Solid-phase synthesis of lipidated peptides

Article abstract of DOI:10.1002/anie.200461150

Blockers, anchors, and reporters: A solid-phase method for the synthesis of lipidated peptides (see scheme) relies on the base-labile Fmoc group to block the N-terminus, an oxidation-labile hydrazide linker for anchoring to the solid support, and lipidate

Full text of DOI:10.1002/anie.200461150

Angewandte
Chemie
Scheme 1. Structure of the C-termini of the N-Ras and the H-Ras
protein.
structures of their parent proteins[2,3] a flexible solid-phase
technology is required. Ideally such a technique would:
1. give access to peptides carrying different combinations of
acid- and base-labile lipid groups; this requires the
application of a set of suitable orthogonally stable
protecting groups and a linker to the solid support, all of
which can be cleaved under the mildest conditions;[3]
2. allow for the introduction of additional reporter and/or
linking groups required for application of the target
peptides in further biological investigations;
3. allow for release of the peptide as a methyl ester, or—if
required—equipped with a different functional group, for
example, a fluorophore at the C-terminus.[2d]
Protein Synthesis
Solid-Phase Synthesis of Lipidated Peptides**
The only method currently available for this purpose[4]
requires a large excess of lipidation reagent, is not readily
automatable, and is not suitable for the preparation of longer
peptides. Thus attempts to prepare peptides with
> 10 amino acids by this method failed in our hands.
Goran Kragol, Maria Lumbierres, Jose M. Palomo, and
Herbert Waldmann*
The Ras proteins serve as central molecular switches in
biological signal transduction cascades regulating cell growth
and differentiation.[1] They incorporate both acid-labile
farnesyl thioethers and base-sensitive palmitic acid thioesters,
which are required for biological activity and terminate in a
cysteine methyl ester (Scheme 1).
Here we describe the successful development of a solid-
phase synthesis method that meets the demands and over-
comes the drawbacks described above. It employs pre-
lipidated amino acid building blocks together with the base-
labile 9-fluorenylmethyloxycarbonyl (Fmoc) group as a
blocking function for the N-terminus, and the oxidation-
labile hydrazide linker for anchoring to the solid support.
The lipidated building blocks required for the new solid-
phase method were synthesized in high overall yields as
shown in Scheme 2 employing in part transformations descri-
bed earlier.[5] Notably only one equivalent of farnesyl chloride
and, in particular, of the N-methylanthraniloyl (Mant)-
functionalized farnesyl analogue GerMantCl were employed.
Building blocks 2 and 4 were then used in the solid-phase
synthesis of H- and N-Ras peptides 9 and 10 (Scheme 3). The
hydrazide unit[6] was employed as a linker to the solid support
since it can be cleaved under mild oxidative conditions and
gives access to lipopeptide esters and acids.[4] Fmoc-hydrazi-
nobenzoic acid functionalized aminomethyl polystyrene resin
is commercially available (NovaBioChem). In order to avoid
racemization of cysteines DIC/HOBt or HBTU/HOBt/TMP
cocktails in CH2Cl2/DMF 1:1 were used for coupling the
cysteine building blocks.[7]
For the efficient and rapid synthesis of tailor-made
lipidated peptides representing the characteristic partial
[*] Dr. G. Kragol,+ Dr. M. Lumbierres, Dr. J. M. Palomo,
Prof. Dr. H. Waldmann
Max-Planck-Institut für molekulare Physiologie
Abteilung Chemische Biologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
and
Universität Dortmund
Fachbereich 3, Organische Chemie
Fax: (+49)231-133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
[+] Current address:
Department of Organic Chemistry and Biochemistry
Rudjer Boskovic Institute
Bijenicka 54, Zagreb (Croatia)
[**] This research was supported by the Deutsche Forschungsgemein-
schaft, the Max-Planck-Gesellschaft, the Fonds der Chemischen
Industrie, the Humboldt Foundation (research fellowship to G.K.),
and the European Molecular Biology Organization (long-term
fellowship to J.M.P.).
Coupling of Fmoc-Cys(Far)OH and Fmoc-Cys(Pal)OH
proceeded with > 90% efficiency if a coupling time of 4 h was
allowed and if for Fmoc-Cys(Pal)OH a double coupling was
performed (monitored by Fmoc determination employing the
established UV-based method). After oxidative cleavage with
Cu(OAc)2 and release from the resin by treatment with
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2004, 43, 5839 –5842
DOI: 10.1002/anie.200461150
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5839

Communications
Scheme 2. Synthesis of lipidated building blocks 2, 4, 6, and 8.
a) 5% TFA, 3% TES, CH2Cl2, 1 h, RT; b) TMSCl (1.1 equiv), 2 h,
reflux, CH2Cl2, then PalCl (3 equiv), Et3N (1.5 equiv) dropwise 3 h,
CH2Cl2, RT; c) FarCl (1 equiv), 4n NH3/methanol, 3 h, 08C, 1 h,
RT;[5a] d) FmocOSu (1.1 equiv), Et3N (1.1 equiv), CH2Cl2, 2 h, RT; e)
GerMantCl (1 equiv), 4n NH3/methanol, 3 h, 08C, 1 h, RT; f)
NBDCl, THF/methanol, 658C, 2 h.[5b] Far =farnisyl, Fmoc=9-fluore-
nylmethyloxycarbonyl, NBD=4-nitrobenzo-2-oxa-1,3-diazole, Pal=
palmitoyl, RT=room temperature, TES=triethylsilane, TFA=tri-
fluoroacetic acid, TMS=trimethylsilyl.
Scheme 3. Solid-phase synthesis of lipidated peptides employing pre-lipidated
amino acid building blocks. a) 1. 20% piperidine/DMF, 2. Fmoc-Cys(Far)-OH
(4 equiv), DIC/HOBt or HBTU/HOBt/TMP, CH2Cl2/DMF (1:1), 4 h; b) Standard
solid-phase synthesis: 1. 20% piperidine/DMF, 2. Fmoc-AA-OH (4 equiv), DIC/
HOBt or HBTU/HOBt/DIPEA, CH2Cl2/DMF (1:1) or DMF, 2 h; c) 1. 20% piperi-
dine/DMF, 2. Fmoc-Cys(Pal)-OH (4 equiv), DIC/HOBt or HBTU/HOBt/TMP,
CH2Cl2/DMF (1:1), 4 h for 10, overnight for 9; d) 1% DBU/DMF, 2”30 s;
e) Fmoc-AA-OH (5 equiv), HATU (5 equiv), DIPEA (20 equiv), CH2Cl2/DMF (4:1),
2 h; f) Cu(OAc)2, pyridine, acetic acid, methanol, CH2Cl2, oxygen atmosphere, 3 h.
DIC=1,3-diisopropylcarbodiimide, DIPEA=N,N-diisopropylethylamine,
DMF=N,N-dimethylformamide, HATU=O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium hexafluorophosphate, HBTU=N-[(1H-benzotriazol-1-yl)-
(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate
N-oxide, HOBt=1-hydroxybenzotriazole, TMP=tetramethylpiperidide.
CH3OH followed by filtration of the crude product
through a short silica cartridge, the peptide methyl
esters corresponding to immobilized peptides 9 and
10 were obtained in overall yields of 69% and
60%, respectively, and with > 90% purity.
For further elongation of the peptide chain the
central problem of the entire method had to be
solved. After removal of the Fmoc group from the
N-terminal S-palmitoylated cysteine a rapid S!N
acyl shift had to be feared,[8] and, in addition, the
base-labile Fmoc group had to be removed under
conditions that leave the thioester intact, although
this group is very sensitive to nucleophilic attack.[2]
Substantial experimentation revealed that the desired
elongated and S-palmitoylated peptides can be obtained in a
very practical and efficient manner if the Fmoc group is
cleaved by treatment with a solution of 1% DBU in DMF[9]
for 2 ” 30 s followed each time by fast washing with DMF (6 ”
5 s) and immediate coupling for 2 h employing five equiv-
alents of preactivated (10 min) amino acid building block, five
equivalents of HATU, and twenty equivalents of diisopropyl-
5840
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Angew. Chem. Int. Ed. 2004, 43, 5839 –5842

Angewandte
Chemie
Table 1: Lipidated peptides synthesized by means of the solid-phase method.
ethylamine in CH2Cl2/DMF 4:1 as solvent.
Under these conditions the desired elon-
gated peptides 12 and the analogue 13
resulting from S!N acyl shift were
formed in a ratio of 95:5.
Entry
Cmpd.
Structure[a]
Yield [%]
28
1
14
In order to demonstrate the scope of the
method, the lipid-modified peptides shown
in Table 1 were prepared. In general, a resin
with a loading of 0.35–0.37 mmolgꢀ1 was
employed, and the peptides were prepared
in 5- to 20-mg amounts. Immediately after
release from the resin and filtration through
a silica cartridge the peptides were > 80%
pure (determined by HPLC analysis; see
the Supporting Information).
The peptides shown in Table 1 display
different lipidation patterns; in other words,
they are mono- or dilipidated and corre-
spond to the C-termini of the H- or N-Ras
protein. In addition to the lipid groups they
2
3
4
5
15
16
17
18
38
30
28
31
6
7
19
20
23
25
incorporate
a fluorescent label (NBD,
Mant) or a photoactivatable group (benzo-
phenone) and/or a maleimidocaproyl group
(MIC), and a biotin group or an unmasked
N-terminal cysteine which can be employed
for coupling to expressed proteins by
expressed protein ligation.[2e] Three pepti-
des (18–20) incorporating the GerMant
analogue of the farnesyl group were synthe-
sized. Due to the laborious preparation of
the GerMant residue (see above) in the
coupling steps employing Fmoc-Cys(Ger-
Mant)OH only 1.5 equivalents of the build-
ing block were employed to ensure efficient
use.
8
21
45
9
22
23
24
25
32
20
10
11
Finally, the applicability of the method
to the synthesis of long lipidated peptides
was investigated. To this end, decapeptide
27 and tetradecapeptide 28 were prepared,
which both mimic the C-terminus of N-Ras.
Both were obtained in milligram amounts
and characterized by 1H NMR spectroscopy
and mass spectrometry (27: ESI-MS: m/zcalcd
for [M+H]+ = 1887.0, m/zfound = 1887.3; 28:
12
13
14
25
26
27
37
25
30
MALDI-MS(DHB):
m/zcalcd
m/zfound = 2481.8;
for
[M+Na]+ = 2481.3,
15
28
12
m/zcalcd for [M+K]+: 2497.3, m/zfound
=
2497.9). These peptides represent the lon-
gest S-palmitoylated and S-farnesylated
peptides synthesized so far. Both were not
accessible by the previously reported solid-
phase method,[4] which demonstrates the
superiority of the method reported in this
article.
[a] Structures to define the abbreviations used:
In conclusion we have developed an
efficient solid-phase method for the syn-
thesis of differently lipidated and addition-
ally modified peptides. This methods meets
the requirements of a widely applicable
Angew. Chem. Int. Ed. 2004, 43, 5839 –5842
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ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5841

Communications
Chem. 2004, 116, 2765 – 2768; Angew. Chem. Int. Ed. 2004, 43,
2711 – 2714; e) A. Rak, O. Pylypenko, T. Durek, A. Watzke, S.
Kushnir, L. Brunsveld, H. Waldmann, R. S. Goody, K. Alexan-
drov, Science 2003, 302, 646 – 650.
potentially automatable methodology and overcomes the
limitations of earlier procedures. In terms of efficiency and
flexibility this solid-phase method is superior to the solution-
phase synthesis. It gives pure peptides more quickly and with
superior overall yield.
[3] D. Kadereit, J. Kuhlmann, H. Waldmann, ChemBioChem, 2000, 1,
144 – 169.
[4] a) B. Ludolph, F. Eisele, H. Waldmann, J. Am. Chem. Soc. 2002,
124, 5954 – 5955; b) B. Ludolph, H. Waldmann, Chem. Eur. J.
2003, 9, 3683 – 3691.
Received: July 1, 2004
Keywords: lipids · proteins · solid-phase synthesis
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Poulter, J. Am. Chem. Soc. 2001, 123, 3176 – 3177; b) C. Peters,
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cysteinyl-peptide esters are oxidatively released from the solid
support without racemization of the C-terminal cysteine.
[7] Y. M. Angell, J. Alsina, F. Albericio, G. Barany, J. Pept. Res. 2002,
60, 292 – 299.
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[1] a) K. Hinterding, D. Alonso-Diaz, H. Waldmann, Angew. Chem.
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[8] Y. Trudelle, A. Caille, Int. J. Pept. Protein Res. 1977, 10, 291 – 298.
[9] J. D. Wade, J. Bedford, R. C. Sheppard, G. W. Tregar, Pept. Res.
1991, 4, 194 – 199.
5842
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 5839 –5842