Expedient synthesis of peptides containing Nε- carboxymethyllysine

Article abstract of DOI:10.1055/s-0033-1378305

Accumulation of advanced glycation endproducts (AGEs) is responsible for the development and progress of diabetes- and age-related complications. Synthesis of specific chemical probes is key for the detailed understanding of biochemical properties of AGEs and their precise roles in the progression of disease. We herein report the expedient synthesis of such probes in the form of peptides site-specifically glycated by the major lysyl AGE, N ^ε-carboxymethyllysine (CML). The facile and economical incorporation of CML into peptide sequences by using the nosyl group has been achieved in a single step on resin. This new method is a substantial improvement over the existing syntheses of CML-containing peptides in that it does not require the use of expensive reagents or elaborate purification techniques. The impact of CML on the proteolytic stability of the host peptide has been investigated using trypsin digest studies. Georg Thieme Verlag Stuttgart. New York.

Full text of DOI:10.1055/s-0033-1378305

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LETTER
▌1835
ε
letter
Expedient Synthesis of Peptides Containing N -Carboxymethyllysine
Expedient Synthesis of Peptides Containing Nε-Carboxymethyllysine
Meder Kamalov,a,b Sunghyun Yang,a Paul W. R. Harris,a Garth J. S. Cooper,b,c,d,e,f Margaret A. Brimble*a,b,c
a
School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland, New Zealand
b
Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, 3 Symonds Street, Auckland, New Zealand
c
School of Biological Sciences, The University of Auckland, 3 Symonds Street, Auckland, New Zealand
Fax +64(9)3737422; E-mail: m.brimble@auckland.ac.nz
d
Centre for Advanced Discovery and Experimental Therapeutics (CADET), Central Manchester University Hospitals, NHS Foundation Trust,
Oxford Road, Manchester, M13 9WL, UK
e
f
Institute of Inflammation and Repair, The University of Manchester, Oxford Road, Manchester, M13 9WL, UK
Department of Pharmacology, Division of Medical Sciences, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK
Received: 11.03.2014; Accepted: 09.05.2014
lagen shows lower levels of solubility and digestibility
with trypsin, which is then assumed to indicate a connec-
tion between AGE accumulation and fibrosis observed in
diabetic heart and kidney diseases. As a result, the precise
Abstract: Accumulation of advanced glycation endproducts
(AGEs) is responsible for the development and progress of diabe-
tes- and age-related complications. Synthesis of specific chemical
probes is key for the detailed understanding of biochemical proper-
ties of AGEs and their precise roles in the progression of disease. impact of particular AGEs on the properties of the host
We herein report the expedient synthesis of such probes in the form
proteins with regards to proteolysis is not well character-
ised. Therefore, peptides site-specifically glycated by
of peptides site-specifically glycated by the major lysyl AGE, Nε-
carboxymethyllysine (CML). The facile and economical incorpora-
AGEs serve as valuable probes for the investigation of
tion of CML into peptide sequences by using the nosyl group has
been achieved in a single step on resin. This new method is a sub-
AGE-impact on host protein proteolysis.
stantial improvement over the existing syntheses of CML-contain-
ing peptides in that it does not require the use of expensive reagents
or elaborate purification techniques. The impact of CML on the pro-
teolytic stability of the host peptide has been investigated using
trypsin digest studies.
Currently, there are two strategies for incorporating CML
into peptide sequences. One strategy uses a preformed
building block and CML-containing peptides have been
synthesised via this building block strategy both by us8
and by others.9 In this approach a suitably protected CML
derivative is assembled via conventional organic synthe-
sis and subsequently incorporated into peptides via auton-
omous solid-phase peptide synthesis (SPPS). This
strategy suffers from poor step economy and low overall
yield as three orthogonal protecting groups are required to
facilitate the SPPS.
Key words: peptides, solid-phase synthesis, protecting groups, ad-
vanced glycation endproducts, Nε-carboxymethyllysine
Advanced glycation endproducts (AGEs) are a structural-
ly diverse family of modified amino acids formed via non-
enzymatic reaction between sugars and proteins.1 Nε-Car-
boxymethyllysine (CML), a key member of the AGE fami-
ly, is known to irreversibly accumulate in organ tissues
over time,2 and this accumulation is significantly acceler-
ated in patients with diabetes mellitus, who also experi-
ence earlier onset of organ damage, such as cataract or
heart disease.3 Considerable interest has been aroused on
the characterisation and use of CML as a biomarker for
disease diagnosis and for evaluation of novel therapeutic
strategies.4
The alternative strategy for synthesis of CML-containing
peptides relies on the site-specific alkylation of the resin-
bound peptide containing a free lysyl ε-amine.10 However,
this procedure has not been documented fully and yields
for CML-peptides made by this approach have not been
reported.
+
NH3
NHFmoc
5 steps
–O
HO
OEt
NH2
N
Ns
O
O
O
O
O
Normal remodelling of the connective tissues requires en-
zymes to degrade fibrillar collagen. Several studies have
highlighted the impact of AGE accumulation on the prop-
erties of collagen with regards to enzymatic proteolysis.5–7
However, in vitro glycation in such studies is often
achieved by incubating the protein in high-glucose solu-
tions over prolonged periods. The resulting ‘glycated’ col-
3
4
NHFmoc
NHFmoc
1 step
HO
HO
NH2
NHNs
5
6
Scheme 1 Building blocks for CML incorporation into peptides
This work reports a straightforward and cost-effective
synthetic procedure to access CML-containing peptides.
Our method uses the 2-nitrobenzenesulfonyl (Ns) protect-
SYNLETT 2014, 25, 1835–1838
Advanced online publication: 06.06.2014
DOI: 10.1055/s-0033-1378305; Art ID: st-2014-d0234-l
© Georg Thieme Verlag Stuttgart · New York
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6

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1836
M. Kamalov et al.
LETTER
OH
O
O
OH
HO
OH
O
NHAc
O
O
O
O
O
H
H
H
N
H
H
H
N
N
N
N
N
N
N
N
N
N
N
N
H
H
H
H
H
H
O
O
O
O
O
O
O
CO2H
HO
OH
H2N
1: R = H
2: R = CH2CO2H
NHR
Figure 1 Structures of the target peptides 1 and 2
ing group for the lysyl ε-amine during SPPS as it is or- In order to demonstrate the practical applicability of the
thogonal to the conditions used for 9- new synthetic approach to append CML directly to pep-
fluorenylmethyloxycarbonyl (Fmoc) solid-phase chemis- tides, a sequence (YGYDEKSTGGISVP) was chosen
try.11,12 Importantly, the Ns group also allows specific from the collagen telopeptide (CTP) region of human type
monoalkylation of the ε-amine.13 We demonstrate that I α1 collagen.14 Such telopeptide regions of collagen are
synthesis of CML-containing peptides via Ns alkylation is common glycation and proteolysis sites in vivo.15,16 It was
facile and can be accomplished both during SPPS on decided to compare the protease digestions of the native
Fmoc-protected intermediate peptide or after completion peptide 1 with the CML-peptide 2, in which the lysine was
of SPPS on the full-length resin-bound peptide. We then substituted for CML (Figure 1).
show the impact of the incorporation of CML on the host
peptide digestibility by the key proteolytic enzyme tryp-
Peptide 1 was first synthesised via automated microwave
SPPS, using HBTU/DIPEA for the coupling steps and
sin, and validate the capacity of this AGE to limit proteol-
20% piperidine for deprotection of the Fmoc groups.17
ysis.
CML building block 4 (Scheme 1) was synthesised in five
Route A
Et
O
Et
O
O
O
Ns
N
HN
a, b
c
P
AcHN
YGYDE
STGGISVP
AcHN
YGYDE
STGGISVP
CONH2
FmocHN
N
N
using 4
H
H
O
O
9
7
8
d
t-Bu
Route B
O
O
Ns
Ns
NH
N
c
a, b
e
2
P
AcHN
YGYDE
STGGISVP
AcHN
YGYDE
STGGISVP
FmocHN
N
N
using 6
H
H
O
O
11
7
10
t-Bu
Route C
O
O
Ns
Ns
a, b
NH
N
a
e
P
STGGISVP
STGGISVP
FmocHN
FmocHN
FmocHN
13
using 6
O
O
7
12
Scheme 2 Synthesis of CML-containing peptide 2 by building block approach (A), on-resin alkylation post-synthesis (B), and on-resin alkyl-
ation mid-synthesis (C). Reagents and conditions: (a) Fmoc-SPPS: (i) Fmoc removal: 20% piperidine in DMF; (ii) coupling: Fmoc-AA-OH,
HBTU, DIPEA; (b) (i) N-terminal Fmoc removal: 20% piperidine in DMF; (ii) Ac2O, DIPEA; (c) (i) Ns removal: 2-mercaptoethanol, DBU,
DMF, r.t., 15 min; (ii) peptide release: TFA–TIS–H2O, r.t., 2 h; (d) 0.1 M aq NaOH, 1 h; (e) tert-butyl bromoacetate, DIPEA, DMF, r.t., over-
night.
Synlett 2014, 25, 1835–1838
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LETTER
Expedient Synthesis of Peptides Containing Nε-Carboxymethyllysine
1837
steps starting from lysine 3 following the conditions we ic strategy but also its orthogonality to Fmoc/t-Bu peptide
described earlier.8
synthesis. Furthermore, comparable yields were obtained
in all cases.
This building block was then incorporated into the CML-
containing peptide 2 via SPPS (Route A, Scheme 2),
where resin-bound peptide 8 was synthesised following
standard conditions from Fmoc-proline 7 attached to ami-
nomethyl resin via a rink amide linker.17 Cleavage of pep-
tide 8 afforded peptide 9, which underwent de-
esterification to give the target CML-containing peptide 2.
The HPLC profile of the crude peptide 2 synthesised via
this traditional building block approach is shown in Figure
SI-1A (see the Supporting Information).
Next, revised conditions for selective CML incorporation
by alkylating the resin-bound peptide sequence were ex-
plored. First, Fmoc-Lys(Ns)-OH 6 (Scheme 1) was syn-
thesised from commercially available Fmoc-Lys-OH 5 in
a single step following conditions reported in literature.18
6 was incorporated into the peptide sequence via automat-
ed SPPS using analogous conditions as for peptides 1 and
2 (Route B, Scheme 2).17
Figure 2 Concentrations of CTP peptides 1 and 2 during the trypsin
digest, extrapolated from HPLC
Upon completion of peptide synthesis but prior to its
cleavage, the N-terminus of the resin-bound peptide was
acetylated with acetic anhydride, and the resulting peptide
10 was subjected to alkylation with tert-butyl bromoace-
tate in the presence of DIPEA to afford peptide 11.19 The
nosyl group on peptide 11 was then smoothly deprotected
in the presence of 2-mercaptoethanol and DIPEA (15
min),20 and the resulting peptide cleaved with TFA–TIS–
H2O (38:1:1). The crude peptide was purified by HPLC to
afford 2 in 30% overall yield with >98% purity. The
HPLC profile of the target peptide (Figure SI-1B) con-
firmed that it was identical to 2 prepared using the original
building block approach (Figure SI-1A).
With CML-modified CTPs in hand, the impact of the
presence of an AGE (CML) on the proteolytic digestibili-
ty of host peptide was investigated. Peptide 2 and the na-
tive peptide 1 were subjected to a bovine trypsin digest.
Solutions of the protease were incubated in the presence
of peptides and aliquots were removed, quenched, and an-
alysed by HPLC.21
Under the experimental conditions, trypsin digested 90%
of the native peptide 1 in 10 minutes (t1/2 = 3 min), while
the concentration of CML-modified peptide 2 remained
unchanged (Figure 2). Because the presence of CML
slows down the digestion of host peptides by trypsin, it is
possible that this AGE is predominantly absorbed in pep-
tide-bound form. Investigation of the subsequent metabol-
ic transit of CML-modified peptides within a suitable
animal model is currently underway.
With a successful method for on-resin alkylation of com-
plete peptide in hand, our attention turned to whether the
Fmoc group was stable to the required alkylation condi-
tions. Stability of the Fmoc group to such conditions
would indicate whether CML could be incorporated effi-
ciently during SPPS, as post-synthetic on-resin modifica-
tion is not always feasible. This is especially the case if
multiple modifications are necessary.
A convenient and economical method for site-specific
glycation of peptides by CML has been developed. This
strategy does not require the use of expensive reagents or
elaborate manipulation of protecting groups, and applica-
bility of this method to standard Fmoc SPPS has been
demonstrated. Trypsin digestion studies of both native
and CML-modified peptides showed that CML dramati-
cally influences the rate of enzymatic proteolysis of the
host peptide.
Peptide 12 was synthesised via standard SPPS (Route C,
Scheme 2) on resin up to the point of attachment of 6, and
then alkylated with tert-butyl bromoacetate in the pres-
ence of DIPEA.19 The resulting peptide 13 underwent fur-
ther SPPS and the Ns group was removed19 prior to TFA-
mediated release of the peptide from the resin. The crude
peptide was analysed by HPLC (Figure SI-1C), which
showed a similar profile to that of the peptide obtained by
post-synthetic alkylation (Figure SI-1B), or the building
block approach (Figure SI-1A). Purification afforded the
target peptide in 26% yield and >96% purity.
Acknowledgment
Authors thank the Maurice Wilkins Centre for Molecular Biodisco-
very for generous financial support.
Examination of HPLC profiles of crude peptide 2 pre-
pared by the traditional building block approach, alkyla-
tion post-synthesis, or alkylation mid-synthesis,
demonstrates not only the reliability of this novel synthet-
Supporting Information for this article is available online
at
http://www.thieme-connect.com/products/ejournals/journal/
10.1055/s-00000083.SunogIpimrfiantoSuIpg
n
fonirtat
ori
© Georg Thieme Verlag Stuttgart · New York
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1838
M. Kamalov et al.
LETTER
precipitated with cold Et2O, isolated by centrifugation,
References and Notes
washed in cold Et2O, dissolved in MeCN–H2O (1:1)
containing 0.1% TFA and lyophilised. The peptides were
analysed for purity by LCMS using a Zorbax C3 column (3.5
μm; 3 × 150 mm; Agilent) at 0.3 mL/min using a linear
gradient. The solvent system used was A (0.1% formic acid
in H2O) and B (0.1% formic acid in MeCN). Purification of
crude peptides was performed by semipreparative HPLC
using a Gemini C18 column (10 μm; 250 × 10 mm;
Phenomenex) at 5 mL/min using a shallow linear gradient.
The solvent system used was A (0.1% TFA in H2O) and B
(0.1% TFA in MeCN). The resulting purified peptides were
analysed by the LCMS system used for crude peptide
analysis. The purities were extrapolated from integrating the
peaks corresponding to peptide 1 (13.1 min) and to peptide
2 (12.7 min).
(1) Kamalov, M.; Harris, P. W. R.; Cooper, G. J. S.; Brimble, M.
A. Chem. Commun. 2014, 50, 4944.
(2) Dunn, J. A.; Patrick, J. S.; Thorpe, S. R.; Baynes, J. W.
Biochemistry (Moscow) 1989, 28, 9464.
(3) Rabbani, N.; Thornalley, P. Amino Acids 2012, 42, 1087.
(4) Beisswenger, P. Amino Acids 2012, 42, 1171.
(5) Fu, M. X.; Requena, J. R.; Jenkins, A. J.; Lyons, T. J.;
Baynes, J. W.; Thorpe, S. R. J. Biol. Chem. 1996, 271, 9982.
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Ramamurthy, N. S. Biochim. Biophys. Acta 1978, 534, 73.
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Brimble, M. Org. Lett. 2012, 14, 5740.
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(11) Demmer, O.; Dijkgraaf, I.; Schottelius, M.; Wester, H.-J.;
Kessler, H. Org. Lett. 2008, 10, 2015.
(12) Monfregola, L.; Leone, M.; Calce, E.; De Luca, S. Org. Lett.
2012, 14, 1664.
(13) Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett.
1995, 36, 6373.
(14) Chu, M.-L.; de Wet, W.; Bernard, M.; Ding, J.-F.; Morabito,
M.; Myers, J.; Williams, C.; Ramirez, F. Nature (London)
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(15) Woodley, D. T.; Yamauchi, M.; Wynn, K. C.; Mechanic, G.;
Briggaman, R. A. J. Invest. Dermatol. 1991, 97, 580.
(16) Paul, R. G.; Bailey, A. J. Int. J. Biochem. Cell Biol. 1996, 28,
1297.
(17) Peptide Synthesis: SPPS was performed via the Fmoc
strategy on Rink Amide resin using a Biotage Alstra peptide
synthesiser on 0.1-mmol scale. The Fmoc group was
deprotected with 20% piperidine in DMF for 2 min + 3 min
at 60 °C. The coupling step was performed with Fmoc-AA-
OH (5 equiv) in DMF (0.2 M), HBTU in DMF (4.5 equiv,
0.45 M) and DIPEA (10 equiv) for 5 min at 75 °C. The final
Fmoc group was removed and the amine was acetylated by
Ac2O in the presence of DIPEA at r.t. for 10 min. The
peptides were released from resin with concomitant removal
of the side-chain protecting groups by treatment with TFA–
TIS–H2O (38:1:1, 5 mL) at r.t. for 2 h. Peptides were
(18) Halpin, D. R.; Lee, J. A.; Wrenn, S. J.; Harbury, P. B. PLoS
Biol. 2004, 2, e175.
(19) Alkylation Conditions: Resin-bound peptide 10 or 12 (0.1
mmol, 1 equiv) was swollen in CH2Cl2 (30 min), then in
DMF (10 min), and drained. A solution of tert-butyl
bromoacetate (70 μL, 0.5 mmol, 5 equiv) with DIPEA (175
μL, 1 mmol, 10 equiv) in DMF (5 mL) was added in one
portion and the reaction mixture was shaken overnight at r.t.
to afford peptides 11 or 13, respectively, which were drained
and washed with DMF.
(20) Ns Deprotection: Resin-bound peptide 8 or 11 (0.1 mmol, 1
equiv) was swollen in CH2Cl2 (30 min), then in DMF (10
min) and filtered. A solution of 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) (90 μL, 0.6 mmol, 6
equiv) and 2-mercaptoethanol (42 μL, 0.6 mmol, 6 equiv) in
DMF (5 mL) was added in one portion and the mixture was
shaken for 15 min, drained, and washed with DMF..
(21) Trypsin Digest: Bovine trypsin (0.3 mg, type XI, 9090
units/mg, Sigma) was dissolved in H2O (1 mL) and 3.3 μL (9
units) of this solution diluted to 1 mL using Tris buffer (pH
8.0) and incubated at 37 °C for 30 min. Substrate peptide
(0.21 μmol) was added in one portion and 50 μL aliquots
removed every minute, quenched with 1 M HCl (50 μL) and
analysed by analytical reverse phase-HPLC using a Luna
C18(2) column (3μ; 150 × 3 mm; Phenomenex) at 0.3
mL/min using linear gradient. The concentrations were
extrapolated from integrating the peaks corresponding to
peptide 1 (14.9 min) and to peptide 2 (15.0 min).
Synlett 2014, 25, 1835–1838
© Georg Thieme Verlag Stuttgart · New York