Solid-phase synthesis of palmitoylated and farnesylated lipopeptides employing the fluoride-labile PTMSEL linker

Article abstract of DOI:10.1016/j.tetlet.2006.02.109

Acid- and base-labile S-palmitoylated and S-farnesylated lipopeptides can be synthesized in high overall yield employing the (2-phenyl-2-trimethylsilyl) ethyl (PTMSEL) linker for anchoring to and release under almost neutral conditions from the solid support.

Full text of DOI:10.1016/j.tetlet.2006.02.109

Tetrahedron Letters 47 (2006) 2671–2674
Solid-phase synthesis of palmitoylated and farnesylated
lipopeptides employing the fluoride-labile PTMSEL linker
Maria Lumbierres, Jose M. Palomo, Goran Kragol and Herbert Waldmann*
Max-Planck-Institut fu¨r Molekulare Physiologie, Abteilung Chemische Biologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany
Universita¨t Dortmund, Fachbereich 3, Chemische Biologie, 44221 Dortmund, Germany
Received 13 January 2006; revised 16 February 2006; accepted 17 February 2006
Abstract—Acid- and base-labile S-palmitoylated and S-farnesylated lipopeptides can be synthesized in high overall yield employing
the (2-phenyl-2-trimethylsilyl) ethyl (PTMSEL) linker for anchoring to and release under almost neutral conditions from the solid
support.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Lipid modified proteins fulfil important roles in various
cellular processes like cell-signalling, vesicular transport,
growth and differentiation.1 Tailor-made lipidated
peptides embodying the characteristic lipidated peptide
sequences of their parent proteins, for example,
sequences incorporating S-palmitoylated and S-farnesy-
lated cysteines, as well as reporter groups and additional
tags, in particular, for coupling to expressed proteins are
efficient tools for the study of the biophysical, biochemi-
cal, structural and biological properties of lipidated
proteins.2 Therefore, the availability of flexible and
high-yielding methods for the solid-phase synthesis of
such lipidated peptides is of substantial interest.
phase if the (2-phenyl-2-trimethylsilyl)ethyl (PTMSEL)
linker is employed for anchoring to the polymeric car-
rier. The PTMSEL linker was recently introduced by
Kunz and co-workers6 for the synthesis of glycopep-
tides. In contrast to other silyl-based linkers which are
cleaved under basic conditions with tetrabutylammo-
nium fluoride (TBAF) in DMF,7 the benzylic C–Si bond
in the PTMSEL linker can be cleaved with TBAF in
CH2Cl2, that is, under nearly neutral conditions
(Scheme 1).
As a model sequence guiding the development of the syn-
thesis method, the C-terminal heptapeptide sequence of
the human N-Ras protein GlyCys(Pal)MetGlyLeuPro-
Cys(Far) (Fig. 1) was chosen. The Ras proteins are criti-
cally involved in cellular signal transduction and belong
to the most important human oncogene products.1–3
Since S-palmitoylated and S-farnesylated peptides are
both acid- and base-labile3 the choice of the linker to
the solid support is crucial. It must be cleavable under
almost neutral conditions and without any harm to the
thioesters and the isoprenyl-thioethers. Currently, only
the hydrazide linker4 and the 4-sulfonamidobutyryl lin-
ker5 are available for this purpose. However, the hydr-
azide linker often yields the desired lipidated peptides
only with moderate yield so that further viable alterna-
tives to these methods are in demand.
O
O
O
O
TBAF*3H2O
CH2Cl2
R
O
R
O
SiMe3
SiMe3
F
PTMSEL
1
Here we report that acid- and base-labile doubly lipi-
dated peptides can be synthesized efficiently on the solid
Me3SiF
O
O
O
H2O
+
Keywords: Lipopeptides; Solid-phase synthesis; Linker groups; Ras
Bu4N
R
OH
R
O
protein.
*
Scheme 1. Structure and fluoride-induced cleavage of the PTMSEL
Corresponding author. Tel.: +49 231 133 2400; e-mail: herbert.
linker.
waldmann@mpi-dortmund.mpg.de
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.02.109

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M. Lumbierres et al. / Tetrahedron Letters 47 (2006) 2671–2674
Fmoc-Gly-Cys-Met-Gly-Leu-Pro-Cys OH
Fmoc-Met-Gly-Leu-Pro-Cys OH
S
S
S
Far
Pal
Far
12, yield: 70%
13, yield: 80%
NO2
N
O
N
H-Met-Gly-Leu-Pro-Cys OH
S
Fmoc-Lys-Leu-Pro-Cys OH
S
Far
15, yield: 83%
14, yield: 82%
Far
O
O
5
Fmoc-Cys-Met-Gly-Leu-Pro-Cys OH
N
Gly-Cys-Met-Gly-Leu-Pro-Cys OH
S
S
Far
O
S
Pal
16, yield: 80%
S
Far
Pal
17, yield: 70%
NO2
N
N
O
O
O
5
N
Gly-Cys-Met-Lys-Leu-Pro-Cys OH
O
S
S
Pal
Far
18, yield: 69%
Figure 1. Lipidated peptides synthesized employing the PTMSEL linker.
The PTMSEL linker 2 was synthesized as described6
and the phenol was alkylated with allyl chloroacetate
to give alcohol 3 (Scheme 2). In order to establish an effi-
cient and flexible synthesis it was planned to develop a
method that allows for sequential introduction of pre-
lipidated building blocks instead of the less efficient
alternative on-resin-lipidation.4 To this end, Fmoc-pro-
tected S-farnesylated cysteine 4 was coupled to alcohol 3
and after Pd(0)-catalyzed cleavage of the allyl ester8 5,
carboxylic acid building block 6 embodying the first lip-
idated amino acid was attached to the solid support. The
carboxylic acid 6 was coupled to the aminomethylpoly-
styrene resin 7 by activation with N-[(dimethylamino)-1-
H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methyl-
methanaminium (HATU) and diisopropylethylamine
(DIPEA) in DMF (Scheme 2) with 98% yield.
O
OH
O
O
HO
HO
SiMe3
SiMe3
a
2
3
b
4
O
O
O
S
O
Fmoc-HN
O
SiMe3
5
c
Elongation of the peptide chain was carried out using
Fmoc chemistry (Scheme 3). The Fmoc group was re-
moved from 8 with 20% piperidine in DMF. Couplings
were performed with 5 equiv of amino acid/HBTU/
HOBt and 10 equiv of diisopropylethylamine in DMF
for 2 h. Palmitoylated cysteine was coupled using N-
[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-
methyl methanaminium hexafluorophosphate N-oxide
(HBTU)/1-hydroxybenzotriazole (HOBt)/trimethylpyri-
dine in CH2Cl2/DMF (1:1) in order to avoid racemiza-
tion9 to give immobilized lipidated peptide 10.
O
O
O
S
OH
Fmoc-HN
O
SiMe3
Far
6
H2N
d
7
O
H
Fmoc N
O PTMSEL
N
In the presence of the base sensitive palmitoyl moiety,
piperidine cannot be employed any more for deprotec-
tion of the Fmoc-group. Furthermore, after removal
of the Fmoc group from the N-terminal S-palmitoylated
cysteine a rapid S!N acyl shift can occur.10 Therefore,
the stronger but nonnucleophilic hindered base 1,8-diazo-
bicyclo[5.4.0]undec-7-ene (DBU) was used. It allows a
fast Fmoc-deprotection (2 · 30 s) which, followed by
immediate addition of a preactivated coupling cocktail
H
S
Far
8
Scheme 2. Synthesis of farnesylated cysteine building block 6 and
attachment to the solid support. Reagents and conditions: (a)
allylchloroacetate K2CO3, KI, acetone; (b) FmocCys(Far)OH (4),
DCC, DMAP, CH2Cl2; (c) Pd(PPh3)4 (5 mol %), PhSiH3, THF/
CH3OH; (d) HATU (1.2 equiv), DIPEA (2.4 equiv), DMF, 24 h.

M. Lumbierres et al. / Tetrahedron Letters 47 (2006) 2671–2674
2673
8
remove the remaining fluoride, dried over MgSO4 and
concentrated in vacuo and the heptapeptide 12 was
obtained in 70% yield without further purification.
a,b
In order to demonstrate the scope of the method, differ-
ent lipidated peptides were synthesized (Fig. 1). In all the
cases, the mono- or dilipidated peptides were obtained
in excellent yields. The successful synthesis of peptide
15 with a free amino group at the N-terminus is remark-
able. Application of the hydrazide linker allowed only
deprotection of N-protected peptides, due to the com-
plexation of the copper salts used for its oxidative cleav-
age with free amino functions.
Fmoc-Met-Gly-Leu-Pro-Cys O
S
PTMSEL
N
H
9
a,c
O
S
The new methodology also gives access to labeled pep-
tides carrying a fluorescent group (14 and 18) and/or a
maleimido group for further ligation to proteins at the
N-terminus (17 and 18).
Fmoc-Cys-Met-Gly-Leu-Pro-Cys
S
O
N
H
PTMSEL
10
In conclusion the PTMSEL linker offers new and advan-
tageous opportunities for the solid-phase synthesis of
differently lipidated peptides. The release of the peptide
from the solid support is efficient in the presence of
TBAFÆ3H2O in CH2Cl2, that is, under almost neutral
conditions. The cleavage conditions are so mild that
acid- and base-labile lipid moieties are not attacked.
d,e
O
S
Fmoc-Gly-Cys-Met-Gly-Leu-Pro-Cys
N
H
O PTMSEL
S
11
Acknowledgements
f
This research was supported by the Max-Planck-Gesell-
schaft and the Fonds der Chemischen Industrie. Jose M.
Palomo would like to thank the European Molecular
Biology Organization (EMBO), for a long term Fellow-
ship and G. Kragol is grateful to the Humboldt Founda-
tion for a Research Fellowship.
O
S
Fmoc-Gly-Cys-Met-Gly-Leu-Pro-Cys-OH
S
12
References and notes
Scheme 3. Solid phase synthesis of the farnesylated and palmitoylated
peptide 12. Reagents and conditions: (a) 20% piperidine in DMF; (b)
5 equiv Fmoc-AA-OH, 5 equiv HBTU/HOBt, 10 equiv DIPEA,
DMF, 2 h (repeat for assembly of the peptide); (c) 4 equiv Fmoc-
Cys(Pal)-OH, 4 equiv HBTU/HOBt/TMP, CH2Cl2/DMF (1:1), 4 h;
(d) 1% DBU in DMF, 30 s (·2); (e) 5 equiv Fmoc-Gly-OH, 5 equiv
HATU, 20 equiv DIPEA, CH2Cl2/DMF (7:1), 2 h; (f) 2 equiv
TBAFÆ3H2O, CH2Cl2, 25 min (·2); DCC: N,N0-dicyclohexylcarbodi-
imide; DMAP: 4-dimethylaminopyridine.
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