Synthesis of monolysyl advanced glycation endproducts and their incorporation into collagen model peptides

Article abstract of DOI:10.1021/ol302745f

The synthesis of advanced glycation endproducts (AGEs), CML, CEL, and pyrraline and their incorporation into collagen model peptides is reported. AGEs are modified amino acids that form on proteins such as collagen and are thought to play a significant role in the pathogenesis of many diseases, particularly diabetes. The synthesis and incorporation of these compounds into synthetic peptides is a key step in developing model systems with which to investigate AGE-modified proteins.

Full text of DOI:10.1021/ol302745f

ORGANIC
LETTERS
2012
Vol. 14, No. 22
5740–5743
Synthesis of Monolysyl Advanced
Glycation Endproducts and Their
Incorporation into Collagen Model
Peptides
Tom M. Woods,† Meder Kamalov,† Paul W. R. Harris,† Garth J. S. Cooper,‡ and
Margaret Brimble*,†
School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland,
New Zealand, and Centre for Advanced Discovery and Experimental Therapeutics
(CADET), Central Manchester University Hospitals NHS Foundation Trust, and
School of Biomedicine, The University of Manchester and Manchester Academic Health
Sciences Centre, Manchester, U.K.
m.brimble@auckland.ac.nz
Received October 4, 2012
ABSTRACT
The synthesis of advanced glycation endproducts (AGEs), CML, CEL, and pyrraline and their incorporation into collagen model peptides is
reported. AGEs are modified amino acids that form on proteins such as collagen and are thought to play a significant role in the pathogenesis of
many diseases, particularly diabetes. The synthesis and incorporation of these compounds into synthetic peptides is a key step in developing
model systems with which to investigate AGE-modified proteins.
Advanced glycation endproducts (AGEs) are a group of
compounds that are formed by nonenzymatic reactions
between reducing sugars and amino acid side-chain resi-
dues in proteins. These post-translational modifications
mainlyinvolve reactions atlysine and arginine residuesand
give rise to a large number of structurally diverse compounds
ranging from simple alkylations to more complex hetero-
cycles and cross-linked structures.1 The formation and accu-
mulation of AGEs in the body is thought to play a significant
role in the pathogenesis of many debilitating diseases includ-
ing diabetes2 and age-related neurodegenerative diseases,
such as Alzheimer’s disease.3 AGE formation and accumu-
lation is most problematic on long-lived proteins such as
collagen and lens-crystallins,4 and these are the most rele-
vant proteins to study when investigating AGEs. However,
the precise molecular mechanisms by which AGEs influence
the progression of these diseases are not clear, and there
remains a need to develop new chemical tools to investigate
them. A key step to achieve this is the site-specific incor-
poration of AGEs into synthetic peptide sequences using
solid phase peptide synthesis (SPPS). This will enable access
to synthetic AGE-modified peptides that will facilitate the
development of suitable model systems to undertake future
detailed molecular studies. The initial step required to
address these fundamental questions is the development of
† The University of Auckland.
‡ The University of Manchester and Manchester Academic Health
Sciences Centre.
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r
10.1021/ol302745f
Published on Web 11/01/2012
2012 American Chemical Society

practical and efficient synthetic strategies for the prepara-
tion of suitably protected AGE building blocks for incor-
poration into SPPS.
incorporation into SPPS has only been reported once,8
while the synthesis of a suitably protected pyrraline build-
ing block has not been reported. Importantly, the existing
syntheses of the CML and CEL Fmoc building blocks
required starting materials that are neither commercially
available nor easy to prepare, and yields for their prepara-
tion were not reported. Therefore, our principal aim was to
develop practical syntheses of suitably protected Fmoc
building blocks of CML, CEL and pyrraline and demon-
strate their use in automated microwave SPPS. The routes
were also designed to provide ready access to the free
amino acid forms of these important AGEs.
Here we report the synthesis of the three monolysyl AGEs,
Nε-(carboxymethyl)lysine (1) (CML), Nε-(carboxyethyl)-
lysine (2) (CEL), and pyrraline (3) (Figure 1) as both Fmoc-
protected building blocks suitable for SPPS and as free amino
acids. Furthermore, the AGE building blocks were readily
incorporated into 21 amino acid residue collagen model
peptide (CMP) sequences thus establishing their utility in
SPPS. The CMPs were subsequently shown to form triple
helices in solution demonstrating their potential as model
systems for investigating AGE-modified collagens.
Our strategy to prepare CML and CEL hinged on the use
of the nosyl (Ns) group alkylation methodology to alkylate
the ε-NH2 group of a lysine derivative.9 Additionally, it was
expected that the Ns group would also serve as an orthogonal
protecting group in SPPS. To facilitate the selective nosyla-
tion of the ε-NH2, the 9-BBN group was used to concomi-
tantly protect the R-NH2 and carboxylic acid group in
lysine.10 We envisaged that when used with other acid labile
protecting groups the BBN group would enable a final global
deprotection step to access the free amino acids while
providing a tractable organic compound prior to this. The
synthesis started with lysine hydrochloride 4, which was
treated with 9-BBN dimer affording BBN-lysine 5. The Ns
group was then introduced affording BBN-Ns-Lys 6 in 88%
yield over two steps. Alkylation of 6 with ethyl bromoacetate
or ethyl bromopropionate afforded compounds 7a (89%)
and 7b (91%), respectively. Selective deprotection of the
BBN-moiety was achieved by treatment of 7a and 7b with
4 M HCl/dioxane. Nonaqueous conditions were essential to
avoid concomitant hydrolysis of the ethyl ester. After re-
moval of the HCl/dioxane under reduced pressure, the crude
products were treated with Fmoc-Cl and K2CO3 in dioxane,
affording the desired Fmoc building blocks 8a and 8b ready
for incorporation into SPPS (Scheme 1).
Figure 1. Three monolysyl AGEs, CML, CEL, and pyrraline.
Although the synthesis of the free amino acid forms of
CML,5 CEL,5f,6 and pyrraline7 are well documented, the
majority of these approaches suffer from lengthy and
difficult procedures and/or poor yields. Furthermore, the
synthesis of CML and CEL as building blocks suitable for
(5) (a) Matsutani, H.; Kusumoto, S.; Koizumi, R.; Shiba, T. Phy-
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Synlett 2012, 23, 531. (h) Csuk, R.; Stark, S.; Barthel, A.; Kluge, R.;
Scheme 1. Synthesis of Fmoc-Protected CML and CEL
Building Blocks 8a and 8b
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Org. Lett., Vol. 14, No. 22, 2012
5741

This strategy also enabled ready access to the free amino
acid forms of CML and CEL. Thus, starting from 9-BBN-
Ns-lysine 6, a one-pot alkylation/Ns deprotection with
either ethyl bromoacetate or ethyl bromopropionate furn-
ished BBN-OEt-CML 9a (68%) and BBN-OEt-CEL 9b
(71%), respectively. Use of the 9-BBN and ethyl ester
protecting groups enabled a final global deprotection to
be carried out by treatment of 9a and 9b with 6 M aq HCl.
Purification of the final compounds using a short reverse-
phase C-18 column eluting with 0.5% MeCN/H2O removed
the organic byproducts (BBN). Finally, CML 10a and CEL
10b, were isolated by lyophilization as their bis-hydrochlor-
ide salts in quantitative yields (Scheme 2). BBN-Lys pre-
cursor 5 also provided the starting material for a more direct
synthesis of CML using an approach similar to that described
by Kihlberg et al.5m Whereas Kihlberg used either formic or
trifluoroacetic acid as solvent we were able to use H2O,
simplifying both the procedure and purification. Hence,
9-BBN-Lys 5 and glyoxylic acid were heated at 40 °C in
H2O for 24 h. The resulting formylated intermediate (not
shown) was then heated at reflux in 1 M aq HCl. Purification
using a C-18 column afforded CML bis-hydrochloride salt
10a in 58% yield over two steps (Scheme 2).
Scheme 3. Synthesis of Pyrraline Derivatives
Scheme 4. Synthesis of CMPs Containing CML and CEL
Scheme 2. Synthesis of the Free Amino Acids of CML and CEL
Fmoc group with 5% piperidine/DCM afforded the free
amino acid form of pyrraline 15 in 62% yield (Scheme 3).
CMPs are synthetic peptides based on the repeat unit
Pro-Hyp-Gly that are able to form triple helices in solution,
thereby mimicking the tertiary structure of collagen. They
have been used extensively as model systems to study the
structure and stability of collagen.12 The synthesis of homo-
trimeric CMPs containing CML, CEL and pyrraline will
establish the general utility of our AGE building blocks
in SPPS while also demonstrating that AGE-modified
CMPs are viable and accessible chemical tools for studying
Our strategy for pyrraline relied on the condensation of a
protected lysine derivative with TBS protected dihydropyr-
anone 12 (Scheme 3). A similar condensation using dihy-
dropyranone 12 has previously been employed within our
group for the synthesis of acortatarin A.11 Hence, a suspen-
sion of Fmoc-lysine 11 and dihydropyranone 12 in THF/
H2O (1:1) was heated at 40 °C for 7.5 h, furnishing the
desired Fmoc protected pyrraline derivative 13 in 67% yield.
Deprotection of the TBS ether in 13 by treatment with dilute
HCl afforded 14 in 88% yield and final deprotection of the
(12) For examples see (a) Holmgren, S. K.; Taylor, K. M.; Bretscher,
L. E.; Raines, R. T. Nature 1998, 392, 666. (b) Erdmann, R. S.;
Wennemers, H. J. Am. Chem. Soc. 2010, 132, 13957. (c) Hodges, J. A.;
Raines, R. T. J. Am. Chem. Soc. 2005, 127, 15923. (d) Persikov, A. V.;
Ramshaw, J. A. M.; Kirkpatrick, A.; Brodsky, B. J. Am. Chem. Soc.
2003, 125, 11501. (e) Yu, S. M.; Li, Y.; Kim, D. Soft Matter 2011, 7, 7927
and references therein.
(10) Dent, W. H., III; Erickson, R.; Fields, S. C.; Parker, M. H.;
Tromiczak, E. G. Org. Lett. 2002, 4, 1249.
(11) Geng, H. M.; Chen, J. L-Y.; Furkert, D. P.; Jiang, S.; Brimble,
M. A. Synlett 2012, 23, 855.
5742
Org. Lett., Vol. 14, No. 22, 2012

the TBS group. This problem was easily circumvented by
removing TIPS from the resin cleavage mixture, which
afforded the desired pyrralineÀCMP 24 in 27% yield and
>95% purity (see the Supporting Information).
A key feature of CMPs is their propensity to form triple
helices in solution, a phenomenon observed by CD spec-
troscopy by the appearance of a small maximum around
225 nm and a large minimum around 200 nm. The forma-
tion and relative stabilities of the triple helices can be
verified by thermal transition curves that show the dena-
turation of the triple helices.14 The CD spectra for the three
AGE-modified CMPs all exhibited a small positive and
large negative peak at the expected wavelengths (see the
Supporting Information). In addition, each AGEÀCMP
showed a clear thermal transition (Tm, 21 = 26 °C, 22 =
25 °C, 24 = 23 °C) upon heating the solutions from 5 to
70 °C (Figure 2), thus demonstrating their potential as
useful model systems to study AGE-modified collagens.
In summary, we have developed efficient syntheses of
the Fmoc-protected and free amino acid forms of the three
monolysyl AGEs: CML, CEL, and pyrraline. Moreover,
we have successfully incorporated each Fmoc-protected
AGE building block site-specifically into a CMP high-
lighting the utility of these building blocks in SPPS.
Importantly, the AGE-modified CMPs were shown to
form triple helices in solution, thus establishing their poten-
tial as model systems for studying AGE-modified collagens.
The synthesis of the free amino acids, Fmoc building blocks,
and AGE-modified peptides will facilitate future investiga-
tions into the roles these AGEs play in the pathogenesis of
diabetes and other relevant diseases thereby enabling the
development of improved therapeutic strategies. The synth-
esis of the CMPs reported herein provides the impetus for the
synthesis of further AGE-modified CMPs and related pep-
tides, thereby facilitating the development of suitable model
systems to probe the chemical, physical and mechanical
properties of AGE modified collagens and related proteins.
Figure 2. CD thermal transition curves for CMPs 21, 22, and 24
measured in 50 mM aqueous AcOH (0.5 mM peptide).
AGE-modified collagens. Surprisingly, given their biological
relevance, there are only two examples in the literature of
synthetic AGE building blocks being incorporated site spe-
cifically into synthetic peptides.8,13
Our aim was to synthesize CMPs comprising seven
repeat units of (Gly-Hyp-Pro) where the central Hyp residue
(position 11) was replaced with our synthetic AGE building
blocks. Thus, aminomethyl polystyrene resin was coupled to
a preloaded Fmoc-Gly-hydroxymethylphenoxypropionic
acid (HMPP) linker affording resin 16. Standard couplings
were then carried out by SPPS furnishing resin 17. The
CML and CEL building blocks were coupled manually
using 2 equiv of building block and HATU affording resin-
bound peptides 18a and 18b. Standard couplings then gave
the desired resin-bound peptides 19 and 20 (Scheme 4).
Pleasingly, the Ns group was readily deprotected on resin
by treatment with 2-mercaptoethanol and DBU without
complication. Cleavage from the resin using a TFA/TIPS/
H2O cocktail and subsequent hydrolysis of the ethyl ester
by treatment with aqueous NaOH afforded CMLÀCMP
21 (19% yield and >95% purity) and CELÀCMP 22
(30% yield and >95% purity).
Acknowledgment. We thank Prof. Catherine Day and
Dr Yoshio Nakatani (University of Otago) and Prof. Tom
Brittain and Kavestri Yegambaram (University of
Auckland) for assistance with CD spectroscopy and the
Maurice Wilkins Centre for Molecular Biodiscovery for
generous financial support.
Using the same protocol, pyrraline building block 13 was
also incorporated into a CMP affording the desired resin
bound peptide 23. In this case, when cleaving the peptide
from the resin using TFA/TIPS/H2O the mass of the major
product was found to be 16 mass units less than the mass of
the desired peptide. This was attributed to a TIPS facilitated
reduction of the benzylic like alcohol following removal of
Supporting Information Available. Experimental pro-
cedures and characterization data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(14) Feng, Y.; Melacini, G.; Taulane, J. P.; Goodman, M. J. Am.
Chem. Soc. 1996, 118, 10351.
(13) Wang, T.; Kartika, R.; Spiegel, D. A. J. Am. Chem. Soc. 2012,
134, 8958.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 22, 2012
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