Open Journal of Synthesis Theory and Applications, 2014, 3, 1-4
Published Online January 2014 (
Microwave Assisted Peptide Synthesis as a New Gold
Standard in Solid Phase Peptide Synthesis:
Phospholamban as an Example
Shadi Abu-Baker1*, Philip Garber1,2, Bryce Hina1, Trevor Reed1, Ghaffari Shahrokh1,
Mohannad Al-Saghir1, Gary Lorigan3
1College of Arts and Sciences, Ohio University, Zanesville, USA
2Genesis Hospital , Ohio University, Zanesville, USA
3Department of Chemistry and Biochemistry, Miami University, Oxford, USA
Email: *
Received October 12, 2013; revised November 16, 2013; accepted December 10, 2013
Copyright © 2014 Shadi Abu-Baker et al. This is an open access article distributed under the Creative Commons Attribution License,
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In this study, we report the microwave assisted synthesis of Phospholamban protein (WT-PLB) as a new gold
standard in solid phase peptide synthesis. Microwave energy offers benefits for both the coupling and deprotec-
tion reactions during peptide synthesis. The use of microwave energy for both the coupling and deprotection
steps makes the microwave peptide synthesizers the most versatile and powerful systems available. It produces
high yield and fast synthesis when compared to conventional peptide synthesizers.
Solid-State Peptide Synthesis; Phospholamban; Microwave Assisted Synthesis
1. Introduction
The use of microwave peptide synthesizers has several
advantages over conventional peptide synthesizers. First,
it allows the use of microwave energy for both the
coupling and deprotection steps using fastest cycle times.
Second, the peptide purity and the yield are comparable
to conventional synthesis. Third, it significantly reduces
purification time and waste. And finally, it allows the
access to peptides impossible to synthesize under con-
ventional conditions. In this study, we compared the
synthesis of WT-Phospholamban (WT-PLB) using both
the conventional and the microwave assisted solid-phase
peptide synthesis. WT-PLB is a hydrophobic 52-amino
acid transmembrane protein that is involved in regu lating
the contraction and relaxation of heart muscle [1-3].
Phosphorylation of PLB by cyclic AMP- and calmodu-
lin-dependent kinases is believed to increase the rate of
calcium re-uptake by the sacroplasmic reticulum and
result in muscle relaxation [1-3]. The isolation and puri-
fication of large quantities of native PLB through mole-
cular biology techniques have not yet been achieved due
to difficulties encountered in the bacterial over expres-
sion of phospholambanc DNA [4,5]. Alternatively, WT-
PLB has been prepared by chemical synthesis using
standard solid-phase peptide synthesis and purification in
organic solvents [6,7]. In addition, this approach gives
the opportunity to synthesize site-specific isotopically
labeled peptides and proteins [8-10]. The biochemical
and biophysical comparison of synthetic PLB and native
PLB revealed that they are both similar in size and fun c-
tionally identical [6,7].
2. Materials and Methods
WT-PLB Synthesis and Purification
WT-PLB was synthesized using both the conventional
solid-phase methods with an ABI 433A peptide synthe-
sizer (Applied Biosystems, Foster city, CA) and with mi-
crowave as sisted liberty peptide synthe sizer (see Figure 1,
*Corresponding a uthor.
Figure 1. New CEM liberty p ept i de synthesi zer.
CEM liberty, Mathew, USA). During the conven-
tional synthesis, we found that the coupling of Leu-7 to
Thr-8 was difficult even after double coupling and ex-
tending the reaction time to six hours. However, this
problem was solved by using the pseudoproline dipeptide
of Fmoc-Leu-Thr (ΨMe,MePro)-OH from Novabiochem
(San Diego, CA). The use of a pseudoproline dipeptide
of Fmoc-Leu-Thr (ΨMe,MeP ro)-OH enhanced the yield (~
25%, ~9 days of synthesis) after lyophylization. Using
the similar protocol the microwave assisted solid-phase
peptide synthesis an enhanced yield compared to of the
conventional method (~35%, ~3 days of synthesis).This
indicates that the microwave assisted method is prefera-
ble as it enhances the yield and reduce the synthesis time.
To synthesize P-PLB, a pre-phosphorylated Fmoc-serine
amino acid was used at amino acid position 16 instead of
the regular Fmoc-serine used in the synthesis of PLB.
The crude peptide was purified on an Amersham Phar-
macia Biotech AKTA explorer 10S HPLC controlled by
Unicorn (version 3) system software. The purified pro-
tein was lyophilized and characterized by matrix-assisted
laser desorption ionization time of flight (MALDI-TOF)
mass spectrometry.
3. Results
3.1. Solid-Phase Peptide Synthesis of WT-PLB
The chemically synthesized form of the full length PLB
(Figure 2(A)) and P-PLB (Figure 2(B)) was used for all
of the solid-state NMR experiments. In general, sol-
id-phase peptide synthesis (SPPS) starts with the
C-terminal amino acid attached to a solid support (res in).
Amino acids are then coupled one at a time till the
N-terminus is reached. Each time an amino acid is added,
Figure 2. Primary sequence of (A) PLB; and (B) P-PLB.
Sites of pseudoproline substitution are highlighted in red.
P-Ser residue highlighted in blue was introduced using
Fmoc-Ser (PO(OBzl)OH)-OH.
the following three steps are repeated: First, deprotection
of the N-terminal amino acid of the peptide bound to the
resin (removal of the Fomc protecting group, see the
aromatic part in Fig ure 2). This step is followed by acti-
vation and coupling of the next amino acid. And finally,
the new N-terminal amino acid is deprotected [11 ].
To control the progress of the synthesis, the deprotec-
tion and coupling steps can be monitored using a UV
detector. Several approaches including switching to dif-
ferent resins and activating reagents as well as using a
pseudoproline dipeptide has been suggested to improve
the yield of poor synthesis [12]. Figure 2 shows the
pseudoproline dipeptide Fmoc-Leu-Thr-(CMe,Mepro)-
OH in red. In this dipeptide, the Thr residue has been
reversibly protected as proline-like TFA-labile oxazoli-
dine [12].
WT-PLB was synthesized using two peptide synthe-
sizers using a procedure developed in the Lorigan’s lab.
Briefly, WT-PLB was synthesized using modified Fmo c -
based solid-phase methods with an ABI 433A peptide
synthesizer (Applied Biosystems, Foster city, CA) and
the microwave assisted liberty peptide synthesizer (CEM
liberty, Mathew, USA). WT-PLB is very hydrophobic;
thus, the synthesis of this peptide is very challenging.
Nevertheless, by using a combination of extended coupl-
ing and deprotection protocols with a single pseudopro-
line dipeptide substitution, we were able to obtain both
purified PLB and P-PLB in a yield of ~25% in ~9 days
using the conventional synthesis and 35% in ~3 days
using the microwave assisted synthesis. Couplings were
performed using 10-fold excess of Fmoc-amino acids
activated with HBTU/DIPEA. The synthesizer was pro-
grammed to use conditional UV feedback monitoring;
coupling and deprotection reactions are extended auto-
matically, and a capping step introduced after the coupl-
ing step, based on the kinetic profile of the Fmoc depro-
tection reaction. The results of the the two methods of
peptide synthesis is summarized in Table 1.
All peptides were cleaved from the resin by treatment
with TFA/EDT/thioanisole/water (10:0.5:0.25:0.5) for
2.5 h, and isolated by centrifugation followed by precipi-
Table 1. Comparing the conventional and microwave as-
sisted solid-phase pepti de synthsis for PLB.
Method Method
Convectional Microwave assisted
~25% yield ~35% yield
~9 days ~3 days
tation with methyl t-butyl ether. PLB consists of a hy-
drophilic N-terminus (residues 1 - 20), a hinge region
(residues 21 - 30) and a hydrophobic
-helical trans-
membrane tail (residues 31 - 52) [1]. From previous
work by Lorigan and co-workers [13], it is known that
the synthesis of the C-terminal transmembrane region of
PLB is extremely difficult, particularly the region from
Cys36 to Cys45. To overcome these difficulties, the Lo-
rigan group developed a strategy involving extended
double coupling together with capping and conditional
repetition of the Fmoc deprotection reaction [13]. Using
this approach, PLB (24 - 52) segment was obtained in a
purified yield of 37 % [13].
Initially, we attempted the synthesis of full length PLB
with standard amino-acid building blocks using the pro-
tocols previously described [13]. A PEG-PS resin (0.22
mmol/g) was selected as the solid support to reduce steric
crowding and aggregation during chain assembly. Using
the conditional feedback monitoring, this synthesis was
completed in 9 days, as compared to 10 days for the
shorter PLB (residues 24 - 52) prepared on polystyrene
resin [13]. And now using the microwave assisted me-
thod the synthesis is completed in about three days only.
UV monitoring of the Fmoc deprotection reactions in-
dicated that the peptide assembly proceeded smoothly
until Leu-7. However, following introduction of this re-
sidue, there was a marked decrease in the height of the
Fmo c deprotection peak, indicating difficulties in the
coupling of Leu-7 to Thr-8. Attempts to improve this
coupling by double coupling or extending the reaction
time to 6 hours had little effect. In view of the problems
with the coupling of Leu-7 to Thr-8, the synthesis was
repeated in exactly the same manner, except that Leu-7
and Thr-8 were introduced simultaneously using the
pseudoproline dipeptide Fmoc-Leu-Thr (CMe, Mepro)-
OH In the presence of this dipeptide, UV monitoring of
the Fmoc deprotection reactions indicated that the pep-
tide assembly proceeded reasonably smoothly until the
end of this synthesis. Similar patterns were seen in both
conventional and microwave assisted synthesis.
3.2. HPLC Purification of WT-PLB
Following global deprotection and cleavage of the pep-
tide from the resin, PLB was purified by preparative re-
verse phase chromatography on a C4 column eluted with
a gradient formed between 0.1% TFA in nanopure water
(solvent A) and MeCN/isopropyl alcohol/water/TFA
(38:57:5:0.1) (Solvent B). After lyophilization and using
standard Fmoc-amino acid building blocks, the purified
peptide was obtained in a yield of only 9% based on ini-
tial resin substitution. Conversely, with the dipeptide and
using the conventional peptide synthesizer, the purified
PLB was obtained in a yield of ~25%, nearly a 3-fold
increase when compared to the synthesis using standard
building blocks. And finally, with the dipeptide and us-
ing the microwave assisted peptide synthesizer, the puri-
fied PLB was obtained in a yield of ~35%, nearly 1.5
folds increase when compared to the conventional me-
3.3. Characterization of WT-PLB Using
When the dipeptide was used to synthesize WT-PLB, a
correct mass of 6080 MU was obtained after the purifica-
tion step. Conversely, when the dipeptide was not used,
MALDI-TOF indicated the presence of an impurity with
a mass of 5144 MU, which could be ascribed to Ac-PLB
(residues 9 - 52).
4. Conclusion
The use of microwave peptide synthesizers has several
advantages over conventional peptide synthesizers as it
increases the peptide purity, yield and reduces the time of
synthesis when compared to conventional synthesis. PLB
was used as an example to emphasize the efficiency of
the microwave assisted synthesis over the conventional
synthesis and it showed better yields (1.5 fold increase)
and faster synthesis( reduce the time from 9 to 3 days).
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