Methods of the site-selective solid phase synthesis of peptide-derived Amadori products

Article abstract of DOI:10.1007/s00726-009-0294-z

Two procedures of glycated peptides' synthesis have been developed. The first method involves reductive alkylation of the ε-amino groups of lysine with 2,3:4,5-di-O-isopropylidene-β-d-arabino-hexos-2-ulo-2,6-pyranose in the presence of sodium cyanoborohydride on solid support. The second one uses a new fully protected lysine derivative, which is a building block designed for direct introduction of the glycated lysine moiety into a peptide, according to the standard solid phase synthesis protocol. The applicability of the proposed methods for the synthesis of peptide-derived Amadori products is discussed. The structure of the synthesized glycated peptides was confirmed by high-resolution mass spectrometry and enzymatic hydrolysis. Circular dichroism studies, performed in water solution, revealed that the formation of the Amadori rearrangement product in the lysine side chain does not influence significantly the conformational preferences of the peptides studied. However, when the solvent was changed to trifluoroethanol, the glycated peptides preferred β-turn conformation.

Full text of DOI:10.1007/s00726-009-0294-z

Amino Acids (2010) 38:881–889
DOI 10.1007/s00726-009-0294-z
ORIGINAL ARTICLE
Methods of the site-selective solid phase synthesis
of peptide-derived Amadori products
Piotr Stefanowicz Æ Monika Kijewska Æ
´
Katarzyna Kapczynska Æ Zbigniew Szewczuk
Received: 12 December 2008 / Accepted: 3 April 2009 / Published online: 19 April 2009
Ó Springer-Verlag 2009
Abstract Two procedures of glycated peptides’ synthesis
have been developed. The first method involves reductive
alkylation of the e-amino groups of lysine with 2,3:4,5-di-
O-isopropylidene-b-D-arabino-hexos-2-ulo-2,6-pyranose in
the presence of sodium cyanoborohydride on solid support.
The second one uses a new fully protected lysine deriva-
tive, which is a building block designed for direct intro-
duction of the glycated lysine moiety into a peptide,
according to the standard solid phase synthesis protocol.
The applicability of the proposed methods for the synthesis
of peptide-derived Amadori products is discussed. The
structure of the synthesized glycated peptides was con-
firmed by high-resolution mass spectrometry and enzy-
matic hydrolysis. Circular dichroism studies, performed in
water solution, revealed that the formation of the Amadori
rearrangement product in the lysine side chain does not
influence significantly the conformational preferences of
the peptides studied. However, when the solvent was
changed to trifluoroethanol, the glycated peptides preferred
b-turn conformation.
Introduction
Amadori compounds—N-substituted (1-deoxy-ketos-1-yl)
amines—are representatives of an important class of
Maillard intermediates. These compounds, formed in
non-enzymatic reactions of proteins’ amino groups with
glucose and other reducing sugars, may decompose and
initiate further reactions. The final result of that process is
the formation of complex mixtures of advanced glycation end
products (AGEs) (Ahmed and Thornalley 2003) involved
in aging and pathological processes such as diabetes and
(Thorpe and Baynes 1996; Mosier et al. 1986) Alzheimer’s
(Moreira et al. 2005) and Parkinson’s (Munch et al. 1998)
diseases. The proteolytic processing of glycated proteins
produces a mixture of glycated peptides, which can be
considered as the markers for diabetes and other diseases.
The defined and analytically pure peptide-derived Amadori
products could be useful as model compounds for studying
the formation of AGEs. Therefore, the search for new
methods of synthesis of Amadori products is intense
(Frolov et al. 2006b, 2007; Stefanowicz et al. 2007).
The first successful methods of peptide-derived Amadori
products’ synthesis were based on the solution phase
approach. The peptides were directly glycated in the pres-
ence of reducing sugars, such as glucose, mannose or gal-
actose (Roscic and Horvat 2006) or, alternatively, a
protected aminofructose moiety was incorporated into the
peptide chain by reductive alkylation of the amino group
by 2,3:4,5-di-O-isopropylidene-b-D-arabino-hexos-2-ulo-2,
6-pyranose (Forrow and Batchelor 1990; Horvat and Jakas
2004). Recently, several new procedures of the site-selec-
tive synthesis of Amadori-modified peptides on solid
support appeared. The protocol developed by Frolov et al.
(2006b) is based on the direct reaction of a deprotected
e-amino group of lysine with the solution of glucose in DMF.
Keywords Solid phase peptide synthesis ꢀ
Amadori rearrangement ꢀ Glycation ꢀ Enzymatic stability
A preliminary result of this work was presented at the 30th European
Peptide Symposium, Helsinki, Finland, 31 August to 5 September
2008, poster 1-01-147.
´
P. Stefanowicz (&) ꢀ M. Kijewska ꢀ K. Kapczynska ꢀ
Z. Szewczuk
Faculty of Chemistry, University of Wrocław,
F. Joliot-Curie 14, 50-383 Wrocław, Poland
e-mail: stp@wchuwr.pl
123

882
P. Stefanowicz et al.
The Frolov’s procedure is simple and general (allows the
synthesis of conjugates of fructose, ribose and other
reducing sugars (Frolov et al. 2006a), and potentially may be
useful for the synthesis of izotopicaly labeled compounds).
However, its low yield (30–35%), difficulties with the
product purification and a serious risk of side reactions limit
its application. Moreover, the process is rather slow and
requires relatively high temperature (70–110°C). Our recent
paper (Stefanowicz et al. 2007) described a new method of
solid phase synthesis of peptide-derived Amadori products
involving the reductive alkylation of e-amino groups of
lysine with 2,3:4,5-di-O-isopropylidene-b-D-arabino-hexos-
2-ulo-2,6-pyranose in the presence of sodium cyanoboro-
hydride on solid support. The reagent used for this procedure
was previously used for solution phase synthesis of Amadori
products.
Sodium cyanoborohydride was purchased from Aldrich.
The 2,3:4,5-di-O-isopropylidene-b-D-arabino-hexos-2-ulo-2,
6-pyranose (1) was prepared according to the procedure
reported by Cubero (1990). The solvents for peptide syn-
¨
thesis (analytical grade) were obtained from Riedel de Haen
(DMF) and J. T. Baker (methanol). Other solvents used in
this work were obtained from Aldrich.
Synthesis1 of Fmoc-Lys(i,i-Fru,Boc)-OH (2)
Fmoc-Lys(Boc)-OH (0.7 g, 1.494 mmol) was dissolved in
trifluoroacetic acid (10 ml) containing 5% of water. After
1 h the solvent was evaporated. Obtained Fmoc-Lys-
OH 9 TFA was dissolved in THF (50 ml) containing
2,3:4,5-di-O-isopropylidene-b-D-arabino-hexos-2-ulo-2,6-
pyranose (1) (3.735 mmol, 2.5 eq). After the addition of
sodium cyanoborohydride (3.735 mmol, 2.5 eq) the solu-
tion was refluxed for 2 h and the mixture was kept for 5 h at
room temperature. After removing THF, the crude product
was acylated overnight with (Boc)2O (3.212 mmol,
2.15 eq) in dioxane (25 ml) containing triethylamine
(5.378 mmol, 3.6 eq) at room temperature. After the
evaporation of the solvent, the crude product was mixed
with water and the mixture was brought to pH 2 using
potassium hydrogen sulfate. Then it was extracted with
ethyl acetate (25 ml). The organic layer was washed with
water, dried over MgSO4 and evaporated to dryness. Finally
the reaction product was purified by chromatography on a
silica gel. After washing the column with chloroform con-
taining 5% of isopropanol, the reaction product was eluted
with 10% isopropanol in chloroform.
A similar approach was also applied by Frolov et al.
(2007). Our recent studies revealed that 2,3:4,5-di-O-iso-
propylidene-b-D-arabino-hexos-2-ulo-2,6-pyranose can be
replaced by its stable and crystalline hydrate, which facil-
itates the procedure.
In this paper we describe the synthesis of a series of
model peptides—the fragments of bovine serum albumine
(BSA).The synthetic fragments of BSA may be used in our
further studies on the formation of the advanced glycation
products. The obtained compounds were characterized by
ESI-MS/MS and CD methods. The structures of glycated
peptides were also confirmed by their proteolytic digestion
combined with direct ESI-MS measurement. This method
of analysis of Amadori modified peptides is efficient for the
determination of the glycation sites and gives more com-
prehensive and easier for interpretation results then those
from a direct MS/MS analysis. We also developed a new
fully protected lysine derivative (Na-9-fluorenylmethox-
ycarbonyl-Ne-tert-butyloxycarbonyl-Ne-N-(2,3:4,5-di-O-
isopropyliden-1-deoxy-b-D-fructopyranose-1-ylo)lysine,
Fmoc-Lys(i,i-Fru,BOC)-OH, which is a building block
useful for incorporating the glycated lysine moiety into the
peptide chain. Application of this derivative allows a facile
synthesis of peptide-derived Amadori products according
to the standard solid phase synthesis protocol.
25
Yield: 42%, mp 79.5–84.5°C, ½aꢁD -14.10 (c 1.0,
MeCN). HPLC: retention time (min) 42.83 (conditions for
HPLC are given in ‘‘Materials and methods’’, ‘‘Purification
1
and characterization of peptides’’). H NMR (CDCl3), d
(ppm) = 1.32–1.52 (12H, m), 1.45 (4H, m), 1.48 (9H, s),
1.74 (1H, m), 1.92 (1H, m), 3.35 (2H, m), 3.50 (2H, s), 3.72
(1H, m), 3.85 (1H, m), 4.19–4.24 (1H, 1H, m two signals
overlap), 4.39 (1H, 2H, m two signals overlap), 4.57 (1H,
m), 5.41 (1H, m), 5.58 (1H, m), 7.19–7.77 (8H, m). HR-
MS: Found 711.3487 calculated for (C38H50N2O11 ? H)?
711.3492; MS/MS (parent 711.35): 369.2, 495.2, 535.2,
553.3, 611.3, 655.3.
Materials and methods
Preparation of peptides
Reagents
Peptides were prepared by manual solid-phase techniques,
on solid support, using the standard Fmoc synthetic pro-
cedure (Chan Weng and White 1999). The following side
chain protecting groups for Fmoc-amino acids were used:
The derivatives of amino acids for peptide synthesis and the
coupling reagent (TBTU) were purchased from NovaBio-
chem. Fmoc-Lys-OH 9 TFA was obtained from Fmoc-
Lys(Boc)-OH by treatment with TFA ? 5% H2O solution
followed by evaporation in vacuo. The preloaded Wang resin
(0.50–0.70 mmol/g) was purchased from NovaBiochem.
1
This procedure should be performed in an efficient fume hood
because of the toxicity of hydrogen cyanide.
123

Methods of the site-selective solid phase synthesis
883
Enzymatic analysis of glycated peptides
t-butyl for Thr, Asp, Glu, and Ser, Mtt for lysine moieties
intended for glycation and Boc for unglycated lysine. The
consecutive amino acid residues were coupled using TBTU
in DMF. After the removal of the Mtt group (using the 1%
solution of TFA in dichloromethane), the e-amino group of
lysine was reductively alkylated with (1) in the presence of
sodium cyanoborohydride. Then the obtained secondary
amino group was protected with Boc2O, and the synthesis
of the peptide was continued according to standard
solid phase synthesis protocol as described previously
(Stefanowicz et al. 2007).
Peptides were dissolved in 10 mM ammonium bicarbonate
buffer to the final concentration of 10 lL. 1 ml samples of
peptide solution, after addition of 20 ll 0.1% trypsin in
water, were incubated for 30 min at room temperature and
then diluted with 5% formic acid in MeCN (1 ml). Obtained
solutions were analyzed directly by the ESI-MS method.
CD spectra
Circular dichroism spectra were recorded on a Jasco J-700
spectropolarimeter. Peptides were dissolved at concentra-
tions of 100 lg/ml and the spectra were recorded in water
and TFE. The pH of water solutions was adjusted to 7 using
0.1 M NaOH. A rectangular quartz cuvette of 1 mm
pathlength was used. Each spectrum represents the average
of eight scans. Data are presented as molar ellipticity [H].
In the second synthetic protocol, which utilizes fully
protected lysine derivative Fmoc-Lys(i,i-Fru,BOC)-OH
(2), the standard Fmoc procedure with TBTU as a cou-
pling reagent was applied. The peptide was cleaved from
the resin using TFA/water/TIS (90:5:5, v/v) for 8 h at
room temperature and precipitated with cold diethyl
ether.
Purification and characterization of peptides
Results and discussion
The crude product was characterized by HPLC on a C18
column. Solvent systems: S1 0.1% aqueous TFA, S2: 80%
acetonitrile ? 0.1% TFA, linear gradient from 0 to 100%
of S2 for 60 min, flow rate 1.0 ml/min, UV detection at
220 nm. The main reaction product was purified by a
preparative reversed-phase HPLC on a Vydac C18 column
(22 mm 9 250 mm), using solvent systems: S1 0.1%
aqueous TFA, S2 40% acetonitrile ? 0.1% TFA, linear
gradient from 50 to 100% of S2 for 60 min, flow rate
7.0 ml/min, UV detection at 220 nm. The fractions were
collected and lyophilized. Their identities were confirmed
on the basis of molecular weights measured with a mi-
crOTOF-Q mass spectrometer equipped with an electro-
spray ionization source.
Synthesis of peptide-derived Amadori products
by reductive alkylation on solid support
In the recent paper we proposed a new method of site-
specific synthesis of glycated peptides on solid support
(Stefanowicz et al. 2007). Briefly, the peptides were
assembled on a Wang resin by the standard Fmoc strategy.
After the removal of the Mtt group, using 1% TFA in
dichloromethane, the obtained free e-amino group of the
lysine side chain was alkylated by 2,3:4,5-di-O-isopropyl-
idene-b-D-arabino-hexos-2-ulo-2,6-pyranose in the pres-
ence of sodium cyanoborohydride. The obtained secondary
amino group was protected using Boc2O and the peptide
synthesis could be continued according to a standard pro-
cedure. After completing the synthesis of the peptide chain,
the N-terminal Fmoc group was removed from the resin
using the solution of piperidine in DMF, and a peptide was
cleaved from the resin using the TFA–water–TIS (90:5:5)
mixture. The cleavage was monitored by ESI-MS. The
deprotection of the hydroxyl groups in the aminofructose
moiety required 8 h at room temperature to remove more
than 95% of diisopropylidene groups. Cleaved peptides
were precipitated using cold diethyl ether, dried in vacuum,
and purified with a preparative HPLC. The fractions con-
taining monoglycated peptides were collected. In some
cases, diglycated compounds (IIb, and IVb) were also
isolated as byproducts. Additionally, the unglycated frag-
ments of BSA were synthesized (not shown in the exper-
imental section) for comparison of their enzymatic stability
and conformational properties with the peptide-derived
Amadori products.
MS/MS spectra
Mass spectrometric measurements were performed on a
quadrupole time-of-flight (micrOTOF-Q) instrument
(Bruker, Germany) equipped with an electrospray source.
Spectra were recorded using aqueous solutions of aceto-
nitrile (50%) and formic acid (1%) at the peptide con-
centration of typically 5 lM. The potential between the
spray needle and the orifice was set to 4.5 kV. In the MS/
MS mode, the quadrupole was used to select the precursor
ions, which were fragmented in the hexapole collision cell
generating product ions that were subsequently mass
analyzed by the orthogonal reflectron TOF mass ana-
lyzer. For collision-induced dissociation (CID) MS/MS
measurements, the voltage over the hexapole collision
cell varied from 25 to 70 V and argon was used as the
collision gas.
123

884
P. Stefanowicz et al.
The obtained compounds are presented in Table 1. Their
lysine. Therefore, we decided to synthesize and test the
novel lysine derivative Fmoc-Lys(i,i-Fru,Boc)-OH (2),
which is compatible with the Fmoc protocol of solid phase
peptide synthesis. The main advantage of this approach is
the possibility of purification of the lysine derivative before
the solid phase synthesis. The compound (2) was obtained
as presented in Scheme 1. The obtained trifluoroacetate,
which in contrast to free Fmoc-Lys-OH is well soluble in
THF, was alkylated using 2,3:4,5-di-O-isopropylidene-b-D-
arabino-hexos-2-ulo-2,6-pyranose in the presence of sodium
cyanoborohydride. After the glycation, the secondary amino
group of lysine was protected using Boc2O. The final product
was purified on a silica-gel column. The purity and identity of
the obtained derivative (2) was confirmed by analytical
HPLC, TLC, ESI-MS, ESI-MS/MS and NMR. Theusefulness
of the obtained building block was tested in the standard
peptide synthesis. A series of the modified fragments of BSA
has been prepared.
purity and identity was tested by HPLC and HR-MS. All
the compounds’ m/z values were consistent with the cal-
culated ones based on their chemical formulas. Selected
compounds (I, Ia, Ic) were subjected for MS/MS analysis,
which confirmed their sequences and allowed the locali-
zation of the site of glycation (Stefanowicz et al. 2007).
Yields (60–95%) and HPLC purities (30–75%) of crude
peptides prepared by the reductive alkylation on solid
support are comparable to those reported in our previous
paper (Stefanowicz et al. 2007). The main side products
were unglycated peptides and peptides containing two
moieties of fructose attached to the e-amino group of lysine
as judged by HR-MS analysis. Unfortunately, the retention
times of these compounds were close. Therefore, a careful
optimization of the separation process was required. This
observation is consistent with the results published by
Frolov (Frolov et al. 2006b; Frolov and Hoffmann 2008).
The purification of peptides glycated at two lysine residues
was even more difficult because numerous byproducts with
very similar chromatographic characteristics were formed.
After the purification, the peptides were characterized in
the same way as compounds prepared by the reductive
alkylation performed directly on the resin. Analytical data
are shown in Table 2. According to the presented data, the
last method gave a purer product. The average purity of
peptides obtained by glycation on the resin (Table 1, only
monoglycated peptides were taken in consideration) is
55%, while the average purity of compounds synthesized
according to the new procedure is 83.5%. The direct
comparison of products IIIa, IIIb and IIIc obtained by two
Synthesis of peptide-derived Amadori products using
the novel lysine derivative: Fmoc-Lys(i,i-Fru,Boc)-OH
The purity of peptide-derived Amadori products can be
improved using a suitable building block containing the
protected fructose residue attached to the e-amino group of
Table 1 Analytical data for glycated peptides obtained by direct reductive alkylation of lysine e-amino group on solid support
Glycated peptide
Rt (min)a Crude
HPLC
Predominant m/z calc/foundc
yield (%) purity (%)b ion
H-Gln-Asp-Thr-Ile-Ser-Ser-Lys-Leu-Lys-Glu-OHe (I)
16.6
17.3
16.8
18.0
18.1
18.6
19.6
12.6
13.1
12.9
65
93
74
78
81
–
62
56
58
63
52
15
50
55
40
53
30
91
80
10
[MH2]2?
[MH2]2?
[MH2]2?
[MH2]2?
[MH2]2?
[MH2]2?
[MH2]2?
[MH3]3?
[MH2]2?
[MH2]2?
[MH3]3?
[MH]?
574.8120/574.8322
655.8385/655.8351
655.8385/655.8361
564.3013/564.3064
645.3277/645.3325
726.3541/726.3864
645.3277/645.3136
406.8950/406.8958
690.8650/690.8623
690.8650/690,8624
514.9302/514.9320
436.2560/436.2563
598.3088/598.3091
760.3616/760.3609
H-Gln-Asp-Thr-Ile-Ser-Ser-Lys-Leu-Lys(Fru)-Glu-OHe (Ia)
H-Gln-Asp-Thr-Ile-Ser-Ser-Lys(Fru)-Leu-Lys-Glu-OHe (Ib)
H-Asp-Asp-Ser-Pro-Asp-Leu-Pro-Lys-Leu-Lys-OH (II)
H-Asp-Asp-Ser-Pro-Asp-Leu-Pro-Lys(Fru)-Leu-Lys-OH (IIa)
H-Asp-Asp-Ser-Pro-Asp-Leu-Pro-Lys(Fru)2-Leu-Lys-OHd (IIb)
H-Asp-Asp-Ser-Pro-Asp-Leu-Pro-Lys-Leu-Lys(Fru)-OH (IIc)
H-Asp-Thr-Glu-Lys-Gln-Ile-Lys-Lys-Gln-Thr-OH (III)
H-Asp-Thr-Glu-Lys(Fru)-Gln-Ile-Lys-Lys-Gln-Thr-OH (IIIa)
H-Asp-Thr-Glu-Lys-Gln-Ile-Lys-Lys(Fru)-Gln-Thr-OH (IIIb)
87
82
70
78
67
80
77
–
H-Asp-Thr-Glu-Lys(Fru)-Gln-Ile-Lys(Fru)-Lys-Gln-Thr-OH (IIIc) 12.8
H-Lys-Ala-Ala-Phe-OHe (IV)
H-Lys(Fru)-Ala-Ala-Phe-OHe (IVa)
H-Lys(Fru)2-Ala-Ala-Phe-OHd (IVb)
a
16.6
16.3
16.0
[MH]?
[MH]?
C18 column Vydac (250 9 4.6 mm): gradient 0–80% B in A in 60 min, A: water ? 0.1% TFA, B: acetonitrile ? 0.1% TFA
Purity based on the integral of the crude product absorption at k 220 nm on RP-HPLC
ESI-MS spectrum, micrOTOF-Q (Bruker Daltonics)
b
c
d
e
Diglycated peptides isolated as byproducts
Synthesis of these peptides was described previously (Stefanowicz et al. 2007)
123

Methods of the site-selective solid phase synthesis
885
Scheme 1 The synthesis of
Fmoc-Lys(i,i-Fru,Boc)-OH
methods and presented in Tables 1 and 2 also revels that
the new procedure provides significantly purer peptides.
The most striking example is diglycated peptide IIIc. The
reductive alkylation on the resin gave a material containing
30% of the desired product, while the synthesis based on
Fmoc-Lys(i,i-Fru,Boc)-OH provided the purity of 83%.
The main advantage of this approach is that the obtained
glycoconjugates are free of unglycated and diglycated
peptide impurities, which are difficult to separate because
of their similar retention times on RP-HPLC (Frolov et al.
2006b). The method utilizing derivative (2) is especially
convenient for the synthesis of long and difficult sequences
or sequences containing more than one glycation site.
Because this new approach does not require any nonstan-
dard reagents and procedures at the stage of peptide syn-
thesis, it is recommended for the automatic synthesis of
glycated peptides.
weakest bonds in the modified side-chain tend to dissociate
preferentially, resulting in a high abundance of ions cor-
responding to various degrees of water loss. A neutral loss
of formaldehyde and of a whole hexose was also observed
(Frolov et al. 2006a; Stefanowicz et al. 2001). The abun-
dance of peptide backbone fragments is low, which makes
the sequencing of glycated peptides difficult, especially
those containing more than one glycation site. Although the
electron transfer dissociation (ETD) method, performed on
specialized ion-trap mass spectrometers only, has recently
been applied to overcome these difficulties (Zhang et al.
2007), the sequencing of the glycated peptides still remains
a challenge.
In our work we decided to use the enzymatic hydrolysis
as a tool to analyze the obtained conjugates. Peptides dis-
solved in ammonium bicarbonate buffer were treated with
the water solution of trypsin, and after a defined time, the
samples were analyzed by ESI-MS. The glycated moiety
remains intact permitting the localization of the carbohy-
drate attachment site. The obtained results are presented in
Fig. 1.
Enzymatic analysis of peptide-derived Amadori
products
Recently, a tandem mass spectrometry (MS/MS) was
applied to the glycated peptide sequencing (Frolov et al.
2006a, b; Stefanowicz et al. 2007). The studies were per-
formed on instruments that utilize CID, in which the
intramolecular vibrational energy redistribution occurs
prior to the bond cleavage (Zhang et al. 2007). Thus, the
There are three groups of isomeric compounds among
peptides subjected to the enzymatic hydrolysis: (Ia; Ib), (IIa;
IIc) and (IIIa; IIIb; IIIc). Those isomers differ by location of
the Lys(Fru) moiety (abbreviated as X) within the peptide
sequences. A comparison of the masses of the hydrolysis
products reveals distinct differences between the isomeric
123

886
P. Stefanowicz et al.
Table 2 Analytical data for glycated peptides obtained using Fmoc-Lys(i,i-Fru,BOC)-OH
Glycated peptide
Rt (min)a Crude
yield (%) purity (%)b
HPLC
Predominant ion m/z calc/foundc
H-Asp-Thr-Ile-Ser-Ser-Lys(Fru)-Leu-Lys-Glu-OH
16.50
17.17
13.00
13.05
12.95
78.9
78
85
88
92
75
78
83
[MH2]2?
[MH2]2?
[MH2]2?
[MH2]2?
[MH2]2?
[MH2]2?
591.8092/591.8110
676.8438/676.8457
690.8650/690.8623
690.8650/690.8630
690.8650/690.8641
771.8914/771.8913
Ac-Gln-Asp-Thr-Ile-Ser-Ser-Lys(Fru)-Leu-Lys-Glu-OH
H-Asp-Thr-Glu-Lys-Gln-Ile-Lys(Fru)-Lys-Gln-Thr-OH
H-Asp-Thr-Glu-Lys(Fru)-Gln-Ile-Lys-Lys-Gln-Thr-OH (IIIa)
H-Asp-Thr-Glu-Lys-Gln-Ile-Lys-Lys(Fru)-Gln-Thr-OH (IIIb)
88.1
81
88.5
87.1
H-Asp-Thr-Glu-Lys(Fru)-Gln-Ile-Lys(Fru)-Lys-Gln-Thr-OH (IIIc) 12.83
a
C18 column Vydac (250 9 4.6 mm): gradient 0–80% B in A in 60 min, A: water ? 0.1% TFA, B: acetonitrile ? 0.1% TFA
Purity based on the integral of the crude product absorption at k 220 nm on RP-HPLC
ESI-MS spectrum, micrOTOF-Q (Bruker Daltonics)
b
c
peptides. For example, peptide Ia (QDTISSKLXE)produced
sequences, glycated in a different way, revealed signifi-
cant similarity. Therefore, in water solution, the influence
of glycation on the conformational preferences of the
investigated peptides determined by the CD method is
negligible. Selected peptides (I, Ia and Ib) were also
investigated in TFE. Results are shown in Fig. 3. In TFE
three bands were observed: the positive at 185 nm and
two negative at 200 and 220 nm, respectively. This type
of CD spectrum (C class) usually is interpreted as a-helix,
but in short peptides may correspond to b-turn confor-
mation of I or III type (Perczel et al. 1991). This influence
of trifluoroethanol on the peptide conformation is well
known in the literature (Woody 1985). The CD spectra of
glycated peptides Ia and Ib are similar. The molar ellip-
ticity at 220 nm for these compounds is significantly
higher in comparison to nonglycated peptide I. This may
suggest that in trifluoroethanol glycated compounds con-
tain more b-turn conformers in the conformational equi-
librium. This result is consistent with previous data (Vass
et al. 2000) evidencing that N-terminally glycated opioid
peptides prefer b-turn conformation. It was also observed,
using the NMR analysis, that the glycation of synthetic
human serum albumin a-helix 28 can influence or change
the local protein secondary structure (Howard and Smales
2005). Similar studies performed on the helical fragment
of lysozyme also revealed that the glycation of lysine
results in the disruption of the local secondary structure
(Povey et al. 2008).
fragments: LXE (m/z 551.3; z ?1) and QDTISSK (m/z 778.4;
z ?1), while Ib (QDTISSXLKE) produced the fragment
QDTISSXLK (m/z 591.3; z ?2). According to the presented
results, the peptide chain was cleaved only at the carboxyl
end of unmodified lysine, but not at lysine with a glycated or
diglycated side chain, even at the enhanced enzyme con-
centration (fivefold higher) and incubation time (up to 5 h).
This observation is consistent with the previous reports
indicating that the enzymatic activity of trypsin is inhibited
by the lysine glycation (Brock et al. 2003; Lapolla et al.
2004). Analysis of the formed fragments allows the identi-
fication of the hydrolysis sites and in a comprehensive way
confirms the localization of Lys(Fru) residues in the peptide
chain. The pattern of tryptic hydrolysis unambiguously
confirmed the regioselectivity of the proposed synthetic
procedures.
Conformational preferences of peptide-derived
Amadori products
Circular dichroism spectra measured in water solution at
pH 7 are presented in Fig. 2. The spectra of peptides I, Ia,
Ib, II, IIa, IIb, IIc, III, IIIa, IIIb, IIIc exhibit a strong,
negative band at 198 nm. The molar ellipticity of the
investigated peptides ranged from -170,000 to -400,000.
These spectra strongly suggest the unordered (open)
conformation of the peptides studied. According to the
literature data this type of conformation is characterized
by a negative band near 200 nm with the mean molar
ellipticity of 40,000 deg cm2/dmol (Woody 1985). The
CD spectra are presented in three groups (Fig. 2a–c). Each
group of spectra is based on the same sequence. The Lys
(Fru) moiety is not present in peptides I, II, III, whereas
peptide IIb has two Fru groups attached to the same side
chain of Lys. Other compounds differ by location of the
glycated lysine. A comparison of spectra of the same
The literature shows examples of strong influence of the
glycation on the peptide conformation as well as examples
(Mendez et al. 2005; Stefanowicz et al. 2001) of non-sig-
nificant influence of the glycation on the conformational
preferences of the peptide. In this study, the effect of the
peptide modification on the conformation is different in
water and in trifluoroethanol. The presented data suggest
that the conformational consequences of the lysine moiety
glycation depend on the sequence and on the solvent.
123

Methods of the site-selective solid phase synthesis
887
Fig. 1 The ESI-MS spectra of
tryptic hydrolysis products of
peptides. Peptide charges were
calculated based on the
distribution of the isotopic
patterns. Cleavage sites together
with m/z values of fragments are
also presented. X:
monoglycated Lys residue, Z:
diglycated Lys residue
123

888
P. Stefanowicz et al.
Fig. 3 CD spectra of the glycated peptides: H-Gln-Asp-Thr-Ile-Ser-
Ser-Lys-Leu-Lys-Glu-OH, H-Gln-Asp-Thr-Ile-Ser-Ser-Lys-Leu-Lys
(Fru)-Glu-OH and H-Gln-Asp-Thr-Ile-Ser-Ser-Lys(Fru)-Leu-Lys-Glu-
OH. Spectra were measured in TFE, details are given in ‘‘Materials and
methods’’
Conclusion
Two efficient procedures for the solid-phase synthesis of
site-selectively glycated peptides were developed. Both
proposed methods are compatible with the Fmoc strategy
of the solid-phase peptide synthesis. The second method
based on the new lysine derivative gives purer products,
which are free from diglycated and unglycated peptides.
This last approach does not require any nonstandard pro-
cedures therefore its application for the automatic synthesis
of glycated peptides is promising.
References
Ahmed N, Thornalley PJ (2003) Quantitative screening of protein
biomarkers of early glycation, advanced glycation, oxidation and
nitrosation in cellular and extracellular proteins by tandem mass
spectrometry multiple reaction monitoring. Biochem Soc Trans
31:1417–1422. doi:10.1042/BST0311417
Brock JW, Hinton DJ, Cotham WE, Metz TO, Thorpe SR, Baynes
JW, Ames JM (2003) Proteomic analysis of the site specificity of
glycation and carboxymethylation of ribonuclease. J Proteome
Res 2:506–513. doi:10.1021/pr0340173
Chan Weng C, White PD (eds) (1999) Fmoc solid phase peptide
synthesis: a practical approach. Oxford University Press, Oxford
Cubero II, Lopez-Espinosa MTP (1990) Enantiospecific synthesis of
spiroacetals. 1. An enantiospecific synthesis of (-)-talaromycin-
A and (-)-talaromycin-B from D-fructose. Carbohydr Res
205:293–304. doi:10.1016/0008-6215(90)80148-V
Forrow NJ, Batchelor MJ (1990) Synthesis of the N-glycopeptide
partial sequence A(1)-A(12) of the b-chain of glycosylated
hemoglobin HBA1C - a new approach to Amadori N-glycopep-
tides. Tetrahedron 31:3493–3494. doi:10.1016/S0040-4039(00)
97431-8
Fig. 2 CD spectra of the glycated albumin fragments: A: peptides
derived from the sequence H-Gln-Asp-Thr-Ile-Ser-Ser-Lys-Leu-Lys-
Glu-OH, B: peptides derived from the sequence H-Asp-Asp-Ser-
Pro-Asp-Leu-Pro-Lys-Leu-Lys-OH and C: peptides derived from
the sequence H-Asp-Thr-Glu-Lys-Gln-Ile-Lys-Lys-Gln-Thr-OH. All
spectra were measured in water at pH 7, details are given in
‘‘Materials and methods’’
123

Methods of the site-selective solid phase synthesis
889
Frolov A, Hoffmann R (2008) Separation of Amadori peptides from
their unmodified analogs by ion-pairing RP-HPLC with hepta-
fluorobutyric acid as ion-pair reagent. Anal Bioanal Chem
392:1209–1214. doi:10.1007/s00216-008-2377-1
Frolov A, Hoffmann P, Hoffmann R (2006a) Fragmentation behavior
of glycated peptides derived from D-glucose, D-fructose, and
D-ribose in tandem mass spectrometry. J Mass Spectrom 41:
1459–1469. doi:10.1002/jms.1117
Perczel A, Hollosi M, Foxman BM, Fasman GD (1991) Conforma-
tional-analysis of pseudocyclic hexapeptides based on quantita-
tive circular dichroism (CD), NOE, and X-ray data - the pure CD
spectra of type-I and type-II b-turns. J Am Chem Soc 113:9772–
9784. doi:10.1021/ja00026a010
Povey JF, Howard MJ, Williamson RA, Smales CM (2008) The effect
of peptide glycation on local secondary structure. J Struct Biol
161:151–161. doi:10.1016/j.jsb.2007.10.004
Frolov A, Singer D, Hoffmann R (2006b) Site-specific synthesis of
Amadori-modified peptides on solid phase. J Pept Sci 12:389–
395. doi:10.1002/psc.739
Frolov A, Singer D, Hoffmann R (2007) Solid-phase synthesis of
glucose-derived Amadori peptides. J Pept Sci 13:862–867. doi:
10.1002/psc.901
Horvat S, Jakas A (2004) Peptide and amino acid glycation: new
insights into the Maillard reaction. J Pept Sci 10:119–137. doi:
10.1002/psc.519
Roscic M, Horvat S (2006) Transformations of bioactive peptides in
the presence of sugars—characterization and stability studies of
the adducts generated via the Maillard reaction. Bioorg Med
Chem 14:4933–4943. doi:10.1016/j.bmc.2006.03.006
´
´
Stefanowicz P, Boratynski J, Kanska U, Petry I, Szewczuk Z (2001)
Evaluation of high temperature glycation of proteins and
peptides by electrospray ionization mass spectrometry. Acta
Biochim Pol 48:1137–1142
´
Stefanowicz P, Kapczynska K, Kluczyk A, Szewczuk Z (2007) A new
Howard MJ, Smales CM (2005) NMR analysis of synthetic human
serum albumin alpha-helix 28 identifies structural distortion
upon amadori modification. J Biol Chem 280:22582–22589.
doi:10.1074/jbc.M501480200
Lapolla A, Fedele D, Reitano R, Arico NC, Seraglia R, Traldi P,
Marotta E, Tonani R (2004) Enzymatic digestion and mass
spectrometry in the study of advanced glycation end products/
peptides. J Am Soc Mass Spectrom 15:496–509. doi:10.1016/j.
jasms.2003.11.014
Mendez DL, Jensen RA, McElroy LA, Pena JM, Esquerra RM (2005)
The effect of non-enzymatic glycation on the unfolding of
human serum albumin. Arch Biochem Biophys 444:92–99. doi:
10.1016/j.abb.2005.10.019
Moreira PI, Smith MA, Zhu XW, Nunomura A, Castellani RJ, Perry
G (2005) Oxidative stress and neurodegeneration. Ann N Y Acad
Sci 1043:545–552. doi:10.1196/annals.1333.062
procedure for the synthesis of peptide-derived Amadori products
on a solid support. Tetrahedron Lett 48:967–969. doi:10.1016/
j.tetlet.2006.12.022
Thorpe SR, Baynes JW (1996) Role of Maillard reaction in diabetes
mellitus and diseases of aging. Drugs Aging 9:69–77. doi:
10.2165/00002512-199609020-00001
Vass E, Hollosi M, Kveder M, Kojic-Prodic B, Cudic M, Horvat S
(2000) Spectroscopic evidence of b-turn in N-glycated pepti-
domimetics related to leucine-enkephalin. Spectrochim Acta A
Mol Biomol Spectrosc 56:2479–2489. doi:10.1016/S1386-1425
(00)00336-X
Woody RW (1985) Circular dichroism of peptides. In: Hruby VJ,
Meienhofer J (eds) The peptides, vol 7. Academic Press, New
York, pp 16–114
Zhang QB, Frolov A, Tang N, Hoffmann R, van de Goor T, Metz TO,
Smith RD (2007) Application of electron transfer dissociation
mass spectrometry in analyses of non-enzymatically glycated
peptides. Rapid Commun Mass Spectrom 21:661–666. doi:
10.1002/rcm.2884
Mosier MA, Occhipinti JR, Burstein NL (1986) Autofluorescence of
the crystalline lens in diabetes. Arch Ophthalmol 104:1340–1343
Munch G, Gerlach M, Sian J, Wong A, Riederer P (1998) Advanced
glycation end products in neurodegeneration: more than early
markers of oxidative stress? Ann Neurol 44:S85–S88
123