An Azo Coupling Strategy for Protein 3-Nitrotyrosine Derivatization

Article abstract of DOI:10.1002/chem.201901828

Herein, a strategy for the selective derivatization of 3-nitrotyrosine-containing proteins using the classic azo coupling reaction as the key step is described. This novel approach featured multiple advantages and was successfully applied to detect picomole levels of protein tyrosine nitration in biological samples.

Full text of DOI:10.1002/chem.201901828

A Journal of
Accepted Article
Title: Azo Coupling Strategy for Protein 3-Nitrotyrosine Derivatization
Authors: Yuxin Liu, Pengcheng Zhou, Honghong Da, Huiyi Jia, Feifei
Bai, Guodong Hu, Baoxin Zhang, and Jianguo Fang
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To be cited as: Chem. Eur. J. 10.1002/chem.201901828
Link to VoR: http://dx.doi.org/10.1002/chem.201901828
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Azo Coupling Strategy for Protein 3-Nitrotyrosine Derivatization
Yuxin Liu‡a, Pengcheng Zhou‡b, Honghong Daa, Huiyi Jiaa, Feifei Baia, Guodong Hua, Baoxin Zhang*a,
Jianguo Fang*a
Abstract: We described here a strategy for selective derivatization
of 3-nitrotyrosine-containing proteins using the classic azo coupling
reaction as a key step. This novel approach featured multiple
advantages and was successfully applied to detect picomole level of
protein tyrosine nitration in biological samples.
condition.[9] Francis and co-workers developed a two-step
chemo-selective aniline-based oxidative coupling strategy to
detect PTN in cell lysates.[10] However, the oxidative condition
might cause undesired modifications of oxidative-sensitive
residues (such as cysteine and methionine). And based on the
mechanism they proposed,[9b, 10b, 11] the benzylamine derivatives
or aniline might also react with 3-Hydroxytyrosine. Ng and Wong
converted 3-AT to 3-azidotyrosine, followed by click chemistry-
based reactions to label 3-NT residues in proteins with a
moderate overall conversion yield.[12]
Protein tyrosine nitration (PTN), i.e., conversion of the
tyrosine residue into 3-nitrotyrosine (3-NT) by reactive nitrogen
species (RNS), is
a
common protein posttranslational
modification, and is a well-established biomarker of nitroxidative
stress.[1] Elevation of PTN level has been observed in multiple
pathological conditions, and is closely associated with aging[2]
and many diseases, including neurodegeneration, cancer and
3]
cardiovascular diseases.[1c,
The occurrence of 3-NT in
proteome and the important role of PTN have simulated
increasing studies to establish specific and sensitive methods for
Scheme 1. The azo coupling strategy for chemoselective
derivatization of protein 3-NT.
1d, 4]
detection of 3-NT in biological samples.[1a,
Traditional
detection methods of 3-NT in biological samples are generally
divided to two types. One is based on instrumental analysis, like
HPLC, GC, HPLC-MS, tandem mass spectrometry.[5] But this
kind of method requires complicated sample pretreatment
process. The other is based on immunoassays, such as western
blot, immunohistochemistry, immunoprecipitation and ELISA, by
using 3-NT antibodies.[6] However, the specificity and affinity of
anti-3-NT antibodies in recognizing different 3-NT-containing
proteins are varying as most of these antibodies are produced
using 3-NT or specific 3-NT containing protein as an immunogen
without considering the surrounding amino acid sequence and
the compatibility of the antibody respectively. In recent years,
selective chemical derivatization of 3-NT for enriching or
detecting PTN has gained increasing interests.[4, 5c] In general, 3-
NT was first transferred to 3-aminotyrosine (3-AT) by reducing
agents, and subsequently different reagents were employed to
selectively capture 3-AT. These 3-AT-reactive partners, such as
N-hydroxysuccinimide (NHS) ester[7] and aldehydes,[8] may
capture the 3-AT-containing proteins to facilitate the following
instrumental analysis. These reagents react with amines
promiscuously, and it thus requires blocking other protein amino
groups prior to capturing 3-AT. Besides, several coupling
reactions that selectively react with 3-AT have been developed.
For example, Schöneich and his colleagues used benzylamine
derivatives to achieve a turn-on labeling of PTN under oxidative
Inspired by the high yield and mild reaction conditions of azo
dyes synthesis, we explored the application of this classic
reaction for selective derivatization of 3-NT-containing proteins
in biological samples. Our results demonstrated that the azo
coupling reaction works under mild conditions and is
biocompatible for protein modifications. After screening the
coupling partners of 3-AT and optimizing reaction conditions, the
azo coupling reaction shows high chemoselectivity and achieves
nearly a quantitative yield in aqueous solution. The easy
modification of the reactant’s structure enables to introduce
various bioorthogonal groups readily, which facilitates the
following detection of as low as picomole level of 3-NT-
containing proteins in biological samples (Scheme 1).
[a]
Prof Jianguo Fang, Dr Baoxin Zhang, Yuxin Liu‡, Honghong Da,
Huiyi Jia, Feifei Bai, Guodong Hu
State Key Laboratory of Applied Organic Chemistry
College of Chemistry and Chemical Engineering
Lanzhou University
No. 222 South Tianshui Road, Lanzhou 730000 (China)
E-mail: zhangbx@lzu.edu.cn
Figure 1 The coupling partner screen. (A). The basic protocol of
coupling partner screen. Reaction conditions: 1 (1 mmol), coupling
partner (5 mmol), NaNO2 (1.2 mmol). Coupling condition: pH~8.5, 60
min. (B). The structure of coupling partner and its isolated yield of
azo-coupling reaction, isolated yields based on 1. The reaction
mixture was too complicated to separate.
a
fangjg@lzu.edu.cn
Pengcheng Zhou‡
[b]
College of Chemistry & Materials Science
South-Central University for Nationalities
No. 708 Minyuan Road, Wuhan, 430074 (China)
The conversion of 3-NT to 3-AT is well established with
almost a quantitative yield.[12] We thus seek for a suitable
reactant as an azo coupling partner of 3-AT. It is known that
electron-rich aromatic molecules readily react with diazonium
‡ These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the
document.
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salts. We chose 2-amino-p-cresol (1) as a model compound of
3-AT, and studied its reactivity towards several potential azo
coupling partners (Figure 1 and Scheme S3 in Supporting
Information (SI)). With a nearly quantitative conversion, 3-
ethylamino-4-methylphenol (2) was demonstrated to be the most
efficient coupling partner for our azo coupling strategy among all
the tested electron-rich phenolic compounds. The following
HPLC analysis showed 2 (50 mM) accomplished quantitative
coupling with 1 (10 mM) within 5 min (Table S7 in SI). In addition,
the resulting azo compound 3 was stable in the exposure to
dithiothreitol (DTT) and tris-(2-carboxyethyl)phosphine (TCEP),
which were usually used in protein reduction (Table S8 in SI).
After optimizing the reaction pH, the time-dependent
conversion of 1 was then studied (Figure 2B, Scheme S5 &
Table S7 in SI). When 1 mM of 1 reacted with large excess of 2
(120 mM) under the optimized pH (9.0), the product gave a more
than 90% yield in 30 min. Further increasing the reaction time to
60 min gave an almost quantitative yield (98%). Considering the
low occurrence of 3-NT in proteins, we thus kept the reaction
time as 60 min in the following experiments.
Having the optimal conditions (reaction pH and reaction time)
of this azo coupling reaction, the coupling partner (ACP Scheme
1) was then prepared (“Synthetic Procedures” in SI). ACP was
designed based on the structure of 2, and a bioorthogonal group
(azide group) was introduced into ACP to facilitate the following
manipulation of the 3-NT-containing proteins, such as analysis
and enrichment of samples.
Figure 2. The optimization of azo coupling condition in small
molecular level. The diazotizing reaction was performed with 45 mM
NaNO2 in 0.1 M HCl for 20 min. The concentration of product (azo
compound 3) was detected by HPLC-PDA (absorption in 471 nm). (A).
The coupling pH optimization. The concentration of 1 was 10 mM and
the coupling reaction was run with 30 mM 2 in different alkaline
solution (Table S5 & Table S6 in SI) for 15 min. (B). The coupling time
optimization. The using concentration of 1 was 1 mM and the coupling
reaction was run with 120 mM 2 under pH=9.0 for different time.
(C).Two steps of the standard protocol of azo coupling strategy in
small molecular model.
Figure 3. Analysis of peptide and protein derivatization by MS. (A).
ESI-MS analysis of derivatization of 3-NT-containing pentapeptide by
azo coupling strategy. The reactions were run with 20 mM peptide,
100 mM ACP. (B). MALDI-TOF-MS analysis of derivatization of 3-NT-
containing protein by the azo coupling protocol. The reactions were
run with 29 μM Trx, 120 mM ACP. Original data were shown in SI.
Then, we focused on optimization of the azo coupling
reaction conditions with 1 and 2 as model compounds (Figure2,
and Schemes S4 & S5 in SI). The diazonium salt was generated
under traditional way (hydrochloric acid, sodium nitrite, 0 ˚C) by
using aniline 1, and the sulfamic acid was used to remove the
extra nitrous acid (“Small Molecular Experimental Procedures” in
SI).
With above results in hand, we established a two-step
standard protocol for selective derivatization of PTN in proteome.
(Figure 2C, detailed description of the procedure was given in
SI). Since the diazotization and azo coupling cannot be
Coupling pH is an important factor of the azo coupling
reaction, and closely related to the product yield and reaction
kinetics. Firstly, we studied the azo coupling reaction under
different pH conditions (Scheme S4 and Table S6 in SI). The
results showed that there was only trace coupling product when
the pH is below 7.5, while it yielded the highest conversion
under pH=9.0 (Figure 2A). This may be understood by the pH-
dependent reactivity of the reactants: With the pH increased, the
coupling activity of 2 was enhanced due to the presence of more
phenolate species. However, the coupling reactivity of
diazonium salt decreased while the pH further increased due to
the formation of diazohydroxide. Combining these two effects,
the coupling reaction reaches the highest conversion at pH=9.0.
performed under reductive conditions,
a buffer exchange
process would be required between the reduction step and azo
coupling step. In the small molecule and peptide experiments,
the buffer exchange was performed by removing the dithionite
using hydrogen peroxide and direct acidification (“The Modified
Protocol for Peptide” in SI). As for the protein experiments, we
chose the protein precipitation (“3-NT-containing protein protocol”
in SI) or ultrafiltration (“cell lysate protocol” in SI) for separating
the protein from the dithionite solution and re-dissolving the
protein into a new buffer.
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Before starting the protein modification experiments, we
studied this azo coupling labeling strategy in modifying a
synthesized 3-NT-containing model pentapeptide (P-NO2), which
has a sequence of Asp-Gly-(3-NT)-Phe-Ala (Figure 3A and
Figure S1 in SI). Detailed description of the experimental
procedure was given in SI. The specificity of the azo coupling
labeling strategy was evaluated by monitoring the mass change
of the peptide in each step. After the peptide was treated by the
standard protocol with ACP (Figure 3A, “The Modified Protocol
for Peptide” in SI), the ESI-MS analysis of the reaction mixture
was performed. The expected peptides (P-NH2 and P-N3) were
clearly observed after the reduction and coupling reactions,
indicating an efficient derivatization of the 3-NT-containing
peptide by our protocol.
BSA (BSA-N3) was labeled and the linkage could be cleaved
under a biocompatible reductive condition (100 mM sodium
dithionite, 10 mM phosphate buffer (pH=7.4), 15 min, 37 ˚C).
This result demonstrated the specificity of our protocol in protein
level and makes it possible to use this strategy for further
derivatization of 3-NT-containing protein in proteome.
After demonstrating the specificity of azo coupling strategy in
peptide level, the application of the protocol in labeling PTN was
investigated. For further test of our strategy’s specificity, a site-
selective 3-NT-labeled protein was prepared by linking two 3-NT
mimics (with maleimide group, Figure 3B and Scheme S2 in SI)
to E.coli thioredoxin (Trx) by the Michael addition reaction with
Cys32 and Cys35 (“Simulation of 3-NT-containing Protein” in SI).
Then the unmodified protein (Trx), the ‘nitrated’ protein (Trx-NO2)
and the azo labeled protein (Trx-N3, labeled by the standard
protocol) were characterized by MALDI-TOF MS (Figure 3B).
Because the Trx contains all kinds of canonical amino acids, it
allowed us to examine the specificity of the azo coupling
strategy to derivatize 3-NT residues. The data showed clearly
that our azo coupling strategy had high selectivity to label the 3-
NT residues (Figure 3B).
A cleavable group like the azo bond is an ideal choice for
protein labeling. In molecular biology studies, many samples
need to be analyzed by antibody-related assays (like Western
Blot, ELISA and etc.) after labeling. The uncleavable labeling
group may block the antibody-antigen interaction site due to
steric hindrance. However, the azo bond can be cleaved under
mild condition which has been reported by multiple studies.[13] It
could hardly interfere with the interaction between the labeled
protein and antibody since the cleavage of azo bond leaves only
an amino group at the ortho position of phenolic hydroxyl group
in tyrosine. Based on these, a preliminary condition was
optimized in small molecular model (Scheme S6 in SI), the
cleavage experiment was added in the following test in protein
experiments (Figure 4A last step, and Figure S5 in SI).
Figure 4. Applications of azo coupling protocol for PTN detection. (A).
The graphic presentation of the protein labeling protocol for 3-NT-
containing protein. (B-E) SDS-PAGE result of protein experiment. All
the samples were visualized using Fluorescence Imaging and
Coomassie Blue Stain. (B). Testing the specificity of the azo coupling
reaction for reduced and diazotized 3-NT residue on BSA. (10 μg
proteins loaded per lane) (C). Detecting different concentration of 3-
NT-BSA in cell lysates (32 μg HepG2 lysate loaded per lane). 3-NT-
BSA was quantified by the absorbance of 3-NT at 438 nm. (D). Testing
the capability of the azo Coupling strategy to label 3-NT in different
proteins in proteome (32 μg HepG2 lysate loaded per lane) (E).
Detecting 3-NT-containing protein in the cell lysate of Nitro-HepG2 cell
(20 μg nitro-HepG2 lysate loaded per lane).
To investigate the performance of the azo coupling strategy
in labeling 3-NT-containing protein, the nitrated bovine serum
albumin (Nitro-BSA) was prepared by tetranitromethane (TNM)
(“The Preparation of Nitro-BSA” in SI) and chosen as a model of
PTN to investigate the specificity of the azo coupling strategy.
The sample of BSA, nitrated BSA (BSA-NO2), reduced nitro-BSA
(BSA-NH2), diazotized nitro-BSA (BSA-N3) and reduced BSA-N3
(BSA-NH2) were treated with the protein protocol described
above (Figure 4A, Figure S5 and Table S14 in SI). After
In previous studies, the PTN level in the biological samples
was observed up to nanomole level[10a] and has significant
differences among different samples (plasma, urine or tissue).[14]
In addition, the nitration in tyrosine usually occurs in different
kinds of proteins. Since the nitration by TNM was non-site-
selective and unquantified, we linked the 3-NT mimic into
unreduced BSA to make 3-NT-BSA which only contained one 3-
NT structure for the following quantitative test (“The preparation
of 3-NT-BSA” in SI). The minimum labeling concentration of this
strategy was explored by analyzing different amount of 3-NT-
fluorescent
labeling
by
Strain-Promoted
Azide-Alkyne
Cycloaddition (SPAAC), all the samples were analyzed by SDS-
PAGE and the labeled protein was visualized using fluorescence
imaging and Coomassie blue (CB) stain (Figure 4B). The result
showed that only the dithionite-reduced and diazotized nitro-
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BSA (0.03-1 mg/mL) in 4 mg/mL HepG2 cell lysate though SDS-
PAGE. All the samples were treated with the standard protocol
and analyzed by SDS-PAGE (Figure 4C). The CB staining
indicated that equal amount of protein was loaded (right panel of
Figure 4C). In fluorescent detection, the signal could be clearly
detected as low as 7.5 picomole of 3-NT-BSA (Figure 4C, Figure
S11 in SI). Moreover, the data showed this method was
accurate with a linear correlation of 0.998 in the range of 0.9 to
15 μM of 3-NT-BSA (Figure S12 in SI). It demonstrates high
sensitivity and great potential of the azo coupling strategy for
detection of the PTN in biological samples.
Foundation of Gansu Province (18JR4RA003, 17JR5RA193)
and the 111 project.
Keywords: azo coupling • bioorthogonal reactions • protein 3-
nitrotyrosine • protein modification •
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very convenient. Although the need of a buffer exchange
process between the reduction and azo coupling steps may limit
it practical use, this strategy provides an effective and
convenience way to specifically introduce bioorthogonal group to
3-NT for the detection and enrichment of 3-NT-containing
proteins, which facilitates to explore the functions of PTN under
physiological and pathological conditions.
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (21572093 & 21778028), Natural Science
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Entry for the Table of Contents
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Yuxin Liu, Pengcheng Zhou, Honghong
Da, Huiyi Jia, Feifei Bai, Guodong Hu,
Baoxin Zhang*, Jianguo Fang*
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Azo Coupling Strategy for Protein 3-
Nitrotyrosine Derivatization
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