Mild bioconjugation through the oxidative coupling of ortho-aminophenols and anilines with ferricyanide

Article abstract of DOI:10.1002/anie.201307386

Using a small-molecule-based screen, ferricyanide was identified as a mild and efficient oxidant for the coupling of anilines and o-aminophenols on protein substrates. This reaction is compatible with thiols and 1,2-diols, allowing its use in the creation

Full text of DOI:10.1002/anie.201307386

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Chemie
DOI: 10.1002/anie.201307386
Proteins
Mild Bioconjugation Through the Oxidative Coupling of
ortho-Aminophenols and Anilines with Ferricyanide**
Allie C. Obermeyer, John B. Jarman, Chawita Netirojjanakul, Kareem El Muslemany, and
Matthew B. Francis*
Abstract: Using a small-molecule-based screen, ferricyanide
was identified as a mild and efficient oxidant for the coupling
of anilines and o-aminophenols on protein substrates. This
reaction is compatible with thiols and 1,2-diols, allowing its use
in the creation of complex bioconjugates for use in biotech-
nology and materials applications.
varied applications from light harvesting[15] to drug delivery[4]
and water remediation.[16] Each of the complex biomolecule
targets in these studies requires bioorthogonal methods that
are compatible with cysteine chemistry.
Recent work in our group has explored the oxidative
coupling reaction of aniline side chains with electron-rich
aromatic coupling partners such as o-aminophenols.[13,17]
Previous studies demonstrated the use of sodium periodate
as an oxidant for highly efficient and rapid coupling. How-
ever, the ability of periodate to oxidize other moieties on
proteins, notably cysteines and 1,2-diols found in glycans, may
limit the scope of these coupling reactions. Additionally, the
periodate-mediated coupling forms a mixture of two prod-
ucts, and complicates analysis and may prevent its use in
applications such as antibody–drug conjugates which require
well-defined linkages.[3] Herein, we report a substantially
milder oxidant, potassium ferricyanide, which is capable of
coupling anilines and o-aminophenols on protein substrates
without oxidizing thiols or 1,2-diols (Figure 1a). Notably,
a single, stable reaction product is obtained with this oxidant.
We demonstrate the use of this reaction in conjunction with
thiol maleimide chemistry as well as on a complex glycopro-
tein substrate. These optimized reaction conditions should
allow the broad adoption of this coupling chemistry to
virtually any bioconjugation target.
Our efforts to identify milder reaction conditions began
with an HPLC-based assay to screen the ability of different
oxidants to couple p-toluidine to electron-rich coupling
partners, such as 2-amino-p-cresol. Reactions were run with
0.1 mm of each coupling partner and 1 mm oxidant at near-
neutral pH (6.5–7.2) to mimic the conditions compatible for
use on most biomolecules. Reactions were quenched with
excess tris(2-carboxyethyl)phosphine (TCEP, 5–10 mm), and
an internal standard, p-toluenesulfonic acid (p-TsOH,
0.2 mm), was added for reliable quantitation. Many oxidants
screened (Figure 1b) were capable of coupling the small
molecules, and more importantly generated the compound
1 as the sole product. The oxidants formed the [A + B]
product with varying levels of conversion, requiring anywhere
from 15 minutes to 18 hours to reach completion (Figure 1b,c
and Figure S1 in the Supporting Information). We found AgI,
CeIV, CuII, and FeIII were particularly promising and poten-
tially suitable for protein substrates.
T
he synthetic modification of proteins is a critical aspect of
chemical biology and biomaterials science. Synthetically
modified proteins are used to study biochemical function,[1]
modulate pharmacokinetics,[2] and construct new materials
with applications in drug delivery[3,4] and targeted imaging.[5]
Many of these applications require consistent, well-defined
modifications that do not perturb the native protein structure
or function. The precise modification of proteins, however,
presents a significant chemical challenge as the modification
must occur at ambient temperature, near neutral pH, and in
the presence of a wide variety of unprotected functional
groups.
A large number of methods have been developed for the
site-selective modification of proteins.[6] These methods either
rely on natural amino acids found in low abundance, typically
targeting the nucleophilic thiolate side chain of cysteine,[6a,7]
or they rely on engineered artificial amino acids.[6c] The latter
approach can result in completely site-selective modification,
but it depends critically on bioorthogonal[8] chemical reac-
tions that can modify the functional groups with exquisite
selectivity. Ketones,[9] azides,[10] strained alkenes,[11] alkynes,[12]
and anilines[13] are commonly targeted in these reactions.
These strategies are particularly useful for preparing proteins
that are modified in multiple locations for use in biophysical
and materials applications. In our own work,[14] the modifi-
cation of proteins at two distinct locations is required for
[*] A. C. Obermeyer, J. B. Jarman, C. Netirojjanakul, K. El Muslemany,
Prof. Dr. M. B. Francis
Department of Chemistry, University of California, Berkeley
Berkeley, CA 94720-1460 (USA)
E-mail: mbfrancis@berkeley.edu
Prof. Dr. M. B. Francis
Materials Sciences Division, Lawrence Berkeley National Laboratory
Berkeley, CA 94720 (USA)
[**] This work was supported by a grant from the NSF (CHE 1059083).
A.C.O was supported by an NSF graduate research fellowship and
the UC Berkeley Chemical Biology Program (NRSA Training Grant
1 T32 GMO66698). J.B.J. was supported by a SURF Rose-Hills
summer research fellowship, and C.N. was supported by an HHMI
International Student Fellowship.
We next determined the applicability of these oxidants for
a protein substrate containing one aniline side chain (p-
aminophenylalanine, pAF) in the primary sequence (Fig-
ure 1d). The aniline side chain (T19pAF) was introduced into
the coat protein of the MS2 viral capsid using amber stop
codon suppression (Figure 2a).[18] The MS2 capsid self-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201307386.
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Figure 2. a) Representation of the MS2 coat protein dimer, indicating
the location of residues 19 and 87 based on PDB ID: 2MS2. b) The
coupling reaction was quenched by the addition of loading buffer (with
DTT) and analyzed by SDS-PAGE. The lower band corresponds to
unmodified MS2 monomers and the upper band corresponds to MS2
monomers modified with 5k-PEG. c) Schematic for the modification of
both cysteine and pAF residues on the interior and exterior surface of
MS2 capsids, respectively. d) MS2 capsids were first treated with either
a fluorescent maleimide for cysteine alkylation (a) or o-aminophenol
PEG for oxidative coupling (b). The modified proteins were then
subjected to the other conditions (a or b) and analyzed by SDS-PAGE
(periodate (p), ferricyanide (f)). e) Coomassie-stained and fluorescent
images of SDS-PAGE gels of MS2 modified with a fluorescent mal-
eimide (a) and a fluorescent o-aminophenol (b) under various
conditions. The oxidative coupling reaction was carried out with
K3Fe(CN)6 (analogous experiments with NaIO4 appear in Figure S10).
Figure 1. a) Schematic for the oxidative coupling of anilines and o-
aminophenols. b) Oxidants were screened by HPLC to determine
suitable alternatives to NaIO4. c) Reverse-phase HPLC traces of the
reactions shown in (a). Asterisks denote peaks that correspond to the
oxidant. d) The same oxidants as in (b) were tested for their ability to
mediate the coupling reaction on proteins using T19pAF MS2 and o-
aminophenol 5k-PEG as model substrates. e) LC-MS analysis of MS2
monomers (top) modified with 2-amino-p-cresol shows that only one
product is formed when ferricyanide is used as the oxidant (middle),
whereas the use of periodate resulted in a mixture of two different
products (bottom).
assembles from 180 sequence identical monomers to form
a hollow spherical structure.[19] Reactions were performed on
the assembled capsid, but analysis was carried out on the
monomers after disassembly. K3Fe(CN)6 and
Ce(NH4)2(NO3)6 (CAN) maintained high reactivity on the
protein substrate, whereas AgCO2CF3 and CuSO4 showed
poor reactivity on proteins. This was likely due to precip-
itation and slower reaction rates.
Ferricyanide [K3Fe(CN)6] was chosen for further develop-
ment, as it was a milder oxidant than CAN and was
compatible with proteins and most buffers.[20] Coupling
a small molecule, 2-amino-p-cresol (80 mm), to the protein
substrate (20 mm) with ferricyanide (1 mm) for 20 minutes
showed near-complete modification to a single product by
LC-MS (Figure 1e). When the reaction was run under the
same reaction conditions with periodate, an equal mixture of
two products was observed.
(see Figure S2). This product presumably arises from the 1,4-
addition of the aniline to the oxidized o-aminophenol.
Further oxidation and hydrolysis of the imine ortho to the
alcohol gives the final product 1. Two-dimensional NMR
analyses and high-resolution mass spectrometry were used to
confirm that ferricyanide formed product 1 (see Figure S3).
Iminoquinone (1) could be reduced to corresponding hydro-
quinone 3 by TCEP.[13] This reduction was observed on small-
molecule and protein substrates. However, over time the
hydroquinone was observed to reoxidize to the iminoquinone
in air.
The selectivity of the reaction was tested using several
mutants of the MS2 coat protein: wild-type (wt), T19Y, and
T19pAF (Figure 2a). Coupling was only observed when the
oxidant, o-aminophenol, and aniline were all present (Fig-
ure 2b). In addition, the yield was comparable to that of the
reaction mediated by periodate. At higher pH, some back-
ground reactivity with native amino acids was observed.
Initial observations from the HPLC reactivity screen
indicated that ferricyanide formed the same iminoquinone as
was the observed minor product (1) of the periodate reaction
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However, the coupling was selective for the pAF residue
when the reaction pH was held between 6.0 and 6.5. This
selectivity is likely because anilines are deprotonated under
these conditions, thus rendering them uniquely nucleophilic.
Additionally, it was found that addition of 1–10 mm imidazole
could further prevent any undesired nonspecific reactivity.
However this additive was not necessary for many of the
protein substrates tested (see Figure S4).
Other iron(III) sources were evaluated for their ability to
perform the coupling (see Figure S5). Most ferric salts tested
were poorly soluble in water or rapidly formed insoluble iron
oxides, and thus resulted in little to no coupling. Optimization
of the equivalents, time, pH, and buffer revealed that high
levels of conversion (> 75%) could be achieved with only 2.5–
5 equivalents of the aminophenol substrate in 15–20 minutes.
In addition, neither the reaction pH nor the buffer salt was
found to have an effect on the efficiency of the reaction (see
Figure S6). However, caution should be taken when running
the reaction at higher pH as the reaction may lose selectivity
for the aniline side chain under more basic conditions. To
reach high levels of conversion, 5 equivalents of o-amino-
phenol (relative to protein) and 10 equivalents of K3Fe(CN)6
(relative to o-aminophenol) should be used. Additionally, it
was critical to purify the o-aminophenol substrate thoroughly
and store the purified substrate at À208C before use to
achieve high levels of modification.
Computational studies of 1 indicated a strong preference
(ca. 10 kcalmolÀ1) for the iminoquinone tautomer shown
relative to the o-quinone structure (Figure 3a). This was
confirmed by NMR spectroscopy, as only 1 was observed.
Despite the presence of the imine moiety, 1 was found to be
resistant to hydrolysis and relatively stable with respect to
nucleophilic attack. The iminoquinone stability was assayed
by subjecting purified, modified protein (ca. 80% modified,
20 mm) to a range of nucleophilic and reducing additives
(10 mm for 18 h at room temperature). Only in the presence
of competing nucleophilic amines, such as p-anisidine, was
appreciable loss of product observed (Figure 3b,c). Exposure
to a wide range of physiologically relevant pH values (4.0–
10.0), glutathione, or increased temperature (37 and 508C) for
18 hours did not result in product loss, thus indicating the
relative stability of the product (see Figure S7). Additionally,
no loss in product was observed after seven days of storage at
room temperature at neutral pH.
Despite the mild nature of ferricyanide, we wanted to
confirm that excess oxidant could be completely removed
from the bioconjugation reaction.[20d,21] Using standard bio-
molecule purification techniques, such as gel filtration and ion
exchange, it was possible to remove all detectable iron
(< 0.1 mm,; see Figure S8).
The ferricyanide-mediated coupling was also evaluated
for its compatibility with cysteine chemistry. In addition to the
important biological role of cysteine, it is frequently a target
for protein modification.[7] We tested the ability of the
coupling to be used in conjunction with cysteine maleimide
chemistry following the scheme outlined in Figure 2c. A
cysteine introduced to the interior surface of MS2 capsids[5]
was first modified with a fluorescent maleimide and then
subjected to the oxidative coupling conditions. Up to 150
copies of PEG and DNA o-aminophenol-containing sub-
strates were coupled to the fluorescently labeled viral capsid
(see Figure S9).
We also confirmed that unmodified cysteines were still
reactive after the oxidative coupling step. T19pAF N87C MS2
capsids were first reacted with o-aminophenol 5k-PEG using
either K3Fe(CN)6 or NaIO4 and were subsequently treated
with a fluorescent maleimide. After exposure to periodate,
the cysteine no longer reacted with the maleimide. However,
after oxidative coupling with ferricyanide, the cysteine was
successfully labeled with the fluorophore (Figure 2d). To rule
out the possibility that this reactivity was seen because the 5k-
PEG substrate was too large to diffuse into the interior of the
capsids, we also verified that the cysteine maintained
reactivity after oxidative coupling with a small-molecule
aminophenol (Figure 2e). A fluorescent rhodamine amino-
phenol (compound S5, see Scheme S2 in the Supporting
Information) was synthesized and reacted with the capsids. A
spectrally separated fluorescent maleimide (Alexa Fluor 680
C2-maleimide) was then used to assay the reactivity of the
cysteine. The modified protein was analyzed by SDS-PAGE
with two-color fluorescence detection. Only when ferricya-
nide was used as the oxidant was the thiol moiety still reactive
after the oxidative coupling step (Figure 2e; see Figure S10
for reactions with periodate).
The mild nature of ferricyanide increases the compati-
bility of the oxidative coupling reaction with a broader scope
of protein targets. Glycosylated proteins are attractive targets
for modification, with antibody–drug conjugates serving as
a prominent example.[3,22] While sodium periodate can be
used to modify glycoproteins, it is also known to oxidize the
1,2-diols found in sugars.[23,24] To test the ability of ferricya-
nide to modify glycosylated proteins without this side
reaction, an aniline moiety was first site-selectively intro-
duced on the N-terminus of an engineered antibody fragment
(Fc). The Fc was transaminated using pyrodixal-5’-phosphate
(PLP), thus generating a uniquely reactive ketone at each N-
terminus (Figure 4a).[24,25] This ketone was then modified with
Figure 3. a) Computational studies and NMR analysis indicated that
product 1 has a strong preference for the tautomer shown. b,c) The
stability of the product was tested on MS2 after modification with o-
aminophenol 5k-PEG. SDS-PAGE, followed by Coomassie staining and
densitometry analysis (Æ5% accuracy) was used to assess the
stability. Additional data on pH and temperature stability is found in
Figure S7.
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ferricyanide-mediated coupling, even at concentrations of 1m
(see Figure S14). Periodate reactivity was significantly
quenched in the presence of a vast excess of 1,2-diols.
However, at lower concentrations (10 mm) these additives
could be used to protect glycoproteins from periodate while
still allowing the desired oxidative coupling to take place, as
previously reported.[24]
In this study, we used a small-molecule-based screen to
identify alternative oxidants for the coupling of o-amino-
phenols and anilines. Ferricyanide efficiently performed the
coupling on both small molecules and proteins. The updated
coupling reaction formed a single product with excellent
conversion using only a few equivalents of the o-aminophenol
coupling partner. The biomolecule compatibility of this
reaction was demonstrated with several protein and amino-
phenol substrates. Importantly, the ferricyanide-mediated
coupling of o-aminophenols and anilines was completely
orthogonal to both thiol and 1,2-diol moieties, thus facilitating
its use with cysteine chemistry and glycosylated substrates.
These attributes make the reaction particularly appropriate
for applications that require installation of two synthetic
components onto a protein. As an example, we demonstrated
the dual modification of a viral capsid using both cysteine-
maleimide chemistry as well as the ferricyanide-mediated
coupling. The oxidative coupling reaction can reliably be used
on glycoproteins, such as antibodies or antibody fragments,
without any undesired oxidation of the glycan. While the
periodate-mediated coupling is both unfailing and useful, the
ferricyanide-mediated reaction can successfully be used in
situations when NaIO4 is not compatible with the substrate.
Taken together, both oxidative coupling strategies offer
valuable new ways to create nearly any complex bioconju-
gation target.
Figure 4. a) Schematic for the modification of anilines introduced by
transamination of the N-terminus. The terminus was first converted
into a ketone with pyridoxal-5’-phosphate (PLP). This ketone was
modified with an alkoxyamine-functionalized aniline. The aniline then
underwent oxidative coupling with o-aminophenol substrates. b) An
engineered antibody fragment (Fc) was subjected to the conditions
shown in (a). Analysis of the modification with o-aminophenol 5k-PEG
by SDS-PAGE demonstrated the ability of ferricyanide to modify
a complex glycoprotein substrate. c) The Fc sample was treated with
either periodate (p) or ferricyanide (f) followed by a fluorescent alkoxy-
amine to probe for reactive, oxidized sugars (full gels from (b) and (c)
appear in the Supporting Information).
Received: August 22, 2013
Published online: December 5, 2013
an alkoxyamine-functionalized aniline. The aniline-Fc, 9, was
successfully PEGylated with a 5 kDa o-aminophenol PEG
using ferricyanide (ca. 40% modified, Figure 4b). The lower
level of modification was likely due to increased sterics as the
N-termini of the two Fc chains are in close proximity of each
other.
Keywords: amines · bioconjugation · iron · oxidation · proteins
.
The undesired glycan oxidation was probed by treating
the Fc (7) with either NaIO4 or K3Fe(CN)6 followed by
a fluorescent alkoxyamine (Figure 4c).[24] After the oxidants
were quenched with TCEP, the oxidant-treated protein was
incubated with an aminooxy dye to probe for reactive
aldehydes formed as a byproduct of sugar oxidation. Expo-
sure to periodate for 2 minutes resulted in a slight amount of
oxidation of the glycan, with extensive oxidation observed
after 1 hour of exposure. No oxidation of the Fc was observed
after treatment with ferricyanide. Glycopeptide standards
were also used to assess the reactivity of the oxidants toward
1,2-diols. Treatment of a glycosylated erythropoietin fragment
(EPO, 117-131) with K3Fe(CN)6 resulted in no oxidation of
the GalNAc residue over the course of 1 hour (see Fig-
ure S13). Incubation of NaIO4 with glycosylated-EPO, how-
ever, resulted in rapid oxidation of the sugar. To investigate
the compatibility of ferricyanide with 1,2-diols further, several
additives were screened for their effect on reactivity. Man-
nose, glucose, and glycerol had little to no effect on the
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