S. ABU-BAKER ET AL.
OPEN ACCESS OJSTA
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
α
-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-
thod.
3.3. Characterization of WT-PLB Using
MALDI-TOF
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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