Radical acylation of L-lysine derivatives and L-lysine-containing peptides by peroxynitrite-treated diacetyl and methylglyoxal

Article abstract of DOI:10.3109/10715762.2013.871386

Highly electrophilic α-dicarbonyls such as diacetyl, methylglyoxal, 3-deoxyglucosone, and4,5-dioxovaleric acid have been characterized as secondary catabolites that can aggregate proteins and form DNA nucleobase adducts in several human maladies, including Alzheimer's disease, rheumatoid arthritis, diabetes, sepsis, renal failure, and respiratory distress syndrome. In vitro, diacetyl and methylglyoxal have also been shown to rapidly add up the peroxynitrite anion (k2 10-10⁵ M^-1 s^-1), a potent biological nucleophile, oxidant and nitrosating agent, followed by carbon chain cleavage to carboxylic acids via acetyl radical intermediate that can modify amino acids. In this study, we used the amino acid derivatives Ac-Lys-OMe and Z-Lys-OMe and synthesized the tetrapeptides H-KALA-OH, Ac-KALA-OH, and H-K(Boc)ALA-OH to reveal the preferential Lys amino group targeted by acyl radical generated by the α-dicarbonyl/peroxynitrite system. The pH profiles of the reactions are bell-shaped, peaking at approximately 7.5; hence, they are close to the pKa values of ONOOH and of the catalytic H2PO4^- anion. RP-HPLC and ESI-MS analyses of reaction products confirmed ^αN- and ^εN-acetylation of Lys by diacetyl as well as acetylation and formylation by methylglyoxal, with preference for the α-amino group. These data suggest the possibility of radical acylation of proteins in epigenetic processes, where enzymatic acetylation of these biomolecules is a well-documented event, recently reported to be as critical to the cell cycle as phosphorylation. Also noteworthy is the observed formylation of L-Lys containing peptides by methylglyoxal never reported to occur in amino acid residues of peptides and proteins.

Full text of DOI:10.3109/10715762.2013.871386

Free Radical Research, March 2014; 48(3): 357–370
© 2014 Informa UK, Ltd.
ISSN 1071-5762 print/ISSN 1029-2470 online
DOI: 10.3109/10715762.2013.871386
ORIGINAL ARTICLE
Radical acylation of L-lysine derivatives and L-lysine-containing peptides
by peroxynitrite-treated diacetyl and methylglyoxal
R. Tokikawa1, C . L o ff redo1, M. Uemi2, M. T. Machini1 & E. J. H. Bechara1,2
1Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, Brazil, and 2Departamento de Ciências Exatas
e da Terra, Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema, SP, Brazil
Abstract
Highly electrophilic α-dicarbonyls such as diacetyl, methylglyoxal, 3-deoxyglucosone, and4,5-dioxovaleric acid have been characterized
as secondary catabolites that can aggregate proteins and form DNA nucleobase adducts in several human maladies, including Alzheimer’s
disease, rheumatoid arthritis, diabetes, sepsis, renal failure, and respiratory distress syndrome. In vitro, diacetyl and methylglyoxal have
also been shown to rapidly add up the peroxynitrite anion (k2 ∼ 104–105 MϪ1 sϪ1 ), a potent biological nucleophile, oxidant and nitrosating
agent, followed by carbon chain cleavage to carboxylic acids via acetyl radical intermediate that can modify amino acids. In this study,
we used the amino acid derivatives Ac-Lys-OMe and Z-Lys-OMe and synthesized the tetrapeptides H-KALA-OH, Ac-KALA-OH,
and H-K(Boc)ALA-OH to reveal the preferential Lys amino group targeted by acyl radical generated by the α-dicarbonyl/peroxynitrite
system. The pH profiles of the reactions are bell-shaped, peaking at approximately 7.5; hence, they are close to the pKa values of
Ϫ
ε
ONOOH and of the catalytic H2PO anion. RP-HPLC and ESI-MS analyses of reaction products confirmed αN- and N-acetylation
of Lys by diacetyl as well as acetyla4tion and formylation by methylglyoxal, with preference for the α-amino group. These data suggest
the possibility of radical acylation of proteins in epigenetic processes, where enzymatic acetylation of these biomolecules is a well-
documented event, recently reported to be as critical to the cell cycle as phosphorylation. Also noteworthy is the observed formylation
of L-Lys containing peptides by methylglyoxal never reported to occur in amino acid residues of peptides and proteins.
Keywords: diacetyl, methylglyoxal, peroxynitrite, acetyl radical, amino acid acetylation
Abbreviations:A,alanine;Ac,acetyl;Boc,tert-butyloxycarbonylgroup;DA,diacetyl;dGuo,2′-deoxyguanosine;DIC,diisopropylcarbodiimide;
ESI-MS, electrospray ionization mass spectrometry; Fmoc, fluorenylmethyloxycarbonyl group; For, formyl; HOBt, 1-hydroxybenzotriazole;
K, lysine; L, leucine; MG, methylglyoxal; MNP, 2-methyl-2-nitrosopropane; OMe, methyl ester; RP-HPLC, reversed-phase high-performance
liquid chromatography; SPPS, solid-phase peptide synthesis; TIS, triisopropylsilane; Wang resin, p-Alkoxybenzyl Alcohol Resin;
Z, benzyloxycarbonyl group
Introduction
hand, DA is reportedly related to alcohol hepatotoxicity
[15] and to the induction of lung disease [16]. Both MG
and DA are reportedly present in many food products,
urban atmospheres, and cigarette smoke [17–19].
α-Dicarbonyl catabolites from biopolymers such as
diacetyl (DA), methylglyoxal (MG) and glyoxal are much
more electrophilic than monocarbonyls (e.g., acetalde-
hyde and acetone) and therefore undergo faster nucleo-
philic additions by virtue of an electron-withdrawing
vicinal carbonyl group. Accordingly, in phosphate buf-
fers, DA, MG, and glyoxal exist in dynamic equilibrium,
mainly in their mono- and dihydrated forms, leaving only
24%, 6%, and 0.005% of the nominal concentration,
respectively, in the free forms, whereas acetone does not
form hydrates in water [1–3]. Of great biomedical interest
are α-oxoaldehyde metabolites such as (i) MG, which
accumulates in diabetes mainly through the metabolism
of triose phosphates, albeit putatively also from the oxi-
dation of acetone and aminoacetone [4–12]; (ii) 4,5-
dioxovaleric acid in intermittent acute porphyria and
lead poisoning as an oxidation product of overproduced
5-aminolevulinic acid [13,14]; and (iii) 3-deoxyglucosone
in age-related degenerative processes [2]. On the other
Due to their high electrophilicity, α-dicarbonyl catab-
olites rapidly attach to amino groups of basic amino acid
residues of proteins and of nucleic acid bases (Schiff base
condensation reactions), ultimately leading to protein
aggregation [20,21] and exocyclic DNA adduct forma-
tion, respectively [22,23]. This poses an almost impos-
sible analytical challenge to evaluate the real in vivo
concentration of α-dicarbonyls in biological tissues [24].
Low molecular weight products, isolated from the hydro-
lysis of carbonylated proteins and DNA, have been mined
as biomarkers of several human disorders. Among them
are the putative aging biomarker pentosidine (a Maillard
reaction product of ribose and Arg and Lys protein resi-
dues; [25]), 2,4-decadienal-2′-deoxyguanosine (dGuo)
adduct (implicated in mutagenesis and cancer; [26]), and
4-oxo-2(E)-nonenal/reduced glutathione adduct (found
in endothelial cells of cardiovascular disorders; [27].
Correspondence: Etelvino Jose Henriques Bechara, Universidade Federal de São Paulo, DCET, Rua Prof. Artur Riedel, 275, Jardim Eldorado,
Diadema 09972-270 SP, Brazil. Tel: ϩ55-11-33193300. Fax: ϩ55-11-4043-6428. E-mail: ejhbechara@gmail.com
(Received date: 11 October 2013; Accepted date: 27 November 2013; Published online: 31 December 2013)

358 R. Tokikawa et al.
Peroxynitrite, a powerful biological oxidant, nitrating
and nucleophilic agent accumulated in inflammatory
sites and other pathological conditions [28–30], has
also been found to promptly add to α-dicarbonyls at
rise to the hypothesis of radical acetylation of proteins
and DNA (mitochondrial) in biological sites favorable
to peroxynitrite and α-dicarbonyl overload in specific
adverse pathogenic conditions. Today, post-translational
enzymatic acetylation, methylation, phosphorylation,
and ubiquitination of nuclear, cytosolic, and mitochon-
drial proteins are widely and unequivocally demon-
strated to govern pivotal cellular events [36].
rates that increase in the following order: monocarbo-
Ϫ1
nyls (k2 Ͻ 1 0 3
M
s
Ϫ1 ; e.g., propionaldehyde, k2 ϭ
Ϫ1
530 M
s
Ϫ1 , pH 7, 25°C) [31], DA (k2 ∼ 1 ϫ 1 0 4
Ϫ1
Ϫ1
M
s
Ϫ1 ), MG (k2 ∼ 1.0 ϫ 1 0 5 M
s
Ϫ1 ) and glyoxal
(k2 ∼ 1 ϫ 1 0 6 M
s
Ϫ1 ) [32–34]. Peroxynitrite is known
In this study, we used Ac-Lys-OMe and Z-Lys-OMe
and synthesized three tetrapeptides (H-KALA-OH, Ac-
KALA-OH, and H-K(Boc)ALA-OH), aiming to identify
the preferential Lys amino group targeted by acyl radical
generated by the α-dicarbonyl/peroxynitrite system. RP-
HPLC and ESI-MS analyses of reaction products con-
Ϫ1
to be generated in cells by the diffusion-controlled com-
bination of nitric oxide radical and superoxide anion
radical (k2 ∼ 3–20 ϫ 1 0 9 MϪ1 sϪ1 ) [28] and is formed by
the dissociation of peroxynitrous acid (ONOOH), whose
pKa is 6.8 [28], tending toward slightly higher values at
increasing ionic strength of the solution. Considering
that the measured concentrations of free dicarbonyls in
biological samples is in or below the micromolar
range ([33] and ref. therein), while that of CO2 in phys-
iological medium is approximately 1.3 mM, the main
ε
firmed N-acetylation of L-Lys derivatives by DA and
both acetylation and formylation by MG. Detection of
formylated peptides, which obviously implies the inter-
mediacy of formyl radical, was initially disregarded
because the peroxynitrite nucleophilic attack was expected
to take place preferentially on the MG aldehydic carbon,
leading to formate and acetyl radical. Accordingly, our
previous work [33] on the MG/peroxynitrite system did
not reveal production of glyoxalate and MNP-•CH3 or
MNP-•COH radical adducts, which are expected from
peroxynitrite nucleophilic attack on MG ketonic C-2.
Schiff conjugates of DA or MG with the Lys derivatives
were not detected in the present work, although these
reactions have implicated in crosslinking of peptides and
proteins [20,37].
biological fate of peroxynitrite has been claimed to be
Ϫ
CO2, since k2 of peroxynitrite addition to HCO3
/
Ϫ1 Ϫ1
CO2 is 2.6 ϫ 1 0 4 M
s
at pH 7.4 [35]. However, to
balance the extent of peroxynitrite reaction with dicar-
bonyls and competitively with CO2, one must take into
account the flow, compartmentalization, diffusibility of
reagents, and extent of these reactions in tissues.
The reaction mechanism of peroxynitrite addition to
carbonyls to form carboxylic acid fragments and nitric
dioxide is believed to be initiated by phosphate-
catalyzed nucleophilic attack of the peroxynitrite anion,
forming a peroxynitrosocarbonyl adduct whose O–O
bond homolysis yields the nitrogen dioxide radical and
an oxylcarbonyl radical [1,31,33] (Figure 1). Further
β-cleavage of the latter radical gives rise to a shorter
chain carboxylic acid and an acyl radical that reacts
with dissolved molecular oxygen ultimately generating
the corresponding carboxylic acid. This mechanism is
corroborated by capillary electrophoretic detection of
only the acetate ion from DA and acetate plus formate
ions from MG and by EPR acetyl radical trapping
(aH ϭ a N ϭ 0.83 mT), in both cases with 2-methyl-
2-nitrosopropane dimer (MNP). Notably, the peroxynitrite/
dicarbonyl reaction can be coupled to the radical acety-
lation of added amino acids such as His [32] or Lys and
to 2′-deoxyguanosine (dGuo) [33]. These findings gave
Methods
Materials
Reagents of the purest quality available, resins, chromato-
graphic grade solvents, and protected amino acids
were acquired from Sigma–Aldrich Chemical Co. (St.
Louis, MO, USA), Fluka (Buchs, Switzerland), Applied
Biosystem (Foster City, CA, USA), Merck (Darmstadt,
Germany), and Bachem California (Torrance, CA, USA)
and used as received. Unless stated otherwise, all the
solutions were prepared with Millipore Milli-Q purified
®
water and the buffers pretreated with Chelex -100 to
eliminate traces of transition metal ions. DA was previously
Figure 1. Reaction mechanism proposed for the aerobic oxidation of MG and DA to acetate (plus formate, in the case of MG), initiated by
nucleophilic addition of peroxynitrite anion to the dicarbonyl compounds [32,33]. EPR spin trapping experiments with MNP and product
analysis supported the intermediacy of acetyl radical in both reactions, which was found to covalently attach to amino acids and dGuo.

Radical acylation of peptides by a-dicarbonyls 359
Figure 2. HPLC and MS analyses of the reaction of Ac-Lys-OMe with the DA/peroxynitrite system. (a) HPLC chromatogram of
Ac-Lys-OMe (0.5 mg/mL) treated with 5.0 mM DA in the presence of 2.0 mM peroxynitrite in 200 mM phosphate buffer, pH 7.2, at 25°C.
Peaks 1 and 2 correspond to Ac-Lys-OMe and the acetyl adduct Ac-Lys(Ac)-OMe. (b, c) MS and MS/MS spectra of Peak 1; (d, e) MS
and MS/MS spectra of Peak 2.
distilled in a Büchi Model B-580 glass oven with drive
unit at 88°C, while commercial MG aqueous solution was
used as purchased. Stock solutions of 50 mM DA and 55
mM MG were prepared in 200 mM phosphate buffer pH
7.2 (or pH mentioned in the experiment), in which all
the α-dicarbonyl/peroxynitrite/peptide reactions were
performed. Peroxynitrite was prepared as described by
Beckman et al. [45] from 0.60 M NaNO2 and 0.70 M
H2O2, in 0.60 M HCl and 1.50 M NaOH, in a quench-flow
reactor. MnO2 was used to remove excess H2O2; however,
Synthesis of the peptides H-KALA-OH, Ac-KALA-OH,
and H-K(Boc)ALA-OH
These tetrapeptides were synthesized manually by con-
ventional stepwise SPPS, using Fmoc chemistry under
atmospheric conditions and previously established pro-
tocols [39–41]. Deprotection, coupling, and acetylation
reactions were performed at room temperature under
shaking, being monitored by ninhydrin test of the grow-
ing peptide-resin [42]. Washes of the growing peptide-
resin employed DMF, MeOH, and DCM. Briefly, 1.5 g
of Fmoc-Ala-Wang resin (0.45 mmol/g), with particle
size of 100–200 mesh, was washed. The Fmoc group was
removed (by treatment with 20% piperidine in DMF for
10 min under shaking) to obtain H-Ala-Wang resin.
Fmoc-Leu-OH (2-fold excess) was allowed to react with
it [1 h in DMF containing equimolar amounts of DIC/
Ϫ
Ϫ
NO2 and NO3 ions resulting from peroxynitrite
decomposition remained as contaminants. This procedure
usually yields 300–450 mM peroxynitrite, whose concen-
tration was determined spectrophotometrically at 302 nm
Ϫ1
(εϭ1670 M cmϪ1 ; [38]).
Stock solutions of peroxynitrite were kept on ice in
the dark during the experiments.

360 R. Tokikawa et al.
Figure 3. HPLC and MS analyses of the reaction of Z-Lys-OMe with the DA/peroxynitrite system. (a) HPLC chromatogram of Z-Lys-OMe
(0.5 mg/mL) treated with 5.0 mM DA in the presence of 2.0 mM peroxynitrite in 200 mM phosphate buffer, pH 7.2, at 25°C. Peaks 1 and
2 correspond to Z-Lys-OMe and the acetyl adduct Z-Lys(Ac)-OMe; (b, c) MS and MS/MS spectra of Peak 1; (d, e) MS and MS/MS spectra
of Peak 2.
HOBt (1:1)]. The resulting Fmoc-Leu-Ala-Wang resin
was washed and the Fmoc group was removed. Fmoc-
Ala-OH was coupled to the dipeptidyl resin H-Leu-Ala-
peptide resins were treated with 95% TFA containing
2.5% TIS/water at 37°C for 2 h [to provide H-Lys-Ala-
Leu-Ala-OH (H-KALA-OH) and Ac-Lys-Ala-Leu-
Ala-OH (Ac-KALA-OH)] or with TFA 0.5% at room
temperature for 8–10 h [to yield H-Lys(Boc)-Ala-Leu-
Ala-OH]. Cold isopropyl ether was added to the reaction
media. The insoluble residues (resin, residual peptide
resin, and free peptide) were washed with ether, decanted
by centrifugation and four times washed with ether.
The free peptides were solubilized with 60% ACN/
0.09% TFA solution and separated from the resulting
suspension by filtration. Peptide solutions were lyo-
philized to yield the solid crude peptides.
Wang
resin
yielding
the
tripeptidyl
resin
Fmoc-Ala-Leu-Ala-Wang resin, which was washed and
the Fmoc group was removed. Fmoc-Lys(Boc)-OH was
coupled to the resulting Ala-Leu-Ala-Wang resin, the
Fmoc group was removed again to give H-Lys(Boc)-
Ala-Leu-Ala-Wang resin. When required, acetylation of
the resulting tetrapeptide-resin was performed with
acetic anhydride: DCM (1:1) for 10 min.
The peptides were released from the resin and fully
or partially deprotected. Briefly, the corresponding

Radical acylation of peptides by a-dicarbonyls 361
Figure 4. HPLC and MS analyses of the reaction of Ac-Lys-OMe with the MG/peroxynitrite system. (a) HPLC chromatogram of
Ac-Lys-OMe (0.5 mg/mL) treated with 5.0 mM of MG in the presence of 2.0 mM peroxynitrite in 200 mM phosphate buffer, pH 7.2,
at 25°C. Peaks 1 and 2 correspond to Ac-Lys-OMe and the acyl-radical adducts; (b) MS spectrum of Peak 1; (c) MS spectrum of Peak 2
(acetyl and formyl adducts with m/z 245 and 231); (d) MS/MS spectrum of the formyl adduct Ac-Lys(For)-OMe.
Characterization of crude peptides by RP/HPLC and
LC/ESI-MS
Peptide purification
The crude synthetic peptides were loaded into a Waters
600E RP-HPLC system [Waters Delta 600 quaternary
pump, Waters 600 gradient controller, and Waters 2487
Dual Absorbance Detector (Milford, MA)], equipped with
a Rheodyne 3725i-119 injector and a Kipp and Zonen
SE124 recorder connected to a Vydac C18 preparative
column (10 μm, 300 Å, 2.2ϫ25 cm). Fractions were ana-
lyzed by RP-HPLC using the aforementioned analytical
system. The fractions containing the desired peptides were
collected, analyzed, pooled, and lyophilized. Identity of the
final compounds was confirmed using LC-MS and full hydro-
lysis followed by amino acid analysis of the hydrolyzates.
A sample of each crude synthetic peptide was dissolved
in 0.1% H2O/TFA to make up a 1.0 mg/mL solution.
Analytical RP-HPLC was performed in an LDC system
composed of an automatic gradient controller, two
pumps (LDC Analytical, a ConstaMetric 3500 and a
ConstaMetric 3200 pump), a UV detector (Milton
Roy SpectroMonitor 3100), a manual sample injector
(Rheodyne 7125), an integrator (Thermo Separation
Products, Data Jet integrator), and an analytical column
(Vydac C18, 5 μm, 300 Å, 46 ϫ 250 mm). The elution
condition employed was 0.1% TFA/H2O as solvent A,
60% CH3CN/H2O containing 0.09% TFA as solvent B,
linear gradient of solvent B, wavelength of 210 nm, and
flow rate of 1 mL/min.
Standard treatment of L-lysine derivatives and
L-lysine-containing peptides with peroxynitrite
After finding the optimized RP-HPLC condition for
analysis, 10 μL of each peptide solution was loaded onto
a C18 RP-HPLC column coupled to a Shimadzu HPLC
system composed of two LC-10AD pumps, a Shimadzu
SDP-10AV detector, a Rheodyne 7125 injector and a
Micromass Quattro II triple quadrupole mass spectrome-
ter, ESI-MS. The mass spectrometry conditions were cap-
illary 3 kV and cone voltage 37 kV in positive ionization
Diacetyl or MG was added at a final concentration of
2.0 mM into the solution containing the L-lysine deriva-
tive or the L-lysine-containing peptide in 200 mM,
phosphate buffer, 7.2, at room temperature. Subsequently,
the peroxynitrite was added under constant stirring at a
final concentration of 2.0 mM to a solution containing
the dicarbonyl compound and the L-lysine-derivative or
L-lysine-containing peptide.
ϩ
mode [MϩH ] .

362 R. Tokikawa et al.
Figure 5. HPLC and MS analyses of the reaction of Z-Lys-OMe with the MG/peroxynitrite system. (a) HPLC chromatogram of Z-Lys-
OMe (0.5 mg/mL) treated with 5.0 mM of MG in the presence of 2.0 mM peroxynitrite in 200 mM phosphate buffer, pH 7.2, at 25°C. Peak
1 corresponds to Ac-Lys-OMe and Peaks 2 and 3 to the acyl-radicals adducts; (b) MS spectrum of Peak 1; (c, d) MS and MS/MS spectra
of Peak 2 (the formyl adduct Z-Lys(For)-OMe, m/z 323); (e) MS spectrum of Peak 3 (the acetyl adduct Z-Lys(Ac)-OMe, m/z 337).
manual sample injector (Rheodyne 7125) for sample puri-
fication. The reactions containing L-lysine-derivatives
Sample purification and quantitation using RP-HPLC
®
®
RP-HPLC analysis were performed using a Waters HPLC
with automatic gradient controller, two binary pumps
(Waters 515), a PDA detector (Waters 996), an automatic
sample injector (Waters 717) for sample analysis and a
were loaded onto a Phenomenex Luna C18(2) column
(5 μm, 100 Å, 4,6ϫ250 mm); elution was done with a
linear gradient of 5–60% mobile phase B (95% CH3CN/
H2O containing 0.09% TFA) in solvent A (0,1% TFA/
Figure 6. Generation of formyl radical intermediate from nucleophilic attack of peroxynitrite to MG C-2. Minor amounts of products derived
from CO-C1 cleavage are favored by previous hydration of MG [3].

Radical acylation of peptides by a-dicarbonyls 363
Figure 7. HPLC and MS analyses of the reaction of peptide H-KALA-OH with the DA/peroxynitrite system. (a) HPLC chromatogram
of H-KALA-OH (0.5 mg/mL) treated with 5.0 mM of MG in the presence of 2.0 mM peroxynitrite in 200 mM phosphate buffer,
pH 7.2, at 25°C. Peaks 1and 2 correspond to the peptide H-KALA-OH and its acetyl radical adduct, respectively. (b) MS spectrum of
Peak 1; (c) MS/MS spectrum of Peak 1; (d) MS spectrum of Peak 2 (Ac-KALA-OH, m/z 444); (e) MS/MS spectrum of Peak 2.
Ϫ1
H2O) with flow rate of 1.0 mL min at 210 nm. The
ESI/MS analyses of purified samples
L-lysine-containing peptides were loaded onto
a
®
After purification and drying, the samples were reconsti-
tuted in 60% CH3CN/H2O containing 0.09% TFA in
preparation for MS analysis. Analyses were performed
by infusing the sample solution into the mass spectrom-
Symmetry C18 column (5 μm, 100 Å, 4.6ϫ250 mm);
elution was done with a linear gradient of 5–95% of
Solvent B (60% CH3CN/H2O containing 0.09% TFA) in
Solvent A for 30 min, at a wavelength of 210 nm and
flow rate of 1 mL/min. Calibration curves were linear
(r2 ϭ0.9995) across the range of 3–300 μM (nϭ2). The
fraction containing the desired products was collected,
Ϫ1
eter at a flow rate of 5.0 μL min using a syringe
pump (Hamilton Co.). Data were processed with
™
DataAnalysis software (Version 3.2, Bruker Daltonics).
The MS and MSn analyses involved an electrospray volt-
age of 4.5 kV, collision energy 20 kV, capillary tempera-
ture of 220°C, 5 micro scans, and an accumulation time
®
lyophilized under a refrigerated vacuum (Modulyo 4K
™
Freeze Dryer, Edwards High vacuum pump) and stored
at Ϫ80°C.

364 R. Tokikawa et al.
Figure 8. HPLC and MS analyses of the reaction of peptide H-KALA-OH with the MG/peroxynitrite system. (a) HPLC chromatogram of
H-KALA-OH (0.5 mg/mL) treated with 5.0 mM DA in the presence of 2.0 mM peroxynitrite in 200 mM phosphate buffer, pH 7.2, at 25°C.
Peaks 1, 2, and 3 correspond to the peptide H-KALA-OH (b), its formyl radical adduct (c), and its acetyl radical adduct (f), respectively;
(b, c) MS spectrum of Peaks 1 and 2; (d, e) MS/MS and MS3 spectra of Peak 2; (f) MS spectrum of Peak 3 (Ac-KALA-OH).
of 200 ms. Nitrogen was used as a nebulizer and sweep
gas and helium for the fragmentation process.
β-cleavage of the latter radical (Figure 1). Accordingly,
acetate and acetylated L-His or dGuo were detected in the
respective spent reaction mixtures, using LC/MS analysis
in the SRM mode. Next, εN-acetylated L-Lys was obtained
by treating this amino acid with MG upon the addition
of peroxynitrite, using the CE/MS technique [33].
Results and discussion
Massari et al. [32] reported that added L-His or dGuo
compete with dissolved oxygen for the acetyl radical
formed during the treatment of DA with peroxynitrite in
phosphate buffered solution. Acetyl radical originates
from homolysis of a peroxynitrosodiacetyl adduct inter-
mediate yielding an oxylcarbonyl radical, followed by
Using pre-desalted samples in RP-HPLC/ESI/
MSn analyses, we demonstrate below that the DA/
peroxynitrite “reagent” is indeed capable of acetylating
ε
αN- and N-amino groups of Lys derivatives and of
L-Lys-containing tetrapeptides, namely H-KALA-OH,
Ac-KALA-OH, and H-K(Boc)ALA-OH, and that both

Radical acylation of peptides by a-dicarbonyls 365
Figure 9. pH profile of the generation of Ac-KALA-OH by radical acetylation of the peptide H-KALA-OH by the DA or MG/peroxynitrite
systems. H-KALA-OH (0.5 mg/mL) treated with 2.0 mM peroxynitrite in the presence of (a) 10.0 mM DA or (b) 10.0 mM of MG in
200 mM phosphate buffer, pH range of 6.2–8.2, at 25°C. The synthesized peptide Ac-KALA-OH was used as standard and the samples
were analyzed by HPLC.
ε
εN-acetyl- and N-formyl-lysine adducts of Ac-Lys-OMe
are formed concomitantly when MG is used as the acyl
radical donor, the α-amino group being preferentially
acylated.
form, CH3CO-CH(OH)2 [30], it is conceivable that a cer-
tain fraction of peroxynitrite may be attacked at C-2,
whose electrophilic character is exacerbated by the induc-
tive effect of two geminal hydroxyl groups (Figure 6).
Accordingly, an additional peak assignable to the
formyl-adduct Ac-Lys(For)-OMe (m/z 231.1), inferred
from a mass increment of 28 units, was observed in the
Ac-Lys-OMe-containing reaction mixture of peroxyni-
trite with MG (Figure 4b). The corresponding formylated
product, Z-Lys(For)-OMe (m/z 323.2), was also obtained
from the Z-Lys-OMe-containing reaction mixture of per-
oxynitrite with MG (Figure 5c). In this case, both acety-
lated and formylated products were found to co-elute, as
illustrated in Figure 5a. The Ac-Lys(For)-OMe and
Z-Lys-(For)-OMe products have m/z 231.1 and m/z 323.2,
respectively (Figures 4b and 5c). The same fragmenta-
tion profiles were observed for formyl against acetyl
adducts. The ion at m/z 112 (Figure 5d) is assignable to
εN-amino group radical acylation in Ac-Lys-OMe and
Z-Lys-OMe by the peroxynitrite/DA or MG systems
ε
Radical acetylation of N-lysine by the peroxynitrite/DA
(Figures 2 and 3) or MG (Figures 4 and 5) systems in the
Ac-Lys-OMe and Z-Lys-OMe are indicated by a mass
increment of 42 u. The singly charged ions at m/z 203.1
and 245.1 thus represent Ac-Lys-OMe and α,εN-Ac-
Lys-OMe, respectively (Figure 2b and d). Conversely, the
native Z-Lys-OMe and its NεH2 acetylated form have
m/z 295.1 and 337.2, respectively (Figure 3b and d).
ϩ
As depicted in Figure 2e, the protonated form [MϩH ]
of the Ac-Lys(Ac)-OMe adduct undergoes methoxyl
loss to yield the fragment at m/z 213, whose decarbonyla-
tion forms the m/z 185 ion. Subsequent loss of the amino-
acyl group originates the m/z 126 fragment. Fragmentation
of Ac-Lys-OMe (Figure 2c) creates the m/z 186 ion by
deamination and the m/z 171 ion by methoxyl loss,
whose subsequent loss of acetyl group generates the
m/z 129 ion. The ion at m/z 126 is assignable to
εN-acetyllysine [43] and those with m/z 171 and 129 are
ε
formylation at NH2 of lysine and the ion at m/z 126
results from NεH2 acetylated lysine ion. These findings
raise the question as to whether protein lysine residues
could be formylated in vivo by a radical reaction mecha-
nism involving MG and a peroxide, whose occurrence
has never been reported in the biomedical literature.
Radical acylation of H-KALA-OH by the peroxynitrite/
DA or MG system
α
attributable to N-acetyllysine [44]. This was confirmed
by MS fragmentation analysis of Ac-Lys-OH and
H-Lys(Ac)-OH standards (data not shown).
Lysine-containing tetrapeptides were synthesized to guide
the peroxynitrite/DA or MG-promoted acylation toward
When inspecting the MG/peroxynitrite reaction for
acetyl and formyl radical formation by EPR spin trapping
with MNP [33], prevalent formation of acetyl radical
due to preferential peroxynitrite addition to the MG C-1
was expected. In fact, MNP-formyl radical adduct
formation was not detected. This failure was later clarified
when studying the reaction of glyoxal with peroxynitrite:
MNP-formyl radical adduct intermediate proved to be so
unstable that it was detectable only when running constant
flow EPR experiments [34]. However, knowing that c.a.
60% of MG exists in aqueous solution in its monohydrated
ε
the αNH2 or NH2 amino acid groups. Stepwise solid-
phase syntheses and RP-HPLC purifications were straight-
forward in our experimental conditions. The purity of the
final tetrapeptides, determined by RP-HPLC and LC/
ESI-MS, exceeded 90%. Their physicochemical properties
were in perfect agreement with the theoretical data. For
example, purified H-KALA-OH, Ac-KALA-OH, and
H-K(Boc)ALA-OH presented retention times and m/z val-
ues of 8.2 min and 402.2, 9.6 min and 444.2, and 16.9 min
and 502, respectively, in similar separation conditions.

366 R. Tokikawa et al.
Figure 10. HPLC and MS analyses of the reaction of peptide Ac-KALA-OH with the DA/peroxynitrite system. (a) HPLC chromatogram
of Ac-KALA-OH (0.5 mg/mL) treated with 5.0 mM DA in the presence of 2.0 mM peroxynitrite in 200 mM phosphate buffer, pH 7.2,
at 25°C. Peaks 1 and 2 correspond to the peptide Ac-KALA-OH and the acetyl radical adduct Ac-K(Ac)ALA-OH, respectively.
(b, c) MS and MS/MS spectra of Peak 1; (d–f) MS, MS/MS and MS3 spectra of Peak 2.
The major product obtained from modification of the
peptide H-KALA-OH (m/z 402; Figure 7b) by the DA/
peroxynitrite system was αNH2 acetylated lysine
(Ac-KALA-OH, m/z 444 Figure 7d). Small amounts of
di-acetylated H-KALA-OH at both NαH2 and NεH2 groups
were detected in the LC-MS experiments (data not
shown). The ions observed at m/z 84 and 129 attest to the
tetrapeptide lysine residue (intense acylium ion) [44]
(Figure 7c).
confirm the acylation sites of the modified peptides. The
fragmentation analysis of the Acyl-KALA-OH adducts
revealed formation of bn-ion types. In addition, acyl, car-
bonyl, and water losses from b-ions, and the internal cleav-
age b1y1, were corroborated by the formation of the
respective fragments (Figures 7c, e, and 8d). Presence of
an αN-acetyllysine adduct is supported by the ions at m/z
171 and 129 (Figure 7e). Substitution of DA for MG in
the reaction mixture containing H-KALA-OH led to the
formation of For-KALA-OH (m/z 430), with formylation
occurring in the αN-lysine residue, as denoted by the ions
at m/z 157 and 129 (Figure 8d). Thus, the DA or MG/
The Ac-KALA-OH adduct was also identified in the
reaction mixture obtained from H-KALA-OH and MG/
peroxynitrite (Figure 8f). MS/MS and MS3 analyses of
both DA and MG acyl radical donors were performed to
α
peroxynitrite system can modify the N-amino group of

Radical acylation of peptides by a-dicarbonyls 367
Figure 11. HPLC and MS analyses of the reaction of peptide Ac-KALA-OH with the MG/peroxynitrite system. (a) HPLC chromatogram
of Ac-KALA-OH (0.5 mg/mL) treated with 5.0 mM of MG in the presence of 2.0 mM peroxynitrite in 200 mM phosphate buffer,
pH 7.2, at 25°C. Peak 1 corresponds to the peptide Ac-KALA-OH (b). Peak 2 refers to the mixture of its formyl (c, m/z 472.3) and acetyl
radical adducts (c, m/z 486.0). (d,e) MS/MS and MS3 spectra of Peak 2, Ac-K(For)ALA-OH. (f) MS/MS spectrum of Peak 2, Ac-K(Ac)
ALA-OH.
α
[45]; (ii) the increase in NH2/εNH2 peptide deprotona-
tion (H-KALA-OH pI ϭ 10.1; peptide property calcula-
tor, Innovagen), both influencing the graphic ascending
portion; and (iii) the decrease of the concentration of
the peptide H-KALA-OH by the addition of acetyl radical
or acetyl plus formyl radicals, respectively. These data
once more endorse the addition of peroxynitrite nucleo-
philic to both carbonyl groups of MG, although mostly at
C-1 (Figures 1 and 6).
H2PO4̵ (pKa ϭ 7.2) depressing the yield of acetylated
product at pH above 7.5–8.0, since the dihydrogen
phosphate anion catalyzes nucleophilic additions to car-
bonyls [1]. Thus, the bell-shaped pH profiles of the reac-
tion of H-KALA-OH with DA and MG peak at
approximately 8.0 (Figure 9a and b, respectively). On
the other hand, the lower yields of acetylated H-
KALA-OH obtained from MG than from those of DA
(Figure 9) may reflect the opposite effect of the higher
degree of hydration of MG, resulting in a lower concen-
tration of its “free” reactive aldehyde form (6%), while
that of the diketone DA is considerably higher (24%),
but much less reactive toward peroxynitrite than MG
[1–3].
Effect of pH on H-KALA-OH radical acetylation
by the DA or MG/peroxynitrite system
The yield of radical acetylation of H-KALA-OH by the
DA or MG/peroxynitrite system in phosphate buffer,
with pH ranging from 6.2 to 8.2 and monitored by RP-
HPLC at 210 nm, revealed a roughly bell-shaped
response (Figure 9) like that observed previously in a
study of the kinetics of peroxynitrite addition to
DA [32]. This scenario may result from at least three
events: (i) the increase of peroxynitrite anion concentra-
tion by raising the pH (peroxynitrous acid pKa ϭ 6.8)

368 R. Tokikawa et al.
Figure 12. (a) Fragmentation pathway suggested for the acetylated and formylated Ac-Lys-OMe through the α-amino-ε-caprolactam
ion at m/z 129. The lysine residue is confirmed by the ion observed at m/z 84 [44] and the fragments with m/z 126 and m/z 112 are
ε
ε
marker ions for N- acetylation [49] and N-formylation of lysine, respectively. (b) Schematic fragmentation diagram of H-KALA-OH
protonated peptide. bn-Ion types predominate over daughter ions formed by loss of water, ammonia, acyl group or CO (an-ions) and by
internal cleavage (b1y1).
Radical acylation of Ac-KALA-OH by the peroxynitrite/
DA or MG system
their cleavage into smaller ones via the acetyl group, car-
bonyl group, and water losses. Internal cleavage the
b1y1 fragment was also observed. Besides acetylation of
The free NεH2 group of Ac-KALA-OH is acetylated by
the peroxynitrite/DA and MG systems. The HPLC chro-
matogram of the reaction mixture of Ac-KALA-OH
(m/z 444.2) treated with peroxynitrite/DA and the mass
analysis of the reaction product (Figure 10b) support
the formation of the acetyl adduct Ac-K(Ac)-ALA-OH
(m/z 486.3, and fragments 126, and 84). The fragments
depicted in Figure 10 correspond to b-ion series and to
ε
the NH2 group of peptide Ac-KALA-OH, the nitrogen
atom was also formylated by the peroxynitrite/MG
system (Figure 11c). Accordingly, the MS/MS experi-
ments of Ac-K(For)-ALA-OH (m/z 472.3) adduct show
the more intense fragments of the b-ion series. Moreover,
the MS3 spectrum shows the ion at m/z 112 (Figure 11e),
thus supporting the presence of an εN-formyllysine
adduct. These findings are schematized by Figure 12a, b,

Radical acylation of peptides by a-dicarbonyls 369
which depicts the fragmentation route of the modified
peptides.
pathophysiology of several disorders notably diabetes
[12,47,48], the reaction of acyl radical-generated
reactions of α-dicarbonyls with peroxynitrite awaits
further investigation in animal models.
Notably, more accurate mass determination should be
used in studies of complex peptide sequences, considering
that GL-, GI-, LG-, IG-, and AV-ion type fragments may
ε
exhibit the same marker ion formed from N-acetyllysine
Declaration of interest
(m/z 126). Additionally, one should keep in mind that
trimethylation of peptides must be proven to be unequivo-
cal to acetylation, since these processes lead to proximate
mass increments (44 vs. 42 u) [43].
The authors report no declarations interest. The authors
alone are responsible for the content and writing of the
paper.
This work was supported by the Brazilian research
funding agencies FAPESP (São Paulo Research
Foundation, Grants numbers 2006/56530-4 to EJHB and
2008/11695-1 to MTM), CAPES (Federal Agency for the
Support and Improvement of Higher Education), CNPq
(National Council for Scientific and Technological
Development), and INCT Redoxoma (National Institute
of Science and Technology of Redox Processes in
Biomedicine). The first author (R.T.) developed this work
for obtaining a PhD degree.
Radical acetylation of H-K(Boc)ALA-OH by the
peroxynitrite/DA or MG system
The peptide H-K(Boc)ALA-OH (m/z 502.3) had the
εN-acetylated by the peroxynitrite/DA and MG systems.
α
The N-acetyllysine adduct, m/z 544, undergoes loss of
Boc protecting group, forming the Ac-KALA-OH with
m/z 444 (data not shown). Subsequent fragmentation
followed the same profile observed for Ac-KALA-OH
(Figure 7d, e). The αN-formyllysine adduct was detected,
α
in addition to the N-acetyl derivative, when H-K(Boc)
ALA-OH was treated with the peroxynitrite/MG system.
The MS/MS fragmentation spectrum of the formylated
adduct (m/z 530) displayed For-KALA-OH with m/z
430 possibly formed from the loss of Boc (data not
shown). Further fragmentation follows the same profile
observed for For-KALA-OH (Figure 8d, e).
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