Formation of arginine modifications in a model system of N α- Tert -butoxycarbonyl (Boc)-arginine with methylglyoxal

Article abstract of DOI:10.1021/jf103116c

The present study deals with the mechanistic reaction pathway of the α-dicarbonyl compound methylglyoxal with the guanidino group of arginine. Eight products were formed from the reaction of methylglyoxal with N ^α-tert-butoxycarbonyl (Boc)-arginine under physiological conditions (pH 7.4 and 37 °C). Isolation and purification of substances were achieved using cation-exchange chromatography and preparative high-performance liquid chromatography (HPLC). Structures were verified by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry. 2-Amino-5-(2-amino-4- hydro-4-methyl-5-imidazolinone-1-yl)pentanoic acid (3) was determined as the key intermediate precursor within the total reaction scheme. Kinetic studies identified N^--(5-methyl-4-oxo-5-hydroimidazolinone-2-yl)-l-ornithine and N⁷-carboxyethylarginine as thermodynamically more stable products from compound 3. Further mechanistic investigations revealed an acidic hydrogen at C-8 of compound 3 to trigger aldol condensations. This reactivity of compound 3 allowed for the addition of another molecule of methylglyoxal to form products, such as N^--(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6- tetrahydropyrimidine-2-yl)-l-ornithine and argpyrimidine.

Full text of DOI:10.1021/jf103116c

394 J. Agric. Food Chem. 2011, 59, 394–401
DOI:10.1021/jf103116c
Formation of Arginine Modifications in a Model System
r
of N -tert-Butoxycarbonyl (Boc)-Arginine with Methylglyoxal
¨
A
NTJE
K
LOPFER, ROBERT
S
PANNEBERG
,
AND
MARCUS A. GLOMB*
Institute of Chemistry, Food Chemistry, Martin-Luther-University Halle-Wittenberg,
Kurt-Mothes-Strasse 2, 06120 Halle/Saale, Germany
The present study deals with the mechanistic reaction pathway of the R-dicarbonyl compound methyl-
glyoxal with the guanidino group of arginine. Eight products were formed from the reaction of methyl-
R
glyoxal with N -tert-butoxycarbonyl (Boc)-arginine under physiological conditions (pH 7.4 and 37 °C).
Isolation and purification of substances were achieved using cation-exchange chromatography and
preparative high-performance liquid chromatography (HPLC). Structures were verified by nuclear magnetic
resonance (NMR) and high-resolution mass spectrometry. 2-Amino-5-(2-amino-4-hydro-4-methyl-5-imida-
zolinone-1-yl)pentanoic acid (3) was determined as the key intermediate precursor within the total reaction
δ
7
scheme. Kinetic studies identified N -(5-methyl-4-oxo-5-hydroimidazolinone-2-yl)-L-ornithine and N -carbox-
yethylarginine as thermodynamically more stable products from compound 3. Further mechanistic investi-
gations revealed an acidic hydrogen at C-8 of compound 3 to trigger aldol condensations. This reactivity
of compound 3 allowed for the addition of another molecule of methylglyoxal to form products, such as
δ
N -(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-ornithine and argpyrimidine.
7
KEYWORDS: Methylglyoxal-arginine modifications; Maillard reaction; N -(1-carboxyethyl)arginine;
-amino-5-(2-amino-4-hydro-4-methyl-5-imidazolinone-1-yl)pentanoic acid
2
INTRODUCTION
described as 2-ammonio-6-({2-[(4-ammonio-5-oxido-5-oxopentyl)-
amino]-4-methyl-4,5-dihydro-1H-imidazol-5-ylidene}-amino)-
hexanoate (MODIC) (11). From the reaction of MGO with the
guanidino group of arginine, six products have been established in
Reactive R-dicarbonyl compounds, such as methylglyoxal
MGO), formed in early stagesof the Maillard reaction are potent
(
protein modifiers. Quantification of MGO was performed in
foods and also in in vivo samples. In coffee, wine, and apple juice,
levels up to 25 ppm were analyzed (1). Very high amounts of
MGO (761 ppm) were measured in honey samples from New
Zealand (2). Blood levels of diabetic patients demonstrate the
physiological relevance of MGO. In contrast to normal controls
the literature. Henle et al. identified protein-bound imidazolinone
δ
N -(5-methyl-4-oxo-5-hydroimidazolinone-2-yl)-
L
-ornithine with
δ
N exocyclic (MG-H1) (2) (12). Two other imidazolinone structures,
-amino-5-(2-amino-5-hydro-5-methyl-4-imidazolon-1-yl)pentanoic
2
acid (MG-H2) and 2-amino-5-(2-amino-4-hydro-4-methyl-5-
δ
imidazolon-1-yl)pentanoic acid (MG-H3) (3) with N endocyclic,
(
256 ( 92 nM), concentrations of 479 ( 49 nM were found (3).
were synthesized and characterized by Ahmed et al. (13). The
Five mechanisms of formation have been elucidated for MGO.
7
open-chain compound N -(1-carboxyethyl)arginine (6) was synthe-
An important source is the Maillard reaction of reducing sugars
with amines. At early stages, dicarbonyl is formed during degra-
dation of fructosamine or the reverse aldol reaction of 3-deoxy-
glucosone (4). In vivo, both the enzymatic and non-enzymatic
elimination of phosphate from glycerine-3-phosphate or di-
hydroxyacetonphosphate is a main step for the generation of
MGO (5, 6). Furthermore, cytochrome P450 2E1 catalyzes the
formation from acetone and represents an additional physiolo-
gical source(7). Another formation pathwayis described fromthe
oxidation of aminoacetone during the catabolism of threonine (8).
As a highly reactive R-dicarbonyl compound, MGO reacts
sized under high hydrostatic pressure by Alt and Schieberle (13).
Products generated from two molecules, MGO and arginine, are
δ
N -(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydro-
pyrimidine-2-yl)-L-ornithine (1) (14) and argpyrimidine (5) (15).
In the literature, little is known about the detailed formation
pathways of MGO-arginine modifications and their exact mechan-
istic relationship. Therefore, we started to reinvestigate this aspect of
the Maillard reaction, including isolation and characterization of the
key players, and separate incubations of isolated structures. With
respect to the many structures and countless different incubation
setups published in the literature and to compare results with
previous investigations of our working group on glyoxal-arginine
reactions, a simple model system of equimolar concentrations of
ε
with the ε-amino group of lysine to form N -(carboxyethyl)lysine
(CEL) (9) and the imidazolium cross-link 2-ammonio-6-[1-(5-
ammonio-6-oxido-6-oxohexyl)-5-methylimidazolium-3-yl]-
hexanoate (MOLD) (10). The reaction of MGO with both
arginine and lysine leads to a cross-link structure that has been
R
N -tert-butoxycarbonyl (Boc)-arginine and MGO was used.
MATERIALS AND METHODS
*
To whom correspondence should be addressed. Fax: þþ49-345-
Materials. Chemicals of the highest quality available were obtained
from Aldrich (Taufkirchen, Germany), Fluka (Taufkirchen, Germany),
5
527341. E-mail: marcus.glomb@chemie.uni-halle.de.
pubs.acs.org/JAFC
Published on Web 12/02/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 59, No. 1, 2011 395
Merck (Darmstadt, Germany), Roth (Karlsruhe), and Sigma (Taufkirchen,
Germany), unless otherwise indicated.
(400 MHz, CD
3
OD) δ: 171.23 (C-1), 52.20 (C-2), 26.63 (C-3), 22.81 (C-4),
38.97 (C-5), 157.43 (C-6), 176.46 (C-7), 66.01 (C-8), 16.91 (C-9), 64.48 (C-10).
Synthesis. Substances 1, 2, 3, 4, 6, 7, and 8 were isolated from
incubations of 220 μL (1.4 mmol) MGO (40% commercial solution)
Accurate mass (mean of eight measurements ( standard deviation)
þ
m/z: [M þ H] 259.1412 ( 0.0010 (259.1414, calcd for C10
19 4 4
H N O ).
and 213 mg (1.2 mmol)
L-arginine in 10 mL phosphate buffer (0.2 M, pH
Model Reactions. In general, experiments were conducted in 0.2 M
7
.4). Reaction mixtures were incubated for 4.5 h at 40 °C. Subsequently,
phosphate buffer.
R
the pH was adjusted to 4.0 with 1 N HCl. A total of 8 mL of the solution
was adjusted to pH 1 with 1 N HCl and subjected to ion-exchange
chromatography. Fractions were collected and analyzed by thin-layer
chromatography (TLC) and analytical high-performance liquid chroma-
tography (HPLC). After removal of solvents, residues were dissolved in
N -t-Boc-Arginine-MGO Incubations. For mechanistic investiga-
tions, incubations of 20 mM MGO (commercial and synthetic) with
R
20 mM N -t-Boc-arginine were performed at pH 5.0, 7.4, and 8.5 at
37 °C. To stop the reaction and for removal of the protection group,
aliquots of a well-defined volume were taken at several times and the same
volume of 6 N HCl was added. After 30 min, the samples were diluted for
HPLC analysis.
2
H O and subjected to preparative HPLC. Desired substances were
collected and freeze-dried (freeze-drying device, VirTis Benchtop SLC,
Warminster, PA). Substance 5 (argpyrimidine) was synthesized according
to Shipanova et al. (15).
Incubation of Isolated Structures. Further information about the
mechanisms was revealed by incubation of the separated compounds at
pH 5.0, 7.4, and 8.5 at 37 °C. Structures were dissolved in phosphate
buffer. To stop the reaction, samples were diluted with 0.05 M HCl and
analyzed by HPLC and post-column derivatization.
MGO. To eliminate side reactions between products of MGO and
arginine with contaminants in the commercial MGO solution, it was
necessary to synthesizehighly purified MGO. The synthesis was based ona
method by McLellan and Thornalley (16). A total of 5 mL (41.31 mmol)
of pyruvic aldehyde dimethyl acetal (97%) was dissolved in 100 mL of
aqueous sulfuric acid (5%, v/v) and heated over 1 h in a boiling water bath.
Subsequently, the mixture was distilled fractionally on a 34 ꢀ 1.5 cm inner
diameter column under reduced pressure (30 mbar) and nitrogen bleed.
Three fractions (boiling point of the azeotrope = 26 °C) werecollected and
filled up to 100 mL. The concentrations were determined by derivatization
with 1,2-phenylenediamine and HPLC analysis.
Incubations of Isolated Structures with Carbonyl and Dicarbonyl
Compounds. Mechanistic insights were obtained from incubation of com-
pounds 2 and 3 with formaldehyde or MGO. Both structures (5 mM) were
incubated at pH 5.0, 7.4, and 8.5 at 37 °C with 2.5 mM formaldehyde
or MGO. The reaction was stopped by diluting the samples for HPLC
analysis with 0.05 M HCl.
Statistics. Data shown for model reactions resulted from at least three
independent incubations and gave coefficients of variations <5%.
Chromatography. TLC was performed on silica gel 60 F254 plates
δ
N -(4-Carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrim-
idine-2-yl)-
with material having R
8.6 min, isocratic elution with 2% solvent B) and the procedure was
L
-ornithine (1). After ion-exchange chromatography, fractions
2
(Merck) with 4:2:3:3 n-butanol/H O/HOAc/pyridine as the mobile phase.
= 0.33 were verified by analytical HPLC (t
R
=
Visualization of separated material was achieved with ninhydrin.
Ion-exchange chromatography was performed on Dowex 50WX4-400
f
1
þ
continued as described above. Preparative HPLC and subsequent freeze-
drying gave a colorless amorphic material (71.55 mg, 14.2%, 1 þ 2HFBA
salt based on elemental analysis). The chemical structure was confirmed by
(2.5 cm inner diameter ꢀ 22 cm, H form). Before conditioning with
pyridinium formiate (0.2 M, pH 5.0), the column was washed with 1 M
NaOH, 1 M HCl, and water.
1
13
H and C nuclear magnetic resonance (NMR), and results were similar
Solvents were all chromatographic-grade. From the individual chro-
matographic fractions, solvents were removed under reduced pressure.
HPLC. Analytical System. A Jasco (Gross-Umstadt, Germany)
ternary gradient unit 980-PU-ND with degasser DG-980-50, autosampler
to those from Oya et al. (14).
Accurate mass (mean of three measurements ( standard deviation)
þ
m/z: [M þ H] 319.1614 ( 0.0006 (319.1612, calcd for C12
23 4 6
H N O ).
δ
8
51-AS, column oven set at 20 °C, and fluorescence detector FP-920 was
N -(5-Methyl-4-oxo-5-hydroimidazolon-2-yl)-
L
-ornithine (2). After
= 0.48
= 72.7 min, isocratic elution with
% solvent B) and the procedure was continued as described above.
used. Chromatographic separations were performed on stainless-steel
columns (YMC Hydrosphere C18, 250 ꢀ 4.6 mm, Dinslaken, Germany)
using a flow rate of 1.0 mL/min. The mobile phase used was water (solvent
A) and MeOH/water (7:3, v/v; solvent B). To both solvents (A and B),
1.2 mL/L heptafluorobutyric acid (HFBA) was added. Samples were
injected at 2% B and run isocratic for 80 min, and then the gradient
changed to 100% B in 5 min and held at 100% for 20 min. For better
separation, the ratio of solvent B was decreased to 2%. Samples were
analyzed isocratic as well. The fluorescence detector was attuned to 340 nm
for excitation and 455 nmfor emission. Prior, a post-column derivatization
reagent was added at 0.5 mL/min. This reagent consisted of 0.8 g of
o-phthaldialdehyde, 24.73 g of boric acid, 2 mL of 2-mercaptoethanol, and
ion-exchange chromatography, fractions with material having R
were verified by analytical HPLC (t
f
R
2
Preparative HPLC and subsequent freeze-drying gave a colorless amor-
phic material (49.22 mg, 11.6%, 2 þ 2HFBA salt based on elemental
1
13
analysis). The chemical structure was confirmed by H and C NMR and
high-resolution mass spectrometry. The results were similar to those from
Henle et al. (12).
Accurate mass (mean of three measurements ( standard deviation)
þ
m/z: [M þ H] 229.1314 ( 0.0007 (229.1312, calcd for C
9 17 4 3
H N O ).
2
-Amino-5-(2-amino-4-hydro-4-methyl-5-imidazolinone-1-yl)pentanoic
Acid (3). After ion-exchange chromatography, fractions with material
having R = 0.47 were verified by analytical HPLC (t = 74.4 and
8.2 min, isocratic elution with 2% solvent B) and the procedure was
continued as described above. A colorless amorphic material (25.67 mg,
1.1%, 3 þ 2HFBA salt based on elemental analysis) was obtained by
preparative HPLC and subsequent freeze-drying. The chemical structure
2
1 g of Brij35 in 1 L of H O adjusted to pH 9.75 with KOH.
f
R
7
For determination of argpyrimidine (5), samples were measured with-
out post-column derivatization after separation by a gradient chromatog-
raphy program (7% B changed in 60 min to 90% B). The fluorescence
detector was set to 320 nm for excitation and 380 nm for emission.
Preparative System. A Besta HD 2-200 pump (Wilhelmsfeld, Germany)
with a Gynkotek fluorescence detector RF-530 and a SERVOGOR 220
pen recorder was used. Chromatographic separations were performed on a
stainless-steel column (VYDAC 218TP1022, 250 ꢀ 25 mm, RP18, 10 μm)
using a flow rate of 15 mL/min. The mobile phase was identical to the
analytical HPLC system, using a composition of 5% solvent B and
isocratic gradient. The effluent was split and adjusted to a post-column
derivatization, as described under the analytical HPLC system. Fractions
with material were collected, combined, and freeze-dried as described
under Synthesis.
1
1
13
was confirmed by H and C NMR and high-resolution mass spectro-
metry. NMR data gave similar results to those from Ahmed et al. (13).
Accurate mass (mean of three measurements ( standard deviation)
þ
m/z: [M þ H] 229.1301 ( 0.0008 (229.1299, calcd for C
9 17 4 3
H N O ).
2
-Amino-5-(2-amino-4-methyl-4-(methyl-ol)-5-imidazolinone-1-yl)-
pentanoic Acid (4). After ion-exchange chromatography, fractions with
material having R = 0.50 were verified by analytical HPLC (t = 89.9
f
R
min, isocratic elution with 2% solvent B) and the procedure was continued
as described above. Preparative HPLC and subsequent freeze-drying gave
a colorless amorphic material (60.00 mg, 12.5%, 4 þ 2HFBA salt based on
1
13
C
elemental analysis). The chemical structure was confirmed by H and
HPLC-Electrospray Ionization-Mass Spectrometry (HPLC-
ESI-MS). As described above, the same analytical HPLC system was
used to separate the different structures. The mass spectra were obtained
from an Applied Biosystems 4000 Q Trap linear ion trap quadrupole
LC/MS/MS with a Turbo Spray source at positive-ion mode. The source
NMR and high-resolution mass spectrometry.
1
3
H NMR (400 MHz, CD
.7-1.9 (m, 2H, H-3), 1.5-1.7 (m, 2H, H-4), 3.56 (t, J = 6.53 Hz,
H, H-5), 1.19 (s, 3H, H-9), 3.67, (t, J = 7.12 Hz, 2H, H-10). C NMR
3
OD) δ (ppm): 3.85 (t, J = 5.89 Hz, 1H, H-2),
3
1
2
3
13

396 J. Agric. Food Chem., Vol. 59, No. 1, 2011
Kl o¨ pfer et al.
The data unequivocally identified compound 1 as N -(4-carboxy-4,6-
dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)- -or-
nithine. Compound 2 was determined as N -(5-methyl-4-oxo-5-
hydroimidazolon-2-yl)- -ornithine. Compounds 3a and 3b are
δ
L
δ
L
two possible diasteriomeric isomers of 2-amino-5-(2-amino-4-
hydro-4-methyl-5-imidazolon-1-yl)pentanoic acid. Isolated com-
pound 4 was characterized as the diastereomeric aldol addition
3
products of compound 3 and formaldehyde. The J
corre-
H-5,C-7
δ
lation indicated a N -endocyclic ring structure. Moreover, corre-
3
lation J
showed the addition of formaldehyde at the C-8
H-10,C-7
position of compound 3. The assumption that this product was
formed from formaldehyde in commercially available MGO
solution was proved by an experiment with independent synthe-
sized MGO, as described by McLellan and Thornalley (16).
R
Incubation of purified MGO with N -t-Boc-arginine led to the
same product profile as shown in Figure 1A, except for compound
4
(Figure 1B). This result led to the conclusion that compound 4 is
an artifact formed by impurities of formaldehyde in the model
reaction system with commercial MGO.
Compound 5 was determined as argpyrimidine by HPLC with
fluorescence detection and a comparison to the synthesized
standard, as described by Shipanova et al. (15). Compound 6
7
was identified as N -carboxyethylarginine by HPLC-ESI-MS
analysis with m/z 247 [M þ 1].
HPLC-ESI-MS analysis revealed m/z 319 for both signals 7
and 8. Isolation of compounds 7 and 8 by ion-exchange chroma-
tography and purification by preparative HPLC was not success-
ful. HPLC of collected fractions always showed a 1:1 equilibrium
of both structures besides other unidentified material. Because
derivatization with o-phthaldialdehyde was reported to lead to a
comparable molar response in fluorometric analysis, quantitative
results for compounds 7 and 8 were obtained using a standard
solution of compound 1 as reference material.
Figure 1. HPLC chromatogram obtained with fluorescence detection
after post-column derivatization with o-phthaldialdehyde of incubation of
N -t-Boc-arginine (20 mM) with MGO (20 mM) after 24 h at 37 °C and
pH 7.4: (A) incubation with commercial MGO and (B) incubation with
synthesized MGO.
R
R
Reaction Time Course Study. Incubation of MGO with N -t-
Boc-arginine at pH 7.4 showed first rapid formation of compounds
1
, 2, 3, 4, and 6 (Figure 2) and then degradation. In contrast,
was maintained at a voltage of 3.5 kV and a nebulizer gas flow of 60 μL/
min with a declustering potential of 50 V. For MS/MS experiments, the
entrance potential of the collision cell was 9 V, the exit potential was 30 V,
and the collision energy was 33 V.
argpyrimidine (5) was formed slowly but continuously accumulated
during time. The unidentified structures 7and 8reached a maximum
at 6 h (1.4 mM) and were slowly degraded to give 0.4 mM at 336 h.
The pH dependency of the reaction was also investigated.
Selected results are given in Table 1. At pH 5.0, the stability of all
products was much higher (data not shown). In contrast to pH
7.4, more compound 3 than compound 2 was generated at lower
reaction rates (Table 1). At pH 8.5, degradation of all compounds
was faster and less compound 3 than compound 2 was formed.
From these results, it can be concluded that the formation of
compound 3 is preferred under acidic conditions, whereas the
generation of compound 2 occurs under basic conditions.
Diasteriomeric isomers of compound 3 were collected sep-
arately, and the stability of these compounds was studied in
separate incubations at pH 5.0, 7.4, and 8.5. Incubation of
enriched compound 3b at pH 7.4 yielded rapid formation of
compound 3a (panels A and B of Figure 3). After 0.5 h, a ratio
of 1:1 of both isomers was reached. At pH 5.0, the reaction was
slower and both isomers reached equilibrium only after 24 h (data
not shown). HPLC monitoring extended incubations of isolated
compound 3 demonstrated the formation of compounds 2 and 6
at pH 7.4 (Figure 4). This can be explained by opening the ring
structure to give compound 6 and the formation of the thermo-
dynamically more stable product 2. At pH 5.0, the reaction was
slow and yielded only low levels of compound 2 (0.4 mM after
144 h). These results were in line with the data presented in Table 1
and underline the higher stability of compound 3 under acidic
conditions, whereas ringopening and the formation of compound
2 occurs under basic conditions.
Accurate Mass Determination. For accurate mass determination, a
Micromass (Manchester, U.K.) VG platform II quadrupole mass spectro-
þ
meter equipped with an ESI interface was employed: ESI ; source
temperature, 80 °C; capillary, 3.0 kV; and cone voltage, 20 V. The data
were collected in the multichannel acquisition (MCA) mode with 128
channels per m/z unit using 13 scans (6 s) with 0.1 s reset time. The
resolution was 650 (10% valley definition). The sample was dissolved for
analysis in water/MeCN (1:1) containing poly(ethylene glycol) (PEG) 200
(
0.1 μg/μL) as the reference material, ammonium formate (0.1%), and
formic acid (0.2%). The sample concentration was similar to that of PEG
00. The solution was introduced into the ESI source at a flow of 5 μL/
2
μmin. With a m/z 190-285 scan range, five reference peaks could be used
for calibration: m/z 195.1234, 212.1498, 239.1495, 256.1760, and 283.1757.
NMR Spectroscopy. NMR spectra were recorded on Varian Unity
Inova-500 and Varian Gemini 2000 instruments (Darmstadt, Germany).
Chemical shifts are given relative to external Me
4
Si.
RESULTS
Isolation and Characterization of Products. In the reaction of
R
MGO with N -t-Boc-arginine, eight products were formed after
24 h (Figure 1A). Compounds 1, 2, 3, and 4 were isolated by ion-
exchange chromatography. Final purification was performed by
ion-pair chromatography on reversed-phase material. Structural
1
13
evidence was achieved by H and C NMR, as well as hetero-
nuclear multiple-quantum coherence (HMQC) and heteronuc-
lear multiple-bond correlation (HMBC) NMR experiments.
Accurate mass detection confirmed the chemical structures.

Article
J. Agric. Food Chem., Vol. 59, No. 1, 2011 397
Figure 2. Concentrations of important structures during the reaction of
R
N -t-Boc-arginine (20 mM) with MGO (20 mM) at 37 °C and pH 7.4: (9)
N -(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-
δ
δ
-ornithine (1), (2) N -(5-methyl-4-oxo-5-hydro-imidazolinone-2-
-ornithine (2), (1) 2-amino-5-(2-amino-4-hydro-4-methyl-5-imidazo-
2
yl)-
-yl)-
L
L
linone-1-yl)pentanoic acid (3), (f) argpyrimidine (5), and (b)
N -carboxyethylarginine (6).
7
Table 1. pH-Dependent Formation of Compounds 2 and 3 in Incubations of
N -t-Boc-Arginine (20 mM) with MGO (20 mM) at Maximum Concentration
R
and at the End of Incubation (336 h) at 37 °C
compound 2
compound 3
concentration (mM)
Figure 3. Incubation of enriched 2-amino-5-(2-amino-4-hydro-4-methyl-5-
imidazolinone-1-yl)pentanoic acid (3b) after (A) 0 h and (B) 0.5 h at 37 °C
and pH 7.4.
pH
time (h)
336
concentration (mM)
time (h)
5
7
.0
.4
0.95
336
1.44
24
2.92
1.90
6
336
2.42
0.09
3
36
8
.5
24
36
2.12
1.09
3
336
0.74
0.05
3
The formation of compound 4 was investigated by incubation
δ
of compound 3 with formaldehyde. Because of the N -endocyclic
ring structure of compound 4, an acidic hydrogen at C-8 of
compound 3 must be responsible for the addition of formalde-
hyde. As shown in Figure 5A, compound 3 was degraded very fast
to reach 0.6 mM after 24 h. In contrast, compound 4 was formed
with a maximum concentration after 6 h (1.9 mM). These findings
prove the hypothesis that the reaction of compound 3 and
formaldehyde leads to the aldol addition product 4. Reaction
products of compound 3 were also compounds 2 and 6, as
expected from the above separate incubation (Figure 4). Interest-
ingly, incubation of compound 2 and formaldehyde did not lead
to any aldol condensation product (Figure 5B). This suggests that
compound 2 does not carry an acidic hydrogen at C-8.
Figure 4. Degradation of compound 3 (6 mM) at 37 °C and pH 7.4:
δ
(
2) N -(5-methyl-4-oxo-5-hydro-imidazolinone-2-yl)-L-ornithine (2), (1)
The exclusive acidic hydrogen reactivity of compound 3 was
confirmed when compounds 3 and 2 were incubated separately
with MGO. As demonstrated in the model reaction with com-
pound 3, structures 1, 2, 5, 6, 7, and 8 were found (Figure 6). At
pH 7.4 and 37 °C, compound 3 was degraded very fast and
reached a minimum level after 24 h (0.4 mM), whereas the
concentration of compound 1 increased immediately and reached
2
(
-amino-5-(2-amino-4-hydro-4-methyl-5-imidazolinone-1-yl)pentanoic acid
7
3), and (b) N -carboxyethylarginine (6).
144 h. Compounds 7 and 8 reached a maximum level after 6 h
(1.1 and 1.2 mM, respectively) and decreased slowly. Their impact
on the total reaction scheme was investigated in separate experi-
ments described below.
2
.5 mM after 24 h. The formation of compounds 6 and 2 can be
Contrary to compound 3, compound 2 did not react with MGO.
The incubation of compound 2 with MGO (data not shown) was
comparable to the reaction of compound 2 with formaldehyde
(Figure 5B). There was a slight decrease of compound 2 and a
explained by preferred ring opening of compound 3 and the
formation of more stable products under these conditions.
Compound 5 was formed slowly, and 0.5 mM was found after

398 J. Agric. Food Chem., Vol. 59, No. 1, 2011
Kl o¨ pfer et al.
Figure 6. Incubation of compound 3 with MGO (2.5 mM) at 37 °C and pH
δ
7.4: (9) N -(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyr-
imidine-2-yl)-
δ
L-ornithine (1), (2) N -(5-methyl-4-oxo-5-hydro-imidazoli-
none-2-yl)- -ornithine (2), (1) 2-amino-5-(2-amino-4-hydro-4-methyl-5-
L
imidazolinone-1-yl)pentanoic acid (3), (f) argpyrimidine (5), and (b)
N -carboxyethylarginine (6).
7
Figure 5. Incubation of MGO-derived hydroimidazolinones with formalde-
hyde (2.5 mM) at 37 °C and pH 7.4: (A) incubation of compound 3 and
δ
B) incubation of compound 2, with (2) N -(5-methyl-4-oxo-5-hydro-
(
imidazolinone-2-yl)-
L-ornithine (2), (1) 2-amino-5-(2-amino-4-hydro-
-methyl-5-imidazolinone-1-yl)pentanoic acid (3), ([) 2-amino-5-(2-
4
amino-4-methyl-4-(methyl-ol)-5-imidazolinone-1-yl)pentanoic acid (4),
7
and (b) N -carboxyethylarginine (6).
small increase of compound 1 (0.2 mM after 24 h), i.e., much
lower compared to the reaction of compound 3 (Figure 6). All
other structures were only negligible. Neither compound 7 nor
compound 8 could be detected at all. Nevertheless, the generation
of compound 1 in this incubation can be explained by the
equilibrium between compounds 2 and 3, which entails ring
opening via compound 6 and allows for the addition of MGO
and ultimately the formation of compound 1. More precise infor-
mation about this reaction mechanism was obtained when iso-
lated compound 6 was incubated with MGO (Figure 7). Here,
compound 6 was elucidated as a direct precursor for compound 1,
because major portions of compound 6 (2.7 mM after 144 h) were
directly transformed into compound 1 (2.4 mM after 144 h).
In contrast to the incubation of compound 3 with MGO, there
were only small amounts of compounds 7 and 8 formed at later
incubation times (0.7 and 0.6 mM, respectively, after 144 h).
Figure 6 shows the generation of compound 5 from the incuba-
tion of compound 3 with MGO. The detailed reaction pathway
was elucidated by incubation of an isolated 1:1 mixture of
compounds 7 and 8. As demonstrated in Figure 8, degradation
of compounds 7 and 8 lead to the formation of compound 5 up to
Figure 7. Incubation of compound 6 with MGO (2.5 mM) at 37 °C and pH
7.4: (9) N -(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyr-
imidine-2-yl)-L-ornithine (1), (2) N -(5-methyl-4-oxo-5-hydro-imidazoli-
none-2-yl)-L-ornithine (2), (1) 2-amino-5-(2-amino-4-hydro-4-methyl-5-
δ
δ
imidazolinone-1-yl)pentanoic acid (3), (f) argpyrimidine (5), and (b)
N -carboxyethylarginine (6).
7
Incubation of isolated compound 1 at 37 °C and pH 7.4 rejected
this hypothesis. Compound 1 was stable under the incubation
conditions, and only traces of compound 5 were detectable (data
not shown).
DISCUSSION
Several MGO-derived arginine modifications were determined
in food and physiological samples (17-19), but little is known
about their mechanistic relationship. This gave us the motivation
to isolate and characterize the key players and pathways of this
reaction.
50% yield. The reaction was significantly slower under deaerated
conditions (data not shown). Therefore, both structures must be
assigned as direct precursors of compound 5.
From the present incubations of an equimolar mixture of
MGO with N -t-Boc-arginine, it can be concluded that argpyr-
R
Theoretically, because of the pyrimidine-based chemical structure,
compound 1 could be another possible precursor of compound 5.
imidine (5) is the single only advanced glycation end product of
the reaction at pH 7.4 accumulating during time. All other

Article
J. Agric. Food Chem., Vol. 59, No. 1, 2011 399
structures are intermediates and were degraded after 336 h
well (13) but could not be detected nor isolated in the present
study. In vitro incubations of MGO with N -t-Boc-arginine (20)
R
(
Figure 2). In general, the stability of generated products was
higher with decreasing pH (data not shown).
and lens protein (17), respectively, confirmed our results. In these
experiments, MG-H2 isomer concentrations were only very small
or not detectable. In contrast, in vivo investigations on glycated
proteins showed a positive correlation between structure 2 and
MG-H2 (17).
Compounds 2 (MG-H1) and 3 (MG-H3) are imidazolinone
structures, which have been published before (12, 13). A third
imidazolinone structure [2-amino-5-(2-amino-5-hydro-5-methyl-
4
-imidazolon-1-yl)pentanoic acid (MG-H2)] was described as
Because compound 3 plays a key role in the generation of all
other products, different experiments were focused on this
compound. As shown in Table 1, the formation and stability of
the imidazolinone structures 2 and 3 were pH-dependent. Under
acidic conditions, the generation of compound 3 was preferred. In
contrast, more compound 2 than compound 3 was formed with
increasing pH. The same result was found in separate incubations
of compound 3 at pH 5.0, 7.4, and 8.5. Under alkaline conditions,
compound 2 was formed from compound 3 via compound 6 by
opening the ring structure, whereas compound 3 was stable under
acidic conditions (data not shown). When these results are taken
together, they verified that first the reaction of MGO and arginine
kinetically controlled leads to rapid and direct formation of com-
pound 3. With a proceeding reaction time, ring opening results in
thermodynamically more stable products 2 and 6 (Figure 9),
which was further supported by an earlier maximum of formation
for compound 3 than for compounds 2 and 6 in MGO reactions
(
Figure 2). This reaction pathway was already suggested in the
literature (13), but no experiment was performed to prove this
assumption. The formation of compound 3 can only be explained
via a dihydroxyimidazolidine precursor. Indeed, in the early
course of arginine-MGO incubations, several short-lived inter-
mediates evolved but could not be isolated because of their
instability. Dihydroxyimidazolidine has already been described
for the reaction of arginine with glyoxal (21). However, in contrast,
Figure 8. Incubation of compounds 7 and 8 under aerated conditions at
δ
3
1
7 °C and pH 7.4: (9) N -(4-carboxy-4,6-dimethyl-5,6-dihydroxy-
δ
,4,5,6-tetrahydropyrimidine-2-yl)-
-ornithine (2), (f) argpyrimidine (5),
b) N -carboxyethylarginine (6), (half-filled diamond) compound 7, and
half-filled circle) compound 8.
L-ornithine (1), (2) N -(5-methyl-4-
oxo-5-hydro-imidazolinone-2-yl)-
(
(
L
7
Figure 9. Formation and reaction pathways of MGO-arginine modifications.

400 J. Agric. Food Chem., Vol. 59, No. 1, 2011
Kl o¨ pfer et al.
here, dihydroxyimidazolidine represents the initial kinetically
controlled intermediate ultimately leading to carboxymethylargi-
nine as the single, stable end product of the reaction. Lo et al. (22)
suggested that the reaction with MGO proceeds via the reversible
formation of glycosylamine and dihydroxyimidazolidine. How-
ever, this alternative pathway was postulated with an exocyclic N
of the arginine residue, comparable to structure 2, which is void
based on our data.
In physiological systems and foods, concentrations of MGO
are often far below the amount of arginine. Therefore, MGO
becomes a rate-limiting reactant, and the product spectrum will
shift toward the formation of modifications involving only one
molecule of the dicarbonyl compound. Another factor that might
influence the product ratio is that, at the high MGO concentra-
tions used herein, MGO monohydrate will be the preferred
reactive species (23). However, this should not change our above
general findings on reactivity and the mechanistic relationship of
specific structures within the total reaction scheme.
δ
Incubations of separated diastereomeric isomers 3 led to an
immediate equilibrium of both isomers at pH 7.4 (Figure 3). This
rapid racemization at neutral to basic pH can only be explained
by an acidic proton at C-8, enabling enolic intermediates. Alter-
native explanations via ring opening and closure were excluded
because no compound 6 could be detected at such early reaction
times. Further support was the identification of aldol product 4,
as an artifact from the reaction with formaldehyde in commer-
cially available MGO solutions. The notion that an acidic
position should also be true for compound 2 was not substan-
tiated by our experiments. Neither racemization nor aldol con-
densations were observed. Also, Henle et al. reported only one
diastereomeric isomer for the two possible tautomeric forms of
compound 2 (12).
The elucidation of the acidic C-8-H bond was a prerequisite to
understand the formation of pyrimidine structure 1. Incubation
of compound 3 with MGO immediately led to compound 1, the
rapid kinetic identical to the formation of compound 4 from
formaldehyde. In contrast, incubation of compound 6 with MGO
produced a much slower but continuous synthesis of compound
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