Light-Controlled Tyrosine Nitration of Proteins

Article abstract of DOI:10.1002/anie.202102287

Tyrosine nitration of proteins is one of the most important oxidative post-translational modifications in vivo. A major obstacle for its biochemical and physiological studies is the lack of efficient and chemoselective protein tyrosine nitration reagents. Herein, we report a generalizable strategy for light-controlled protein tyrosine nitration by employing biocompatible dinitroimidazole reagents. Upon 390 nm irradiation, dinitroimidazoles efficiently convert tyrosine residues into 3-nitrotyrosine residues in peptides and proteins with fast kinetics and high chemoselectivity under neutral aqueous buffer conditions. The incorporation of 3-nitrotyrosine residues enhances the thermostability of lasso peptide natural products and endows murine tumor necrosis factor-α with strong immunogenicity to break self-tolerance. The light-controlled time resolution of this method allows the investigation of the impact of tyrosine nitration on the self-assembly behavior of α-synuclein.

Full text of DOI:10.1002/anie.202102287

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Protein Nitration
Hot Paper
Light-Controlled Tyrosine Nitration of Proteins
Tengfang Long+, Lei Liu+, Youqi Tao, Wanli Zhang, Jiale Quan, Jie Zheng, Julian D. Hegemann,
Motonari Uesugi, Wenbing Yao, Hong Tian,* and Huan Wang*
Abstract: Tyrosine nitration of proteins is one of the most
important oxidative post-translational modifications in vivo. A
major obstacle for its biochemical and physiological studies is
the lack of efficient and chemoselective protein tyrosine
nitration reagents. Herein, we report a generalizable strategy
for light-controlled protein tyrosine nitration by employing
biocompatible dinitroimidazole reagents. Upon 390 nm irra-
diation, dinitroimidazoles efficiently convert tyrosine residues
into 3-nitrotyrosine residues in peptides and proteins with fast
kinetics and high chemoselectivity under neutral aqueous
buffer conditions. The incorporation of 3-nitrotyrosine resi-
dues enhances the thermostability of lasso peptide natural
products and endows murine tumor necrosis factor-a with
strong immunogenicity to break self-tolerance. The light-
controlled time resolution of this method allows the inves-
tigation of the impact of tyrosine nitration on the self-assembly
behavior of a-synuclein.
bovine tubercular bacteria (Figure 1A).[2] In addition to small
molecule metabolites, nitration of cellular biomolecules,
including oligonucleotides, lipids and proteins, are common
biological events in living systems as a consequence of
elevated oxidative stress.[3] Tyrosine (Tyr) nitration represents
one of the most important oxidative protein posttranslational
modifications (PTMs) in many organisms and is closely
related to human physiology, pathology and aging.[3a,4]
Tyrosine residues are converted to 3-NT residues by nitric
oxide-derived oxidants in vivo, and the accumulation of 3-NT
in cellular proteins is usually considered as a biomarker for
inflammation-associated conditions and various diseases in-
cluding cancer and neurological disorders.[3b,5]
Nitration of tyrosine dramatically alters its physicochem-
ical properties, including the pKa value of the phenol moiety,
the hydrophobicity, redox potential and steric bulkyness,
thereby impacting the structure and function of proteins.[3a]
For example, enzymatic inactivation by tyrosine nitration is
well-established for human manganese superoxide dismutase
(MnSOD), where the bulky side chain of 3-NT close to the
active site shuts down the entrance of reactive oxygen
species.[6] Protein–protein recognition and self-assembly be-
havior of proteins could also be affected by tyrosine nitration,
as demonstrated by the altered aggregation tendency of a-
synuclein, b-amyloid and the microtubule-associated tau
protein upon nitration.[7]
Introduction
Bioactive nitroarene natural products are widely distrib-
uted in all life domains including plants, fungi, bacteria and
mammals.[1] For example, cyclic peptide natural product
ilamycin B2 contains a 3-nitrotyrosine (3-NT) residue which
contributes to its antibiotic bioactivity against human and
The unique property of 3-NT allows its application as
chemical probe in biochemical research. 3-NT can be
employed as a fluorescence quenching unit for investigating
protein folding, conformational change and ligand-protein
interactions.[8] Furthermore, 3-NT residues are highly immu-
nogenic and implicated in the pathology of autoimmune
diseases.[9] As an immunogenic epitope, the incorporation of
3-NT in proteins is applied to trigger autoimmune T cell
responses that can break self-tolerance.[10] Schultz and co-
workers demonstrated that a murine TNF-a mutant contain-
ing a single 3-NT is able to induce a strong antibody response
and breaks the self-tolerance toward wild-type mTNF-a.[11]
Overall, tyrosine nitration as an oxidative PTM is of high
relavence to human biology, and the resulting 3-NTresidue is
a useful molecular tool for biochemical research.
Despite the biological importance of tyrosine nitration in
proteins, related studies have been impeded by the lack of
convenient methods to incorporate 3-NT residues into
proteins. Genetic-code expansion platforms provide a power-
ful tool to generate proteins with 3-NTat a specific position in
vivo.[11,12] However, such methods often suffer from low
protein yields, especially when multiple 3-NT incorporation is
desired. Furthermore, reductive modification of 3-NT to 3-
aminotyrosine residues in heterologous expression hosts, such
[*] T. Long,[+] L. Liu,[+] Y. Tao, Dr. J. Zheng, Prof. Dr. H. Wang
State Key Laboratory of Coordination Chemistry, Chemistry and
Biomedicine Innovation Center of Nanjing University, Jiangsu Key
Laboratory of Advanced Organic Materials
School of Chemistry and Chemical Engineering, Nanjing University
No. 163 Xianlin Ave, Nanjing 210093 (China)
E-mail: wanghuan@nju.edu.cn
W. Zhang, J. Quan, Dr. W. Yao, Prof. H. Tian
Jiangsu Key Laboratory of Druggability of Biopharmaceuticals,
School of Life Science and Technology
China Pharmaceutical University, Nanjing 211198 (China)
E-mail: tinahew@139.com
Dr. J. D. Hegemann
Institute of Chemistry, Technische Universitꢀt Berlin
Straße des 17. Juni 124, 10623 Berlin (Germany)
Prof. Dr. M. Uesugi
Institute for Chemical Research and Institute for Integrated Cell-
Material Sciences (WPI-iCeMS), Kyoto University
Uji, Kyoto 611-0011 (Japan),
and
School of Pharmacy, Fudan University
Shanghai 201203 (China)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202102287.
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Figure 1. Light-induced nitration of phenol derivatives and tyrosine residues in proteins by 5-methyl-1,4-dinitroimidazole (DNIm). A) 3-NT residues
in bioactive peptides, engineering therapeutic and cellular proteins. The structures of mTNF-a and a-synuclein are modified from their crystal
structures (PDB codes 2TNF and 2KKW). B) Current methods for tyrosine nitration of proteins. C) Light-induced nitration of phenol derivatives
and tyrosine residues proteins by DNIm.
as E. coli, poses additional challenges to its application.[13]
Chemical nitration of proteins serves as an important alter-
native. Currently used reagents for protein nitration include
peroxynitrite/CO2,[14] H2O2/sodium nitrite/myeloperoxida-
se,[14c] 3-morpholinosydnoimine (SIN-1),[15] tetranitromethane
(TNM)[16] and NaNO2 (Figure 1B).[17,22] However, these
chemical methods usually suffer from the requirement of
excess amount of nitration reagents, prolonged reaction time
(usually hours), and low-to-moderate modification yields.[13]
More importantly, these protein nitration reagents usually
lack chemoselectivity toward tyrosine, leading to undesired
side reactions such as hydroxylation of tyrosine, oxidation of
cysteine/methionine/tryptophan residues and protein oligo-
merization via 3,3’-dityrosine crosslinking.[13] Herein, we
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report the development of 5-methyl-1,4-dinitroimidazole
(DNIm) as a light-controlled and biocompatible protein
nitration reagent. Upon 390 nm irridiation, DNIm efficiently
converts tyrosine residues into 3-NT residues in peptides and
proteins with fast kinetics and high chemoselectivity under
aqueous buffer conditions. We demonstrate that the incorpo-
ration of 3-NT residues enhances the thermostability of lasso
peptide natural products and endows murine tumor necrosis
factor-a with strong immunogenicity to break self-tolerance.
Furthermore, the light-controlled time-resolution of this
method allows the investigation of the impact of tyrosine
nitration on the self-assembly behavior of a-synuclein.
radical (1) could recombine with a nitro radical at the C2
position to generate a stable and light-insensitive product 2,4-
DNIm (Figure 2B, Figure S2).
Next, we explored the applicability of DNIm in the
nitration of small molecules, peptides and proteins. Phenol
derivatives bearing electron-donating and electron-withdraw-
ing groups at para- and ortho-positions were well accepted for
the reaction, yielding corresponding nitration products in
high yields (Figure 2C, entries 2a–2i). Naphthol and 7-
hydroxy-2H-chromen-2-one derivatives were also nitrated
by DNIm in 47–63% yields (Figure 2C, entries 2j–2m).
Bioactive phenol derivative naringenin was smoothly nitrated
in 63% yield as well (Figure 2C, entry 2n). Tyrosine nitration
of peptides is efficiently accomplished by DNIm under
aqueous buffer conditions. Tripeptide P-1 was nitrated by
DNIm under light in PB buffer at pH 6.0 with more than 95%
conversion (Figure 3A). No dimerization of P-1 by tyrosine
crosslinking was detected by HPLC and ESI-MS (Figure S8).
The nitration of peptides by DNIm was chemoselective
toward tyrosine residues under aqueous conditions, as
tripeptides P-2–P-4 containing Phe, His and Met were not
modified by DNIm (Figure 3B, Figure S9–S11). As an
exception, Cys-containing tripeptide P-5 was quantitatively
converted into disulfide-linked product Di-(P-5) (Fig-
ure S12). Peptides P6–P8, as well as endomorphin and Leu-
enkephalin, were all nitrated specifically at tyrosine residues,
demonstrating the utility of this reaction in generating 3-NT-
containing bioactive peptides (Figure 3B, Figure S13–18).
We further challenged DNIm to modify complex tyrosine-
containing lasso peptide natural products, which are riboso-
mally synthesized and posttranslationally modified peptides
with unique lariat knot topologies.[19] The thermostability of
lasso peptides mainly depends on the bulky residues (the plug
residues) above and below the ring structures to prevent the
unthreading of the C-terminal tails (Figure 3C).[20] Using
microcin J25 as a lasso peptide model substrate, DNIm
efficiently introduced two 3-NT residues in this molecule,
including the sterically hindered Y20 residue (Figure 3C,
Figure S19). Caulonodin IV was selected as an example of
heat-sensitive lasso peptide, which contains two tyrosine
residues (Y15 and Y16) as the upper plug residues and one
phenylalanine as the lower plug residue (Figure 3D).[21] These
results show that DNIm is capable of nitrating Tyr residues in
crowded local environments. While it is known that the
thermal stability of lasso peptides is often primarily dictated
by characteristics of their lower plug residues, the alteration
of other residues can also affect the thermal stability of these
compounds. We envisioned that nitration of Y15 and/or Y16
residues would improve the thermostability of caulonodin IV
by increasing the bulkiness of the upper plug residues
(Figure 3D). Under standard nitration conditions, DNIm
successfully converted Y15 and Y16 into 3-NT residues, as
determined by LC-MS/MS analysis (Figure S20). The result-
ing (3-NT)2-caulonodin IV exhibited significantly enhanced
resistance against thermally induced unthreading than wild-
type caulonodin IV under various temperatures (Figure 3E,
Figure S21). For example, after incubation in aqueous buffer
at 958C for 10 min, 56% of (3-NT)2-caulonodin IV remained
in the lasso fold, whereas only 29% wild-type caulonodin IV
Results and Discussion
Nitroarene compounds, such as 2-nitrophenyl and 2-
nitroveratrole, are often photo-responsive and utilized as
photo-cleavable structural units.[18] We therefore explored the
light-responsive property of DNIm, which is stable toward
biological nucleophiles such as lysine. DNIm has an absorb-
ance spectrum up to 400 nm, and thus 390 nm LED was
selected as a light source (Supporting Information, Fig-
ure S1). Upon light irradiation in acetonitrile, DNIm was fully
converted into 4-nitro-5-methyl-imidazole (NIm) and 5-
methyl-(2,4)-dinitroimidazole (2,4-DNIm) in 6 min (Fig-
ure S2). Treatments with heat (508C) under dark conditions
failed to induce the decomposition of DNIm, indicating that
this reaction is triggered specifically by light (Figure S3).
When p-methylphenol (1a) and tyrosine derivative 1b were
supplied as substrates, the ortho-positions of 1a and 1b were
readily nitrated by DNIm with concomitant products 2a and
2b in about 70% isolated yield (Figure 2A, Figure S4).
Radical trapping reagents, including 2,2,6,6-tetramethylpiper-
idinyloxy (TEMPO), butylated hydroxytoluene (BHT) and
1,1-diphenylethylene, significantly inhibited the nitration of
1a, suggesting that the reaction follows a radical mechanism
(Figure 2A, Figure S4). Indeed, when DNIm was irradiated
by 390 nm LED in the presence of 1,1-diphenylethylene,
adducts of 1,1-diphenylethylene conjugated with a nitro
group or a NIm moiety were observed by LC-MS and GC-
MS analysis (Figure S5), indicating the generation of nitro
and NIm radical species. When compound 1b was reacted
with DNIm in the presence of BHT, nitro-BHT 3b and
tyrosine-BHT conjugate 4b were detected by both GC-MS
and LC-MS analysis, indicating the presence of nitro and
tyrosine radical species during the reaction (Figure S6).
Kinetic analysis of the DNIm-mediated nitration reaction
revealed that the light-induced cleavage of N(1)-NO2 bond is
the rate-limiting step, as the initiate rate of DNIm consump-
tion and nitrophenol product 2a formation were both
independent of the concentration of substrate 1a (Figure S7).
Thus, we propose that light irradiation cleaves the N(1)-NO2
bond in DNIm by generating a NIm radical (1) and a nitryl
radical (Figure 2B). The NIm radical (1) subsequently
abstracts a hydrogen atom from the phenol substrate (2) to
generate a radical species (3), which could couple with a nitro
radical to generate the desired ortho-nitrophenol product (4)
(Figure 2B). In the absence of phenol derivatives, the NIm
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Figure 2. Light-induced nitration of phenol derivatives by DNIm. A) Nitration of p-methylphenol and tyrosine by DNIm. Reaction conditions:
substrate (1a) or (1b) (2.0 mM) and DNIm (2.4 mM) are reacted in acetonitrile under 390 nm LED irradiation for 1 h. B) Proposed reaction
mechanism of DNIm-mediated nitration of phenol derivatives. C) Substrate scope of DNIm-mediated nitration of phenol derivatives.
retained their lasso topology (Figure 3E, right panel). Thus,
by simple late-stage nitration of lasso peptides by DNIm, we
can generate these unique interlocked structural units with
enhanced thermostability. Together, these data demonstrate
that DNIm is a biocompatible and robust nitrating reagent
highly specific towards tyrosine residues in complex peptides.
To examine the ability of DNIm to introduce 3-NT into
proteins, lysozyme was selected as a model protein with three
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Figure 3. DNIm-mediated nitration of tyrosine-containing peptides. A) Light-induced nitration of tripeptide (P-1) by DNIm. The conversion was
>95% based on three independent assays, as determined by HPLC analysis. B) DNIm-mediated tyrosine nitration of peptides. C) DNIm-
mediated tyrosine nitration of microcin J25; D) DNIm-mediated tyrosine nitration of caulonodin IV; E) Thermostability of caulonodin IV and (3-
NT)2-caulonodin IV in aqueous conditions.
Tyr residues. Incubation of lysozyme with 14 equiv. of DNIm
(to the total protein) in PB buffer at pH 6.0 under 390 nm
irradiation generated lysozyme with a single nitration, (3-
NT)-lysozyme, as the major product in 10 min (Figure 4).
Further MS analysis of trypsin digested (3-NT)-lysozyme
indicated that Tyr23 was the dominant nitration site, which is
in agreement with previous studies that Tyr23 is the most
accessible tyrosine residue of lysozyme (Figure S22, Ta-
ble S4).[22] In addition, RNase A and histone-H2A were also
nitrated with high tyrosine specificity (Figure S22, Table S5).
Together, these data demonstrate that DNIm is a highly
efficient and chemoselective tyrosine nitration reagent for
proteins under both native and denaturing conditions.
The high chemoselectivity and fast reaction rate of DNIm-
mediated tyrosine nitration prompted us to explore its utility
in investigating the impact of this oxidative PTM on the
biophysical properties of proteins. a-Synuclein (ASN) is of
particular interest to study because its tyrosine nitration is
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addition of (3-NT)4-ASN into wild-type ASN samples result-
ed in delayed aggregation in a dose-dependent manner and
yielded overall lower levels of ThT fluorescence (Figure S31).
The aggregation of ASN in vivo is a highly complex
process, which is further complicated by Tyr-nitration of these
proteins. At any given time point, ASN proteins in the forms
of monomers, oligomers, protofibrils and fibrils, along with
a mixture of their Tyr-nitration products, all participate and
impact the dynamic balance of aggregation and disassembly.
Previous studies usually utilize synthetic ASN proteins
bearing 3-NT residues at specific positions or nitrated ASN
proteins generated by conventional protein nitrating re-
agents,[7b–d] which must be purified as monomers to remove
any crosslinked byproducts before subjected to in vitro
agitation assays to monitor their self-assembly behavior. Such
over-simplified systems failed to mimic the in vivo scenario
and hardly provide any information regarding the impact of
tyrosine nitration on ASNꢀs biophysical behavior in real time.
Due to the high Tyr-specificity of DNIm-mediated ASN
nitration, the purification step is not necessary, and all
oligomerization forms of ASN proteins can be well reserved.
Furthermore, this method is light-controlled and fast, allow-
ing us to monitor the impact of tyrosine nitration on the self-
assembly behavior of ASN during the course of aggregation
with time-resolution. We designed an assay where wild-type
ASN was first agitated in vitro for a given period of time to
allow the progressive formation of b-sheet-rich ASN oligo-
mers, protofibrils or fibrils (Figure 5A). At selected time
points (0, 1, 2 and 5th day), ASN samples were treated with 2.0
or 10.0 equiv. DNIm for 10 min under 390 nm irradiation,
which yielded partial or full tyrosine nitration of ASN
proteins, respectively. The resulting ASN samples were then
subjected to further agitation, and the aggregation was
monitored by the ThT fluorescence assay every 24 hours
until the 8th day. Results showed that, without the DNIm
treatment, the aggregation of ASN reached the maximum
level in 5 days (Figure 5B and C). When the samples were
treated with DNIm at the early stage of agitation (day-0 and
day-1), nitrated ASN proteins completely lost its tendency to
assemble into b-sheet-rich oligomers via agitation afterwards
(Figure 5B and C, day-0 and day-1 nitration samples).
Tyrosine nitration on day-2, when significant amount of b-
sheet-rich ASN oligomers have accumulated, decreased the
intensity of ThT fluorescence by 26–42% instantly upon the
DNIm treatment, and inhibited further agitation-induced
increase of ThT fluorescence (Figure 5B and C, Figure S32).
These results indicate that ASN nitration at this stage induced
partial dissociation of ASN oligomers and more importantly,
halts the progress of further ASN assembly. In contrast,
fibrillous constructs of ASN (samples of day-5) were highly
resistant to the DNIm treatment with only 3–12% ThT
fluorescence loss, and retained their assembly status after-
wards (Figure 5B and C, Figure S32). Transmission electron
microscopy (TEM) analysis of ASN samples with or without
DNIm-mediated nitration was in agreement with results of
ThT assays. ASN samples that were nitrated at day-0
displayed small spherical-shaped oligomeric intermediates
without any fibril structures after agitation (Figure S33). In
contrast, ASN samples that were nitrated at day-8 retained
Figure 4. Light-triggered tyrosine nitration of proteins by DNIm. Modi-
fication conditions: protein (50 mM) and DNIm of indicated concen-
trations are incubated in 50 mM phosphate buffer at pH 6.0 under
390 nm for 10 min.
associated with neurodegenerative diseases, including Par-
kinsonꢀs disease.[3a,5] DNIm exhibits superior capability in
nitrating ASN proteins, compared to NaNO2 and peroxyni-
trite solutions, by cleanly converting them into (3-NT)4-ASN
with tetra-nitration in only 10 min, as determined by LC-MS/
MS analysis (Figure 4, Figure S23–S27). The ASNY_F mutant
protein with all four Tyr residues mutated into Phe was not
modified by DNIm, further demonstrating the high Tyr-
specificity of this method (Figure S28). TNM is one of the
commonly used tyrosine nitration reagents, and treatment of
ASN proteins by TNM resulted in > 80% proteins as Tyr-Tyr
crosslinked oligomers instead of Tyr-nitrated proteins.[7c] In
sharp contrast, SDS-PAGE analysis of a Tyr-nitrated ASN
sample generated by DNIm indicated that only less than 5%
ASN dimer was generated, further demonstrating the supe-
riority of this Tyr-nitration reagent (Figure S22). (3-NT)4-
ASN generated by our method retains the same secondary
structure as wild-type ASN, as shown by CD spectroscopy
(Figure S29). However, (3-NT)4-ASN completely lost the
tendency to aggregate through in vitro agitation, as indicated
by a ThT fluorescent assay (Figure S30). Furthermore,
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Figure 5. The impact of tyrosine nitration on ASN aggregation during the course of in vitro agitation. A) Illustration of the experimental design.
ASN sample was first agitated for a period of time (0, 1, 2 and 5 days) to allow aggregation to occur before DNIm-mediated tyrosine nitration.
After nitration, ASN samples were agitated in vitro until the 8th day. The formation of b-sheet-rich oligomers was monitored by ThT fluorescence
assay at intervals of every 24 hours. B–C) Impact of tyrosine nitration on the progress of ASN aggregation by 2.0 equiv. and 10 equiv. DNIm.
characteristic fibril structures under TEM (Figure S33).
Together, our data indicates that tyrosine nitration suppresses
the ability of ASN monomers to form b-sheet-rich aggregates
or be recruited into existing aggregates. Nitration of ASN had
limited impact on the b-sheet-rich aggregates that are already
stably formed. Thus, our tyrosine nitration method provides
a significantly improved platform to mimic and investigate
how this oxidative PTM affects the biophysical behavior of
proteins with time-resolution compared with traditional
protein nitration methods.
Finally, we applied this method to prepare (3-NT)-
containing proteins with therapeutic potential. Incorporation
of nitroaryl groups, such as p-nitrophenylalanine and 3-NT
residues, often enhances the immunogenicity of peptides and
proteins (Figure 6A). DNIm-mediated Tyr nitration may
provide a facile method to prepare (3-NT)-containing pro-
teins as epitopes. As an alternative method to the genetic
code expansion strategy, introduction of multiple 3-NT
residues in one protein by DNIm may also allow us to
investigate the correlation between the average number of 3-
NT residues in a protein and the resulting immunogenicity.
We selected murine tumor necrosis factor-a (mTNF-a) as an
example. Site-specific incorporation of a single 3-NT residue
in mTNF-a has been reported to break its self-tolerance in
mice, and such effect is not dependent on the site of 3-NT
incorporation.[11,23] We prepared nitrated mTNF-a proteins
with 2.0 to 25 equiv. DNIm under standard nitration con-
ditions. As a result, nitrated mTNF-a proteins carrying 0.5 to
3.5 3-NT residues on average were acquired. The nitration
sites of these mTNF-a proteins were analyzed by LC-MS
after trypsin digestion. For mTNF-a proteins carrying one
nitration on average, Tyr86, Tyr114/118 and Tyr140/150 were
determined as the major nitration sites (Figure 6B, Table S3).
The immunogenicity of the resulting nitrated mTNF-a pro-
teins was evaluated in vivo. 36 C57BL/6 mice were random-
ized into six groups and injected with PBS buffer, wild-type
(WT) mTNF-a and nitrated mTNF-a samples by using
RIMMS (repetitive immunization at multiples sites) on day-
0, day-7 and day-14. Antibody titers against WT mTNF-
a were determined by ELISA using a horseradish peroxidase
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Conclusion
We have developed a simple yet effective dinitroimida-
zole reagent for light-controlled tyrosine nitration in peptides
and proteins. Our nitration method is featured by its high
chemoselectivity towards tyrosine residues and fast reaction
kinetics, which is superior to conventional protein nitration
reagents. The applicability of the reagent was demonstrated
by tyrosine nitration of various phenol derivatives, complex
peptides and proteins. In addition to providing a new tool for
tyrosine nitration, our light-controlled approach may offer
a general strategy for investigating the impact of tyrosine
nitration on biophysical and immunogenic properties of
proteins. The light-controlled chemical strategy described in
the present study paves the way for developing a new class of
chemical tools to investigate the role of protein tyrosine
nitration and may offer opportunities to photo-manipulate
protein nitration.
Acknowledgements
This work is supported by NSF of China (Grant 21922703 and
91953112, 91753112, and 81973222), the Natural Science
Foundation of Jiangsu Province (Grant BK20190004 and
BK20202004) and the Fundamental Research Funds for the
Central Universities (Grant 14380131), and SPIRITS 2019 of
Kyoto University. T.L. is supported by the co-mentorship
program of the JSPS Core-to-Core Program, “Asian Chem-
ical Biology Initiative”. We thank Dr. Qunfeng Luo for his
contribution at the early stage of this project and Prof. Jie Li
(NJU) for inspiring discussion. We thank Prof. Chuanzheng
Zhou (Nankai University) and Prof. Chengwu Zhang (Nanj-
ing Tech University) for providing the expression plasmids for
the histone H2A and the a-synuclein proteins. This work was
inspired by the international and interdisciplinary environ-
ments of JSPS A3 Foresight Program “Asian Chemical Probe
Research Hub.”
Figure 6. Preparation of tyrosine-nitrated mTNF-a as a protein epitope
to break immunological self-tolerance. A) (3-NT)11-mTNF-a prepared
by genetic incorporation of an unnatural amino acid. B) Major tyrosine
nitration sites for DNIm-mediated nitration in mTNF-a. C) Serum titer
for C57BL/6 mice immunized with PBS, WT mTNF-a and mTNF-
a carrying various numbers of 3-NT residues. The data indicate
autoantibody responses of mTNF-a at a 25600 titer measured by
ELISA. One-way ANOVA followed by Tukey’s multiple comparison test,
***p<0.01 compared with PBS group, ###p<0.01, ##p<0.05
compared with WT mTNF-a group. (n=6 mice per group).
conjugate of goat anti-mouse IgG secondary antibody on day
7, 14, 21, 28 and 35. As expected, PBS buffer and WT mTNF-
a induced no significant serum IgG titers against WT mTNF-
a as a self-protein (Figure 6C). Although incorporation of
0.5 equiv. 3-NT in mTNF-a had no observable impact on its
immunogenicity, the (1.0*3-NT)-mTNF-a sample induced
significantly high serum IgG titers against WT mTNF-a,
indicating the generation of antibodies that are highly cross-
reactive with WT mTNF-a. Interestingly, increasing the
average number of 3-NT incorporation in mTNF-a proteins
strongly inhibited or completely abolished their ability to
stimulate the generation of antibodies against WT mTNF-a,
as indicated by the cases of (2.0*3-NT) and (3.5*3-NT)-
mTNF-a (Figure 6). Together, these results demonstrate that
DNIm-mediated Tyr nitration is a robust method in the
preparation of (3-NT)-containing proteins, although as a mix-
ture, with increased immunogenicity. Importantly, our study
provides the first evidence that the immunogenicity of mTNF-
a is highly dependent on the number of 3-NT residues
incorporated and demonstrated that an increased number of
3-NT residues does not necessarily lead to stronger immuno-
genicity.
Conflict of interest
The authors declare no conflict of interest.
Keywords: dinitroimidazole · light-induced reaction ·
nitration of phenol · protein nitration · tyrosine nitration
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Manuscript received: February 14, 2021
Revised manuscript received: March 31, 2021
Accepted manuscript online: April 12, 2021
Version of record online: && &&
,
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These are not the final page numbers!

Angewandte
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Research Articles
Protein Nitration
A simple yet effective dinitroimidazole
reagent for light-controlled nitration of
tyrosine residues in peptides and pro-
teins was developed.
T. Long, L. Liu, Y. Tao, W. Zhang, J. Quan,
J. Zheng, J. D. Hegemann, M. Uesugi,
W. Yao, H. Tian,*
H. Wang*
&&&& — &&&&
Light-Controlled Tyrosine Nitration of
Proteins
Angew. Chem. Int. Ed. 2021, 60, 2 – 11
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!