Rapid, hydrolytically stable modification of aldehyde-terminated proteins and phage libraries

Article abstract of DOI:10.1021/ja5023909

We describe the rapid reaction of 2-amino benzamidoxime (ABAO) derivatives with aldehydes in water. The ABAO combines an aniline moiety for iminium-based activation of the aldehyde and a nucleophilic group (Nu:) ortho to the amine for intramolecular ring closure. The reaction between ABAO and aldehydes is kinetically similar to oxime formations performed under stoichiometric aniline catalysis. We characterized the reaction by both NMR and UV spectroscopy and determined that the rate-determining step of the process is formation of a Schiff base, which is followed by rapid intramolecular ring closure. The relationship between apparent rate constant and pH suggests that a protonated benzamidoxime acts as an internal general acid in Schiff-base formation. The reaction is accelerated by substituents in the aromatic ring that increase the basicity of the aromatic amine. The rate of up to 40 M^-1 s ^-1 between an electron-rich aldehyde and 5-methoxy-ABAO (PMA), which was observed at pH 4.5, places this reaction among the fastest known bio-orthogonal reactions. Reaction between M13 phage-displayed library of peptides terminated with an aldehyde moiety and 1 mM biotin-ABAO derivative reaches completion in 1 h at pH 4.5. Finally, the product of reaction, dihydroquinazoline derivative, shows fluorescence at 490 nm suggesting a possibility of developing fluorogenic aldehyde-reactive probes based on ABAO framework.

Full text of DOI:10.1021/ja5023909

Communication
pubs.acs.org/JACS
Rapid, Hydrolytically Stable Modification of Aldehyde-Terminated
Proteins and Phage Libraries
Pavel I. Kitov, Daniel F. Vinals, Simon Ng, Katrina F. Tjhung, and Ratmir Derda*
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
S
* Supporting Information
antibody-glycan conjugates,9 the labeling of the proteins in
cells,10 protein ligation,11,12 and the diversification of genetically-
ABSTRACT: We describe the rapid reaction of 2-amino
benzamidoxime (ABAO) derivatives with aldehydes in
water. The ABAO combines an aniline moiety for
iminium-based activation of the aldehyde and a
nucleophilic group (Nu:) ortho to the amine for
intramolecular ring closure. The reaction between ABAO
and aldehydes is kinetically similar to oxime formations
performed under stoichiometric aniline catalysis. We
characterized the reaction by both NMR and UV
spectroscopy and determined that the rate-determining
step of the process is formation of a Schiff base, which is
followed by rapid intramolecular ring closure. The
relationship between apparent rate constant and pH
suggests that a protonated benzamidoxime acts as an
internal general acid in Schiff-base formation. The reaction
is accelerated by substituents in the aromatic ring that
increase the basicity of the aromatic amine. The rate of up
to 40 M−1 s−1 between an electron-rich aldehyde and 5-
methoxy-ABAO (PMA), which was observed at pH 4.5,
places this reaction among the fastest known bio-
orthogonal reactions. Reaction between M13 phage-
displayed library of peptides terminated with an aldehyde
moiety and 1 mM biotin-ABAO derivative reaches
completion in 1 h at pH 4.5. Finally, the product of
reaction, dihydroquinazoline derivative, shows fluores-
cence at 490 nm suggesting a possibility of developing
fluorogenic aldehyde-reactive probes based on ABAO
framework.
encoded libraries.13
The formation of oximes and hydrazones is the most widely
used chemical modification of aldehyde-displaying proteins. The
relatively slow kinetics of these ligations can be accelerated by
aniline catalysis.14,11,12 We used this approach for the rapid
modification of phage-displayed peptides in the presence of 100
mM aniline (Figure 1A).13 The resulting oxime and hydrazone
Figure 1. (A) Ligation of AOB to phage-displayed aldehydes is
accelerated by aniline, but preincubation of phage with aniline in the
absence of AOB leads to rapid loss of aldehyde and loss of reactivity
toward AOB. (B) In anilinium acetate, pH 4.7, the aldehyde disappears
with t1/2 ∼40 min; however, aldehydes alone are stable at the same pH in
sodium acetate buffer.
apid, site-specific, and hydrolytically stable modifications of
Rbiomolecules are critical for unnatural modification of
proteins, labeling of cells, synthesis of antibody-drug conjugates,
and other applications. Aldehydes are versatile functional groups
for protein functionalization. The popularity of aldehyde
chemistry is rapidly growing due to continuous expansion of
methods for incorporation of this functional group into proteins.
Important examples include: periodate oxidation of the N-
terminal Ser/Thr residues,1 pyridoxal-5′-phosphate-mediated N-
terminal transamination,2 and periodate oxidation of sialic acids
displayed on proteins,3,4 oxidation of glycoproteins by galactose
oxidase,5 incorporation through protein farnesyltransferase,6 and
enzymatic oxidation of proteins by formylglycine-generating
enzyme.7 Aldehyde reactions on proteins were used in a number
of therapeutic products, such as proteins PEGylated through N-
terminal glyoxal.8 Prominent new applications of aldehyde
ligations developed in the past 10 years are the generation of
linkages, however, suffer from susceptibility to hydrolysis.15 To
overcome this problem, Bertozzi16 and Rabuka17 modified the
Pictet−Spengler reaction to yield hydrolytically stable six-
membered rings starting from protein-displayed aldehydes in
water at pH 4−7. Here, we expand the scope of bio-orthogonal
aldehyde chemistry by introducing hydrolytically stable linkages
in model aldehydes, peptides, and bacteriophage-displayed
peptide libraries.
Formation of aniline Schiff bases at the N-terminal glyoxal
motif displayed on phage provides 100-fold acceleration in the
reaction with α-nucleophiles, such as aminooxybiotin (AOB)
(Figure 1A).13 Interestingly, this Schiff base rapidly and
irreversibly degrades when glyoxal is incubated in aniline buffer
Received: March 9, 2014
© XXXX American Chemical Society
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Journal of the American Chemical Society
Communication
in the absence of any α-nucleophiles (Figure 1A,B). Imine-
activated N-terminal aldehydes in proteins, therefore, are
susceptible to attack by a variety of nucleophiles, whereas the
aldehyde is stable under these conditions. We could not isolate a
definitive product of this reaction, but based on the evidence
from the literature, we hypothesize that the intermediate
aromatic imine could be quenched either by amines (Lys) or
reactive aromatic size chains (Tyr, Trp). To further explore the
reactivity of glyoxal Schiff bases with nucleophiles, we prepared a
model water-soluble aldehyde (1) that mimics the N-terminal
glyoxal in a protein and tested its reaction with five commercially
available anilines containing a weak nucleophilic group at the
ortho position (2−6, Scheme 1). The reactions of 1 with 2−6 at
pH 4.5 were monitored by HPLC (Figure S1).
Scheme 1. Reaction of Aldehyde 1 with Aniline-Containing
Nucleophilic Substituents in the ortho Position
Figure 2. Kinetics of the reaction between 6 and 1. (A) Reaction scheme
and conditions. Block arrows indicate protons used for monitoring
reaction progress by 1H NMR spectroscopy. (B) Rate constants
a
1
measured by H NMR spectroscopy. Reactions were conducted in
CD3COONa buffer (100 mM) at pD 4.5 at ∼10 fold excess of either 6 or
1, and the resulting data were fitted to first-order kinetics. We used first-
order expression A(t) = A0(1 − exp(−k1t)) to fit the data, and the
second-order rate constant was derived as k2 = k1/Cex, where Cex is
concentration of the excess reagent (here ABAO). (C) Disappearance of
the reactants and concurrent appearance of the product as determined
by 1H NMR spectroscopy. Spectra were acquired every 17 s (∼230 data
points are in this graph). Ratio of product resonance and the sum of
product and starting material resonance were used to determine %
conversion.
a
In brackets shown is % conversion after 1 h.
Anthranilamide 2 is known to react with aldehydes in highly
acidic conditions to yield stable quinazolinones; however, at pH
= 4.5, more than 24 h was required to achieve 50% conversion. 2-
(Aminomethyl)aniline (3) reacted rapidly with the aldehyde, but
the product was hydrolytically unstable. Diaminobenzene (4)
reacted slower than 3 due to the suboptimal “anti-Baldwin” 5-
endo-trig geometry required for ring closure. An amino-
thiobenzamide (5) yielded a single product in 1 h, which,
however, degraded into a complex mixture of products upon
incubation for 24 h (Figure S1C). The reaction of 2-amino
benzamidoxime (ABAO, 6) was fastest and yielded a single stable
product. A similar reaction in ethanol was previously described.18
We isolated the product of the reaction and confirmed its
structure as a 1,2-dihydroquinazoline-3-oxide (7, Figures 2, S2).
The product 7 formed irreversibly and did not undergo
hydrolysis upon storage in acetate buffer (pH 4.5) over 13 days,
as monitored by HPLC (Figure S3). This observation suggested
that the ABAO−aldehyde reaction can be applied for the
introduction of hydrolytically stable modifications into proteins.
We investigated the kinetics by 1H NMR spectroscopy (Figures
2, S4). The rate of this reaction was moderate at acidic pH. The
rate constant of the reaction between ABAO and N-terminal
glyoxyl derivative 1 was 0.086 M−1 s−1 at pH 4.5. The reaction
was first order with respect to ABAO and the aldehyde and
exhibited weak pH dependence in the range of 4−6 (Figure 2B),
which is characteristic of general-acid-catalyzed formation of the
Schiff base.19 No accumulation of Schiff base was observed in
NMR experiments. Consumption of the reactants was
accompanied by concomitant appearance of the final product
(Figure 2C). This observation suggested that the formation of
Schiff base is the rate-determining step, while the attack of
amidoxime is a relatively rapid process.
Figure 3. Relationship between the measured reaction rates and pKa of
the corresponding protonated anilines for seven ABAO analogues. (A)
Structures of ABAOs and corresponding anilines. (B) Reaction rates
measured by NMR in CD3COONa buffer (100 mM) at pD 4.5. (C)
Scatter plot showing relationship between log k and pKa. (D−E)
Structures and reaction rates for 12 and 13. Basicity of aniline nitrogen
in 12′ was estimated by an ab initio calculation (see Figure S8).
derivatives of ABAO (Figure 3). The reactivity of derivatives 6−
12 exhibited an apparent linear free-energy relationship (LFER)
with the pKa of the corresponding anilinium cations and
suggested that a high basicity of the nitrogen is necessary to
accelerate the Schiff base formation. As a result, we identified
para-methoxy ABAO (PMA) (11) as the most reactive derivative
of the tested analogues. Deviation from the LFER was observed
for the analogue with alkyl substitution at the aromatic amine
(13, Figure 3E). It provided a mechanistic insight: reaction of N-
alkylated ABAO proceeds through an obligatory positively
charged Schiff base, whereas nonalkylated ABAO could avoid
To design ABAO derivatives with enhanced reaction rates, we
explored the rate of reaction of the model aldehyde 1 and
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Communication
significant positive charge buildup in the corresponding
transition state by engaging reaction pathways catalyzed by
general base and acid (Figure S5).
aldehyde in the same conditions (see Figure 3 in ref 16). Most
importantly, glucose reacts with PMA very slowly with the rate
≪0.1 M−1 s−1 (Figure 4, black square), thus suggesting that this
reaction can be conducted in biological medium in the presence
of large excess of reducing carbohydrates.
To identify the nature of general acid and base catalysts, we
measured the reaction at different pH and buffer compositions.
The formation of the 1,2-dihydroquinazoline-3-oxide derivatives
from PMA or ABAO was accompanied by a visible change in
color, which permitted characterization of the reaction by UV
spectroscopy. Reaction rates in deuterated buffer were similar
when measured by UV and NMR spectroscopy. Upon
replacement of the deuterated buffer with protic buffer we
observed a significant inverse solvent kinetic isotope effect (sKIE,
Figure S6); the rate constant of the reaction was up to 2-fold
higher in deuterated acetate buffer. An inverse sKIE was
previously observed for other nucleophilic additions to aldehydes
in aqueous solutions.20 The expanded pH profile measured by
UV (Figure 4) suggested that reaction is catalyzed by general acid
with pKa ∼ 4−5.
To confirm that ABAO derivatives are useful for protein
bioconjugation, we modified functionality-rich peptide WY-
DANHSKPL displaying an N-terminal glyoxyl. Reaction yielded
two products with identical mass (diastereomers resulting from a
new chiral center at the aldehyde carbon, Figure S12). It did not
modify any of the reactive chains as confirmed by MS/MS
fragmentation analysis of the products (Figure S13). UV
monitoring confirmed that kinetics of the reaction of peptide
and iso-electronic model aldehyde (1) are nearly identical
(Figure S12). To monitor conjugation on intact proteins, we
oxidized glycans on horseradish peroxidase (HRP) with NaIO4
and added biotin-displayed ABAO derivative (BABAO, Figure
5A) to yield biotinylated HRP. ELISA assay (Figure S14) and
Figure 4. The pH dependence on rates of the reaction between 11 and
aldehydes. Reaction rates were measured by monitoring the change in
UV absorbance at 400 nm. For the representative raw traces see Figure
S16.
We identified acetate (pKa = 4.7) as a weak catalyst: at pH =
4.5, the rate increased by a factor of <2 with an increase in buffer
concentration from 50 to 400 mM (Figure S7). We hypothesized
that the main general acid catalysis is performed in an
intramolecular fashion by the benzamidoxime (BAO) function-
ality (calculated pKa = 4.2, Figure S8). Catalysis of imine
formation by intramolecular general acids is well-known from
original mechanistic investigations by Hine21 and recent reports
by Dawson22 and Kool.23 Based on the above observations, we
proposed a three-step reaction mechanism, which satisfies the
observed order in reagents and pH profile (see Figure S9 and
kinetic equations on page S20−S21). It starts from formation of a
carbinolamine catalyzed by protonated BAO functionality
(general-acid), followed by a general acid and general-base-
catalyzed elimination of the carbinolamine to form a neutral
Schiff base, and ends with a rapid intramolecular ring closure.
PMA reacted remarkably fast with electron-rich aldehydes
(Figures 4, S10−S11). At pH 4.5, the rate constant was 30−40
M−1 s−1 for isobutyraldehyde (iBuA), which was used by Bertozzi
et al. as a model of a formyl-glycine residue.16 The reaction was
four times faster than Pictet−Spengler cyclization with iBuA at
pH = 4.5; PMA ligation was ∼2-fold faster than Pictet−Spengler
cyclization in near neutral buffer (pH 6−7).16 Reaction with
another biologically relevant aldehyde, electron-deficient N-
terminal glyoxal 1, proceeded with the rate of ∼1 M−1 s−1. This
rate is comparable to Pictet−Spengler reactions of the same
Figure 5. (A) Structures of ABAO derivatives that contain propargyloxy
group and biotin. (B) Quantification of the reaction of BABAO with the
M13 phage displaying a library of peptides with N-terminal glyoxal and
wt phage displaying no aldehyde (1011 PFU/mL each). (C) Phage
mixture was exposed to BABAO for the specified time. (D) To measure
the reaction rate, we exposed the mixture of phage to 1 mM BABAO and
measured biotinylation as a function of time by avidin capture. Error bars
represent standard deviations from four independent kinetics experi-
ments.
MALDI mass spectrometry confirmed modification albeit
heterogeneity of glycosylation precluded identification of exact
stoichiometry. Bovine serum albumin, which was present in the
same solution, lacks glycosylation and was not modified under
these conditions (Figure S14). Modification with BABAO had
no effect on enzymatic activity of HRP, and it turned over its
substrate in ELISA assay (Figure S14).
We then confirmed reaction of ABAO with glyoxal at the N-
terminus of phage-displayed peptide library. An indirect biotin
capture13 confirmed that the rate of disappearance of the
aldehyde on phage in 1 mM PMA in sodium acetate buffer (pH
4.8, Figure S15) was similar to the reactions observed on purified
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Communication
substrates (Figure 4). To rule out the degradation of aldehyde
observed in aniline buffers (Figure 1), we measured the rate of
direct biotinylation (Figure 5). Again, we observed that phage
was biotinylated by BABAO at a rate similar to aforementioned
rates (Figures S15, 4). Phage retained its ability to replicate,
confirming that reaction conditions are mild enough to maintain
the integrity of the M13 virion. Wild-type M13 phage particles
displaying no aldehyde, which were present in the same solution,
were not biotinylated, confirming the specificity of the reaction.
The change in π-conjugation upon reaction and high rigidity of
the product opens an opportunity to design fluorogenic ABAO
derivatives. Indeed the PMA derivative exhibited a dramatic
increase in fluorescence upon reaction with aldehydes (Figure 6,
will serve as a platform for development of new bioconjugation
strategies, fluorogenic probes, and post-translational diversifica-
tion of genetically-encoded libraries. We envision that it will aid
applications where long-term stability and built-in quality control
by UV spectrometry are desired (e.g., long-term protein labeling
and synthesis of antibody-drug conjugates).
ASSOCIATED CONTENT
* Supporting Information
Experimental details and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org
■
S
AUTHOR INFORMATION
Corresponding Author
Ratmir.derda@ualberta.ca
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Research was supported by the Alberta Glycomics Centre. We
thank Tiffany Lai, Jae Choi, and Shailesh Ambre for the assistance
in synthesis of intermediates and measurement of reaction rates.
We thank Prof. Todd Lowary and Prof. Dennis Hall for critical
review of this manuscript.
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