Microwave-assisted 18O labeling of Fmoc-protected amino acids

Article abstract of DOI:10.1002/psc.2682

Recently, there has been an increased interest in isotopical labeling of peptides. Although there are several techniques allowing for a complete labeling of all carboxyl groups in peptides, regioselective labeling would be beneficial in many situations. Such labeling requires the use of ¹⁸O-labeled Fmoc amino acids. We have designed a method for such labeling that is an improvement on a technique proposed earlier. The new procedure is suitable for microscale synthesis and could be used in peptide and proteomics laboratories. Although for the majority of tested amino acids our method gives good labeling efficiency, it is time consuming. Therefore, we have decided to use microwave-assisted procedure. This approach resulted in reduction of reaction time to 15 min and increased reaction efficiency.

Full text of DOI:10.1002/psc.2682

Research Article
Received: 7 May 2014
Revised: 8 July 2014
Accepted: 11 July 2014
Published online in Wiley Online Library: 6 August 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2682
18
Microwave-assisted O labeling of
Fmoc-protected amino acids
Maciej Modzel, Halina Płóciennik, Alicja Kluczyk, Piotr Stefanowicz*
and Zbigniew Szewczuk
Recently, there has been an increased interest in isotopical labeling of peptides. Although there are several techniques allowing
for a complete labeling of all carboxyl groups in peptides, regioselective labeling would be beneficial in many situations. Such
labeling requires the use of 18O-labeled Fmoc amino acids. We have designed a method for such labeling that is an improvement
on a technique proposed earlier. The new procedure is suitable for microscale synthesis and could be used in peptide and
proteomics laboratories. Although for the majority of tested amino acids our method gives good labeling efficiency, it is time
consuming. Therefore, we have decided to use microwave-assisted procedure. This approach resulted in reduction of reaction
time to 15 min and increased reaction efficiency. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: 18O labeling; quantitative proteomics; peptide synthesis; stable isotopes; microwave-assisted labeling
synthesis both for coupling reactions and deprotection and
cleavage [15,16].
Introduction
Peptides labeled with stable isotopes are a useful research tool
in biochemistry, molecular biology, and analytical chemistry.
They can be used as internal standards for quantitative mass
spectrometry (MS) analysis of peptides that makes them useful in
proteomics [1–3]. The labeling of peptides also influences the
nuclear magnetic resonance, MS, and infrared spectra, which helps
in studies on peptide structure, interactions (both intermolecular
and intramolecular), and fragmentation pathways [4–7]. Such
approach can be also relevant to protein analysis. The labeling with
18O is convenient and often used in proteomic research because
the heavy oxygen atom can be directly incorporated into a peptide
molecule [8]. Currently, there are many protocols for labeling
peptides with 18O, both by acid catalysis and by treatment with
proteolytic enzymes in H128O, as well as for labeling of carboxylic
acids, for example, iodoacetic acid used for tagging cysteine
containing peptides [9–13]. Some of those methods require harsh
conditions or long reaction times; therefore, they are of limited
use as standard laboratory procedure. Moreover, they offer only
limited selectivity in distribution of the heavy oxygen. A direct
peptide labeling can affect either the C-terminal carboxyl group
or side chain carboxyl groups or all of them. A method of synthesis
of labeled building blocks for selective incorporation of 18O dur-
ing peptide synthesis has been developed by Marecek et al. [14].
It involves the use of HCl in H128O:1,4-dioxane solution, with 20 ml
of labeled water for 3–5 g of Fmoc amino acid. The synthesis is
carried out under reflux, and the products are isolated by
evaporation of the reaction medium. This procedure requires
significant reaction times and in some cases repetition of
equilibration, particularly for isoleucine (34 h for each equilibra-
tion). However, for solid-phase synthesis of labeled peptide
standards for MS, a quick small-scale procedure for preparation
of protected amino acids might be useful.
Taking this into account, we have designed a new method
suitable for preparation of small portions of labeled Fmoc amino
acids, which can then be easily incorporated into peptides
according to the standard Fmoc strategy. The application of a mi-
crowave synthesizer reduced the reaction time to 15min. To the
best of our knowledge, this is the first efficient microwave-assisted
method of 18O labeling, applicable for milligram quantities of Fmoc
derivatives, consuming 20 μl of H128O per 5 mg of material.
Materials and Methods
Reagents
1,4-Dioxane was purchased from POCh (Gliwice, Poland), concen-
trated sulfuric acid (96% H2SO4) from Stanlab (Lublin, Poland),
sodium chloride from Chempur (Piekary Śląskie, Poland). H128O
(97% 18O), DABSYL chloride, LCMS grade acetonitrile, and metallic
sodium were bought from Sigma-Aldrich (St Louis, MO, USA). TFA
was bought from Merck (Darmstadt, Germany) and Fmoc-protected
amino acids and preloaded Wang resins from NovaBiochem (Merck,
Darmstadt, Germany). Water was purified using a Hydrolab purifica-
tion system (Hydrolab, Poland).
Fmoc Amino Acid Labeling
Fmoc amino acid samples (5mg) were placed in 2 ml ampoules. An
amount of 2 M HCl in dioxane (380 μl, prepared earlier by saturating
*
Correspondence to: Piotr Stefanowicz, Faculty of Chemistry, University of Wrocław,
F. Joliot-Curie 14, 50-383 Wrocław, Poland. E-mail: piotr.stefanowicz@chem.uni.
wroc.pl
Microwave-assisted synthesis is a well-known way of increasing
the rates of organic reactions. It is frequently used in peptide
Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383, Wrocław,
Poland
J. Pept. Sci. 2014; 20: 896–900
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

MICROWAVE-ASSISTED 18O LABELING
1,4-dioxane with dry HCl) was added, followed by H128O (97% 18O)
(20 μl). The ampoules were flame sealed and left rotating for 24 h
at room temperature. The solvent was then evaporated in a stream
of dry nitrogen. The product was analyzed by high-performance liquid
chromatography (HPLC) and MS (data presented in supporting
material) and used for peptide synthesis without further purification.
phenylalanine, valine, leucine, isoleucine, proline, and methio-
nine. Other amino acids, if necessary, can be labeled by our
method by using different protecting groups or introducing
the acid-labile protecting group after labeling.
The labeling procedure without microwave assistance
required 24 h at room temperature. Some of Fmoc derivatives
(notably glycine) did not dissolve completely despite vigorous
mixing, which could have impaired the labeling efficiency. After
24 h, the ampoules were opened, and their contents evaporated
in a stream of dry nitrogen and subsequently analyzed by HPLC
and MS. No degradation of Fmoc derivatives was observed;
the obtained products were practically pure according to HPLC
chromatograms. We also verified that no deprotection
occurred using the ninhydrin reaction. However, the efficiency
of labeling – determined by comparing the intensities of the
peaks corresponding to unlabeled, monolabeled, and bilabeled
species in the mass spectra – was not uniform. The 24 h incuba-
tion at room temperature resulted in 85–90% labeling for some
of the amino acids (alanine, phenylalanine, and leucine);
whereas for valine and isoleucine, the exchange was 45% and
27%, respectively. The difficulty of isoleucine labeling has been
reported in literature [14] and attributed to steric hindrance.
The results are summarized in Table 1.
In order to improve the efficiency and shorten the reaction time,
we have decided to use a CEM microwave synthesizer. As the
volume of our sample was low (less than 0.5ml), we had to use a
sheath liquid to fill the vial and act as thermal buffer for better
control of microwave energy. In standard operation mode, the
CEM synthesizer adjusts the microwave energy to maintain the
specified temperature. A small volume of the reaction medium in
a typical 10 ml reaction vessel may lead to complete evaporation
of the sample; therefore, we developed a method of vial insertion
[7] Water was chosen as the sheath liquid because of its high
specific heat and boiling point similar to that of dioxane (the pic-
ture of reaction vessel containing sealed ampoule and water is pre-
sented in supplementary material). As expected, reaction in a
microwave reactor proceeded rapidly. The maximum efficiency
(over 85% for all examined Fmoc amino acids) was obtained in
15min – extension of the reaction time (to 30 and 60 min) did
not affect the labeling yield. With the exception of methionine,
the protected amino acids did not undergo any noticeable decom-
position, as verified by HPLC and negative ninhydrin reaction.
Moreover, no amino acid repetitions were observed during peptide
synthesis. Only in the case of methionine that a significant
Microwave-assisted Labeling
An ampoule, prepared as described in Results and Discussion, was
placed in a microwave vial partially filled with water acting as
sheath liquid. The CEM Discover synthesizer (Matthews, NC, USA)
equipped with gas cooling system and magnetic stirring was
operated in standard mode, in which the temperature and time
of irradiation are set, and the synthesizer adjusts the power to
maintain the temperature. The temperature was set to 110°C, and
the reaction times were set to 15, 30, and 60 min. The sample was
then evaporated to dryness in a stream of dry nitrogen and
analyzed by HPLC and MS (data presented in supporting material).
Peptide Synthesis
The synthesis was carried out in syringe reactors (Intavis, Koeln,
Germany) according to standard Fmoc strategy [17]. For each
synthesis, 5 mg of Wang resin loaded with corresponding Fmoc
amino acid (0.55–0.65 mmol/g) was used. In each coupling step,
four equivalents of unlabeled Fmoc amino acids were used with
TCTU as coupling agent. In the case of 18O-labeled derivatives, only
two equivalents (from 2 to 3 mg) were used to reduce the cost of
synthesis. The peptides were then cleaved with TFA:H2O mixture
(95 : 5, v:v), lyophylised, and analyzed by HPLC and MS (exemplary
data presented in supporting material).
MS Analysis
Mass spectra were recorded on a Bruker micrOTOF-Q mass
spectrometer and apex ultra 7 T FT-ICR mass spectrometer
(Bremen, Germany) equipped with electrospray ion source in the
Laboratory of Mass Spectrometry at the Faculty of Chemistry,
University of Wrocław. The samples were dissolved in a 50 : 50
acetonitrile–water mixture containing 0.1% HCOOH
HPLC Analysis
The analysis was performed on Dionex chromatography system,
using
a Jupiter Proteo column 90A, 250 × 4.6mm, 4 μm
(Phenomenex, Torrance, CA, USA). The eluents were as follows:
(A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile:water
(80 : 20, v:v). The gradient was from 0% to 80% B in A in 40min, flow
rate 1 ml/min. Dionex variable wavelength detector operating at
210, 254, and 277 nm was used. In the case of DABSYL-tagged
samples, 210, 254, and 473 nm absorption were analyzed. In order
to observe all the possible products, the data registration was
extended to include the column regeneration and re-equilibration.
Table 1. Labeling efficiency of obtained Fmoc-protected amino acids
calculated by comparing the intensity of the peaks corresponding with
unlabeled, monolabeled, and bilabeled species in mass spectra
Amino
acid
Labeling efficiency –
regular method (%)
Labeling efficiency – microwave-
assisted method (%)
Ala
Phe
Gly
Ile
90
86
65
27
88
45
70
84
90
91
92
88
92
88
92
86a
Results and Discussion
We studied the 18O incorporation to standard Fmoc amino acid
derivatives using a solution of HCl in dioxane to catalyze the
isotope exchange reaction. These reaction conditions excluded
from our experiments the derivatives containing acid-labile side
chain protecting groups (Boc and Trt). For this reason, we
have chosen the following amino acids: glycine, alanine,
Leu
Val
Pro
Met
aPartial decomposition observed.
J. Pept. Sci. 2014; 20: 896–900
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
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MODZEL ET AL.
decomposition was revealed as two chromatographic peaks of
roughly equal intensities. However, methionine is known to
decompose under acidic peptide hydrolysis conditions [18]. The re-
sults are summarized in Table 1, and representative results for va-
line are shown in Figure 1.
synthesis brings a two units shift to the final peptide mass, as
could be seen in Figures 2 and 3. According to MS data, the in-
corporation of the 18O label into the peptides was nearly quanti-
tative. The location of the heavy oxygen was verified by MS/MS
method. It showed unambiguously that the 18O stays in the car-
bonyl group of the labeled amino acid.
We have also synthesized a series of peptides using the la-
beled amino acids directly after the exchange procedure. The se-
quences of model peptides were selected from proteolytic
digests of various proteins, investigated in our research team
[19]. Fmoc-AKAAF is a model peptide, containing phenylalanine
to facilitate ultraviolet (UV) detection, whereas Fmoc-VGSK and
Fmoc-QIK are tryptic fragments of human serum albumin
(HSA), dabsyl-AKADLAK, prepared for UV–visible studies, con-
tains ADLAK – a tryptic fragment of HSA; Fmoc-GVFR and
Fmoc-IETMR are tryptic fragments of HSA propeptide and of bo-
vine serum albumin, respectively. To increase the sensitivity of
UV–visible detection, the peptides were treated with dabsyl
chloride or left with N-terminal Fmoc group. The synthesis was
carried out on solid support, according to a standard Fmoc pro-
tocol, with four equivalents of regular Fmoc derivatives and only
two equivalents of the labeled derivatives, in order to reduce the
cost of the synthesis. Each labeled amino acid used in peptide
To summarize, we have developed an improved scale-down
procedure of Fmoc amino acid labeling protocol, which gives
good results for almost all tested Fmoc-protected amino acids.
Our method is suitable for microscale synthesis (3–5 mg of
amino acid derivative) using commercially available reagents.
We have used 20 μl of H128O to obtain 5 mg of labeled amino acid,
which is comparable with the consumption described by
Marecek et al. [14]; however, our method is much faster and
practically quantitative, which is especially important in case of
small amounts of special Fmoc derivatives. Moreover, our
method does not require a reflux setup, so the whole apparatus
is less complex and less prone to contamination with atmo-
spheric water. We have also highlighted that microwaves signif-
icantly accelerate and increase the yield of the reaction. The
method is effective for all the examined amino acids except for
methionine, which undergoes decomposition. Although the
Figure 1. Fmoc valine-labeled without (panels A and B) and with microwave assistance (panels C and D). MS spectra (panels A and C) and chromatograms
registered at 254 nm (panels B and D). The peaks in panel A at 362.1336, 364.1384, and 366.1431 correspond to [M + Na]+ ions with no, 1 and 2 heavy oxygen
atoms, respectively. In panel B, peaks at 362.1295, 364.1347, and 366.1437 correspond to [M+ Na]+ ions with no, 1 and 2 heavy oxygen atoms, respectively.
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J. Pept. Sci. 2014; 20: 896–900

MICROWAVE-ASSISTED 18O LABELING
Figure 2. MS spectrum of Fmoc-AKAAF synthesized with one alanine labeled without microwave assistance. The region of peak at 731.3673 (corresponding
to [M + H]+ ion) was enlarged to show the isotopic pattern. The peak at 1461.7207 corresponds to a noncovalent dimer.
enable a cost-efficient synthesis of peptides permanently la-
beled with stable isotope as standards for quantitative
proteomics.
Acknowledgements
This work was financially supported by grant no. 1236/M/WCH/13
at the Faculty of Chemistry, University of Wrocław, from the Ministry
of Science and Higher Education of Poland.
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Additional supporting information may be found in the online ver-
sion of this article at the publisher’s web site.
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J. Pept. Sci. 2014; 20: 896–900