Improved Synthesis of a Difficult Peptide by UV Monitoring

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Application Note BIO-0011
Improved Synthesis of a Difficult Peptide by UV Monitoring
INTRODUCTION
The AKR/Gross murine leukemia viruses (MuLV)
are C-type retroviruses that cause the
development of leukemia in AKR (H-2 k ) mice. 1
Type-specific antiretroviral cytotoxic Tlymphocyte
(CTL) responses have been
reported that allow differentiation between
infection by the AKR/Gross MuLV and other
MuLV. 1 A 10-mer derived from the C-terminal
portion of the AKR/Gross MuLV CTL epitope has
been used as a model “difficult” peptide for this
study because it cannot be synthesized under
standard conditions. 2,3
UV monitoring of Fmoc deprotection allows the
optimization of difficult syntheses. Typically,
Fmoc removal is accomplished with piperidine,
although other reagents such as piperazine may
also be used. Piperidine reacts with the Fmocprotected
amino acid to remove the Fmoc
group (which is converted to dibenzofulvene)
yielding the free amine following
decarboxylation (Figure 1). The dibenzofulvene
can then react with piperidine to form the
dibenzofulvene-piperidine adduct (DBF-pip).
Figure 1. Fmoc removal by piperidine.
The DBF-pip molecule shows a distinct UV
absorbance at 301 nm that is easily discernible
from the background absorbance of the other
reagents present in the deprotection reaction.
Thus, by monitoring the UV absorbance at 301
nm, the relative efficiency of a given
deprotection reaction can be measured.
When the UV spectrometer is connected to the
Liberty/Liberty1, all liquid waste from the
deprotection step flows through the UV flow
cell, and the absorbance is measured. If the
absorbance detected after the second (3 min)
deprotection is above the set threshold, the
deprotection is considered failed, and
additional 3 minute deprotections are
performed until either a passing value is
obtained or a maximum number of
deprotections has been reached.
In theory, a difficult deprotection will
correspond to a difficult coupling. When using
the UV Monitoring Option, the coupling
conditions can be modified (double coupling,
extended coupling time, and/or capping) on
failure to compensate for this difficulty.
MATERIALS AND METHODS
Reagents
All Fmoc amino acids and O-(Benzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate
(HBTU) were obtained from CEM
Corporation. PAL-PEG-PS resin was obtained
from Applied Biosystems (Carlsbad, 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-methylpyrrolidone (NMP),
anhydrous diethyl ether, acetic acid, HPLC grade
water and acetonitrile were obtained from VWR
(West Chester, PA).
Peptide Synthesis:
WFTTLISTIM-NH2
The peptide was prepared using the CEM
Liberty automated microwave peptide

synthesizer equipped with the UV Monitoring
option, on 0.500 g of PAL-PEG-PS resin (0.20
meq/g substitution). Deprotection (20%
piperidine) was performed in two stages with
an initial deprotection of 30 s followed by 3 min
at 75 °C. Coupling reactions were performed
with 5 fold excess Fmoc-AA-OH with 1:0.9:2
AA/HBTU/DIEA for 5 min at 75 °C. For syntheses
with UV monitoring, deprotection and coupling
conditions were modified on failure as shown in
Table 1.
Table 1. UV monitoring conditions.
Synthesis
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Number of
Deprotections (Max)
30 s 3 min
Double
Couple
on Fail
Extended
Coupling
on Fail
1 1 1 No No
2 1 3 No 15 min
3 1 3 Yes 15 min
Cleavage was performed using 92.5% TFA/2.5%
H2O/2.5% TIS/2.5% DODT for 30 min at 38°C.
Following cleavage the peptide was precipitated
and washed in diethyl ether. Peptides were
lyophilized in 10% acetic acid prior to analysis.
Peptide Analysis
The peptides were analyzed on a Waters
Atlantis C18 column (2.1 ×150 mm) at 214 nm
with a gradient of 5 – 70% MeCN with 0.1% TFA,
0 – 20 min, with the column heated to 40 °C.
Mass analysis was performed using an LCQ
Advantage ion trap mass spectrometer with
electrospray ionization (Thermo Electron).
RESULTS
The MuLV CTL peptide was first synthesized
under standard microwave conditions, yielding
a crude purity of 37%. Deletions of F, W, T, and
WT were observed (Figure 2).
Application Note BIO-0011
Figure 2. Synthesis 1 (Standard Conditions)
The peptide was resynthesized with UV
monitoring, with a maximum of three 3 min
deprotections, and extended coupling time (15
min) on failure.
Failures were observed for Thr 3 and Phe 2 (Figure
3a), accounting for the deletions of F and W
respectively. Target was obtained at a crude
purity of 33%. The deletion products observed
without UV monitoring were still present
(Figure 3b).
Figure 3. Synthesis 2 (15 min coupling on failure).
a. UV absorbance data (failures indicated by *).
b. HPLC data.
-WT -W -T
-F
-W
-WT
-F -T
Target
* *
Target

Finally, the peptide was synthesized with a
maximum of three 3 min deprotections, and
double coupling and extended coupling time
(15 min) on failure. Failures were observed for
Leu 5 , Thr 3 , and Phe 2 (Figure 4a), accounting for
the deletions of T, F, and W respectively. Target
was obtained at a crude purity of 88%. The F
and W deletions were minimized, and the T and
WT deletions were eliminated (Figure 4b).
Figure 4. Synthesis 3 (15 min double coupling on
failure).
a. UV absorbance data (failures indicated by *).
b. HPLC data.
CONCLUSION
UV monitoring is a powerful tool for the
optimization of peptide synthesis, allowing
access to peptides that were previously difficult
to synthesize at high purity.
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-W -F
*
* *
Target
Application Note BIO-0011
REFERENCES
1. Green, W. R. Immun. Rev. 1999, 168, 271-
286.
2. Redemann, T., Jung, G. Peptides 1996. In
Proceedings of the 24 th European Peptide
Symposium; Ramage, R., Epton, R., Eds.;
Mayflower Scientific Ltd: Kingswinford, UK,
1998; p 749.
3. Carpino, L. A., et al. Tetrahedron Lett. 2004,
45, 7519-7523.