Site-selective solid phase synthesis of carbonylated peptides

Article abstract of DOI:10.1007/s00726-015-1967-4

Abstract The aim of our research was to design an efficient method for the solid phase synthesis of carbonylated peptides. For this purpose, we designed and synthesized a fully protected derivative Fmoc-amino(2,5,5-trimetyhyl-1,3-dioxolan-2-yl)acetic acid

Full text of DOI:10.1007/s00726-015-1967-4

Amino Acids
DOI 10.1007/s00726-015-1967-4
ORIGINAL ARTICLE
Site‑selective solid phase synthesis of carbonylated peptides
Mateusz Waliczek1 · Monika Kijewska1 · Piotr Stefanowicz1 · Zbigniew Szewczuk1
Received: 2 December 2014 / Accepted: 17 March 2015
© The Author(s) 2015. This article is published with open access at Springerlink.com
Abstract The aim of our research was to design an effi-
cient method for the solid phase synthesis of carbonylated
peptides. For this purpose, we designed and synthesized a
fully protected derivative Fmoc-amino(2,5,5-trimetyhyl-
1,3-dioxolan-2-yl)acetic acid (Fmoc-Atda-OH) of a novel
unnatural amino acid (Thr(O)-2-amino-3-oxo-butanoic
acid). To obtain the mentioned derivative, two synthetic
strategies were investigated using different reagents for
carbonyl protection, ethane-1,2-diol and 2,2-dimethyl-pro-
pane-1,3-diol. The racemization of oxidized threonine was
also analyzed and discussed. We successfully carried out
the solid phase synthesis of peptides containing a Thr(O)
moiety using Fmoc-Atda-OH according to the standard
Fmoc strategy. The application of the designed building
block allows the synthesis of peptides containing D,L‑
Thr(O) residue, which may be used as models of oxida-
tively modified peptides which occur in biological systems
and are related to many diseases.
Introduction
The Reactive Oxygen Species (ROS) formation is the con-
sequence of a redox regulation in cells (Madian and Reg-
nier 2010a). The impaired balance between production
and removing ROS is an undesirable phenomenon that
can lead not only to oxidation of proteins but also other
biomolecules, like DNA, fatty acids, and carbohydrates.
The proteins, however, are the most susceptible to ROS
impact. Many changes in amino acid residues resulting
from oxidation reactions have been characterized, such as
carbonylation or disulfide formation (Madian and Regnier
2010b). The selected examples are presented in Table 1
(Madian and Regnier 2010a). These post-translational
modifications can be divided into three groups distin-
guishable by mass spectrometry. One involves oxidative
cleavage in either the protein backbone or amino acid side
chains, with Pro, Arg, Lys, Thr, Glu or Asp residues most
likely to undergo oxidative cleavage. The Cys and Met are
most susceptible to oxidative modification. Other moieties,
especially Lys, Arg, Pro and Thr incur formation of car-
bonyl groups (aldehydes and ketones) in the side chains.
This modification is based on the addition of lipid oxida-
tion products such as 4-hydroxy-2-nonenal to proteins.
Finally, carbonyl groups in proteins can be generated by
advanced glycation.
Keywords Oxidative stress · Oxidation · Carbonylation ·
Solid phase peptide synthesis · Carbonylated peptides ·
Posttranslational modification
Handling Editor: J. Bode.
Some of these oxidations are reversible and can play a
role in metabolism, the other—irreversible—often lead to
protein inactivation. Protein carbonylation is considered to
be the most common protein oxidation reaction, therefore,
it has become an indicator of the oxidative stress (Moller
and Rogowska-Wrzesin´ska 2011; Roe et al. 2010). It has
been proven that metal-catalyzed oxidation (MCO) is the
main mechanism of protein carbonylation. The other path-
ways leading to formation of carbonyl moieties involve
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-015-1967-4) contains supplementary
material, which is available to authorized users.
* Monika Kijewska
monika.kijewska@chem.uni.wroc.pl
1
Faculty of Chemistry, University of Wrocław, F. Joliot-Curie
14, 50-383 Wrocław, Poland
1 3

M. Waliczek et al.
Table 1 List of the different types of oxidative modifications
(Madian and Regnier 2010a)
The resulting isotopic signature observed in a mass spec-
trum allows detection of carbonylated peptides (Roe et al.
2010). Similarly, it was proven that treating the sample
containing glycated peptides with H128O under microwave
activation, results in the isotopic exchange of anomeric
oxygen which allows detection of the Amadori products by
MS techniques (Kijewska et al. 2011). DNPH derivatized
carbonylated peptides were also analyzed by ESI–MS/MS
in the negative ion mode allowing a differentiation of vari-
ous types of carbonyl compounds through a specific frag-
mentation (Bollineni et al. 2011a).
Amino acids
Oxidative modification
T
2-Amino-3-oxo-butanoic acid
Hydroxylation
Y, D, K, F, P
K
Aminoadipic semialdehyde, Amadori products,
3-Deoxyglucosone adducts, glyoxal adducts,
methylglyoxal adducts, hydroxynonenal,
malonodialdehyde, N-homocysteinylation
R
C
Glutamic semialdehyde
Sulfonic acid, sulfinic acid, sulfenic acid,
S-homocysteinylation
In view of the reasons given above, there is a need for
new methods of carbonylated peptide synthesis, since they
are useful as standards for quantitative analysis and help in
development of enrichment methods. Actually, the avail-
able methods allow only for synthesis of model unnatural
carbonylated peptides. Marceau (2005) and co-workers
(Buré et al. 2012; Bollineni et al. 2011b) reported synthesis
of the C-terminal alpha-oxo aldehyde peptides and pyru-
vic acid-containing peptides using solid phase synthesis.
Another approach, based on periodate oxidation of 2-ami-
noalcohol (for example serine or threonine), results in for-
mation of the aldehyde group at the N-terminus of a pep-
tide chain.
The objective of our current work is a synthesis of pep-
tides containing oxidized threonine using a fully protected
building block. This approach allows preparation of ana-
lytically pure product containing Thr(O) residue. Accord-
ing to literature reports (Madian and Regnier 2010b), such
peptides were detected in products of enzymatic hydrolysis
of oxidized proteins. This synthetic approach is similar to
that applied in our previous paper, focused on the reaction
of a lysine moiety with reducing sugars (Stefanowicz et al.
2010). In the current project, we extended our research to
the physiologically relevant oxidative modifications result-
ing in carbonylation of proteins. It may be expected that
these model compounds will be useful for development
and testing affinity-based procedures of preconcentration of
carbonylated peptides. In addition, isotopically labeled ana-
lytically pure peptides containing carbonyl groups may be
used for the quantitative determination of this modification
in enzymatic digests.
M
P
Sulfoxide, sulfon
Glutamic semialdehyde, pyroglutamic
either oxidative cleavage of proteins by α-amidation
pathway or oxidation of glutamyl side chains (Stadtman
and Levine 2003; Requena et al. 2001). An addition of
4-hydroxynoneal, the main product of lipid peroxidation,
to nucleophilic groups in the side chains of amino acids
is another mechanism leading to formation of the car-
bonylated proteins (Dalle-Donne et al. 2006). This post-
translational modification (PTM) is linked to numerous
diseases, including diabetes (Telci et al. 2000), cancer or
Parkinson and Alzheimer diseases (Smith et al. 1991; Utt-
ara et al. 2009).
Characterization of carbonylated proteins has been a
challenge for contemporary proteomics due to their low
abundance in the biological material as well as a diversity
of possible modifications resulting from various pathways
leading to the carbonyl compound formation. Because of
the requirements imposed by proteomics, many useful
methods allowing selecting and recognizing the carbon-
ylated proteins have emerged. Most of these techniques
are based on formation of Schiff bases (Dalle-Donne et al.
2005; Grimsrud et al. 2008) ranging from the classic dini-
trophenylhydrazine derivatization (DNPH) (Palmese et al.
2011), through biotin hydrazide (Mirzaei et al. 2008)
method to Girard’s P reagent (Mirzaei and Regnier 2006)
and Oxidation-Dependent Element Coded Affinity Tags
(O-ECAT) (Lee et al. 2006). The widely used enrichment
approach consists reaction of the carbonyl group with bio-
tin hydrazide and a further selection, by means of immobi-
lized avidin or streptavidin on an affinity column (Mirzaei
et al. 2008). The additional advantage of Girard’s P reagent,
which is also based on hydrazone formation, is an increase
in ionization efficiency resulting from the presence of a
permanent positive charge in the quaternary ammonium
moiety (Mirzaei and Regnier 2006). The high-resolution
mass spectrometry combined with stable isotope labeling
involving specific incorporation of 18O into the carbonyl
moieties is also used for studying carbonylated proteins.
Experimental
Reagents
Z-Thr-OBzl (>98 %) was purchased from Alfa Aesar
(Karlsruhe, Germany). The oxidizing agents: pyridinium
chlorochromate (PCC) was obtained from Sigma-Aldrich
(St. Louis, MO, USA) and sulfur trioxide-pyridine com-
plex was obtained from Fluka (Steinheim, Germany). The
1 3

Site-selective solid phase…
solvents used for Fmoc-Amda-OH synthesis were: dichlo-
romethane (Chempur, Piekary Sla˛skie, Poland), dimethyl
HPLC: retention time (min): 35.6 (conditions for HPLC
are given in the “Experimental” section). HR-MS m/z:
found 364.127 calculated for (C19H19NO5 + Na)+ 364.116.
´
sulfoxide (Scientific Limited Park, Northapton, U.K),
methanol (POCh, Poland), ethanol (Eurochem BGD), ace-
tone (POCh, Poland), benzene (Eurochem BGD, Tarnów,
Poland) and diethyl ether (Chempur, Poland). Ethylene gly-
col, 2,2-dimethyl-propane-1,3-diol and p-toluenesulfonic
acid as a catalyst were obtained from Sigma-Aldrich (St.
Louis, MO, USA). The protecting group 9-fluorenylme-
thyl succinimidyl carbonate (Fmoc-OSu) was purchased
from Iris Biotech GmbH (Marktredwitz, Germany). Ethyl
acetoacetate as a starting substrate in Method 2 was from
Sigma-Aldrich (St. Louis, MO, USA). The zinc dust and
N,N-diisopropylethylamine were purchased from Fluka
(Steinheim, Germany). Sodium nitrite was obtained from
POCh and glacial acetic acid from J. T. Baker (Deventer,
The Netherlands). The inorganic salts such as potassium
hydrogen sulfate and anhydrous magnesium sulfate were
purchased from Chempur. The solvents for peptide synthe-
sis (analytical grade) were obtained from Riedel de Haën
(Seelze, Germany) (DMF) and J. T. Baker (methanol).
Synthesis of Z‑Thr(Amda)‑OBzl (2) The crude, oxidized
product (0.51 g, 1.5 mmol) was dissolved in benzene
(60 ml). To this mixture, ethylene glycol (0.36 g, 5.8 mmol)
and p-toluenesulfonic acid (120 mg) (as a catalyst) were
added (Green and Wuts 1999). The resulting mixture was
refluxed for 2.5 h using a Dean-Stark apparatus to remove
water. After the reaction completion, the solvent was evapo-
rated under reduced pressure. HR-MS m/z: found 408.141
calculated for (C21H23NO6 + Na)+ 408.142.
Synthesis of Fmoc‑Thr(Amda)‑OH (3) To remove protect-
ing groups, the obtained crude product was subjected to
hydrogenolysis in methanol, using 5 % Pd on charcoal as a
catalyst. The reduction was carried out for 4 h and the reac-
tion progress was controlled with TLC (eluent: E2). Then,
the mixture was filtered through a paper filter and evapo-
rated to dryness.
TLC: retention time Rf: 0.19 (conditions for TLC are
given in the “Experimental” section). HR-MS m/z: found
162.083 calculated for (C6H11NO4 + H)+ 162.076.
The last step included introduction of the fluorenylmeth-
oxycarbonyl group (Fmoc). For this purpose, the crude
product (1.5 mmol) was dissolved in water (15 ml) and
brought to pH 8 with NaHCO3. After determination of the
pH, Fmoc-OSu (0.49 g, 1.5 mmol) was added. Due to the
precipitation, the stoichiometric amount of DIEA (0.25 ml)
in acetone (15 ml) was added. The reaction progress was
controlled using TLC (eluent: E1). After the reaction
completion, acetone was removed using a rotary evapora-
tor. The remaining solution was acidified using potassium
hydrogen sulfate and then extracted with ethyl acetate
(30 ml), and washed with water. Finally, the combined
organic layers were dried over anhydrous magnesium sul-
fate and evaporated to dryness.
Synthesis of Fmoc‑amino(2‑metyhyl‑1,3‑dioxolan‑2‑yl)
acetic acid (Method 1A)
and Fmoc‑amino(2,5,5‑trimetyhyl‑1,3‑dioxolan‑2‑yl)
acetic acid (Method 1B)
Method 1A
Synthesis of Z‑Thr(O)‑OBzl (1) Oxidation 1 Z-Thr-
OBzl (0.5 g, 1.5 mmol) was dissolved in dichloromethane
(40 ml). After the addition of PCC (0.69 g, 0.32 mmol) and
molecular sieves (3 g), the resulting mixture was stirred for
24 h. After this time, the mixture was diluted with diethyl
ether (100 ml) and filtered through a short column contain-
ing silica gel and evaporated to dryness.
HPLC: retention time (min): 35.7 (conditions for HPLC
are given in the “Experimental” section). HR-MS m/z:
found 364.128 calculated for (C19H19NO5 + Na)+ 364.116.
Oxidation 2 Z-Thr-OBzl (0.5 g, 1.5 mmol) was dissolved
in dichloromethane (5 ml) and then triethylamine (0.6 ml.
4.3 mmol) was added. The mixture was cooled to −10 °C
in the ice bath. The next step included addition of the SO3/
pyridine complex in freshly distilled DMSO (5 ml). The
resulting mixture was stirred for 2.5 h. The progress of the
reaction was controlled using TLC (eluent: E2). After the
reaction completion, water (20 ml) with potassium hydro-
gen sulfate was added to increase the salting-out effect.
Then, the mixture was extracted with ethyl acetate (15 ml)
and the organic layer was washed with water and brine.
The combined organic extracts were dried over anhydrous
MgSO4 and evaporated to dryness.
Fmoc‑Amda‑OH yield: 25 %; HPLC: retention time
(min): 33.3 (conditions for HPLC are given in the “Experi-
mental” section). HR-MS m/z: found 406.126 calcu-
lated for (C21H21NO6 + Na)+ 406.126; MS/MS (parent
406.126): 362.148.
Fmoc‑βAla‑OH HPLC: retention time (min): 32.5
(conditions for HPLC are given in the “Experimen-
tal” section). HR-MS m/z: found 334.105 calculated
for (C17H18NO4 + Na)+ 334.105; 1H NMR (CDCl3) δ
(ppm) = 7.74 (d, J = 7.5 Hz, 2H), 7.56 (d, J = 7.4 Hz,
2H), 7.38 (t, J = 7.4 Hz, 2H), 7.29 (t, J = 7.3 Hz, 2H),
5.25 (s, 1H), 4.42 (m, 2H), 3.42 (m, 2H), 2.50 (m, 2H); 13
C
NMR (CDCl3) δ (ppm) = 176.93, 156.58, 143.96, 141.55,
127.93, 127.27, 125.29, 120.22, 67.03, 47.43, 36.55, 34.30.
1 3

M. Waliczek et al.
Method 1B
used directly in the next step. The obtained ether solution
containing acetic anhydride (2.1 g, 20.6 mmol) and glacial
acetic acid (5.78 g, 96.2 mmol) was placed in two-necked,
round bottom flask equipped with thermometer. Then, the
zinc dust (1.92 g, 293 mmol) was added in small portions
over a period of 1.5 h with vigorous stirring at temperature
in the range of 40–50 °C. The exothermic reaction was
cooled in a water bath. After completion of the metal addi-
tion, the mixture was stirred for an additional 30 min. The
excess zinc powder was filtered and washed thoroughly
with three 10 ml portions of glacial acetic acid. The result-
ing filtrate was evaporated under reduced pressure and
finally, thick oil was obtained.
HPLC: retention time (min): 8.8 (conditions for HPLC
are given in the “Experimental” section). HR-MS m/z:
found 210.081 calculated for (C8H13NO4 + Na)+ 210.074;
1H NMR (CDCl3) δ (ppm) = 1.29 (t, J = 7.1 Hz, 3H), 2.04
(s, 3H), 2.36 (s, 3H), 4.25 (qd, J = 6.0 Hz, 3.0 Hz, 2H),
5.22 (d, J = 6.5, 1H); 13C NMR (CDCl3) δ (ppm) = 14.19,
28.33, 62.88, 63.37, 166.30, 170.09, 198.87.
Synthesis of Z‑Thr(Atda)‑OBzl (2) This procedure
included the same synthetic step as described in Method 1A.
As a protecting group, 2,2,-dimetylopropan-1,3-diol was
used. The crude, oxidized product (0.51 g, 1.5 mmol) was
dissolved in toluene (60 ml) and then, 2,2,-dimetylopropan-
1,3-diol (0.60 g, 5.8 mmol) was added to this mixture with
p-toluenesulfonic acid (120 mg) as a catalyst. The resulting
mixture was refluxed for 2.5 h using a Dean-Stark apparatus
to remove water. After the reaction completion, the solvent
was evaporated under reduced pressure. HR-MS m/z: found
450.193 calculated for (C24H29NO6 + Na)+ 450.188.
Synthesis of Fmoc‑Thr(Atda)‑OH (3) The last two steps
consisted of hydrogenolysis and introduction of the Fmoc
group as described in Method 1A. Finally, the reaction prod-
uct was purified by chromatography on a silica gel. Impuri-
ties were eluted first using chloroform containing 3 % of
methanol, then, the reaction product was eluted using chlo-
roform containing 3 % of methanol and 0.15 % of acetic
acid.
Synthesis of Ac‑Thr(Atda)‑OEt (2*)
Yield: 60 %; HPLC: retention time (min): 31.5 (con-
ditions for HPLC are given in the “Experimental” sec-
tion). HR-MS m/z: found 448.172 calculated for
(C24H27NO6 + Na)+ 448.173; MS/MS (parent 448.171):
404.182; 1H NMR (CDCl3) δ (ppm) = 7.74 (d, J = 7.5 Hz,
2H), 7.58 (t, J = 7.2 Hz, 2H), 7.37 (t, J = 7.4 Hz, 2H), 7.29
(td, J = 7.4 Hz, 0.9 Hz, 2H), 5.62 (d, J = 9.1 Hz, 1H), 4.63
(d, J = 9.3 Hz, 1H), 4.39 (p, J = 10.6 Hz, 2H), 4.22 (t,
J = 7.1 Hz, 1H), 3.67 (dd, J = 45.9 Hz, 11.5 Hz, 2H), 3.55
(dd, J = 45.9 Hz, 11.5 Hz, 2H), 1.51 (s, 3H), 1.10 (s, 3H),
0.82 (s,3H); 13C NMR (CDCl3) δ (ppm) = 171.01, 156.55,
143.97, 141.50, 127.32, 127.29, 125.38, 120.17, 70.26,
70.23, 67.65, 59.87, 47.31, 30.24, 23.13, 22.56, 16.57.
The carbonylated product (0.5 g, 2.6 mmol) was dissolved
in toluene (60 ml) and 2,2-dimethyl-propane-1,3-diol
(1.08 g, 10.3 mmol), and p-toluenesulfonic acid as a cat-
alyst (150 mg) were added. The resulting mixture was
refluxed for 2.5 h using a Dean-Stark apparatus. After the
reaction completion, the solvent was evaporated under
reduced pressure (Green and Wuts 1999). HR-MS m/z:
found 296.141 calculated for (C13H23NO5 + Na)+ 296.147.
Synthesis of Fmoc‑Thr(Atda)‑OH (3*)
To remove the C and N protecting groups, the alkaline
hydrolysis with 3.5 M NaOH was carried out for 48 h. The
reaction was controlled with a ninhydrin test. After the
hydrolysis was completed, the resulting strongly alkaline
mixture was used in the next step. It was brought to pH 8
with concentrated hydrochloric acid and treated with Fmoc-
OSu (0.90 g, 2.56 mmol) in acetone, to reach the conditions
for introducing the Fmoc-protecting group described in this
paper (Method 1A or 1B). Finally, the reaction product was
purified as described in Method 1B.
Synthesis
of Fmoc‑amino(2,5,5‑trimetyhyl‑1,3‑dioxolan‑2‑yl)
acetic acid (Method 2)
Synthesis of Ac‑Thr(O)‑OEt (1*)
Ethyl acetoacetate (1 g, 7.63 mmol) was placed in two-
necked, round bottom flask equipped with a thermometer
and a magnetic stirrer. After cooling in the ice bath, gla-
cial acetic acid (1.4 ml) and water (2 ml) were added with
stirring. Sodium nitrite (1.58 g, 45.8 mmol) was added in
portions over 1.5 h with the temperature kept constant at
about 5 °C. Then, the ice bath was removed and the stir-
ring was continued for 4 h. At that time, the temperature
increased to 34–38 °C within 2 h and then decreased to
about 29 °C. The reaction product was extracted with die-
thyl ether (3 × 10 ml). The combined organic layers were
Yield: 60 %; HPLC: retention time (min): 31.5 (conditions
for HPLC are given in the Experimental section). HR-MS
m/z: found 448.173 calculated for (C24H27NO6 + Na)+
448.173; MS/MS (parent 448.173) 404.180.
Peptide preparation
The model peptides were prepared on a solid support
according to the standard Fmoc protocol (Chan and White
1 3

Site-selective solid phase…
2000). The coupling of respective amino acid residues was
carried out using TCTU in DMF. The acetal protection
of the carbonyl group was removed simultaneously with
the peptide cleavage from the resin, using TFA/H2O/TIS
(95:2.5:2.5, v/v) for 3 h at room temperature, resulting in a
carbonylated peptide.
reflectron TOF mass analyzer. For the collision-induced
dissociation (CID) MS/MS measurements, the voltage
over the hexapole collision cell varied from 15 to 30 V and
argon was used as a collision gas.
LC–MS
Purification and characterization of peptides
The LC–MS analysis was performed on an Agilent 1200
HPLC system coupled to a micrOTOF-Q system mass
spectrometer (Bruker Daltonics, Germany). Separation was
carried out on an RP-Zorbax (50 × 2.1 mm, 3.5 µm) col-
umn with a gradient elution of 0–80 % B in A (A, 0.1 %
HCOOH in water; B, 0.1 % HCOOH in acetonitrile) at
room temperature (flow rate: 0.1 ml/min) over 40 min.
The crude peptide products after releasing from the resin
were analyzed using a Thermo Separation HPLC system
with a UV detection (210 nm) on a Vydac Protein RP C18
column (4.6 × 250 mm, 5 μm), with a gradient elution
of 0–80 % S2 in S1 (S1 = 0.1 % aqueous TFA in H2O;
S2 = 80 % acetonitrile + 0.1 % TFA) for 40 min (flow rate
1 ml/min). The main carbonylated product was purified
using preparative reversed-phase HPLC on a TOSOH Bio-
science TSKgel ODS 120T column (21.5 mm × 300 mm;
10 μm), using eluent systems: S1 0.1 % aqueous TFA, S2
80 % acetonitrile + 0.1 % TFA, linear gradient from 50 to
100 % of S2 for 40 min, flow rate 7.0 ml/min, UV detec-
tion at 220 nm. The resulting fractions were collected and
lyophilized. The identities of the products were confirmed
by MS analysis using a microTOF-Q mass spectrometer
equipped with an electrospray ionization source.
Circular dichroizm
The CD spectrum was recorded on a Jasco J-600 spectropo-
larimeter. Fmoc-Atda-OH was dissolved in methanol at a
concentration of 0.07 mg/ml. The spectrum of the solvent
was recorded under identical conditions and subtracted
during data analysis. A rectangular quartz cell of 1 mm
pathlength was used. The data are presented as a mean resi-
due molar ellipticity [ϴ]. The sample was measured in the
range from 260 to 195 nm.
TLC analysis
Results and discussion
The reactions described in this paper were controlled using
TLC. This procedure was performed using TLC plates (Pre-
coated TLC plates ALUGRAM® SIL G/UV254, 5 × 10 cm)
and eluent systems: n-buthyl alcohol:acetic acid:water
(4:1:1) as an eluent E1; chloroform:methanol (95:5) as an
eluent E2. The chromatograms were visualized using a UV
lamp (LAMAG) at 254 nm.
Synthesis of Fmoc‑Amda‑OH (Fmoc‑amino(2,5,5‑
trimethyl‑1,3‑dioxolan‑2‑yl)acetic acid)
Herein we propose a new method for synthesis of an unnat-
ural fully protected amino acid for the solid phase synthesis
of carbonylated peptides according to the standard Fmoc
protocol. So far, only one product, protected aminoadi-
pic semialdehyde, which is a derivative of the oxidized
lysine moiety, is commercially available. However, to the
best of our knowledge, there are no reports on successful
application of this derivative in the peptide synthesis. We
performed the synthesis of a modified amino acid (fully
protected, oxidized threonine), according to the scheme
presented in Fig. 1a. After protecting the N-amino and car-
boxylic groups, the side chain of threonine was oxidized
using pyridinium chlorochromate (PCC) (Corey and Suggs
1975) for 24 h at room temperature. The alternative reagent
that allows obtaining the same carbonylated compound
was the SO3/pyridine complex in DMSO. Our attempts at a
direct synthesis of Fmoc-Amda-OH by oxidation of Fmoc-
Thr-OH by PCC failed because of fast decarboxylation
of the reaction product. Therefore, we examined the pro-
tection of the carbonyl group using ethylene glycol. After
deprotection of the α-amino and carboxylic groups by
Mass spectrometry measurements
Mass spectrometric measurements were performed on
a quadrupole time-of-flight (micrOTOF-Q) instrument
(Bruker, Germany) equipped with an electrospray ioniza-
tion source. The instrument was operated in the positive
or negative ion mode and calibrated before each analysis
with the Tunemix mixture (Bruker Daltonics) by a quad-
ratic method. In the MS/MS experiments, the collision
energy (5–20 eV) was optimized for the best fragmenta-
tion. The solvent used for recording the mass spectra was
acetonitrile:water:formic acid (50:50:0.1) mixture or meth-
anol. 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
1 3

M. Waliczek et al.
CH3
O
O
O
CH3
CH3
HO
O
HO
OH
PCC
HO
Cs2CO3, PhCh2Br
DMF, 48h
OH
O
Z-Cl
NH2
HN
DCM
O
HN
O
O
CH3
O
O
CH3
CH3
O
O
O
O
O
OH
O
O
O
O
1. H2/Pd
2. Fmoc-OSu
HN
O
Pg
HN
HN
O
O
H3O+
O
O
1
3
2
O
O
CH3
CH3
Fmoc-Amda-OH
(Method 1A)
OH
O
O
Pg:
or
HO
HO
OH
O
H3C
H3C
Fmoc-Atda-OH
(Method 1B)
O
Fig. 1 Synthesis of Fmoc-Amda-OH (Method 1A) and Fmoc-Atda-OH (Method 1B)
catalytic hydrogenation in methanol, the Fmoc group was
introduced. Our results show that synthesis of the oxidized
derivative of threonine carried by the proposed method is
feasible. In the ESI–MS spectrum of crude Fmoc-Amda-
OH, a signal derived from the desired product is observed
(Fig. S1A Supplementary Materials). The spectrum is dom-
inated by two peaks. Next to the peak at m/z 406.126, cor-
responding to the sodium adduct of a carbonylated amino
acid, there is another peak at m/z 334.105. Our further stud-
ies revealed that this signal corresponds to Fmoc-βAla-OH.
The yield of the desired product is relatively low while its
retention time is close to that of the by-product (Fig. S1B
Supplementary Materials).
The reaction product was purified by liquid chro-
matography on a silica gel, using 5 % isopropanol in
chloroform as an eluent. Unfortunately, separation of
Fmoc-Amda-OH and Fmoc-βAla-OH on a preparative
scale was not efficient because of coelution of these com-
pounds (Fig. 2b). The ESI–MS spectrum (Fig. 2a) of the
crude product is dominated by two peaks corresponding
to Fmoc-βAla-OH and Fmoc-Amda-OH. The presented
chromatograms (Fig. 2b) confirmed only the purity of the
by-product.
Our investigations confirmed that the by-product
obtained is Fmoc-βAla-OH. In the ESI–MS spectrum
(Fig. S2A Supplementary Materials), only one peak corre-
sponding to this product is observed. The structure of this
compound was confirmed by NMR (Fig. S2B, S3, S4 Sup-
plementary Materials). Obkircher et al. (2008) suggested
previously that β-alanine may be formed by the Lossen
rearrangement during the introduction of the Fmoc-protect-
ing group.
As the yield of the synthesis was low and the separation
of Fmoc-Amda-OH and Fmoc-βAla-OH on a preparative
scale was not feasible, we decided to change the carbonyl
group protection, therefore, a second synthetic strategy was
proposed (Fig. 1b). This procedure included the same syn-
thetic steps, but that time the carbonyl group was protected
by 2,2-dimethylpropane-1,3-diol. According to the litera-
ture, this protecting group should be more labile at acidic
conditions (Newmann and Harper 1958). We also expected
that the new, more hydrophobic protection will facilitate
chromatographic purification of the reaction product.
The result of the synthesis is presented in Fig. 3. In the
ESI–MS spectrum, the signal of the desired product is
observed. The product was successfully purified and only
1 3

Site-selective solid phase…
[M+Na]+
Fmoc-Amda-OH
33.5
[M+Na]+
A
100
B
1+
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
334.105
1+
Fmoc-βAla-OH
406.125
OH
O
NH
32.1
O
O
CH
O
3
O
O
OH
O
HN
O
Fmoc-βAla-OH
Fmoc-Amda-OH
0
10
20
30
40
Rt [min]
1+
1+
428.108
356.087
0
150
200
250
300
350
400
450
500
550 m/z
Fig. 2 ESI–MS spectrum (a) and chromatogram (b) of the purified product (Synthesis of Fmoc-Amda-OH Method 1A)
Fmoc-Atda-OH
B
crude
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
31.3
26
CH
HN
O
O
O
3
H
H
C
C
3
A
OH
O
[M+Na]+
3
24
1+
100
448.172
O
0
10
20
30
40
Fmoc-Atda-OH
Rt [min]
pure
C
2.5
2.0
1.5
1.0
0.5
0.0
31.5
1+
404.181
1+
-CO2
470.153
0
0
10
20
30
40
200
250
300
350
400
450
500
550
600
m/z
Rt [min]
Fig. 3 ESI–MS (a) of pure Fmoc-Atda-OH; chromatogram of crude (b) and pure (c) product obtained by Method 1B
1 3

M. Waliczek et al.
O
O
Fig. 4 Synthesis of Fmoc-Atda-
OH (Method 2)
O
NaNO2
Zn, AcOH
N
O
H3C
O
CH3
HO
O
CH3
Ac2O, 45 oC
aq. AcOH
CH3
CH3
CH3
Na+
O
O
CH
3 O
O
O
H3C
H3C
OH
3.5 M NaOH
O
CH3
H3C
O
CH3
HO
100 oC, reflux, 48h
HN
CH3
HN
O
O
CH3
2*
1*
CH
HN
3 O
O
O
H3C
H3C
CH
3 O
O
O
H3C
H3C
Fmoc-OSu
pH 8
OH
O
OH
NH2
O
3*
Fmoc-Atda-OH
CH
HN
O
O
O
Fig. 5 ESI–MS of crude prod-
uct Fmoc-Atda-OH obtained by
Method 2
3
100
H
H
C
C
[M+Na]+
3
3
OH
O
1+
448.1728
O
Fmoc-Atda-OH
1+
404.1796
-CO2
1+
179.0798
1+
353.2564
1+
545.1757
1+
129.0880
0
100
200
300
400
500
600
700
m/z
one peak corresponding to fully protected oxidized threo-
nine is present in the chromatogram of the purified frac-
tion. The structure of this building block was confirmed by
NMR (Fig. S5, S6 Supplementary Materials). During the
synthesis of Fmoc-Atda-OH using 2,2-dimethylpropane-
1,3-diol as a protecting group, the amount of obtained
1 3

Site-selective solid phase…
Fmoc-β-Ala-OH was negligible. These data may be
explained basing on the literature (Obkircher et al. 2008).
The authors investigated the amount of Fmoc-βAla-OH
formed from Fmoc-OSu, in relation to the amino acid resi-
due being protected. It seems that the branched amino acids
influence the formation of Fmoc-βAla-OH by-product
during Fmoc protection reaction. The content of impurity
depended on the amino acid structure and reaction condi-
tions. The by-product was formed as result of Lossen rear-
rangement of succinimidyl moiety; however, the influence
of amino acid structure on the Fmoc- β-Ala-OH content in
the crude product was not completely resolved.
In addition, we designed another synthetic strategy uti-
lizing ethyl acetoacetate. After nitrosylation of ethyl ace-
toacetate, the reduction in the presence of zinc dust and
acetic acid combined with a direct acetylation was per-
formed. It allows obtaining the ethyl ester of N-acetylated
2-amino-3-ketobutyric acid with a high yield and purity.
The carbonyl group in the obtained product was protected
using 2,2-dimethyl-propan-1,3-diol. Then, alkaline hydrol-
ysis (3.5 M NaOH for 48 h) was performed. Finally, the
N-amino group was protected by the Fmoc group. This syn-
thetic pathway is presented in Fig. 4. Purity and identity of
the product were confirmed by HPLC and HR-MS (Fig. 5).
All the m/z values were consistent with the calculated ones
based on chemical formulas of the expected compounds.
Based on the obtained analytical data, we proved that the
desired product was obtained.
Chirality
1H NMR experiments using Ac-Thr(O)-OEt were car-
ried out in D2O and CDCl3, to check the dependence of
the exchange rate of α-hydrogen on solvent and time. The
study revealed that the hydrogen–deuterium exchange was
relatively fast. The peak corresponding to α-hydrogen was
1
not present in the H NMR spectra obtained 3 min after
dissolving the sample in D2O, while the sample dissolved
in de-acidified chloroform showed a doublet correspond-
ing to α-hydrogen (Fig. 6, S7 Supplementary Materials).
This result reveals a lability of the α-carbon proton which
may be explained by formation of enolic form characteris-
tic for dicarbonyl systems. Fmoc-Atda-OH was subjected
to CD study. The CD spectrum presented in Fig. S8 (Sup-
plementary Materials) did not show any ellipticity in the
range from 260 to 190 nm. Observed racemization is likely
a result of planar enol formation.
Synthesis of carbonylated peptides
Pure, fully protected derivative of oxidized threonine
(Fmoc-D,L-Atda-OH) was applied in the solid phase syn-
thesis of carbonylated peptides. Peptides were prepared
by manual solid phase technique using the standard Fmoc
synthetic strategy with TBTU as a coupling reagent. The
peptide was cleaved from the resin using TFA/water/TIS
(95:2.5:2.5, v/v).
A
2
B
1
α- hydrogen
5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8
f1 (ppm)
Fig. 6 1H NMR spectra of Ac-Thr(O)-OEt measured in: a D2O after 3 min; b CDCl3
1 3

M. Waliczek et al.
A
1.0
0.8
14.82
14.50
b3
b4
O
O
CH
O
CH
3
3
0.6
0.4
0.2
0
NH
NH
H C
3
NH
NH
O
NH
O
CH
O
3
HO
O
y4
y3
CH
3
0
10
20
30
40
Rt [min]
b3
C
100
1+
[M+H]+
1+
B
b4
284.124
1+
100
520.2396
355.161
[M+Na]+
1+
[M+H]+
542.2218
1+
[M+K]+
520.240
1+
477.2818
1+
558.1959
1+
355.1613
y3
1+
308.161
y4
1+
213.087
1+
1+
1+
118.394
1+
403.198
502.229
338.3421
1+
1+
1+
443.124
256.130
173.741
0
0
100 150 200 250 300 350 400 450 500 m/z
200 250 300 350 400 450 500 550 600 650 m/z
Fig. 7 Analytical data of Ac-Thr(O)-Ala-Ala-Ala-Phe-OH (Method 2). a Chromatogram; b ESI–MS spectrum; c ESI–MS/MS spectrum
A series of synthetic model peptides containing the build-
ing block (H-Thr(O)-Ala-Phe-OH, H-Thr(O)-Ala-Ala-Ala-
Phe-OH, Ac-Thr(O)-Ala-Ala-Ala-Phe-OH, H-Gly-Thr(O)-
Ala-Ala-Ala-Phe-OH, H-Leu-Val-Asn-Glu-Val-Thr(O)-Glu-
Phe-Ala-Lys-OH) were synthesized. To make this model
more realistic, we based the sequence of one peptide on a
fragment of naturally occurring protein (tryptic fragment [66–
75] HSA containing Thr residue). The oxidized threonine res-
idue replaces a threonine residue in a natural protein giving
the sequence: H-Leu-Val-Asn-Glu-Val-Thr(O)-Glu-Phe-Ala-
Lys-OH. The influence of position of the unnatural amino
acid in the peptide chain on efficiency of deprotection of
the carbonyl group was studied. In the case of application of
2,2-dimethylpropane-1,3-diol as a protecting group, we found
that incorporation of a carbonylated amino acid at the N-ter-
minus does not allow removal of the masking group even
after 24 h incubation. However, the elongation of carbon-
ylated peptide (H-Gly-Thr(O)-Ala-Ala-Ala-Phe-OH) or acet-
ylation of the N-terminus (Ac-Thr(O)-Ala-Ala-Ala-Phe-OH)
allowed a complete removal of the protecting group from car-
bonyl in 3 h (Fig. 7a). Application of this derivative resulted
in obtaining of the carbonylated peptides with a 90 % yield.
A signal of the protonated, carbonylated peptide is observed
in the MS spectrum of the crude product (Fig. 7b). The parent
ion at m/z 520.239, was subjected to MS/MS fragmentation.
The fragmentation spectrum is dominated by a series of b and
y ions covering the whole sequence of the peptide (Fig. 7c,
S9 Supplementary Materials). In the chromatogram of a pure
carbonylated peptide, two signals corresponding to diastere-
omers were observed because of racemization.
We also performed a successful synthesis of a tryptic
fragment [66–75] HSA containing carbonylated threonine
(H-Leu-Val-Asn-Glu-Val-Thr(O)-Glu-Phe-Ala-Lys-OH).
The ESI–MS and ESI–MS/MS spectra, and the HPLC
chromatogram of pure H-Leu-Val-Asn-Glu-Val-Thr(O)-
Glu-Phe-Ala-Lys-OH are presented (Fig. S10 Supplemen-
tary Materials).
In the synthesis, we applied a racemic derivative Fmoc-
D,L-Atda-OH, obtaining a mixture of diastereoisomeric
peptides. However, the composition of this product is a
matter of equilibrium and does not depend on the chirality
of applied substrate (Fmoc-Atda-OH).
1 3

Site-selective solid phase…
11.1
B
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
-0.02
10.5
A
11.1
10.5
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
6
8
10
12
14
Rt [min]
11.1
C
0.12
0.10
0.08
0.06
0.04
0.02
0.00
-0.02
-0.04
10.5
6
8
10
12
14
Rt [min]
6
8
10
12
14
Rt [min]
Fig. 8 a Chromatogram of pure H-Leu-Val-Asn-Glu-Val-Thr(O)-
Glu-Phe-Ala-Lys-OH; b chromatograms of fractions corresponding
to each peak reanalyzed 30 min after separation (solid line the frac-
tion eluted at 10.5 min; dotted line the fraction eluted at 11.1 min);
c chromatograms of fractions corresponding to each peak reanalyzed
24 h after separation (solid line the fraction eluted at 10.5 min; dotted
line the fraction eluted at 11.1 min
We tested a progress of racemization as a function of
time for peptide H-Leu-Val-Asn-Glu-Val-Thr(O)-Glu-
Phe-Ala-Lys-OH using HPLC. Two signals corresponding
to two isomeric forms of the pure peptide are observed in
the chromatogram (Fig. 8a). The fractions corresponding
to both peaks were collected and subjected to re-analysis
of purity at the same conditions. The obtained chromato-
grams showed that two isomeric forms appeared after less
than 30 min (Fig. 8b). The HPLC analysis executed after
24 h for the separated signals showed the same peak areas
(Fig. 8c).
LC–MS analysis confirmed that two peaks in the chro-
matogram correspond to compounds with the same molec-
ular weight (Fig. 9). The resolution of the column used
for LC was not sufficient to completely separate these
two signals. However, the presented spectra (Fig. 9a, b)
revealed unquestionably the same m/z for the appropriate
parts of peaks. Additionally, the extracted ion chromato-
gram was generated by Data Analysis program and exactly
the same profile of the chromatogram was obtained. In the
chromatogram (Figs. 8, 9), the peaks corresponding to two
diastereoisomeric forms of modified peptides are charac-
terized by broadening and tailing. These features addition-
ally confirmed our results concerning the fast equilibrium
between both stereoisomeric peptides. Based on the NMR
study, we confirmed the exchange rate of α-hydrogen.
Theoretically, the effect may result from the presence of
an enol form. Additionally, the cis-/trans-isomerizatiom on
the peptide bond may contribute to a further peak boarding;
however, a thorough investigation is needed to confirm this
speculation.
We also applied a crude, fully protected amino acid
containing ethylene glycol as a protection of the carbonyl
group (Fmoc-Amda-OH) for the solid phase synthesis of
carbonylated peptides. Peptides were prepared as previ-
ously described. Our results indicate that Fmoc-Amda-OH
was also successfully incorporated into model peptides. The
ESI–MS spectrum of crude product Ac-Thr(O)-Ala-Ala-
Ala-Phe-OH is presented (Fig. S11 Supplementary Mate-
rials). In the spectrum, three main peaks correspond to the
protonated peptides containing β-alanine, Thr(O), and Amda
[protected Thr(O)]. After extending the time of cleavage of
1 3

M. Waliczek et al.
Intens.
6
x10
B
[66-75] HSA TIC +All MS
EIC 574.3 0.05 +All MS
1.5
1.0
0.5
A
0.0
15.25
15.50
15.75
16.00
16.25
16.50
16.75
17.00
17.25
Time [min]
2+
+MS, 15.7-15.8 min
100
A
574.308
0
100
+MS, 16.0-16.1 min
2+
B
574.322
0
300
400
500
600
700
800
900
m/z
Fig. 9 LC–MS of pure HSA [66–75]; a ESI–MS of the peptide eluted at 15.7–15.8 min; b ESI–MS of the peptide eluted at 16.0–16.1 min
the peptide, the deprotection was complete (see below) and
the signal at m/z 564.267 was not observed. We found that
incorporation of a carbonylated amino acid at the N-terminus
does not allow removing the acetal group even after 24 h of
incubation, in the mixture used for cleavage or microwave-
assisted TFA (Kluczyk et al. 2010) cleavage from the resin.
The probable reason is the proximity of the amino group
which easily gets protonated. The positive charge on the
nitrogen prevents protonation of acetal oxygen because of
possible electrostatic repulsion. The elongation of peptide at
the N-terminus allowed the removal of the acetal group after
8 h of incubation. Acetylation of the N-terminal amino group
decreased the time of complete cleavage to 4 h. The purity
of synthesized peptides was checked by HPLC. The chro-
matograms of the crude product after various times of cleav-
age are presented (Fig. S12A Supplementary Materials). As
one can see, after 4 h of incubation, the acetal group is com-
pletely removed from the carbonyl group. Two main prod-
ucts of synthesis were successfully separated using HPLC.
The chromatograms of pure compounds are presented (Fig.
S12B and C Supplementary Materials).
We observed the racemization also in the case of
Fmoc-Amda-OH synthesis. The racemization is a result
of chemical structure of oxidized threonine derivative. The
proton at α carbon is activated by two carbonyl groups and
undergoes fast exchange. The chromatograms of crude
and pure carbonylated peptides (Ac-Thr(O)-Ala-Ala-Ala-
Phe-OH) prepared by solid phase synthesis using crude
Fmoc-Amda-OH were placed in Supplementary Materials
(Fig. S12A and C Supplementary Materials). Two fractions
corresponding to these signals were collected and analyzed
by mass spectrometry, showing the same molecular mass,
which is in good agreement with assumption that Thr(O)
residue is susceptible to racemization.
Conclusions
We designed a straightforward and convenient method
of synthesis of the fully protected carbonylated build-
ing block Fmoc-Atda-OH. Two different protections
of the carbonyl group were tested. The application of
1 3

Site-selective solid phase…
Kluczyk A, Rudowska M, Stefanowicz P, Szewczuk Z (2010) Micro-
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2,2-dimethylpropane-1,3-diol results in a better yield and
purity of the fully protected building block as compared
to ethylene glycol. The crude Fmoc-Amda-OH and pure
Fmoc-Atda-OH were successfully applied in the solid
phase synthesis of carbonylated peptides. The kinetics of
the carbonyl group deprotection with respect to the posi-
tion of carbonylated amino acid residue in the peptide
chain was investigated, revealing that peptides with N-ter-
minal Thr(O) are not susceptible to deprotection with trif-
luoroacetic acid. In addition, the main by-product (Fmoc-
βAla-OH) formed during Fmoc-Amda-OH was identified.
Acknowledgments This work was supported by a Grant No UMO-
2012/07/D/ST5/002324 from the Polish National Science Centre.
Conflict of interest The authors declare that they have no conflict
of interest.
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution License which permits any use, distribu-
tion, and reproduction in any medium, provided the original author(s)
and the source are credited.
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1 3