Microwave Assisted Peptide Synthesis as a New Gold Standard in Solid Phase Peptide Synthesis: Phospholamban as an Example

doi:10.4236/ojsta.2014.31001

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Open Journal of Synthesis Theory and Applications, 2014, 3, 1-4

Published Online January 2014 (http://www.scirp.org/journal/ojsta)

http://dx.doi.org/10.4236/ojsta.2014.31001

OPEN ACCESS OJSTA

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: *[email protected]

Received October 12, 2013; revised November 16, 2013; accepted December 10, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-

ABSTRACT

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.

KEYWORDS

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.

S. ABU-BAKER ET AL.

OPEN ACCESS OJSTA

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-

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

-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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