Synthesis of peptides containing 5-hydroxytryptophan, oxindolylalanine, N-formylkynurenine and kynurenine

Article abstract of DOI:10.1002/psc.1322

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5-HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the ${bf alpha}$-amino group of both amino acids, and the commercially available 5-HTP, with Fmoc-succinimide. High yields of Fmoc-Kyn were obtained by acid hydrolysis of Fmoc-NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M-type), and ${bf beta}$-enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5-HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion-pair reagent on a C₁₈-phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan.

Full text of DOI:10.1002/psc.1322

Research Article
Received: 16 July 2010
Revised: 17 September 2010
Accepted: 4 October 2010
Published online in Wiley Online Library: 19 January 2011
(wileyonlinelibrary.com) DOI 10.1002/psc.1322
Synthesis of peptides containing
5-hydroxytryptophan, oxindolylalanine,
N-formylkynurenine and kynurenine
Toni Todorovski,a Maria Fedorova,a Lothar Hennigb and Ralf Hoffmanna∗
ROS,continuouslyproducedincells,canreversiblyorirreversiblyoxidizeproteins,lipids,andDNA.Attheproteinlevel,cysteine,
methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis
of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins
in vitro and in vivo: 5-HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then
protected the α-amino group of both amino acids, and the commercially available 5-HTP, with Fmoc-succinimide. High yields
of Fmoc-Kyn were obtained by acid hydrolysis of Fmoc-NFK. All four Fmoc derivatives were successfully incorporated, at high
yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M-type), and β-enolase. The correct
structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing
5-HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion-pair reagent on a C18-phase. Such
c
synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan. Copyright ꢀ
2011 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: ROS; oxidative stress; tryptophan; oxidation; solid phase peptide synthesis
Introduction
pathogenesis of several age-related diseases, such as Alzheimer’s
disease, Parkinson’s disease, atherosclerosis, and diabetes mellitus
[12–16]. Kyn derivatives and Oia appear also to play a key role in
cataractogenesis [17–19]. Furthermore, 5-HTP, DiOia, Oia, and
Kyn are formed in the brains of patients with eosinophilia-
myalgia syndrome [20]. More recently, elevated levels of oxidized
tryptophan residues in plasma proteins were reported for diabetes
[21] and for skeletal muscles in an in vivo model of acute oxidative
stress [22,23].
Despite the importance and often studied effects of tryptophan
oxidation in proteins by ROS, general strategies to specifically
synthesize suitably modified peptide models for functional and
structural studies, or to improve analytical strategies, have, to the
best of our knowledge, not yet been reported. In this study, we
ROS can be produced by many different chemical reactions. These
reactions can originate from either cellular events, e.g. metal-
catalyzed reactions, mitochondrial electron transport reactions,
and neutrophil or macrophage activation during inflammation,
or external events, e.g. UV light, X-rays, or pollutants. ROS as
natural by-products of the oxygen metabolism play important
roles in cellular signaling [1,2], but can also modify and
often functionally deactivate other biomolecules, such as lipids,
DNA, and proteins. For this reason, elevated ROS levels are
prevented by effective scavenging systems based on enzymes
(e.g. superoxide dismutase, catalase, glutathione peroxidase,
thioredoxin, thioredoxin reductase, and peroxiredoxin) or small
antioxidants (e.g. ascorbic acid (vitamin C), carotenes, glutathione,
lipoic acid, tocopherol (vitamin E), ubiquinol, or uric acid (2,6,8-
trioxypurine). The potential damage from ROS depends on several
factors, including the following: cell type, absolute level and
duration of ROS production, ROS species generated, site of
generation, and proximity of the oxidants to a possible target.
Usually, proteins as major components in most environments
represent the major targets [3–5]. Among the 20 proteinogenic
amino acid residues, methionine, cysteine, tyrosine, tryptophan,
histidine, and phenylalanine are especially prone to oxidation [6].
In tryptophan residues, the indole ring is easily oxidized by
different ROS, forming several well-known tryptophan oxidation
products: 5-HTP, Oia, DiOia, NFK, Kyn, OH-Kyn, and OH-NFK [7,8].
As these oxidation products cannot be reduced by the cellular
machinery, and often change the protein structure and function,
they are usually degraded by enzymes or the proteasome [9,10],
but they can also accumulate and aggregate in the cells [11].
These processes may link oxidation processes directly to the
∗
Correspondence to: Ralf Hoffmann, Institute of Bioanalytical Chem-
¨
istry, Biotechnologisch-Biomedizinisches Zentrum, BBZ, Universitat Leipzig,
Deutscher Platz 5, 04103 Leipzig, Germany.
E-mail: Hoffmann@chemie.uni-leipzig.de
a
Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedi-
¨
cine (BBZ), Faculty of Chemistry and Mineralogy, Universitat Leipzig, Leipzig,
Germany
b
Institute of Organic Chemistry, Faculty of Chemistry and Mineralogy, Univer-
¨
sitat Leipzig, Leipzig, Germany
Abbreviations used: CID, collision-induced dissociation; DiOia, dioxin-
dolylalanine; Fmoc-OSu, N-(9-fluorenylmethoxycarbonyl)-succinimide; Kyn,
kynurenine; MSTFA, N-methyl-N-(trimethylsilyl)trifluoroacetamide; NFK, N-
formylkynurenine; 5-HTP, 5-hydroxytryptophan; OH-Kyn, hydroxykynurenine;
OH-NFK, hydroxy-N-formylkynurenine; Oia, oxindolylalanine; PSD, post-
source decay; ROS, reactive oxygen species; RP-HPLC, reversed phase high-
performance liquid chromatography; SPE, solid phase extraction.
c
J. Pept. Sci. 2011; 17: 256–262
Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd.

PEPTIDES WITH OXIDIZED TRYPTOPHAN RESIDUES
report the synthesis of Fmoc-protected 5-HTP, Oia, Kyn and NFK
derivatives. These building blocks were successfully incorporated
into three peptide sequences, recently shown to be oxidized
in vivo [22,23], with standard procedures on solid phase. The
correct structures were confirmed for all peptides by tandem mass
spectrometry. Interestingly, all isobaric peptides containing 5-HTP
and Oia were well separated by RP-HPLC.
Table 1. Reaction conditions used to couple the Fmoc-group to 5-
HTP, Oia, and NFK and the yields obtained for the purified compounds
MSTFA Reaction time of Reaction time for
Compound
(eq.)
silylation (min) Fmoc coupling (h) Yield (%)
Fmoc-5-HTP
Fmoc-Oia
3
3
3
30
30
24
48
26
40
35
30
Fmoc-NFK
210
Materials and Methods
Reagents
Fmoc-Protection of Tryptophan Derivatives
DMSO (99.9%, water free), acetic anhydride (>99%), formic acid
(98–100%), TFA (spectrophotometric grade, >99%), and sodium
hydroxide (NaOH, >99%) were purchased from Sigma-Aldrich
Chemie GmbH (Taufkirchen, Germany), while potassium chlo-
ride (laboratory reagent grade, >99% purity) was obtained from
Fisher Scientific GmbH (Schwerte, Germany). Hydrochloric acid
(37%), acetic acid (100%), and methanol (>99.9%) were pur-
chased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany).
Citric acid (analytical grade) was purchased from SERVA Elec-
trophoresis GmbH (Heidelberg, Germany). Tryptophan (>99%)
and 5-HTP (>99%) were obtained from Fluka (Sigma-Aldrich
Chemie GmbH). DMF (peptide synthesis grade), DCM (99.9%), and
acetonitrile (HPLC-S gradient grade) were obtained from Biosolve
(Valkenswaard, Netherlands). Standard Fmoc-amino acid deriva-
tives were purchased either from MultiSynTech GmbH (Witten,
Germany) or from ORPEGEN Pharma (Heidelberg, Germany), con-
taining tert-butyl-based protecting groups at serine, threonine,
tyrosine aspartic acid, and glutamic acid; the triphenylmethyl
protecting group at asparagine, glutamine, and histidine; and
the 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) pro-
tecting group for arginine. Water was purified in-house with a
PURELAB ultra analytic system (ELGA, Berkefeld GmbH, Ransbach,
Germany).
5-HTP, Oia, Kyn, and NFK were protected at the α-amino group
with Fmoc by a standard procedure [25]. Briefly, each amino acid
derivative (1 g) was suspended in methylene chloride (10 ml) and
treated with three equivalents of MSTFA at room temperature
(RT). The mixture was refluxed (50 ◦C) until a clear solution was
obtained. The solution was then cooled to RT, one equivalent
of Fmoc-OSu was added, and the resulting mixture was stirred
at RT until the reaction was complete (Table 1), as monitored by
TLC. After completion of the reaction, the carboxyl group was
deprotected with methanol (2.25 ml, RT, 0.5 h) and the solvent
evaporated on a rotary evaporator. Water (30 ml) was added to
the crude product, stirred for 30 min, and filtered. The residue was
thenwashedthreetimeswithamixtureofaqueouscitricacid(10%)
andmethanol(1 : 1byvolume, 5 ml)andwateruntilthefiltratewas
neutral. The combined filtrates were dried under vacuum (40 ◦C)
and then lyophilized [25]. The obtained Fmoc-amino acids were
finally purified by RP-HPLC on a C18-column (internal diameter
21.2 mm, length 250 mm, particle size 10 µm, pore size 30 nm,
Phenomenex) using a linear gradient from 40.5 to 42.3% aqueous
acetonitrile (0.1% TFA) over 1 min, followed by a linear gradient
from 42.3 to 69.3% over 30 min. The flow rate was 10 ml/min. The
correct syntheses were confirmed by NMR on a Varian Mercury
plus (Darmstadt, Germany) (Supporting information, Table S1).
The monoisotopic masses were determined by MALDI-TOF-MS
in positive-ion mode using a 4700 proteomic analyzer (MALDI-
TOF/TOF-MS, Applied Biosystems GmbH, Darmstadt, Germany).
Thematrixwasα-cyano-4-hydroxy-cinnamicacid(BrukerDaltonics
GmbH, Bremen, Germany). The recorded monoisotopic masses
for Fmoc-5-HTP (yield 40%), Fmoc-Oia (yield 35%), and Fmoc-
NFK (yield 30%) were m/z 443.18 (theoretical 443.16), 443.16
(theoretical 443.16), and 459.12 (theoretical 459.15), respectively.
Fmoc-Kyn was released from Fmoc-NFK (20 mg) by hydrolysis
(10% aqueous TFA, 10 ml, 8 h, RT) using the conditions described
for the free amino acid [26]. On the basis of the peak areas
obtained on RP-HPLC, more than 90% of Fmoc-NFK was converted
to Fmoc-Kyn. TFA was removed (rotary evaporator, 50 ◦C), and
the solid product was then washed several times with water
and dried under vacuum. The obtained product (yield 85%) was
characterized by NMR (Supporting information, Table S1) and
MALDI-MS (m/z 431.15, theoretical 431.16).
Synthesis of Oia
The synthesis of Oia relied on a published strategy of Trp oxidation
by DMSO (yield 65%) [24]. The crude product was dissolved in 3%
(v/v)aqueousMeCNcontaining0.1%(v/v)TFAataconcentrationof
20 g/l. Aliquots of 800 mg were loaded on a Strata C18-E cartridge
(internal diameter 27 mm, length 135 mm, particle size 50 µm,
pore size 6.5 nm, Phenomenex Inc., Aschaffenburg, Germany). The
column was washed with the same eluent composition (80 ml)
before Oia was eluted [50 ml, 12% (v/v) aqueous MeCN containing
0.1% (v/v) TFA].
Synthesis of Kyn and NFK
Kyn was synthesized by oxidation of Oia [20]. Oia (1 g, 4.54 mmol)
was dissolved in NaOH solution (0.15 mol/l, 100 ml) containing KCl
(0.05 mol/l, pH12.75). Whileairwascontinuouslybubbledthrough
the solution, the reaction was monitored by RP-HPLC. The highest
yield (>90%) of Kyn was obtained after 2 h, and was contaminated
by only a small amount (≈10%) of DiOia. For longer reaction
times the content of DiOia did not increase further, but several, as
yet uncharacterized, by-products appeared. The reaction mixture
was neutralized by addition of hydrochloric acid (1 mol/l) and the
solvent was removed on a rotary evaporator. The solid product
was purified by SPE using the same conditions described above for
Oia. The overall yield was 55%. NFK was synthesized from purified
Kyn by selective formylation (yield 60%) and used without further
purification [20].
Solid Phase Peptide Synthesis
Peptides were synthesized by Fmoc/tBu-chemistry on a Syro2000
multiple peptide synthesizer (MultiSynTech GmbH) using eight
equivalents of the amino acid derivatives activated with 1,3-DIC
in the presence of DIC/HOBt in DMF [27] at the 26-µmol scale.
The modified tryptophan derivative was incorporated manually
by activating the corresponding Fmoc derivative (5 eq.) with HATU
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J. Pept. Sci. 2011; 17: 256–262 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

TODOROVSKI ET AL.
1
1
NH2
2 NH2
3′
6′
2
3′
6′
A
1′
8′
1′
8′
2′
7′
2′
7′
4′
5′
4′
5′
HO
COOH
COOH
O
N
N
H
H
Oia (65%)
5-HTP
NH2
COOH
N
H
Trp
O
OH1
NH2
O
NH2
2 NH2
3′
6′
3′
6′
COOH
3′
2′
2′
7′
2
COOH
4′
5′
4′
5′
2′
4′
2
1′
1′
1′
8′
O
HCOOH, (CH3CO)2O
2 h, RT
1
1
8′
COOH
+
7′ NH2
5′
7′ NHCHO
N
6′
H
DiOia
Kyn (55%)
NFK (60%)
8
7
B
9
6
O
O
O
COOH
O
10
11
H2N
+
O
DCM, MSTFA, RT
5
3
COOH
2
4
O
N
HN
H2C
R
1
9
6
H2C
O
R
8
7
Figure 1. Reaction schemes showing the synthetic strategies used to obtain the different oxidation products of tryptophan (A) and the corresponding
Fmoc-protected derivatives (B). Tryptophan was oxidized to Oia with DMSO in the presence of hydrochloric acid and glacial acetic acid [24] and then
further to Kyn, which was then formylated to obtain NFK. The numbering of the C-atoms in each structure indicates the assigned signals of the NMR
spectra, as presented in Materials and Methods. R indicates the side chain of Trp and its analogs, e.g. indole for Trp and oxindole for Oia.
(4.9 eq.) in the presence of DIPEA (10 eq.) [28]. The efficiency of the
manual coupling was verified by a Kaiser test [29] and RP-HPLC.
The resin-bound peptides were cleaved and deprotected
with TFA containing a scavenger mixture (12.5%, v/v) of water,
thioanisol, m-cresol, and ethandithiol (2 : 2 : 2 : 1 by vol.) at RT for
2 h. The peptides were then precipitated in cold diethyl ether and
centrifuged (1620 g). The pellet was washed twice with cold ether,
dried under air, dissolved in 3% aqueous acetonitrile containing
0.1% TFA, and stored at −20 ◦C.
of 0.2 ml/min. The monoisotopic masses of all peptides were
recorded in positive ion reflector TOF (re-TOF) mode with the
matrix α-cyano-4-hydroxy-cinnamic acid, as described above. In
addition, the peptide sequences, including the modified positions,
were confirmed in TOF/TOF mode in the absence (PSD mode) or
presence of air in the collision cell (CID mode).
Results and Discussion
SynthesisofFmoc-5-HTP,Fmoc-Kyn,Fmoc-NFK,andFmoc-Oia
Peptide Purification
Oia and NFK were obtained in reasonable yields by oxidation
of tryptophan and in high purities after SPE. The NMR data
confirmed the expected structures, including the L-configuration
ofthechiralcenterattheα-C-atom(Figure 1).TheFmoc-groupwas
incorporated into both compounds at high levels of around 70%.
Following purification with RP-HPLC, the overall yields obtained
were between 30 and 40% (Table 1) and purity levels were above
95%. Fmoc-Kyn-OH was obtained by acid hydrolysis from Fmoc-
NFK incorporated without side chain protection.
Crude peptides were initially analyzed on a Jupiter C18-column
(internal diameter 2 mm, length 150 mm, particle size 5 µm, pore
size 30 nm, Phenomenex) using a linear gradient from 3 to 57%
aqueous acetonitrile (0.1% TFA) for 30 min at a flow rate of
¨
0.2 ml/min using an Akta HPLC System (GE Healthcare GmbH,
Munich, Germany). All peptides were purified on a Jupiter C18
-
column (internal diameter 10 mm, length 250 mm, particle size
5 µm, pore size 30 nm, Phenomenex) by a linear gradient starting
at 3% aqueous acetonitrile (slope of 1% acetonitrile per minute)
and containing 0.1% TFA as the ion-pair reagent at a flow rate
of 5 ml/min. The lyophilized fractions were characterized on the
microbore Jupiter C18-column using a linear gradient from 3 to
27% aqueous acetonitrile (0.1% TFA) over 10 min, followed by
a gradient of 27–48% over the next 35 min with a flow rate
Peptide Synthesis
On the basis of our previous work on the analysis of tryptophan
oxidation of skeletal muscle proteins in a rat model for oxidative
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PEPTIDES WITH OXIDIZED TRYPTOPHAN RESIDUES
1541.65
28.2*
A
2.4
2.0
1.6
1.2
0.8
0.4
1428.75
27.6
0
0
5
10
15
20
25
30
35
Retention time (min)
C
B
1557.78
25.6*
1557.77
26.5*
1557.77
26.8*
1.2
1.2
1.0
0.8
1.0
0.8
0.6
0.4
0.2
0
0.6
0.4
0.2
0
1573.74
24.8
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
Retention time (min)
Retention time (min)
1545.81
26.7*
1573.81
26.4*
E
D
1.4
1.2
1.0
1.2
1.0
0.8
1545.81
26.7
0.8
0.6
0.4
0.2
0
1355.74
23.5
0.6
1355.74
23.6
0.4
0.2
0
0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
Retention time (min)
Retention time (min)
Figure 2. Reversed-phase chromatogram of the crude H-LAQSNGXGVMVSHR-OH peptides containing Trp (A), 5-HTP (B), Oia (C), Kyn (D), and NFK (E) in
position seven of the sequence. Peaks marked with an asterisk contain the correct sequence. The peptides were analyzed on the microbore Jupiter
C18-column. Numbers on top of the peaks indicate the retention time (lower) and m/z values (upper) recorded by MALDI-MS.
stress, we selected three sequences containing a single trypto-
phan residue prone to oxidation [23]. Peptides IWHHTFYNELR,
SFLVWVNEEDHLR, and LAQSNGWGVMVSHR, which correspond
to skeletal muscle actin (residues 87–97), creatin kinase M-type
(residues 224–236), and β-enolase (residues 359–372), respec-
tively, were all identified, along with the oxidized Trp-residues,
in a tryptic digest of the rat skeletal protein extract using tan-
dem mass spectrometry. These 11- to 14-residue-long peptides
should also provide a good separation of potential by-products by
RP-HPLC, and an easier characterization of the targeted structure
and potential by-products by MALDI-MS. All modified peptides
containing 5-HTP, NFK, Kyn, or Oia were obtained in high yields
and with high purities, without any indication of reduced cou-
pling or Fmoc-cleavage efficiencies at the Trp-positions. The crude
peptides typically contained the desired product at the following
levels:35%(NFK), 50%(Kyn), 55%(Oia), and65%(5-HTP), asjudged
from the RP-chromatograms (Figure 2 and Table 2), independent
of the Trp derivative. Thus, all four oxidation products were suc-
cessfully incorporated by standard protocols, which should also
allow the synthesis of longer peptides. Importantly, all derivatives
appeared to be stable during Fmoc deprotection with piperidine
and the final cleavage with TFA. Only the formamide of NFK was
partially hydrolyzed during TFA cleavage. At prolonged cleavage
times (200 min), the hydrolysis rate was approximately 15%, as
judged by RP-HPLC. The resulting Kyn-containing peptides eluted
0.2–1.0 min later (0.1–0.6% acetonitrile), which allowed reliable
purification. Shorter cleavage times or lower temperatures should
further reduce this side reaction. At first view, it appears surpris-
ing that the NFK-containing peptide, with its formylated amino
group, elutes earlier than the corresponding Kyn-peptide, with
the free amino group. The relatively strong retention of the Kyn-
containing peptides is probably attributable to the formation of
ion-pairadductsbetweentheTFAanionandthepositivelycharged
amino group of the Kyn-residues.
Besides the targeted peptides, the RP-chromatograms con-
tained only minor by-products with deleted or partially protected
sequences. This indicated that neither of the Trp derivatives was
irreversibly modified by reactive intermediates formed during the
TFA cleavage and that the regular scavenger mixtures can effec-
tively protect them. Moreover, the next N-terminal amino acid
derivatives were efficiently coupled. Importantly, the aromatic
amino group in the side chain of Kyn was not acylated during the
solid phase synthesis within the detection limits, indicating that
it is not necessary to protect it for HATU- and DIC-activation. For
longer peptides containing Kyn at the C-terminal end, it might still
be useful to incorporate fully protected Kyn derivatives. The same
might also be true for syntheses performed at elevated tempera-
tures, such as those that are microwave assisted. In conclusion, all
peptideswereeasilypurifiedbyRP-HPLCanddisplayedthecorrect
c
J. Pept. Sci. 2011; 17: 256–262 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

TODOROVSKI ET AL.
Table 2. Peptide sequences used in this study, their monoisotopic masses (MALDI-TOF-MS), retention times (RP-HPLC), and corresponding yields
of purified peptides
Peptide sequence
Theoritical m/z
Experimental m/z
Retention time (min)
Yield (%)
SFLVWVNEEDHLR
1643.81
1659.81
1659.81
1675.81
1647.81
1541.76
1557.76
1557.76
1573.76
1545.76
1515.74
1531.74
1531.74
1547.74
1519.74
1643.76
1659.84
1659.82
1675.82
1647.84
1541.65
1557.78
1557.77
1573.81
1545.81
1515.70
1531.78
1531.78
1547.78
1519.59
28.5
26.4
49
30
29
16
26
52
38
32
20
30
53
39
38
16
31
SFLV[5-HTP]VNEEDHLR
SFLV[Oia]VNEEDHLR
SFLV[NFK]VNEEDHLR
SFLV[Kyn]VNEEDHLR
LAQSNGWGVMVSHR
LAQSNG[5-HTP]GVMVSHR
LAQSNG[Oia]GVMVSHR
LAQSNG[NFK]GVMVSHR
LAQSNG[Kyn]GVMVSHR
IWHHTFYNELR
27.1/27.7
27.0
28.0
23.2
21.1
21.7/21.9
21.8
22.1
24.0
I[5-HTP]HHTFYNELR
I[Oia]HHTFYNELR
22.9
23.3/23.4
23.6
I[NFK]HHTFYNELR
I[Kyn]HHTFYNELR
23.8
All Oia-containing peptides yielded two signals in the chromatograms with identical masses, which are most likely caused by tautomerization of the
Oia-residue.
Figure 3. MALDI reflector TOF mass spectra of the purified H-LAQSNGXGVMVSHR-OH peptides containing Trp (A) 5-HTP (B), Oia (C), Kyn (D), and NFK
(E) in position seven of the sequence. The main signals at m/z 1541.65, 1557.78, 1557.77, 1573.81, and 1545.81 correspond to the monoisotopic signals
of the expected [M+H]+ ions. Sodiated ions are labeled with an asterisk. The mass spectra were recorded in positive-ion mode with the matrix,
α-cyano-4-hydroxy-cinnamic acid.
c
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PEPTIDES WITH OXIDIZED TRYPTOPHAN RESIDUES
Figure 4. MALDI-PSD mass spectrum of the purified H-LAQSNG-Oia-GVMVSHR-OH peptide. The mass spectrum was recorded in positive-ion mode with
the matrix, α-cyano-4-hydroxy-cinnamic acid.
masses in the MALDI mass spectra, as shown for the β-enolase
sequence (Figure 3).
in LC-MS techniques. Finally, isotope-labeled synthetic peptides
(e.g. 13C or 15N) will enable the quantification of all four oxidation
products relative to the Trp content. Thus, the oxidation levels of
Trp-containing proteins can be quantified for in vivo studies.
All Oia-containing peptides eluted in two peaks with similar
retention times (Figure 2 and Table 2). Both fractions always
showed the correct peptide mass in MALDI-MS and identical
fragmentation patterns in MALDI-PSD and CID mode (Figure 4).
Reinjection of either fraction again yielded the same two peaks
with identical retention times and area ratios (data not shown).
Most likely, these peaks represented the lactam–lactim tautomers
of the Oia residue [20,30], although a sterically hindered cis–trans
configuration of the peptide backbone cannot be ruled out.
Owing to the isomeric nature of Oia and 5-HTP, it is not
possible to distinguish the corresponding isobaric peptides by
MS, not even after fragmentation, as both derivatives can form
the same fragment ions with respect to the m/z values. Thus, it
is necessary to separate them first, e.g. by liquid chromatography
(LC), such as RPC, which can be favorably coupled on-line to a
mass spectrometer. For the three sequences studied here, the
isobaric compounds were separated with the 5-HTP-containing
peptides always eluting about 0.5–1 min (0.3–0.6% acetonitrile)
earlier (Table 2 and Figure 2).
Supporting information
Supporting information may be found in the online version of this
article.
References
1 D’Autreaux B, Toledano MB. ROS as signalling molecules:
mechanisms that generate specificity in ROS homeostasis. Nat.
Rev. Mol. Cell Biol. 2007; 8: 813–824.
2 Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling.
Am. J. Physiol. Lung Cell Mol. Physiol. 2000; 279: L1005–L1028.
3 Davies MJ. Singlet oxygen-mediated damage to proteins and its
consequences. Biochem. Biophys. Res. Commun. 2003; 305: 761–770.
4 DaviesKJA,DelsignoreME,LynSW.Proteindamageanddegradation
by oxygen radicals. II. Modification of amino acids. J.Biol.Chem. 1987;
262: 9902–9907.
5 Stadtman ER, Levine RL. Free radical-mediated oxidation of free
amino acids and amino acid residues in proteins. Amino Acids 2003;
25: 207–218.
6 Berlett BS, Stadtman ER. Protein oxidation in aging, disease and
oxidative stress. J. Biol. Chem. 1997; 272: 20313–20316.
7 Simat TJ, Steinhart H. Oxidation of free tryptophan and tryptophan
residues in peptides and proteins. J. Agric. Food Chem. 1998; 46:
490–498.
8 Garrison WM. Reaction mechanism in the radiolysis of peptides,
polypeptides and proteins. Chem. Rev. 1987; 87: 381–398.
9 Chang TC, Chou WY, Chang GG. Protein oxidation and turnover.
J. Biomed. Sci. 2000; 7: 357–363.
10 Louie JL, Kapphahn RJ, Ferrington DA. Proteasome function and
protein oxidation in the aged retina. Exp.Eye.Res. 2002; 75: 271–284.
11 SquierTC.Oxidativestressandproteinaggregationduringbiological
aging. Exp. Gerontol. Mol. Aging 2001; 36: 1539–1550.
12 Butterfield DA. Oxidative stress in neurodegenerative disorders.
Antioxid. Redox. Signal. 2006; 8: 1971–1973.
Conclusions
Theoxidizedtryptophanderivativeswereincorporatedindifferent
peptide sequences by the Fmoc/tBu-strategy using either HATU
or DIC activation in high yields without major side reactions.
Only the formyl group of NFK was slowly released during the
final TFA-cleavage step, but the Kyn-containing peptides obtained
were separated by RP-HPLC and therefore did not contaminate
the purified peptides. With this study, we have identified widely
applicable strategies for the synthesis of 5-HTP, Kyn, NFK, and
Oia peptides that will be useful for the detailed study of their
fragmentation behavior in tandem mass spectrometry, and which
could even enable the distinction of isobaric derivatives like 5-HTP
and Oia by characteristic fragmentation patterns. Moreover, such
peptides could serve as standards to confirm suggested oxidative
modifications by their retention times and fragmentation pattern
13 Beckman KB, Ames BN. The free radical theory of aging matures.
Physiol. Rev. 1998; 78: 547–581.
14 Hsiai T, Berliner JA. Oxidative stress as a regulator of murine
atherosclerosis. Curr. Drug Targets 2007; 8: 1222–1229.
c
J. Pept. Sci. 2011; 17: 256–262 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

TODOROVSKI ET AL.
15 Hamblin M, Friedman DB, Hill S, Caprioli RM, Smith HM, Hill
MF. Alterations in the diabetic myocardial proteome coupled
with increased myocardial oxidative stress underlies diabetic
cardiomyopathy. J. Mol. Cell Cardiol. 2007; 42: 884–895.
16 Norris EH, Giasson BI. Role of oxidative damage in protein
aggregation associated with Parkinson’s disease and related
disorders. Antioxid. Redox Signal. 2005; 7: 672–684.
22 Fedorova M, Kuleva N, Hoffmann R. Identification of cysteine,
methionine and tryptophan residues of actin oxidized in vivo during
oxidative stress. J. Proteome Res. 2010; 9: 1598–1609.
23 Fedorova M, Todorovski T, Kuleva N, Hoffmann R. Quantitative
evaluation of tryptophan oxidation in actin and troponin I from
skeletal muscles using a rat model of acute oxidative stress.
Proteomics 2010; 10: 2692–2700.
17 Rousseva LA, Gaillard ER, Paik DC, Merriam JC, Ryzhov V, Garland DL,
Dillon JP. Oxindolealanine in age-related human cataracts. Exp. Eye
Res. 2007; 85: 861–868.
24 LabrooRB, CohenLA. Preparativeseparationofthediastereoisomers
of dioxindolyl-L-alanine and assignment of stereochemistry at C-3.
J. Org. Chem. 1990; 55: 4901–4904.
18 Vazquez S, Aquillina JA, Jamie JF, Sheil MM, Truscott RJW. Novel
protein modification by kynurenine in human lenses. J. Biol. Chem.
2002; 277: 4867–4873.
25 Raillard SP, Mann AD, Baer TA. An efficient procedure for the
preparation of Fmoc-amino acids. Org. Prep. Proced. Int. 1998; 30:
183–186.
19 Snytnikova OA, Kopylova LV, Chernyak EI, Morozov SV, Kolosova
NG, Tsentalovich YP. Tryptophan and kynurenine levels in lenses
of Wistar and accelerated senescence OXYS rats. Mol. Vis. 2009; 15:
2780–2788.
20 Simat T, MeyerK, Steinhart H. Synthesis and analysis of oxidation and
carbonyl condensation compounds of tryptophan. J. Chromatogr., A
1994; 661: 93–99.
21 Jain SK. Can tryptophan oxidation lead to lower tryptophan level
in diabetes? A commentary on ‘‘Propagation of protein glycation
damage involves modification of tryptophan residues via reactive
oxygen species: Inhibition by pyridoxamine.’’ Free Radical Biol. Med.
2008; 44: 1273–1275.
26 Savige WE. New oxidation products of tryptophan. Aust. J. Chem.
1975; 28: 2275–2287.
27 Frolov A, Singer D, Hoffmann R. Solid-phase synthesis of glucose-
derived Amadori peptides. J. Pept. Sci. 2007; 13: 862–867.
28 Chan WC, White PD. Fmoc Solid Phase Peptide Synthesis. Oxford
University press: New York, 2003.
29 Kaiser E, Colescott RL, Bossinger CD, Cook PI. Color test for detection
of free terminal amino groups in the solid-phase synthesis of
peptides. Anal. Biochem. 1970; 34: 595–598.
30 Van de Weert M, Lagerwerf FM, Haverkamp J, Heerma W. Mass
spectrometric analysis of oxidized tryptophan. J. Mass Spectrom.
1998; 33: 884–891.
c
wileyonlinelibrary.com/journal/jpepsci Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2011; 17: 256–262