Synthesis of peptides containing C -terminal methyl esters using trityl side-chain anchoring: Application to the synthesis of a-factor and a-factor analogs

Article abstract of DOI:10.1021/ol302592v

A new cysteine anchoring method was developed for the synthesis of peptides containing C-terminal cysteine methyl esters. This method consists of attachment of Fmoc-Cys-OCH₃ to either 2-ClTrt-Cl or Trt-Cl resins (via the side-chain thiol) followed by preparation of the desired peptide using Fmoc-based SPPS. We applied this method to the synthesis of the mating pheromone a-factor and a 5-FAM labeled a-factor analog. The peptides were obtained with high yield and purity and were shown to be bioactive in a growth arrest assay.

Full text of DOI:10.1021/ol302592v

ORGANIC
LETTERS
2012
Vol. 14, No. 22
5648–5651
Synthesis of Peptides Containing
C‑Terminal Methyl Esters Using Trityl
Side-Chain Anchoring: Application
to the Synthesis of a‑Factor and
a‑Factor Analogs
Veronica Diaz-Rodriguez,† Daniel G. Mullen,† Elena Ganusova,‡ Jeffrey M. Becker,‡
and Mark D. Distefano*,†
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455,
United States, and Department of Microbiology, University of Tennessee, Knoxville,
Tennessee 37996, United States
diste001@umn.edu
Received September 20, 2012
ABSTRACT
A new cysteine anchoring method was developed for the synthesis of peptides containing C-terminal cysteine methyl esters. This method
consists of attachment of Fmoc-Cys-OCH3 to either 2-ClTrt-Cl or Trt-Cl resins (via the side-chain thiol) followed by preparation of the desired
peptide using Fmoc-based SPPS. We applied this method to the synthesis of the mating pheromone a-factor and a 5-FAM labeled a-factor analog.
The peptides were obtained with high yield and purity and were shown to be bioactive in a growth arrest assay.
Due to the importance of peptides in studies of biologi-
cal processes, new, more efficient, synthetic methods are
needed.1 Peptides containing methyl esters at the C-termi-
nus can beusedastools for the studyofproteinprenylation
due to the presence of this modification in fully processed
farnesylated and geranylgeranylated proteins.2 These
peptides can also be useful for a variety of biological
applications since the presence of the alkyl ester at the
C-terminus increases the hydrophobicity of the peptide making
them membrane permeable.3,4 Although such ester-modified
(3) Turner, R. A.; Weber, R. J.; Lokey, R. S. Org. Lett. 2010, 12,
1852.
(4) Doh, H. J.; Cho, W. J.; Yong, C. S.; Choi, H. G.; Kim, J. S.; Lee,
C. H.; Kim, D. D. J. Pharm. Sci. 2003, 92, 1008.
(5) Xue, C. B.; Caldwell, G. A.; Becker, J. M.; Naider, F. Biochem.
Biophys. Res. Commun. 1989, 162, 253.
(6) Marcus, S.; Caldwell, G. A.; Miller, D.; Xue, C. B.; Naider, F.;
Becker, J. M. Mol. Cell. Biol. 1991, 11, 3603.
† University of Minnesota.
‡ University of Tennessee.
(1) Grauer, A.; Koenig, B. Eur. J. Org. Chem. 2009, 5099.
(2) Pechlivanis, M.; Kuhlmann, J. BBA-Proteins Proteom. 2006,
1764, 1914.
r
10.1021/ol302592v
Published on Web 11/02/2012
2012 American Chemical Society

peptides can be useful in a variety of ways, only a handful of
reports in the literature describe their synthesis.3,5À9
Fmoc-Cys-OCH3 (or a related ester if desired), a compound
obtainable in one step from Fmoc-Cys-OH. After growing
the desired peptide using Fmoc-based SPPS, the peptide of
interest is cleaved from the resin by deprotection of the Cys
sulfur under acidic conditions. While a trityl-based resin has
been used to synthesize peptides containing a C-terminal
cysteamine linker (via resin linkage through the thiol), that
work did not describe the use of such resin for the prepara-
tion of C-terminal cysteine esters.13 Thus, to the best of our
knowledge, this is the first report on the use of cysteine side-
chain anchoring to trityl-based resins for the synthesis of
peptides containing methyl esters at the C-terminus.
The first peptide prepared with this method was the
yeast mating pheromone a-factor from Saccharomyces
cerevisiae. This dodecapeptide has attracted considerable
attention in the field of protein prenylation due to its
similarity with the C-terminal portion of larger, farnesy-
lated proteins. Moreover, in common with these proteins,
the farnesyl moiety and the methyl ester group incorpo-
rated at the C-terminal Cys of the a-factor peptide have
been shown to be critical for its bioactivity.14,15 Fluores-
cently labeled analogs of a-factor would be particularly
useful for studying their binding to cell-surface receptors.16
Unfortunately, most of the existing methods described in
the literature for peptide C-terminal methyl ester synthesis
are not convenient or suitible due to their use of aforemen-
tioned acidic or oxidative conditions.
One previously used method to obtain peptides with
C-terminal esters is the Merrifield Boc/Bzl protecting
group strategy that involves attachment of the peptide to
the resin througha benzylester linkageand requiresthe use
of hydrofluoric acid (HF) for the release of the peptide
from the solid support.5 Another method, used by Waldmann
and co-workers, utilizes a hydrazine containing resin and
consists of producing prenylated peptides containing C-term-
inal methyl esters directly from the solid support.8 This
hydrazine procedure requires an oxidation step with copper
with concomitant methanolysis in order to cleave the peptide
from the resin. In general, the use of such strong acidic/
oxidative conditions makes these methods less expeditious
from an experimental point of view and less versatile in their
scope.
Scheme 1. Attachment of Fmoc-Cys-OCH3 onto Trityl-Based
Resinsa
The method reported here began with the attachment of
Fmoc-Cys-OCH3 to commercially available 2-ClTrt-Cl
resin via its thiol functionality in order to obtain 2a
(Scheme 1). The loading of the first amino acid was
determined by Fmoc absorbance after deprotection with
20% piperidine in DMF. The a-factor sequence was then
assembled using Fmoc-based SPPS to obtain 4a (Scheme 2).
Since the linkage between the cysteinyl thiol and 2-ClTrt
resin is acid labile, the peptide was cleaved from the resin
upontreatment withReagent K (TFA/thioanisole/phenol/
water/ethanedithiol, 82.5:5:5:5:2.5) along with simulta-
neous deprotection of the acid-labile amino acid side-chain
protecting groups. Using this concise procedure, it was
possible to obtain the a-factor precursor peptide 5 with a
crude purity of 82% (Figure 1a). This peptide was then
purified and obtained in 42% overall yield. Farnesylation
of5wasperformedusing conditionsdevelopedbyNaider17
and previously reported by Mullen et al. for a-factor
synthesis to yield 6.18
a * = stereogenic center of the cysteine R carbon.
A different approach for solid phase peptide synthesis
(SPPS) consists of anchoring the peptide to a solid support
through the side chain of a trifunctional amino acid. Reports
on the synthesis of peptides, using cysteine anchoring
methods, have shown that they assist in reducing the risk
of racemization and formation of byproducts commonly
found in SPPS.10,11
The method described herein consists of the attachment
of Fmoc-Cys-OCH3 to a trityl-based resin to form a thio-
ether bond. An important feature of such resins is that they
are commercially available; in contrast, the XAL handle
developed by Barany and co-workers requires a multistep
sequence for its synthesis.12 Hence, the sole requirement
necessary to implement this new method is accessibility to
(13) Rietman, B. H.; Smulders, R.; Eggen, I. F.; Vanvliet, A.;
Vandewerken, G.; Tesser, G. I. Int. J. Pept. Protein Res. 1994, 44, 199.
In this work, the authors coupled cysteamine hydrochloride onto trityl
resin in the presence of TFA followed by elongation via standard SPPS.
Subsequent on-resin treatment with iodine led to resin cleavage and the
formation of cyclic disulfides or disulfide-linked dimers depending on
the peptide sequences employed. No data were provided concerning the
synthesis of peptides containing C-terminal cysteine residues although
the authors suggested that the synthesis of peptides containing a
C-terminal cysteine amide could be accomplished using this approach.
(14) Dawe, A. L.; Becker, J. M.; Jiang, Y.; Naider, F.; Eummer, J. T.;
Mu, Y. Q.; Gibbs, R. A. Biochemistry 1997, 36, 12036.
(7) Millington, C. R.; Quarrell, R.; Lowe, G. Tetrahedron Lett. 1998,
39, 7201.
(8) Ludolph, B.; Eisele, F.; Waldmann, H. J. Am. Chem. Soc. 2002,
124, 5954.
(9) O’Reilly, N.; Charbin, A.; Lopez-Serra, L.; Uhlmann, F. Yeast
2012, 29, 233.
(10) Barany, G.; Han, Y. X.; Hargittai, B.; Liu, R. Q.; Varkey, J. T.
Biopolymers 2003, 71, 652.
(11) Huang, Z.; Derksen, D. J.; Vederas, J. C. Org. Lett. 2010, 12,
2282.
(15) Sherrill, C.; Khouri, O.; Zeman, S.; Roise, D. Biochemistry 1995,
34, 3553.
(16) Khouri, O.; Sherrill, C.; Roise, D. Biochemistry 1996, 35, 14553.
(17) Xue,C. B.;Becker,J.M.;Naider,F.Tetrahedron Lett. 1992,33, 1435.
(12) Han, Y. X.; Bontems, S. L.; Hegyes, P.; Munson, M. C.; Minor,
C. A.; Kates, S. A.; Albericio, F.; Barany, G. J. Org. Chem. 1996, 61,
6326.
Org. Lett., Vol. 14, No. 22, 2012
5649

Scheme 2. Synthesis of the Mating Pheromone a-Factor Using
the Cysteine Anchoring Methoda
Scheme 3. Strategy Used for the Synthesis of 5-FAM-Labeled
a-Factor Precursor Peptide 9 and 5-FAM Labeled a-Factor (10)
a The a-factor precursor peptide was synthesized on resin, and
farnesylation of peptide 5 was performed in solution to obtain peptide 6.
Having demonstrated the success of this simple method
for the preparation of a-factor, we next undertook the
synthesis of a fluorescently labeled analog of a-factor. The
5-FAM fluorophore was selected due to its high fluores-
cence, low cost, and ease of attachmenttolysine side chains
during SPPS.19 Importantly, studies have shown that amino
acid substitution20 or truncation21 of the a-factor lysine
residue does not significantly affect the bioactivity of the
pheromone; we envision that fluorescently labeled forms of
a-factor will be useful for monitoring the binding of the
pheromone to its cognate receptor as well as for photo-
affinity labeling applications. It should be noted that earlier
efforts to prepare such an analog using hydrazine resin were
unsuccessful presumably due to overoxidation of the fluoro-
phore in the Cu-catalyzed cleavage step.
The synthesis of 5-FAM labeled a-factor was analogous
to that of the standard a-factor (Scheme 3) with slight
modifications. After attachment of Fmoc-Cys-OCH3 to
2-ClTrt-Cl resin, the peptide was extended using SPPS.
However, the lysine residue incorporated an ivDde-pro-
tected ε-amino group resulting in the formation of inter-
mediate 7. This protecting group was orthogonally cleaved
in the presence of hydrazine followed by coupling of the
5-FAM fluorophore to obtain 8. Deprotection of side chains
and cleavage of the peptide from the resin were achieved
simultaneously in the presence of Reagent K to give an 81%
crude purity for peptide 9as shown in Figure 1b. Purification
of the crude material produced the peptide in 36% yield
which was then farnesylated to give peptide 10.
Because the yields of these peptides were somewhat
lower than anticipated, we chose to repeat the synthesis
shown in Scheme 2 using a different trityl-based resin.
Thus, peptide 5 was synthesized using a commercially
available Trt-Cl resin in order to determine if the absence
of the electon-withdrawing chloride would have an effect
onthe finalyield (see Supporting Information (SI)). Toour
surprise, the yield of peptide 5 increased from 45% to 82%
by simply changing the resin to Trt-Cl (Table 1). We
hypothesized that the presence of the electron-withdraw-
ing chloride in the 2-ClTrt containing resin increases the
riskof β-elimination by increasingthe leavinggroup ability
of the protected thiol resulting in peptide loss from the
resin during each piperidine deprotection cycle. Accord-
ingly, we treated resin-bound model tripeptides 11a and
12a with piperidine for varying times and determined the
amount of peptide remaining on the solid support via
quantitative ninhydrin analysis; interestingly, no signifi-
cant loss of peptide occurred except after very long (24 h)
exposure (Table S1). In contrast, when resin bound 2a or
3a was treated with piperidine, some loss of peptide from
the solid support was observed (Table S2). The loss was
greater using the 2-ClTrt-Cl resin (26%) than with the
(18) Mullen, D. G.; Kyro, K.; Hauser, M.; Gustavsson, M.; Veglia,
G.; Becker, J. M.; Naider, F.; Distefano, M. D. Bioorg. Med. Chem.
2011, 19, 490.
(19) Wollack, J. W.; Zeliadt, N. A.; Mullen, D. G.; Amundson, G.;
Geier, S.; Falkum, S.; Wattenberg, E. V.; Barany, G.; Distefano, M. D.
J. Am. Chem. Soc. 2009, 131, 7293.
(20) Huyer, G.; Kistler, A.; Nouvet, F. J.; George, C. M.; Boyle,
M. L.; Michaelis, S. Eukaryot. Cell 2006, 5, 1560.
(21) Caldwell, G. A.; Wang, S. H.; Xue, C. B.; Jiang, Y.; Lu, H. F.;
Naider, F.; Becker, J. M. J. Biol. Chem. 1994, 269, 19817.
5650
Org. Lett., Vol. 14, No. 22, 2012

Trt-Cl resin (9%), consistent with the hypothesis that
β-elimination is responsible for the reduction in yield ob-
served with the former. The observation that some loss
occurs with 2a and 3a but not with 11a and 12a suggests
that this β-elimination reaction is context dependent. Im-
portantly, Trt-Cl resin appears to be the support of choice
for this application.
Table 1. Comparison of % Yield Obtained with 2-ClTrt-Cl and
Trt-Cl Resins for the Synthesis of 5
entry
resin
purity of crudea (%)
isolated yieldb (%)
1
2
2-ClTrt-Cl
Trt-Cl
82
81
45
82
a Purity of peptide 5 in the crude mixture after deprotection and cleavage
from the resins but prior to HPLC purification. b After purification.
a
Scheme 4. Synthesis of Model Peptide H-Gly-Phe-Cys-OCH3
Figure 1. HPLC of (a) crude peptide 5 and (b) crude peptide 9
after cleavage from the 2-ClTrt resin.
Since the procedure reported here uses a side-chain
cysteine anchoring method, and the Cys is not attached
to the solid support via an ester linkage, the risk of race-
mization of the C-terminal Cys is minimal.10 However, since
it has been previously reported that repetitive treatment
with piperidine can sometimes cause racemization,22 a study
was carried out to examine whether this method leads to a
loss in chiral integrity of the Cys residue. Model tripeptides
13a and 13b containing a methyl ester at the C-terminus
were synthesized using 2-ClTrt-Cl (Scheme 4) and Trt-Cl
resin. The C-terminal acid forms of these tripeptides have
been previously reported to manifest baseline resolution
when separated by HPLC.11 Peptides 13a (Rt = 15.5 min)
and 13b (Rt = 14.9 min) were analyzed by HPLC indivi-
dually and via coinjection, and the data showed negligible
(<1%) racemization product for both resins (see SI).
To verify that peptides 6 and 10, synthesized with the
cysteine anchoring method described herein, are biologi-
cally active, we tested their ability to cause growth arrest of
cells expressing the Ste3p a-factor receptor. In that assay,
previously characterized, a-factor with wild-type potency
stimulated growth arrest with an end point of 0.12 ng;5 the
synthetic material produced in this study (6) yielded an
identical end point. In contrast, the 5-FAM a-factor (10)
manifested an end point of 8.0 ng indicating that while the
fluorescent analog has somewhat lower activity, it still
possesses significant (12.5%) bioactivity and hence should
be useful for future studies.
a * = stereochemistry of the cysteine R carbon.
two trityl-based resins for the synthesis of the a-factor
precursor peptide showed that the yield of the desired
peptide almost doubledwhenusing the Trt-Cl resin instead
of the 2-ClTrt-Cl resin. Model tripeptides were synthesized
using both resins, and HPLC analysisshowedformation of
the desired peptide without significant racemization. The
bioactivities of the synthesized a-factor and 5-FAM la-
beled a-factor were confirmed by a growth arrest assay.
The 5-FAM a-factor analog showed significant although
somewhat reduced bioactivity. The success of this simple
method for the synthesis of peptides containing C-terminal
Cys methyl esters using commercially available resins opens
the door to the synthesis of a wide variety of C-terminal ester
modified peptides that can be used to study protein pre-
nylation and other structurally related biological processes.
Acknowledgment. This research was supported by the
National Institute of Health Grants GM008700 (L.Q.),
GM084152 and GM058842 (M.D.D.), and GM22087
(J.M.B.)
In summary, a new method has been developed for
the synthesis of peptides containing methyl esters at the
C-terminus. This method was applied to the synthesis of
a-factor and a fluorescently labeled a-factor analog produ-
cing crude materials of high purity. A comparison between
Supporting Information Available. Experimental pro-
cedures, HPLC and MS data. This material is available
free of charge via the Internet at http://pubs.acs.org.
(22) Han, Y. X.; Albericio, F.; Barany, G. J. Org. Chem. 1997, 62,
4307.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 22, 2012
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