Intact glycation end products containing carboxymethyl-lysine and glyoxal lysine dimer obtained from synthetic collagen model peptide

Article abstract of DOI:10.1016/j.bmcl.2004.08.044

Glycation reactions using a model peptide of collagen and glucose or ribose resulted in detection of carboxylmethyl-lysine in the peptide; and treatment with glyoxal provided a dimer of the peptide linked with glyoxal lysine dimer (GOLD). Advanced glycation end products (AGE) are accumulated in human tissues when long-lived proteins are glycated due to hyperglycemia and/or aging. In this study, we synthesized a collagen model peptide, Ac-(Pro-Hyp-Gly) ₅-Pro-Lys-Gly-(Pro-Hyp-Gly)₅-Ala-NH₂ to investigate intact AGEs in peptides. The peptide formed a stable triple helix structure, and was subjected to glycation reactions with glucose, ribose and glyoxal. Besides carboxymethyl-lysine in the peptide, a conjugated form linked with glyoxal lysine dimer (GOLD) was detected upon treatment with glyoxal. This is the first example of intact glycation-derived dimers of peptides retaining intrinsic protein structures.

Full text of DOI:10.1016/j.bmcl.2004.08.044

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5677–5680
Intact glycation end products containing carboxymethyl-lysine and
glyoxal lysine dimer obtained from synthetic collagen model peptide
Hiroaki Yamada,a Tomoko Sasaki,a Sachiko Niwa,a Tohru Oishi,a Michio Murata,a,*
Toru Kawakamib and Saburo Aimotob
aDepartment of Chemistry, Graduate School of Science, Osaka University, 1–16 Machinakeyama, Toyonaka, Osaka 560-0043, Japan
bInstitute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Received 13 June 2004; revised 18 August 2004; accepted 18 August 2004
Available online 15 September 2004
Abstract—Advanced glycation end products (AGE) are accumulated in human tissues when long-lived proteins are glycated due to
hyperglycemia and/or aging. In this study, we synthesized a collagen model peptide, Ac-(Pro-Hyp-Gly)5-Pro-Lys-Gly-(Pro-Hyp-
Gly)5-Ala-NH2 to investigate intact AGEs in peptides. The peptide formed a stable triple helix structure, and was subjected to
glycation reactions with glucose, ribose and glyoxal. Besides carboxymethyl-lysine in the peptide, a conjugated form linked with
glyoxal lysine dimer (GOLD) was detected upon treatment with glyoxal. This is the first example of intact glycation-derived dimers
of peptides retaining intrinsic protein structures.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Nonenzymatic glycation reactions of proteins lead to
formation of advanced glycation end products (AGEs).
Hyperglycemia inherent to diabetes patients accelerates
accumulation of AGE, which consequently causes dia-
betes complications. Among long-lived proteins suscep-
tible to glycation, collagen, the most abundant protein
family in human tissues, is potentially implicated in
AGE-related illnesses such as osteoarthritis,1,2 cardio-
vascular failures,3,4 and renal disorders.5,6 Chemical
and biochemical investigations have been carried out
to identify the structural entities of AGEs and their for-
mation mechanisms. In the last two decades, the struc-
tures of glycation moieties and their linkages between
proteins have been extensively studied to accumulate
the chemical knowledge on this complicated reactions
and their resultant products. As seen for the diagnostic
use of hemoglobin A1c, the initial glucose conjugation
to an amino group in proteins triggers a further glycat-
ion reaction sequence. In addition, other carbonyl
equivalents are known to play a crucial role in forming
AGE in human tissues. Dicarbonyl agents represented
by glyoxal and methylglyoxal can be generated through
the primary metabolic pathway of carbohydrates as well
as the oxidative degradations of unsaturated fatty
acids.7–9 Glyoxal-derived AGEs have therefore been
extensively investigated, and some of them turned out
to be heavily involved in formation of AGEs. Among
these, carboxymethyl-lysine (CML, 1)10 is one of the
best known AGE, and often utilized as a diagnostic
marker for protein glycation in patients. Besides these
N-substituted lysine and arginine, crosslinkers of pro-
teins comprising glycated lysine/lysine or lysine/arginine
have been attracting much attention since protein cross-
linking greatly damages physiological functions, and
potentially causes AGE-related complications. There
are several crosslinkers reported from glycated proteins
such as crossline, DOGDIC, GODIC, GOLD, MOLD,
pentosidine, and so on. In particular, GOLD (2),
glyoxal lyse dimer, generated from two molecules of
lysine and glyoxal, has been found from human tissues
as one of the aging-related AGEs.11
Most of AGEs including CML10 and GOLD12 were ini-
tially discovered from reactions between amino acids
and sugars; that is, the detailed structure studies of
AGEs have so far been based on products obtained
from N-protected lysine and N-protected arginine in
the presence of glucose, glyoxal or other glycating
agents. Some of these AGEs such as CML, pentosidine,
and GOLD7,11,13,14 have been also detected from human
tissues. Despite a great advance brought by these
biochemical studies, little data are available as to how
Keywords: Advanced glycation end products; Collagen.
*
Corresponding author. Tel./fax: +81 66850 5774; e-mail: murata@
ch.wani.osaka-u.ac.jp
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.044

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H. Yamada et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5677–5680
closely glycation reactions of proteins in human can be
reproduced by model systems using an amino acid–su-
gar mixture. Previous studies have demonstrated that
acid treatments such as hydrolysis of peptides results
in degradation of some AGEs.15–17 To know what is
exactly happening in protein glycation under physiolog-
ical conditions, intact AGEs should be examined with-
out chemical or enzymatic degradation. Glycation of
natural proteins, however, results in an extremely com-
plexed mixture of AGEs, making their structure elucida-
tion practically impossible. To address these problems,
we attempted to prepare a simple and small model pep-
tide of collagen that is expected to provide a limited
number of glycation products. In this letter, we report
the synthesis of lysine-containing collagen model pep-
tide 3 and its glycation products.
Boc-Ala-MBHA resin
1) Boc-Pro-Hyp(Bzl)-Gly-OBt/DIEA (5 times)
2) Boc-Gly, Boc-Lys(Cl-Z), Boc-Pro
3) Boc-Pro-Hyp(Bzl)-Gly-OBt/DIEA (5 times)
Ac-(Pro-Hyp(Bzl)-Gly) -Pro-Lys(Cl-Z)-Gly-
5
(Pro-Hyp(Bzl)-Gly) -Ala-MBHA resin
5
4) HF treatment
5) RP-HPLC
Ac-(Pro-Hyp-Gly) -Pro-Lys-Gly-(Pro-Hyp-Gly) -Ala-NH
2
5
5
Scheme 2. Conditions for coupling between Boc-amino acid and
peptide on resin for (1)–(3): (a) washed with N-methylpyridone and
then CH2Cl2; (b) 50% TFA/CH2Cl2, rt, 5min and then 25min; (c)
washed with CH2Cl2 and then N-methylpyridone; (d) dicyclohexyl-
carbodiimide-activated Boc-amino acid or Boc-Pro-Hyp(Bzl)-Gly,
N-methylpyridone, rt, 90min; (e) washed with CH2Cl2 and then N-
methylpyridone; (f) Ac2O, N-methylpyridone, rt, 5min. (4) Anhydrous
HF, anisole, 0ꢁC, 90min. (5) See Fig 1.
A collagen model was designed as 3, which possesses
several features to facilitate AGE analysis. First, the
model peptide of 34 residues comprises the repeated
tripeptides of Pro-Hyp-Gly that stabilize the triple
helix structure of collagen.18 Secondly, one lysine resi-
due is introduced in place of Hyp 17 as a single site
for glycation, which should simplify the profile of
AGEs. Thirdly, the N- and C-termini of the peptide
are substituted with N-acetyl and amide groups, respec-
tively, to reduce ionic repulsion.
Boc proline and Hyp(Bzl)-OFm (O-benzyl-hydroxypro-
line fluorenylmethyl ester) followed by deprotection
with piperidine gave Boc-Pro-Hyp(Bzl)-OH. Further
coupling with Gly-OMe provided Boc-Pro-Hyp(Bzl)-
Gly-OMe. After hydrolyzing methylester, the tripeptide
was purified by crystallization as a dicyclohexylamine
salt. The solid-phase synthesis of peptide 3 using Boc-
Pro-Hyp(Bzl)-Gly-OH was carried out as depicted in
Scheme 2. Starting from Boc-Ala-MBHA resin (MBHA
resin: 4-methylbenzhydrylamine resin), Boc-Pro-Pro-
Gly was condensed five times according to a standard
Boc solid-phase method and then Boc-Gly, Boc-
Lys(Cl-Z), and Boc-Pro were successively condensed to
furnish 19-mer peptide. Further coupling with Boc-
Pro-Hyp(Bzl)-Gly five times provided the protected pep-
tide resin, which was then treated with anhydrous HF
containing 10% anisole to furnish collagen model 3.19
The HPLC elution profile of peptide 3 showed two
peaks, implying that 3 undergoes association/dissocia-
tion between monomeric and trimeric forms under the
HPLC conditions; although one at 18.5min may proba-
bly be due to the triple helix and the other at 21.1min
due to monomers, their assignment is not completed.
Collagen model peptide was prepared by the combined
use of liquid-phase and solid-phase syntheses. Tripep-
tide Boc-Pro-Hyp(Bzl)-Gly was obtained by a solution
phase method as shown in Scheme 1. Coupling between
H
HOOC
N
COOH
NH
2
Carboxymethyl-lysine (CML) 1
HOOC
N
N
COOH
NH
NH
2
2
Glyoxal lysine dimer (GOLD) 2
The transition temperature (Tm) from triple helix to
monomeric forms was determined by a differential scan-
ning calorimeter (DSC) to be 69.8ꢁC, which significantly
exceeded that of (Pro-Hyp-Gyl)10, 60.1ꢁC.20 This high
Tm of 3 assured the stability of triple helix during a long
glycation period, typically three months at 37ꢁC. The
molecular weight of the triple helix is about 9.2 kDa,
which can be regarded as a small protein. Collagen mod-
el 3 was subjected to glycation reactions using three dif-
ferent agents, glucose, ribose, and glyoxal,21 where
incubation times were varied depending on their reactiv-
ity; three month at 37ꢁC for glucose, four weeks at 37ꢁC
for ribose, 24h at 40ꢁC for glyoxal. In the MALDI-TOF
spectra of their glycation products, an ion peak corres-
ponding to CML-containing 3 was commonly observed
at m/z 3142 (Fig. 2a and c); a spectrum for ribose is not
shown since it is similar to Figure 2a except for a lack of
Ac-(Pro-Hyp-Gly)5-Pro-Lys-Gly-(Pro-Hyp-Gly)5-Ala-NH2
Collagen Model Peptide 3
b
a
Boc-Hyp(Bzl)-OH
c
H-Hyp(Bzl)-OFm
Boc-Hyp(Bzl)-OFm
d
Boc-Pro-Hyp(Bzl)-OFm
Boc-Pro-Hyp(Bzl)-OH
f
e
Boc-Pro-Hyp(Bzl)-Gly-OMe
Boc-Pro-Hyp(Bzl)-Gly-OH
Scheme 1. Reagents and conditions: (a) FmOH, WSCl/HCl, DMAP,
CH2Cl2, rt, 12h; (b) HCl, dioxane–CH2Cl2, rt, 1h (89% for two steps);
(c) Boc-Pro-OH, WSCI/HCl, HOBt, DIEA, DMF, rt, 12h (80%); (d)
piperidine, DMF, rt, 3h (78%); (e) Boc-Gly-OCH3, WSCI/HCl, HOBt,
DIEA, DMF, rt, 40min (84%); (f) KOH aq THF/H2O 5:1 rt, 1h (98%).

H. Yamada et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5677–5680
5679
natural collagen23 was estimated to be less than 0.1%
of total lysine residues under the same glycation condi-
tions although a trace amount of CML was detected
by LC–MS. This low yield is probably because further
conversion of CML proceeded in natural collagen to
generate more complicated AGEs. AGE with 162/168
mass-unit higher ion was detected (Fig. 2a/b), which
may correspond to a fructosyl derivative of 3, an Ama-
dori product of glucose-conjugated 3. Among the AGEs
obtained with glyoxal, ion peaks corresponding dimers
of 3 appeared at m/z 6200–6300 (Fig. 2c). In particular,
an ion peak at m/z 6229 corresponds to dimer of 3 linked
with GOLD. The generation of a GOLD moiety was
confirmed by LC–MS analysis.24 To our knowledge,
the present result provides the first example of observa-
tion of an intact glycation-derived peptide dimer with
intrinsic higher structures. In the glycation reactions
with glucose and ribose, AGEs in early stage such as
CML and fructosyl-lysine were formed, which suggested
that a longer incubation period is necessary for more ad-
vanced AGEs including crosslinkers. In contrast, gly-
oxal elicited dimerization of the peptide due to its
higher reactivity.
Figure 1. HPLC elution profile of peptide 3. Column: Cosmosil 5C18-
MS (10 · 250mm) at a flow rate of 2.5mL/min. Eluent: aqueous
acetonitrile containing 0.1% TFA with linear gradient elution as
depicted in figure. Peaks A and B gave rise to an identical molecular
mass in MALDI-TOF, suggesting one of them in the triple helix form.
As Reiser et al. have reported,25 the glycation of type I
collagen occurs in a sequence specific manner, which evi-
dently indicates that AGE formation reactions can never
be reproduced with amino acid derivatives. The present
model peptide 3 is much smaller than collagen, and may
not replicate the higher structure such as the super coil
and long fibrous assemblage. It does, however, form
the triple helix characteristic of the natural protein.
The dominant formation of GOLD in the peptide may
be in parallel with the higher content of GOLD (and
MOLD) than other dicarbonyl-derived crosslinkers in
human plasma and tissues.11,14,26 Moreover, we have
observed some differences between amino acid deriva-
tives and model peptide 3 in preliminary experiments;
the formation rate of CML appears higher with 3 than
with N-Boc-lysine.27 The present findings suggest the
utility of protein mimics for questing true AGEs occur-
ring in human tissues. Further glycation reactions using
collagen model peptides are currently underway.
Acknowledgements
Figure 2. MALDI-TOF spectra of peptide 3 after glycation treatment.
(a) Glycation products from 3 and glucose; (b) those from 3 and 13C6-
glucose; (c) those from 3 and gyoxal. An ion peak at m/z 3142 in a or
inset spectra of c corresponds to CML-bearing 3, and that at m/z 6229
corresponds to a dimer of 3 linked with GOLD. An ion peak at 6287 in
c matches a further glyoxal adduct of the GOLD-linked dimer.
We are grateful to Dr. Nobuaki Matsumori in authorsÕ
laboratory (M.M.) for discussion; and to Dr. Katsumasa
Iijima, Nippi Research Institute of Biomatrix for infor-
mation on collagen glycation. This work was supported
in part by a Grant-In-Aid for Scientific Research on
Priority Area (A) (No. 12045243) from MEXT,
Japan.
ion at m/z 3246. The presence of CML 1 in 3 was further
confirmed by amino acid analysis for its hydrolysates.22
When, in lieu of glucose, 13C6-glucose was used for gly-
cation reactions, the products gave rise to ion peaks at
m/z 3144 (Fig. 2b), clearly indicating the incorporation
of C2 unit from glucose. The amount of CML formed
in glucose-treated 3 was estimated to be 10% of lysine
based upon HPLC quantification23 for acid hydrosates,
which roughly corresponds to that by MALDI-TOF
(Fig. 1a). On the other hand, the yield of CML from
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