Oxidative deamination of benzylamine by glycoxidation

Article abstract of DOI:10.1016/S0968-0896(02)00584-9

In the present study, model reactions for the oxidative deamination by glycoxidation using benzylamine were undertaken to elucidate the detail of the reaction. Glucose, 3-deoxyglucosone (3-DG), and methylglyoxal (MG) oxidatively deaminated benzylamine to

Full text of DOI:10.1016/S0968-0896(02)00584-9

Bioorganic & Medicinal Chemistry 11 (2003) 1411–1417
Oxidative Deamination of Benzylamine by Glycoxidation
Mitsugu Akagawa, Takeshi Sasaki and Kyozo Suyama*
Department of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science,
Tohoku University, Sendai, 981-8555, Japan
Received 15 October 2002; accepted 14 November 2002
Abstract—In the present study, model reactions for the oxidative deamination by glycoxidation using benzylamine were undertaken
to elucidate the detail of the reaction. Glucose, 3-deoxyglucosone (3-DG), and methylglyoxal (MG) oxidatively deaminated
benzylamine to benzaldehyde in the presence of Cu2+ at a physiological pH and temperature but not glyoxal. 3-DG and MG were
more effective oxidants than glucose. We have determined the effects of metal ions, pH, oxygen, and radical scavengers on the
oxidative deamination. The formation of benzaldehyde was greatest with Cu2+, and was accelerated at a higher pH and in the
presence of oxygen. EDTA, catalase, and dimethyl sulfoxide significantly inhibited the oxidation, suggesting the participation of
reactive oxygen species. From these results, we propose a mechanism for the oxidative deamination by the Strecker-type reaction
and the reactive oxygen species-mediated oxidation during glycoxidation.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
with aging1,7À10 and the long-term complications of
The nonenzymatic glycation reaction (Maillard reaction)
is thought to contribute to the pathogenesis of diabetic
complications1À6 and the aging process.1,7À10 In this
reaction, free amino groups of protein react slowly with
the carbonyl groups of reducing sugars to yield Schiff base
intermediates, which then undergo Amadori rearrange-
ment to yeild stable ketoamine derivatives (Amadori pro-
ducts). Subsequently, the Schiff bases and Amadori
products degrade into a-dicarbonyl such as 3-deoxy-
glucosone (3-DG), methylglyoxal (MG), and glyoxal
(GO).11 These compounds are more reactive than the
parent sugars with respect to their ability to react with
amino groups of proteins. These a-dicarbonyl com-
pounds irreversibly modify lysine and arginine residues
in proteins at physiological conditions, leading to the
formation of various advanced glycation end products
(AGEs) which frequently have chromophores, fluoro-
phores, and protein cross-links.1,3À6,12,13 Recent researches
have demonstrated that the formation and accumulation
of AGEs in plasma and tissue proteins are associated
diabetes.1À6 Therefore, it has been recently proposed
that a-dicarbonyl stress is among the major factors in
the pathogenesis of diabetic complications. Moreover,
glycation can give rise to oxygen free radicals in the
presence of O2 and transition metal ions,14À17 and the
formation of some AGEs has been shown to require
oxygen free radicals.17À19 The production of oxidants by
the oxidation of glucose-protein adducts has been
termed glycoxidation.
Recently, we found that the level of a-aminoadipic-d-
semialdehyde, which is an oxidative deamination pro-
duct of lysine residue, in rat plasma protein is sig-
nificantly higher in streptozotocin-induced diabetic rats
compared with normal controls. Furthermore, we
demonstrated that the lysine residue of bovine serum
albumin (BSA) is oxidatively deaminated during the
incubation with various carbohydrates in the presence
of Cu2+ at a physiological pH and temperature.20 This
experiment showed that 3-DG and MG are the most
efficient oxidants of the lysine residue. When the reac-
tion was initiated from glucose, a significant amount of
a-aminoadipic-d-semialdehyde was also formed in the
presence of Cu2+. The reaction was significantly inhib-
ited by deoxygenation, catalase, and a hydroxyl radical
scavenger, suggesting the participation of the reactive
oxygen species in the reaction. From these results, we
Abbreviations: BSA, bovine serum albumin; 3-DG, 3-deoxyglucosone;
DMSO, dimethyl sulfoxide; EDTA, ethylendiaminetetraacetic; MG,
methylglyoxal; GO, glyoxal; HPLC, high performance liquid chroma-
tography.
*Corresponding author. Tel.: +81-22-717-8818; fax: +81-22-717-
8820; e-mail: suyama@bios.tohoku.ac.jp
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00584-9

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M. Akagawa et al. / Bioorg. Med. Chem. 11 (2003) 1411–1417
proposed the mechanism for the oxidative deamination
by the a-dicarbonyl-mediated Strecker-type reaction
and the reactive oxygen species-mediated oxidation
during glycoxidation. Furthermore, we hypothesized
that the formation of the highly reactive a-aminoadipic-
d-semialdehyde is involved in the development of dia-
betic complications.
explored. Figure 3 shows the results obtained when a
fixed amount of benzylamine (5.0 mM) and glucose, 3-
DG, or MG (1.0 mM) were incubated with various
concentrations of Cu2+ (0, 125, 250, and 500 mM) for 7
days. The formation of benzaldehyde was apparently
dependent on the concentration of Cu2+
.
To find the other factors governing the formation of
benzaldehyde, we examined the effect of pH on the
oxidative deamination. Reaction mixtures with pH
values varying from 6.0 to 10.0, containing benzylamine
(5.0 mM), CuSO4 (250 mM), and glucose, 3-DG, or MG
(1.0 mM) were incubated at 37 ꢀC for 7 days. As shown
in Figure 4, benzaldehyde was formed even faster in
basic solutions. Nevertheless, at pH 6, the oxidation was
In this research, to characterize the oxidative deami-
nation by glycoxidation, we carried out a model reac-
tion using benzylamine under a quasi-physiological
condition. A model glycoxidation between benzylamine
and carbohydrates in the presence of Cu2+ produced
significant amount of benzaldehyde. To determine the
participation of reactive oxygen species, we also investi-
gated the effect of radical scavengers. On the basis of
these data sets, we made a mechanistic interpretation.
Results
The oxidative deamination by glycoxidation was asses-
sed by the reaction of benzylamine with glucose, 3-DG,
MG, and GO. Benzylamine (5.0 mM) was incubated in
phosphate buffer (50 mM) with each carbohydrate (1.0
mM) under a physiological pH and temperature (pH
7.4, 37 ꢀC). The time course of the oxidative deamina-
tion of benzylamine was monitored by the formation of
benzaldehyde. As shown in Figure 1A, MG oxidatively
deaminated benzylamine to benzaldehyde but not glu-
cose, 3-DG, or GO. There have been various investi-
gations of transition metal ions showing the stimulation
of the formation of AGEs1,21 and a-dicarbonyls11 in
vitro. We examined the effect on the oxidation by incu-
bation of benzylamine with each carbohydrate in the
presence of Cu2+ . As shown in Figure 1B, in the pre-
sence of 250 mM Cu2+, a significant amount of alde-
hyde was produced by the reaction with glucose, 3-DG,
and MG. There was an increase in the concentration of
benzaldehyde over the incubation period (14 days).
Nevertheless, GO produced only a small amount of
benzaldehyde. The final formation of benzaldehyde by
glucose, 3-DG, MG, and GO after 14 days was
11.5Æ0.2 mM, 31.3Æ1.1 mM, 48.6Æ2.1, and 0.7Æ0.1
mM (meanÆSEM, n=3), respectively.
Because the oxidation was apparently stimulated by the
addition of Cu2+, we investigated the effects of physio-
logically important metals on the reaction. Benzylamine
(5.0 mM) was incubated with 1.0 mM glucose, 3-DG, or
MG in the presence of each metal ion (250 mM) at pH
7.4 and 37 ꢀC for 7 days. As shown in Fig. 2, the for-
mation of benzaldehyde by glucose, 3-DG, and MG was
the greatest with Cu2+. MG significantly oxidized
benzylamine irrespective of the presence or absence of
each metal ion. 3-DG also underwent the oxidation in
the presence of VO2+ and Ag+. Nevertheless, only
Cu2+ catalyzed the oxidation by glucose. No metal ions
other than Cu2+ catalyzed the oxidation by GO (data
not shown).
Figure 1. Time course of oxidative deamination of benzylamine by
glucose, 3-DG, and MG. Benzylamine (5.0 mM) was incubated with
1.0 mM of each carbohydrate in 50 mM phosphate buffer (pH 7.4) in
the presence or absence of Cu2+ (250 mM) at 37 ꢀC. After the reaction
was terminated, benzaldehyde was measured by HPLC. A, without
Cu2+. B, with Cu2+ . The values are shown as meansÆSEM (n=3).
The effect of Cu2+ concentration on the oxidative de-
amination by glucose, 3-DG, and MG was also

M. Akagawa et al. / Bioorg. Med. Chem. 11 (2003) 1411–1417
1413
Figure 2. Effect of various kinds of metal ions on oxidative deamination of benzylamine by glucose, 3-DG, and MG. Benzylamine (5.0 mM) was incu-
bated with 1.0 mM of each carbohydrate in 50 mM phosphate buffer (pH 7.4) in the presence or absence of indicated metal ions (250 mM) at 37 ꢀC for 7
days. After the reaction was terminated, benzaldehyde was measured by HPLC. The values are shown as meansÆSEM (n=3).
hardly observed in the reaction with glucose and 3-DG.
Interestingly, at pH 10, glucose produced the greatest
amount of benzaldehyde.
benzaldehyde, clearly illustrating the involvement of
oxygen in the oxidative deamination (Table 1).
The possibility was considered that one-electron-
reduced oxygen intermediates may be generated in the
reaction, possibly including hydrogen peroxide, super-
oxide, and hydroxyl radical which in turn might con-
tribute to the oxidative reaction. Actually, reactive
oxygen species have been reported to be generated in
the early and the advanced glycation processes,14À16 and
these species have been shown to participate in the for-
The presence of oxygen is important in the Maillard
reaction,21 and, actually, oxygen is required for the for-
mation of some AGEs.17,22 To assess the role of oxygen
in the reaction, we incubated benzylamine with glucose,
3-DG, and MG in the presence of Cu2+ under a nitro-
gen atmosphere. Indeed, the reaction under a nitrogen
atmosphere markedly inhibited the production of
Figure 3. Oxidative deamination of benzylamine by glucose, 3-DG,
and MG as a function of Cu2+ concentration. Benzylamine (5.0 mM)
was incubated with 1.0 mM of each carbohydrate in 50 mM phosphate
buffer in the presence or absence of Cu2+ (0–500 mM) at 37 ꢀC for 7
days. After the reaction was terminated, benzaldehyde was measured
by HPLC. The values are shown as meansÆSEM (n=3).
Figure 4. Effect of pH on oxidative deamination of benzylamine by
glucose, 3-DG, and MG. Benzylamine (5.0 mM) was incubated with
1.0 mM of each carbohydrate in 50 mM phosphate buffer (pH 6–10) in
the presence of Cu2+ (250 mM) at 37 ꢀC for 7 days. After the reaction
was terminated, benzaldehyde was measured by HPLC. The values are
shown as meansÆSEM (n=3).

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M. Akagawa et al. / Bioorg. Med. Chem. 11 (2003) 1411–1417
mation of some AGEs.17À19 Furthermore, we have
recently demonstrated that various primary amines,
including the lysine residue of BSA, are oxidatively
deaminated to the corresponding aldehyde in the Fenton-
type system.23 Thus, benzylamine and Cu2+ were incu-
bated with 1.0 mM of glucose, 3-DG, MG, and H2O2 in
the presence or absence of catalase, a radical scavenger,
and a metal ion chelator. As shown in Table 1, addition
of catalase (100 U/mL) completely inhibited the benzal-
dehyde formation by glucose and H2O2 and significantly
inhibited it by 3-DG and MG. The oxidation of
benzylamine by glucose, 3-DG, and MG was also sig-
nificantly inhibited in the presence of 50 mM dimethyl
sulfoxide (DMSO) which is a hydroxyl radical sca-
venger. Furthermore, marked inhibition was observed
in the incubation with H2O2 in the presence of DMSO
(71.8% inhibition). Addition of a copper chelator (250
mM), ethylenediaminetetraacetic acid (EDTA), effec-
tively inhibited the oxidation by glucose, 3-DG, and
MG as well as H2O2 (78.7–100% inhibition). These
results suggest that the hydroxyl radical is produced by
the Fenton-type reaction and is responsible for the
benzaldehyde formation.
was initiated from glucose, a significant amount of
benzaldehyde was also formed in the presence of Cu2+
,
probably due to the generation of a-dicarbonyls. We
have determined the effects of metal ions, pH, oxygen,
and radical scavengers on the oxidative deamination.
The formation of benzaldehyde by glucose, 3-DG, and
MG was greatest with Cu2+, and the oxidation was
accelerated at a higher pH, and required oxygen. Fur-
thermore, EDTA, catalase, and DMSO significantly
inhibited the oxidation of benzylamine. These data are
consistent with our previous results using BSA.20 From
these results, once again we propose that the oxidative
deamination during glycoxidation is caused by the
a-dicarbonyls-mediated Strecker-type reaction and
reactive oxygen species as shown in Figure 5.
The decarboxylation/deamination of amino acids to
carbonyls is known as the so-called Strecker degra-
dation in food science. In the Strecker degradation,24,25
a number of carbohydrate-derived a-dicarbonyls as well
as glucose are able to degrade a-amino acids at high
temperatures, thus generating an aldehyde with one
carbon atom less than a-amino acids. On the other
hand, o-quinone compounds, which have an a-dicarbonyl
group, are known to catalyze the oxidative deamination
of primary amines to form the corresponding aldehydes
under physiological conditions.26,27 Recent researches
have shown that a-dicarbonyls are formed in early gly-
cation from the degradation of glucose and Schiff base
adducts in the presence of trace metal ions.11 Therefore,
the a-dicarbonyls derived from glycoxidation poten-
tially may serve as adventitious oxidants of amines and,
in the course of the oxidative deamination, generate
aldehydes. In practice, a model experiment showed that
3-DG and MG are more efficient oxidants of benzyl-
amine than glucose. Although GO produced only a small
amount of benzaldehyde, assuming its reactivity, GO
may preferentially form stable cross-links or carboxy-
methyllysine type compounds during the incubation
with benzylamine.28,29 On the basis of these facts, Fig-
ure 5 shows the proposed mechanism of the formation
of benzaldehyde from benzylamine by the Strecker-type
reaction. The formation of a-dicarbonyls is induced
through the autoxidation of glucose and the degra-
dation of Amadori products or Schiff bases adduct by
metal ion-catalysis.11 Subsequently, the resulting MG
and 3-DG could condense with benzylamine to form a
Schiff base adduct, an iminoketone (I). Then the a-pro-
ton of benzylamine would be abstracted by basic media,
and the enolization might give an iminoenaminol (II).
In the electron transfer process, it is assumed that Cu2+
serves as the electron-pair acceptor and stabilized II
through the formation of a coordination complex
because the requirement of Cu2+ was observed in model
studies. Finally, spontaneous hydrolysis of II can lead
to the release of an enaminol (III) and the formation of
benzaldehyde. The mechanism of the Strecker-type
reaction is not accompanied by decarboxylation and is
consistent with the o-quinone-mediated mechanism
previously proposed.27 The action of transition metal
ions in this mechanism is in agreement with the experi-
mental observation that the oxidative deamination by
glucose required transition metal ions.
Discussion
In a previous paper, we reported that glycoxidation
caused the oxidative deamination of the lysine residue in
BSA and that 3-DG and MG were the most efficient
oxidants.20 In that study, we characterized the long-
term process of the glycoxidative modification of
BSA.20 This report shows that the reaction is likewise
caused in a short incubation time, offering for the first
direct proof of glycoxidation-induced oxidative deami-
nation. In the present study, model experiments using
benzylamine were undertaken to elucidate the details of
the oxidative deamination reaction by glycoxidation.
Measurements of benzaldehyde allowed us to define
further the characteristics and mechanism of the reac-
tion. 3-DG and MG oxidatively deaminated benzyl-
amine to benzaldehyde at a physiological pH and
temperature in the presence of Cu2+. When the reaction
Table 1. Effect of N2, EDTA, and scavengers on the oxidative dea-
mination of benzylamine by carbohydrates
Condition
% Inhibition
3-DG MG
Glucose
H2O2
—
a
N2
60Æ1.5 83.7Æ0.260.8 Æ1.4
Catalase (100 U/mL)b 100.0Æ0.0 68.9Æ6.7 24.8Æ4.1 100.0Æ0.0
DMSO (50 mM)
EDTA (250 mM)
33.2Æ1.3 29.1Æ0.7 22.5Æ4.4 71.8Æ1.4
100.0Æ0.0 85.5Æ1.5 78.7Æ0.0 81.4Æ0.3
Benzylamine (5.0 mM) was incubated with each carbohydrate (1.0
mM) or H2O2 (1.0 mM) in 50 mM phosphate buffer (pH 7.4) in the
presence of Cu2+ (250 mM) and indicated materials at 37 ꢀC for 7
days. After the reaction was terminated, benzaldehyde was measured
by HPLC. The values are shown as meansÆSEM (n=3).
aIncubation under N2 atmosphere.
bIn the control experiment, benzylamine (5.0 mM) was incubated with
each carbohydrate (1.0 mM) or H2O2 (1.0 mM) in 50 mM phosphate
buffer (pH 7.4) in the presence of Cu2+ (250 mM) and heat-inactivated
catalase at 37 ꢀC for 7 days.

M. Akagawa et al. / Bioorg. Med. Chem. 11 (2003) 1411–1417
1415
Figure 5. Proposed mechanism of oxidative deamination of lysine residue by the Strecker-type reaction.
Another possible pathway of the oxidative deamination
by glycoxidation is reactive oxygen species-mediated
oxidative deamination. The fact that the aerobic gly-
cation in the presence of transition metals is accom-
panied by radical-generating reactions supports this
possibility.14 Furthermore, 3-DG, MG, and GO also
produce superoxides during the reaction with lysine and
10 arginine.16 In the model reaction, catalase and
DMSO significantly inhibited the oxidation of benzyla-
mine to benzaldehyde by glucose, 3-DG, and MG,
indicating the participation of hydroxyl radicals.
The dependency of the oxidative deamination on
both oxygen and Cu2+ is also consistent with a
metal ion-catalyzed mechanism for the production of
hydroxyl radicals, probably through the intermediacy of
superoxide and H2O2. Recently it has been demon-
strated that lysine residue is oxidatively deaminated to a-
aminoadipic-d-semialdehyde residue by reactive oxygen
species.23,30,31 In addition, we have found that various
primary amines are converted to the corresponding
aldehydes in the presence of H2O2 and transition metal
ions, and the oxidation is effectively prevented by cata-
lase, DMSO, and EDTA.23 Therefore, the hydroxyl
radical generated by the Fenton-type reaction is also
likely to contribute to the oxidative deamination by
glycoxidation.

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M. Akagawa et al. / Bioorg. Med. Chem. 11 (2003) 1411–1417
The results presented here suggest that the oxidative de-
amination is one of the major reactions in glycoxidation.
Once generated, the aldehydes can condense with each
other through aldol condensation or with other amines
through Schiff base formation, leading to formation of
the complex products. Therefore, the oxidative deami-
nation may play an important part in the formation of
AGEs by glycoxidation.
3-DG, MG, and GO (100 mL). The reaction mixtures
were incubated at 37 ꢀC with shaking in the dark. After
incubation for 7 days, the reaction was terminated by
the addition of acetic acid (100 mL). Then the produc-
tion of benzaldehyde was measured by HPLC as
described above.
Effect of pH. Reaction mixtures (100 mL) in a micro test
tube contained 13 mM benzylamine, 500 mM CuSO4
and 100 mM sodium phosphate buffer at various pH
values (6, 7.4, 8, 9, and 10). The reaction was started by
the addition of 2.0 mM glucose, 3-DG, and MG (100
mL). The reaction mixtures were incubated at 37 ꢀC with
shaking in the dark. After incubation for 7 days, the
reaction was terminated by the addition of acetic acid
(100 mL). Then the production of benzaldehyde was
measured by HPLC as described above.
Conclusion
The data presented above clearly demonstrate that gly-
coxidation induces the oxidative deamination. Glucose,
3-DG, and MG oxidatively deaminated benzylamine to
benzaldehyde in the presence of Cu2+ at a physiological
pH and temperature. 3-DG and MG were much effec-
tive oxidants compared with glucose. Deoxygenation
and radical scavengers significantly inhibited the reac-
tion. From these results, we propose the mechanism for
the oxidative deamination by the Strecker-type reaction
and the reactive oxygen species-mediated oxidation
during glycoxidation.
Incubation under nitrogen. Reaction mixtures (100 mL)
in a Pyrex test tube contained 10 mM benzylamine, 500
mM CuSO4, and 100 mM sodium phosphate buffer (pH
7.4). After the addition of 2.0 mM glucose, 3-DG, and
MG, the test tube was tightly fitted with a silicone rub-
ber cap. The tube was immediately evacuated and then
filled with N2 gas through a hypodermic needle. After
another hypodermic needle was inserted in the tube to
serve as an outlet port, gas was passed through the
incubation mixture for 10 min and charged until the
pressure of 0.05 MPa inside the tube was reached. Then
the reaction mixture was incubated at 37 ꢀC for 7 days
with shaking in the dark.
Experimental Materials
Acetonitrile was of HPLC grade from Nacalai Tesque
Co., Kyoto, Japan. Catalase from bovine liver was from
Tokyo Kasei Co., Tokyo, Japan. 3-DG was from
Dojindo Laboratories Co., Kumamoto, Japan. All
other chemicals were from Nacalai Tesque Co.
Effect of catalase. Reaction mixtures (100 mL) in a
micro test tube contained 10 mM benzylamine, 500 mM
CuSO4, and 100 mM sodium phosphate buffer (pH 7.4)
in the presence of catalase (200 U/mL) or heat-inacti-
vated catalase (200 U/mL at 100 ꢀC for 20 min). The
reactions were started by the addition of 2.0 mM glu-
cose, 3-DG, and MG (100 mL). The reaction mixtures
were incubated at 37 ꢀC with shaking in the dark. After
incubation, the reaction was terminated by the addition
of acetic acid (100 mL).
Detection of benzaldehyde in incubation of benzylamine
with glucose, 3-DG, MG, and GO
General procedure. Reaction mixtures (100 mL) in a
micro test tube contained 10 mM benzylamine, 500 mM
CuSO4, and 100 mM sodium phosphate buffer, pH 7.4.
Typical reactions were started by the addition of 2.0
mM glucose, 3-DG, MG, and GO (100 mL). The reac-
tion mixtures were incubated at 37 ꢀC with shaking in
the dark. After incubation, the reaction was terminated
by the addition of acetic acid (100 mL). We confirmed
that this procedure completely stopped the reaction.
After centrifugation at 7740 g for 5 min at room tem-
perature, the samples (20-mL aliquots) were chromato-
graphed with acetonitrile/distilled water (4:6, v/v)
containing 0.1% phosphoric acid. High performance
liquid chromatography (HPLC) was performed on a
Perkin Elmer Liquid Chromatograph Integral 4000 sys-
tem (Norwalk, CT, USA) using a reverse-phase HPLC
column (Cosmosil 5C18-AR- II, 150Â4.6 mm, Nacalai
Tesque Co.) with detection at 245 nm. The column oven
was maintained at 30 ꢀC. Benzaldehyde was eluted at
5.2min at a flow rate of 1.0 mL/min.
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