
1
mm. The reactions were followed by HPLC, the ligation
product 3 was quantitated by integration (214 nm), and the
resulting data were fit to the second-order rate equation. In
the absence of a catalyst, the half-time (t =2) of the reaction,
1
Figure 1. a) Formation of ligation product 3 over time at a reactant
concentration of 1mm in 0.1m ammonium acetate buffer (pH 4.5) in
the absence (&) and in the presence of 10mm aniline (!), and in
1
00mm anilinium acetate buffer (pH 4.6;
b) RP HPLC traces recorded after 1 h for the three ligation reactions
gradient: 5–15% 9:1 v/v MeCN/H O in H O, 0.1 % TFAin 10 min).
&
) ([1]=[2]=1mm; RT).
(
2
2
The solubility of large macromolecules is frequently
limited to 0.1mm concentrations owing to their high molec-
ular weight, low availability, and tendency to aggregate. The
enhanced ligation rates achieved through catalytic transimi-
nation suggested that oxime bonds could be formed effi-
ciently at these low concentrations. Indeed, when the ligation
is performed at pH 4.5 and at 0.1mm of each peptide, the t
1
=
2
for oxime formation is reduced 30-fold from 8200 minutes
À1 À1
(
2
5.7 d; kobs = 0.020 Æ 0.001m s ) in the absence of aniline to
À1 À1
70 min (kobs = 0.62 Æ 0.03m s ) in the presence of 10mm
aniline (Figure 2). The ligation rate was further increased to a
total of more than 400-fold using a 100mm anilinium acetate
buffer (pH 4.6); thus the peptides could be ligated with a t of
1
=
2
À1 À1
just 19 min (kobs = 8.6 Æ 2.0m s ) (Figure 2).
Since many biological macromolecules are conformation-
ally unstable at low pH, it is often desirable to perform
ligation reactions at pH 7. As shown in Figure 3, the
uncatalyzed ligation is very slow under these conditions; t
1
=
2
À1 À1
is 11000 min (7.6 days; kobs = 0.0015 Æ 0.0001m s ), and
competition is observed with the slow intramolecular ring
closure of the glyoxylyl-functionalized peptide 1 to its
Scheme 2. Oxime ligation between glyoxylyl-LYRAG 1 and aminooxy-
acetyl-GRGDSGG 2 performed in either the absence or the presence of
aniline.
[
12]
corresponding cyclic isomers. In contrast, oxime ligation
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À1 À1
2
80 min (kobs = 0.061 Æ 0.006m s ; Figure 3), similar to the
rate observed for the uncatalyzed oxime ligation at pH 4.5.
These reaction conditions could find utility in the ligation or
labeling of folded protein domains.
The application of aniline as a nucleophilic catalyst
extends the scope of oxime ligations to new conditions
highly relevant for bioconjugation. The rate enhancement can
be attributed to the in situ generation of a comparatively high
concentration of a reactive electrophile, the protonated
aniline Schiff base, without affecting the equilibrium for
oxime formation. It has been established that imine formation
involves the nucleophilic attackof a free amine on the
protonated
Scheme 1a).
carbonyl
followed
by
dehydration
[
13,14]
(
Since the pK of a protonated carbonyl is
a
[
15]
in the range of À4 to À10, the concentration of this highly
reactive electrophile is extremely low at pH 4.5. Although
lowering the pH would lead to an increase in protonated
carbonyl, the concomitant protonation of the reacting amine
counteracts this effect.
Aniline and its derivatives meet the unique requirements
[
11]
Figure 2. a) Formation of ligation product 3 over time at a reactant
concentration of 0.1mm in a 0.1m ammonium acetate buffer (pH 4.5)
in the absence (&) and in the presence of 10mm aniline (!), and in a
for nucleophilic catalysis of oxime ligations. Aniline (pK =
a
[11a]
4
.6; p-methoxyaniline: pK = 5.3)
has a pK similar to that
a
a
of a-effect nitrogens, such as the amine of the aminooxy
1
00mm anilinium acetate buffer (pH 4.6;
b) RP HPLC traces recorded after 1 h for the three ligation reactions
gradient: 0–33.5% 9:1 v/v MeCN/H O in H O, 0.1% TFAin 15 min).
&
) ([1]=[2]=0.1mm; RT).
[
16]
group (pK ꢀ 4.6). However, the pK of an imine formed
a
a
with an a-effect nitrogen is typically 5–6 units below the pK
(
a
2
2
of the parent amine, whereas the pK of the aniline Schiff base
a
[
17]
is only 2 units below that of aniline.
Consequently, the
aniline Schiff base is significantly protonated under ligation
conditions, whereas the imine formed with the a-effect
nitrogen is almost completely deprotonated. In addition, the
small equilibrium constant (K ) for the formation of the
eq
[
11]
aniline Schiff base allows the use of an excess of aniline
without having it compete with the formation of the oxime. At
neutral pH, aniline derivatives with a high pK should be
a
more effective catalysts, establishing higher concentrations of
the protonated aniline Schiff base. The ability to generate a
reactive species without competing with the desired product is
a key property of any nucleophilic catalyst.
Nucleophilic catalysis using aniline or other appropriate
amine derivatives should be applicable to any reaction for
which imine formation or transimination is the rate-deter-
mining step. Aniline is a mild, fairly unreactive nucleophile
and is therefore compatible with many functional groups as
demonstrated here by the use of unprotected peptides.
Furthermore, aniline is soluble in a range of solvents and
can be used as a convenient aqueous buffer with acetic acid,
and both its solubility and nucleophilicity can be tuned
through the introduction of substituents on the aromatic ring.
Aniline catalysis will have a major impact on the numerous
Figure 3. a) Formation of ligation product 3 over time at a reactant
concentration of 1mm in a 0.1m sodium phosphate buffer (pH 7) in
the absence (&) and in the presence of 100mm p-methoxyaniline (!)
[1–8]
applications of imine chemistry including bioconjugation,
[
18,19]
[20]
cellular labeling,
surface immobilization, and dynamic
(
[1]=[2]=1mm; RT). b) RP HPLC traces recorded after 22 h for the
[
21,22]
combinatorial libraries.
two ligation reactions (gradient: 5–15% 9:1 v/v MeCN/H O in H O,
2
2
0
.1% TFAin 10 min); the peaks marked with an asterisk correspond
to two isomers of the cyclization product of 1 (confirmed by ESI-MS).
Received: July 19, 2006
Published online: October 19, 2006
at neutral pH can be achieved by using 100mm p-methoxyani-
line as a catalyst (selected because of its elevated pK of 5.3).
Keywords: bioconjugation · nucleophilic catalysis · peptides ·
Schiff bases · transimination
a
.
In the presence of the p-methoxyaniline catalyst, the t =
1
is
2
Angew. Chem. Int. Ed. 2006, 45, 7581 –7584
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7583

Communications
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