Application Note BIO-0001 Microwave Synthesis of ACP 65-74

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Microwave Synthesis of ACP 65‐74
INTRODUCTION
The application of microwave energy has
proved to be a major enabling tool for many
chemical reactions requiring energy input,
and recently microwave irradiation has
been applied to solid phase peptide
synthesis. Peptide synthesis often suffers
from long reaction times and low yields due
to problems such as aggregation.
Microwave energy can help break up these
aggregates improving the synthesis quality
while speeding up the synthesis rate. The
acyl carrier protein (ACP) fragment 65‐74,
H‐Val‐Gln‐Ala‐Ala‐Ile‐Asp‐Tyr‐Ile‐Asn‐Gly‐
OH, is a widely used standard model for
demonstrating peptide synthesis efficiency.
The presence of several hydrophobic
residues, and in particular the hindered Val
and Ile residues, can lead to poor coupling
and deprotection reactions resulting in low
yield and purity of the peptide.
In this application note ACP 65‐74 is
synthesized in good yield and purity using
microwave irradiation for the deprotection,
coupling, and final cleavage steps.
MATERIALS AND METHODS
Reagents
All Fmoc amino acids were obtained from
CEM Corporation and contained the
following side chain protecting groups:
Asn(Trt), Asp(OtBu), Gln(Trt), and Tyr(tBu).
O‐(Benzotriazol‐1‐yl)‐N,N,N′,N′‐
tetramethyluronium hexafluorophosphate
(HBTU) and 1‐hydroxybenztriazole hydrate
(HOBt) were also obtained from CEM
Corporation. Fmoc‐Gly‐Wang resin was
Application Note BIO‐0001
obtained from Novabiochem (San Diego,
CA). Diisopropylethylamine (DIEA),
piperidine, trifluoroacetic acid (TFA),
triisopropylsilane (TIS), and 3,6‐dioxa‐1,8‐
octanedithiol (DODT) were obtained from
Sigma Aldrich (St. Louis, MO).
Dichloromethane (DCM), N,N‐
dimethylformamide (DMF), N‐
methylpyrrolidine (NMP), anhydrous diethyl
ether, acetic acid, HPLC grade water, and
HPLC grade acetonitrile were obtained from
VWR (West Chester, PA).
Peptide Synthesis
The peptide was prepared using the CEM
Liberty automated microwave peptide
synthesizer on 0.164 g of Fmoc‐Gly‐Wang
resin (0.61 meq/g substitution).
Deprotection (20% piperidine with 0.1 M
HOBt) was performed in two stages with an
initial deprotection of 30 seconds followed
by 3 minutes at 75 °C. Coupling reactions
were performed with 5 fold excess Fmoc‐
AA‐OH with 1:0.9:2 AA/HBTU/DIEA for 5
minutes at 75 °C. Cleavage was performed
using 92.5:2.5:2.5:2.5 TFA/water/TIS/DODT
for 30 minutes at 38 °C. Following cleavage
the peptide was precipitated and washed
with diethyl ether.
Peptide Analysis
The peptide was analyzed on a Waters
Atlantis C18 column (2.1 x 150 mm) at 214
nm with a gradient of 5 – 95% MeCN (0.1%
formic acid), 0 – 20 minutes. Mass analysis
was performed using an LCQ Advantage ion
trap mass spectrometer with electrospray
ionization (Thermo Electron, Waltham, MA).

RESULTS AND DISCUSSION
ACP 65‐74 was synthesized in 5 hours using
microwave irradiation to accelerate the
deprotection, coupling, and the peptide
cleavage steps to give the peptide in 96%
isolated yield and about 87% crude purity
(Figure 1), and the product was confirmed
by mass spectrometry (Figure 2).
Figure 1: ACP 65‐74 crude chromatogram
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Application Note BIO‐0001
Figure 2: ACP 65‐74 mass spectrum
CONCLUSIONS
The application of microwave energy to
solid phase peptide synthesis allows for
much faster cycle times while driving
deprotection and coupling reactions to
completion. This leads to peptides with
higher crude purity and improved yields
including peptides with “difficult”
sequences.