Solid-phase synthesis of lipidated Ras peptides employing the Ellman sulfonamide linker

Article abstract of DOI:10.1002/chem.201001642

A detailed study on the solid-phase synthesis of lipidated peptides of the Ras family employing the Ellman sulfonamide linker is reported. Using the C-terminal N-Ras sequence, critical issues such as lipidated amino acid resin loading, peptide elongation in the presence of labile groups and optimized conditions for release of the peptides were investigated. A versatile methodology for the synthesis of peptides with diverse lipid motifs and C-terminal methyl esters has accordingly been established.

Full text of DOI:10.1002/chem.201001642

FULL PAPER
DOI: 10.1002/chem.201001642
Solid-Phase Synthesis of Lipidated Ras Peptides Employing the
Ellman Sulfonamide Linker
Gemma Triola,[a] Marc Gerauer,[a] Kristina Gçrmer,[a] Lucas Brunsveld,[a, b] and
Herbert Waldmann*[a]
Abstract: A detailed study on the solid-phase synthesis of lipidated peptides of the
Ras family employing the Ellman sulfonamide linker is reported. Using the C-ter-
minal N-Ras sequence, critical issues such as lipidated amino acid resin loading,
peptide elongation in the presence of labile groups and optimized conditions for
release of the peptides were investigated. A versatile methodology for the synthe-
sis of peptides with diverse lipid motifs and C-terminal methyl esters has accord-
ingly been established.
Keywords: lipopeptides · peptides ·
racemization · solid-phase synthesis ·
sulfonamide linker
Introduction
Typically the small GTPase is lipidated via S-alkylation and/
or S-acylation of cysteines in its C terminus. Post-transla-
tional prenylation (farnesylation or geranylgeranylation)
and palmitoylation together with C-terminal methylation
and specific phosphorylations are the typical fine-tuning
mechanisms that are required for controlled and reversible
membrane association of the Ras proteins.[7]
The importance of the membrane affinity of the post-
translationally modified Ras proteins is balanced by an elab-
orate biochemical generation of these fully functionalized
and modified lipidated proteins. Their biological synthesis
and purification are challenging, time-consuming and in
many cases not practical or applicable. Studies with Ras pro-
teins lacking the post-translationally modified C terminus,
are significantly easier performed, but lack the important in-
formation dictated by membrane association. Therefore
chemical biology approaches have been developed that pro-
vide access to fully functional lipidated peptides and pro-
teins. Together with the insertion of additional non-natural
modifications, these chemical biology approaches have en-
Intracellular signaling and transport strongly depend on pe-
ripheral membrane proteins specifically anchored to one
face of a given membrane. The functioning of these mem-
brane proteins relies on the correct protein localization and
affinity for specific internal cell membranes and this is gov-
erned by post-translational lipidations.[1–3] Representative
examples of this process can be found in proteins of the Ras
superfamily. These small guanosine triphosphatases
(GTPases) play critical roles in controlling cellular process-
es, such as signal transduction, cell proliferation and differ-
entiation, and transport.[4] As a result, mutations in Ras pro-
teins can lead to uncontrolled cell growth and cancer.[5,6]
[a] Dr. G. Triola, Dr. M. Gerauer, K. Gçrmer, Prof. Dr. L. Brunsveld,
Prof. Dr. H. Waldmann
Max-Planck-Institut fꢀr molekulare Physiologie
Abteilung Chemische Biologie, Otto-Hahn-Strasse 11
44227 Dortmund (Germany)
AHCTUNGTREGaNNNU bled the study of the complete functional proteins in the
context of cellular membranes.[3,7–13]
and
We have previously shown that fully lipidated and modi-
fied Ras proteins can be generated by a combination of bio-
logical and chemical processes based on the connection of a
truncated GTPase core to a synthetic lipidated peptide C
terminus.[8,14] The development and application of solid-
phase lipidated peptide synthesis techniques has played a
major role in the synthesis of these proteins.[7,15,16] The
major challenges of a fully solid-supported synthesis of lipi-
dated peptides are the identification of orthogonally stable
Technische Universitꢁt Dortmund, Fakultꢁt Chemie (Germany)
Fax : (+49)231-133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
[b] Prof. Dr. L. Brunsveld
Technische Universiteit Eindhoven
Department of Biomedical Engineering
Laboratory of Chemical Biology, Den Dolech 2
Eindhoven (The Netherlands)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201001642.
Chem. Eur. J. 2010, 16, 9585 – 9591
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9585

protecting groups, a solid-support linker that allows selective
introduction of lipid functionalities as well as additional
functional groups for tracing such peptides in biological sys-
tems (fluorescence and photoactivatable tags), specific C-
terminal modifications, and functional groups for ligation to
the protein core. Additionally, specific conditions typical for
normal peptide synthesis have to be avoided due to incom-
patibilities with the lipid functionalities. Examples are the
incompatibility of high acid concentrations, typically used to
remove Boc-protecting groups, with the acid-labile prenyl
groups, and of strong nucleophiles, used, for example, in the
removal of the Fmoc-protecting group, with labile lipid thio-
esters. The development of new linker strategies for solid-
phase lipidated peptide synthesis has therefore been attract-
ing significant attention.[7,17] Ideally, these linkers provide a
flexible and rapid entry for a wide variety of different types
of lipidated peptides. The hydrazide linker and the sulfon-
ied for the formation of several lipidated peptides
(Scheme 1, route A).
ACHTUNGTRENNUNGamide linker are the two most versatile linkers that current-
ly meet the majority of these criteria.[17–19]
The hydrazide linker has been extensively studied and ap-
plied.[20] It provides a flexible tool for the synthesis of sever-
al differently modified lipopeptides.[19,21] An alternative for
solid-phase lipidated peptide synthesis is the safety-catch
sulfonamide linker.[19] This linker was originally developed
by Kenner[22] and later modified by Ellman,[23] who im-
proved the loading efficiency and the cleavage conditions by
using haloacetonitriles for activation and release. This linker
has been successfully used in the synthesis of different pep-
tide derivatives and other compound classes on solid sup-
port,[24] and has been shown by us in an initial report to also
provide an advantageous platform for lipidated peptide syn-
thesis.[19] The acyl sulfonamide functionality is stable to
treatment with acid or base, but can be activated for release
of the lipidated peptide under very mild conditions by selec-
tive N-alkylation of the N-acyl sulfonamide, and by subse-
quent attack of different types of nucleophiles. In general,
the alkyl sulfonamide linker thus meets the requirements
listed above for the synthesis of a variety of lipidated pep-
tides.
Scheme 1. Alternative concepts (general scheme) for the synthesis of lipi-
dated N-Ras peptides with C-terminal modifications using the acyl sulfo-
namide linker.
Results and Discussion
Loading of the acyl sulfonamide linker: It has previously
been shown that a high acyl sulfonamide loading with mini-
mal racemization may pose a problem for the acyl sulfon-
AHCTUNGTRENNUNG
amide linker strategy.[24] Ellman et al. screened different
loading conditions and reported the low reactivity of this
linker and the concomitant racemization occurring during
the loading step. Coupling conditions such as N,N’-diisopro-
pylcarbodiimide (DIC) with HOBt failed and the addition
of a catalytic amount of DMAP to a symmetric anhydride
typically led to 10% racemization.[23] The optimal conditions
found for loading of for example Boc-Phe-OH, being
PyBOP with DIPEA in CH2Cl2 or CHCl3 at À208C, are,
however, relatively unpractical when using a solid-phase
peptide synthesis reactor. As an alternative, Fmoc-Leu-OH
has been, for example, loaded on the alkyl sulfonamide
linker using DIC and methylimidazole (MeIm) leading to
low levels of racemization.[25] Fmoc-protected amino acid
fluorides have been extensively used, in solution and solid-
phase synthesis, especially for the coupling of hindered
amino acids.[26,27] N-Protected (Boc, Z or Fmoc) acyl fluo-
rides can be prepared in solution by reaction of the corre-
sponding free acid with DAST or cyanuric fluoride and sub-
sequently they can be purified by recrystallisation in good
enantiopurities.[26–30] In situ conversion of the acid into the
fluoride and subsequent coupling has also been reported by
using TFFH and DIPEA.[31] These conditions were, for ex-
ample, successfully applied for the synthesis of Z-phenylgly-
cine fluoride (Z-Phg-F) and coupling to H-Pro-OH without
Here we report in detail on the use of the alkyl sulfon-
ACHTUNGTRENNUNGamide linker for the synthesis of lipidated Ras peptides. The
N-Ras sequence was used as a suitable platform to investi-
gate the scope and limitations for this linker, as it embodies
both a prenyl functionality and a palmitoyl group in its C
terminus and additionally is post-translationally modified to
a C-terminal methyl ester. Using the C-terminal N-Ras se-
quence critical issues such as lipidated amino acid resin
loading, peptide elongation in the presence of labile groups
and optimized cleavage conditions are reported. Two gener-
al approaches have been investigated to generate the C-ter-
minal N-Ras sequences, differing in concept by the incorpo-
ration of the C-terminal functionality via peptide cleavage
from the resin. On the one hand the characteristics of the
cleavage of the complete peptide with methanol were ex-
plored (route B, Scheme 1), while on the other hand, the
cleavage of the peptide from the resin with the completely
post-translationally modified C-terminal cysteine was stud-
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Lipidated Ras Peptides
FULL PAPER
racemization. However, it has also been reported that Bz-
Phe-OH was fully racemized when coupled to NH2-Ala-
CO2Me using these conditions, thus indicating that this
methodology finds its limitations, depending on the protect-
ed amino acid.[31] Recently, acyl fluorides have been used for
loading the acyl sulfonamide linker.[29]
zation of the cysteine, both Fmoc-d- and Fmoc-l-CysACHTUNGTRENNUNG(Trt)-
OH were loaded on the acyl sulfonamide linker using the
TFFH/DIPEA (1:2). To avoid a possible racemization of the
activated cysteine during cleavage with the weaker nucleo-
phile methanol, release of the cysteine was performed using
H2N-Phe-CO2Me as nucleophile, yielding dipeptide
(Scheme 3). Even though a high loading of the Fmoc-Cys-
AHCTUNGTREG(NNUN Trt)-OH amino acid was obtained, these conditions resulted
2
Most proteins of the Ras superfamily feature a C-terminal
lipidated cysteine. Protected cysteines are one of the most
critical amino acids regarding racemization and thus can
pose a limitation to solid-phase peptide synthesis. Systematic
studies performed by Barany et al.,[32] showed that most of
the standard coupling protocols led to unacceptable epimeri-
zation levels for cysteines. In the same work, several im-
proved methods for the incorporation of cysteines into pep-
tides were reported. Basically, the use of DIC in combina-
tion with HOBt or HOAT, the use of BOP (or HATU) in
combination with HOBt (or HOAT) and trimethylpyridine
(TMP) as a base in a 4:4:4 ratio in CH2Cl2/DMF (1:1), or
preformed pentafluorophenyl (Pfp) esters, led to minimal
racemization levels. Racemization of cysteines had, howev-
er, not been studied for the loading conditions described
above for the acyl sulfonamide linker. Only for the prepara-
tion of Fmoc-cysteine fluoride in solution with cyanuric fluo-
ride, it was shown that synthesis and recrystallization did
not affect cysteine enantiopurity. Loading of Fmoc-cysteine
fluoride to the sulfonamide linker and nucleophilic displace-
ment with H2N-Phe-CO2Me, led to a tolerable level of 3%
of epimerization.[29] However, loading of lipidated cysteines
and other loading conditions had not been studied.
in approximately 25% of racemization, as could be detected
using normal-phase chiral HPLC (Table 1, entry 1). Clearly
the in situ generation of the cysteine fluoride leads to epi-
merization.
Table 1. Loading conditions, loading efficiency and cysteine racemization
detected by cleavage with H2N-Phe-CO2Me and subsequent chromato-
graphic evaluation (n.d.=not determined).
Entry Conditions
t
Loading d-Cys
[%]
[%]
1
2
3
4
5
6
7
H-l-Cys
H-l-Cys
H-l-Cys
H-l-Cys
H-l-Cys
H-l-Cys
H-l-Cys
N
4 h
16 h
1 h
1 h
4 h
70
60
30
30
70
11
2
25
30
2.5
3
3
n.d.
n.d.
40 min
Different approaches for the loading of cysteine deriva-
To investigate the characteristics of the linker loading re-
quired for typical lipidated peptides, we focused on the load-
ing of cysteine derivatives as these frequently constitute the
C-terminal amino acid in fully post-translational modified
tives to the acyl sulfonamide linker were therefore investi-
gated. The use of DIC with MeIm resulted in a similar
degree of racemization (Table 1, entry 2). Loading of Fmoc-
CysACTHUNRTGNEUNG(Far)-OH was not efficient when using PyBOP/DIPEA
Ras proteins. First the loading of Fmoc-Cys
linker via the in situ generation of Fmoc-CysACHTUNGTRENNUNG
by using TFFH and DIPEA (1:1:2), linker activation with
iodoacetonitrile, and cleavage using methanol as a nucleo-
phile was investigated. Diastereomeric dipeptide Ac-Pro-
G
(Table 1, entry 6) or DIC/HOBt (not shown). Stronger acti-
vation using DAST as a fluorinating agent resulted in side
reactions with the farnesyl moiety and led to unsatisfying
low loading efficiencies (Table 1, entry 7). Fmoc-CysACHTUNGTRENNUNG(Trt)-F
could, however, be prepared by reaction of cyanuric fluoride
Cys
terminus of N-Ras, was therefore prepared on the sul-
fonamide linker using the conditions described above. To
ACHTUNGTRENNUNG(Far)-CO2Me (1) (Scheme 2), corresponding to the C
and pyridine.[29] Loading of the sulfonamide linker was
AHCTUNGTREGaNNUN chieved in 30% and after cleavage only 3% of d-cysteine
T
could be detected (Table 1, entry 3). These conditions could
also be successfully applied to the synthesis of farnesylated
Fmoc-cysteine fluoride, without side-reactions with the far-
nesyl chain, in 70% loading yield after longer reaction times
(Table 1, entry 5). Cleavage with H2N-Phe-CO2Me resulted
in dipeptide 3 (Scheme 3) with only low racemization.
probe for racemization, two dipeptide diastereomers (l,l
and l,d) were prepared in enantiomerically pure form on
solid support using the hydrazine linker strategy. Analysis of
the dipeptides obtained from the sulfonamide linker by
1H NMR and chiral HPLC measurements showed that sig-
1
nificant racemization of the cysteine had occurred. H NMR
spectra of l,l- and l,d-peptides synthesized on the hydra-
zine linker show characteristic signals corresponding to the
methyl ester singlet as well as the NH proton. The doubling
of the peaks of the enantiomerically pure samples results
from rotamers. The spectra corresponding to the dipeptide
synthesized on the acyl sulfonamide linker showed a mixture
of two diastereomers, comparable to the spectra obtained
with a known mixture of both diastereomers (Scheme 2A).
In order to determine the exact contribution of both the
loading and the cleavage conditions to the observed racemi-
Peptide chain elongation: Initial peptide synthesis protocols
on the sulfonamide linker employed HBTU/HOBt/DIPEA
(1:1:2) for coupling of model N-Ras peptides yielded only
incomplete couplings. Gluszok et al. recently studied the re-
action between a sulfonamide and HBTU and found that
under basic conditions tetramethylsulfonylguanidines are
formed.[33] The interception of HBTU through this pathway
might explain the inefficient couplings observed. In our
hands the combination of DIC/HOBt or PyBOP/HOBt for
the elongation of the peptide sequence was more effective.
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9587

H. Waldmann et al.
tide chain elongations. Hence,
peptide synthesis was per-
formed using four equivalents
of the corresponding amino
acid and DIC/HOBt in a ratio
1:1:1 and proceed with com-
plete coupling. The same condi-
tions were applied when per-
forming the synthesis in an au-
tomated microwave peptide
synthesizer (Liberty, CEM) and
gave similar results.
Peptide release from the sul
ACHTUNGTRENNUNGfon-
AHCTUNGTERGaNNUN mide linker: The safety-catch
principle incorporated into the
sulfonamide linker is based on
the high stability of the N-acyl
sulfonamides under acidic and
basic conditions. This stability is
lost when the NH-acyl sulfon-
AHCTUNGTRENNUNG
a
attack by a nucleophile at the
C-terminal acyl functionality of
the peptide and release of the
peptide from the resin. Alkyla-
tion of the N-acyl sulfonamide
has been reported with tri
ylsilyldiazomethanes or halo-
acetonitriles under basic condi-
ACHTUNGTRENNUNGmeth-
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
tions. Although Flavell et al.
proved by NMR that e-cyano-
methylation of methionine oc-
curred under these condi-
tions,[34] Backes and Ellman al-
ready reported in 1999 that pro-
tected cysteine and methionine
were not methylated during the
Scheme 2. A) Characteristic 1H NMR signals of NH and methyl ester signals of Ac-l-Pro-l-Cys
(1a) and Ac-l-Pro-d-Cys(Far)-OCH3 (1b) after cleavage from the hydrazide linker and of a mixture of 1a and
1b (NMR spectra represents rotamers). B) 1H NMR signals of 1 after cleavage from acyl sulfonamide linker.
a) Fmoc-l- or Fmoc-d-Cys(Far)-OH, HOBt, HBTU, DIPEA in DMF/CH2Cl2, b) 20% piperidine in DMF; c)
Fmoc-Pro-OH, DIC, HOBt in DMF; d) acetic acid, DIC, HOBt in DMF; e) Cu(OAc)2, MeOH, Pyridine,
(Far)-OH, TFFH, DIPEA in DMF/CH2Cl2; g) ICH2CN, DIPEA in NMP;
ACHTUNGTRNEN(UNG Far)-CO2Me
release.[23] Similarly, we did not
AHCTUNGTRENNUNG
observe any S-alkylation of me-
thionine or protected cysteines
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
during the synthesis of the N-
acetic acid in CH2Cl2; f) Fmoc-l-CysACTHUNGTRENNNUG
Ras sequences.[19] The second
h) MeOH, DMAP in CH2Cl2/THF.
step of the cleavage is the nu-
cleophilic attack on the N-alky-
lated sulfonamide. Different nucleophiles (amines, amino
acid methyl esters and thiols) have been successfully used
for this purpose.[23,24,35] As mentioned above, N-Ras proteins
are post-translationally modified and this includes the meth-
ylation of the C-terminal cysteine. For that reason peptide
release from the solid support using methanol, thus provid-
ing the peptide methyl ester, would be highly desirable. Al-
cohols can be used for the nucleophilic attack after activa-
tion of the linker, but due to their low nucleophilicity a cata-
lytic amount of DMAP should be added. This approach has
already proven suitable for the release of the peptides from
Scheme 3. Resin loading with cysteine fluorides and cleavage with H2N-
Phe-CO2Me to afford dipeptides 2 and 3.
Because of the higher cost of PyBOP and lower stability,
the DIC/HOBt combination was chosen for all further pep-
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Lipidated Ras Peptides
FULL PAPER
the solid support as glycine ester derivatives.[36] Racemiza-
tion was not an issue in this case as glycine, the C-terminal
amino acid in this study bears no stereogenic center.
In order to determine the influence of different cleavage
conditions on cysteine racemization Fmoc-l- or Fmoc-d-
CysACHTUNGTRENNUNG(Trt)-F was generated using the cyanuric fluoride ap-
proach (see above) to avoid racemization and loaded on the
acyl-sulfonamide linker. Two dipeptide diastereomers (l,l
and l,d Fmoc-Pro-Cys) were then assembled on the solid
support. The linker was subsequently activated with
ICH2CN and DIPEA, and finally a solution of methanol
with catalytic amounts of DMAP was used for the nucleo-
philic cleavage of the dipeptides 4a/4b, that were obtained
in 80% yield (Scheme 4A). The released dipeptides 4a/4b
were analyzed by normal-phase chiral HPLC what showed
that 35% of epimerization had occurred under these cleav-
age conditions (Scheme 3 and Table 2, entry 2). Similar epi-
merization results were obtained by loading Fmoc-Leu-OH
using either DIC/MeIm or Ellman’s conditions (PyBOP,
DIPEA in CH2Cl2 at À208C). In both cases a Boc-Phe-Leu
dipeptide was assembled and after cleavage with a solution
of methanol and DMAP, dipeptide 6 was obtained in 44%
yield and with 35% of epimerization (Table 2, entry 4). In
contrast, no epimerization was observed in the loading of
Fmoc-Pro and in the release of dipeptides ll/ld-Leu-Pro
(7) with MeOH and catalytic amounts of DMAP (Table 2,
entry 5). Release of the Boc-Phe-Leu dipeptide with thio-
phenol as activated ester 5 (Table 2, entry 3) and subsequent
transesterification with methanol to give compound 6 also
led to racemization after stirring 5 in methanol. Thus, this
procedure is not suitable for further use.
Scheme 4. Dipeptides synthesized using the Ellman sulfonamide linker.
A) Synthesis of dipeptides 2 and 4; B) synthesis of dipeptides 5, 6 and 7.
a) H-CysACHTNUGRTNE(UGN Trt)-F, DIPEA in CH2Cl2; b) ICH2CN, DIPEA in NMP; c)
H2N-Phe-CO2Me in CH2Cl2; d) 20% piperidine in DMF; e) Fmoc-Pro-
OH, DIC; HOBt in DMF; f) MeOH, DMAP in CH2Cl2; g) Fmoc-Leu-
OH, DIC, MeIm, in CH2Cl2/DMF; h) Boc-Phe-OH, DIC; HOBt in
DMF; i) thiophenol in CH2Cl2; j) MeOH; k) Fmoc-Pro-OH, DIC, MeIm
in CH2Cl2/DMF.
Table 2. Racemization of the model dipeptides upon release from the
Ellman linker.
These results demonstrate that release of the peptides
with methanol leads to racemization of amino acids, includ-
ing the important cysteines. In order to obtain access to N-
Ras peptides with a C-terminal methyl ester, a different
strategy was therefore followed. This strategy was based on
the release of the peptide from the resin using the amine
functionality of the C-terminal amino acid already function-
alized as methyl ester. To estab-
Entry
Cleavage
Yield [%]
d-AA [%]
Peptide
1
2
3
4
5
PheCO2Me
MeOH/DMAP
thiophenol
MeOH/DMAP
MeOH/DMAP
47
80
85
44
31
2.5
35
<1
35
2
4
5
6
7
<1
lish this approach first Fmoc-
Pro-OH was loaded onto the
resin and after Fmoc deprotec-
tion and acetylation was subse-
quently released with the farne-
sylated cysteine methyl ester
H2N-CysACHTUNGTRENNUNG(Far)-CO2Me
(Scheme 5). Following reported
conditions, that is, 24 h shaking
for activation with ICH2CN and
subsequent shaking for 6 h with
the corresponding amine, the
dipeptide product was obtained
in low yields only. The yield
could not be improved using
longer reaction times in the
cleavage step. Microwave-as-
sisted chemistry, however, al- Scheme 5. Peptides corresponding to N-Ras termini synthesized using this strategy.
Chem. Eur. J. 2010, 16, 9585 – 9591
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9589

H. Waldmann et al.
lowed to accelerate the release reaction significantly. Using
five equivalents of the farnesylated cysteine methyl ester in
a solution of CH2Cl2/THF (1:1), the reaction was fast and
the desired dipeptide 1 could be obtained after 10 min mi-
crowave irradiation at 508C with an initial microwave power
of 40 W. The dipeptide 1 was isolated from the reaction mix-
ture in 40% yield, high purity, and without racemization by
preparative HPLC (Scheme 5).
ACHTUNGTRENNUNG
Lipidated Ras peptide synthesis employing the sulfonamide
linker: After having established a suitable protocol for
amino acid loading, peptide elongation, and peptide release,
the applicability of the synthesis of the N-Ras sequences 8–
11 was evaluated. The peptides correspond to the mono-
and double-lipidated N-Ras C terminus, featuring for exam-
ple labile palmitoylated cysteines 11 and stable thioether
mimics thereof 9. These peptides were obtained following
the procedure detailed above in high purities and without
racemization. Farnesylated N-Ras 8 could be obtained in
high purity and in 18% overall yield after preparative
HPLC purification. The same strategy was used for the syn-
thesis of the double lipidated N-Ras peptide 9, bearing a far-
nesyl and a hexadecyl moiety as a non-hydrolysable mimic
of the fully lipidated N-Ras sequence (farnesylated and pal-
mitoylated), and was obtained in 16% yield. Palmitoyl
groups are known to be highly sensitive to nucleophiles.
Therefore, a modified strategy was used for the synthesis of
the fully lipidated N-Ras sequence 11, bearing a farnesyl
and a palmitoyl group. In this case, the release step was per-
formed in a buffered mixture containing farnesylated cys-
teine methyl ester as well as acetic acid and pyridine. Pep-
tide 11 could be obtained following this protocol as deter-
mined by LCMS of the crude mixture, but unfortunately
separation of 11 from excess amine could not be achieved
by reverse-phase preparative HPLC, probably due to the
high hydrophobicity of the product. Therefore, trityl-pro-
tected peptide 10 was synthesized and isolated, and palmi-
toylation was performed in solution, affording peptide 11 in
7% overall yield.
Experimental Section
General protocol for the synthesis of lipopeptides 8–11: The resin was
loaded with Fmoc-l-Pro-OH (DIC, MeIm). In general a resin with an ini-
tial loading of 0.40–0.45 mmolgÀ1was employed. Peptide elongation was
performed using DIC/HOBt on an automated microwave peptide synthe-
sizer. After washing several times with DMF/CH2Cl2/DMF, 0.1–
0.15 mmol of the resin were activated with ICH2CN/DIPEA and nucleo-
philic displacement was carried out with
a solution of NH2-Cys-
AHCTUNGTRENNUNG
8 to 20 mg amounts after purification by preparative HPLC. For detailed
descriptions and HPLC conditions see the Supporting Information.
Ac-Gly-CysACHTUNTGRENN(UG StBu)-Met-Gly-Leu-ProCysACHTUGNRTEN(NUGN Far)-CO2Me (8): 18% yield;
1
[a]2D0 = À51.42 (c = 0.14, CHCl3); H NMR (400 MHz, [D6]DMSO): d=
0.88 (t, 6H, J=6.83 Hz, 6H, Leu-CH3), 1.28 (s, 9H, tBu), 1.44 (m, 2H,
Leu-Hb), 1.53 (s, 6H, J=1.61 Hz, Far-CH3), 1.64 (d, 6H, J=4.11 Hz, Far-
CH3), 1.66 (m, 1H, Leu-Hg), 1.85 (s, 3H, Ac), 1.92 (m, 4H, Far-CH2,
Pro-Hga, Met-Hba), 2.01 (m, 8H, 3 Far-CH2, Pro-Hgb, Met-Hbb), 2.02
(s, 3H, Met-CH3), 2.64–2.72 (m, 1H, Cys
Cys(Far)-Hba), 2.81–2.76 (m, 1H, Cys(StBu)-Hbb), 2.86 (dd, 1H, J=
13.89, 5.38 Hz, Cys(Far)-Hba), 2.94 (m, 1H, Cys(Far)-Hbb), 3.17 (tdd,
2H, J=19.76, 12.99, 8.08 Hz, Cys(Far)-CH2), 3.50 (m, 1H, Pro-Hda),
3.62, 3.65 (s, 3H, OCH3), 3.71 (m, 5H, Pro-Hda, Gly-Ha), 4.23–4.32 (m,
1H, Met-Ha), 4.39 (m, 2H, Pro-Ha, Cys(StBu)-Ha), 4.50 (m, 2H, Cys-
(Far)-Ha), 4.57 (m 1H, Leu-Ha), 5.07 (q, 2H, J=5.36 Hz, Far), 5.15 (m,
ACHTUNGTNER(NUNG StBu)-Hba), 2.75 (m, 0.5H,
G
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
1H, Far), 7.97 (m, 2H, Leu-NH, Gly-NH), 8.09 (d, 1H, J=7.84 Hz, Met-
NH), 8.16 (t, 1H, J=5.66 Hz, Gly-NH), 8.21 (d,1H, J=7.84 Hz, Cys-
ACHUTNGREN(NUG StBu)-NH), 8.25 ppm (d, 1H, J=7.84 Hz, 1H, CysACHTUNGTNER(NUGN Far)-NH); LC-MS
(ESI): m/z: calcd for C48H84N8O9S4: 1028.51 [M+H]+; found: 1027.84
[M+H]+, 1044.18 [M+NH4]+; tR = 10.57 min; ES MS: m/z: calcd for
C48H84N8O9S4: 1028.50514; found 1028.50645 [M+H]+, 1050.48756
[M+Na]+.
Conclusion
Ac-Gly-Cys(Hd)-Met-Gly-Leu-ProCysACTHNUTRGNEUNG
(Far)-CO2Me (9): 16% yield; [a]D20
Cysteine is one of the amino acids most prone to racemiza-
tion. This puts restraints on the synthetic methodology for
peptide synthesis in general, and for synthesis of lipidated
peptides with C-terminal cysteine esters in particular. The
sulfonamide linker in principle offers an attractive linker
system to generate lipidated peptide esters, due to its ortho-
gonality to the different functional groups in the peptide,
and thus is a good alternative to the hydrazide linker. The
direct generation of C-terminal cysteine methyl esters, how-
ever, by direct release from solid support turned out to be
challenging. Optimization of amino acid loading, peptide
elongation and peptide release conditions was required to
successfully generate lipidated N-Ras peptides. Loading of
either trityl protected or farnesylated cysteines could be
= À34.64 (c = 0.28, CHCl3).1H NMR (400 MHz, [D6]DMSO): d=0.85
(m, 9H, Leu-CH3, HDCH3), 1.21 (m, 24H, CH2HD), 1.45 (m, 3H,
CH2HD, Leu-Hg), 1.54 (s, 6H, Far-CH3), 1.61 (d, 6H, J=1.34 Hz, Far-
CH3), 1.67 (m, 2H, Leu-Hb), 1.80–2.06 (m, 14H, Far-CH2, HD-CH2, Pro-
Hg, Met-Hb), 1.83 (s, 3H, Ac), 2.02 (s, 3H, Met-CH3), 2.53–2.35 (m, 4H,
Pro-Hb, Met- Hg), 2.69–2.61 (m, 2H, Cys
2.84–2.74 (m, 2H, Cys(Far)-Hbb, Cys(HD)-Hbb), 3.14 (ddd, J=25.39,
12.93. 7.63 Hz, 2H, Cys(Far)-CH2), 3.50 (m, 1H, Pro-Hda), 3.60 (s, 3H,
OCH3), 3.71–3.58 (m, 3H, Pro-Hdb, Gly-Ha), 4.25 (dt, J=8.68, 4.80 Cys-
(Far)-Ha), 4.43–4.34 (m, 3H, Met-Ha, Pro-Ha, Cys(HD) Ha), 4.56 (dt,
ACHTUNGERTN(NUNG Far)-Hba, Cys(HD)-Hba),
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
1H, J=8.64, 5.87 Hz, Leu-Ha), 5.05 (dd, 2H, J=12.54, 5.98 Hz, Far),
5.14 (t, 1H, J=7.57 Hz, Far), 7.93–8.21 ppm (m, 6H, Gly-NH, Leu-NH,
Cys-(Far)-NH, Cys(HD)-NH, Met-NH); LC-MS: m/z: calcd for
C60H105N7O9S3: 1164.72; found: 1164.33 [M+H]+, 1181.04 [M+NH3]+; tR
= 11.98 min; ES MS: m/z: calcd for C60H105N7O9S3: 1164.72087 [M+H]+;
found: 1164.72168 [M+H]+, 1186.70339 [M+Na]+.
9590
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ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 9585 – 9591

Lipidated Ras Peptides
FULL PAPER
Ac-Gly-Cys
(Trt)-Met-Gly-Leu-ProCys
E
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2.36–2.41 (m, 2H, Pro-Ha), 2.63–2.69 (m, 1H, Cys
(m, 2H, Cys(Far)-Hbb, Cys(Trt)-Hba), 2.79–2.92 (m, 1H, Cys
3.15 (ddd, J=19.53, 12.10, 5.72 Hz, 2H, Cys(Far)-CH2), 3.49 (m, 1H, Pro-
Hda), 3.60 (s, 3H, OCH3), 3.63 (m, 5H, Pro-Hdb), 3.68 (t, 2H, J=
6.03 Hz, Gly-Ha, 4.17–4.22 (m, 1H, Cys(Far)-Ha), 4.30 (m, 1H, Met-
Ha), 4.37 (m, 2H, Pro-Ha, Cys(Trt)-Ha), 4.55 (dd, 1H, J=13.88,
8.68 Hz, Leu-Ha), 5.05 (d, 2H, J=5.25 Hz, Far), 5.15 (dd, 1H, J=15.67,
8.01 Hz, Far). 7.19–7.71 (m, 15H, Ar), 7.90–7.95 (m, 2H, Gly-NH, Leu-
NH), 8.01 (m, 1H, Cys-Far-NH), 8.11 (t, 1H, J=5.47 Hz, Gly-NH), 8.17
G
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R
G
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
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(Trt)-
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Ac-Gly-Cys
(Pal)-Met-Gly-Leu-ProCys
E
(11):
1H NMR
(400 MHz, [D6]DMSO): d=0.85 (m, 9H, Leu-CH3, PalCH3), 1.19 (m,
24H, CH2Pal), 1.35 (m, 3H, CH2HD, Leu-Hg), 1.52 (s, 6H, Far-CH3),
1.60 (s, 6H, Far-CH3), 1.67 (m, 2H, Leu-Hb), 1.82–2.01 (m, 14H, Far-
CH2, Pal-CH2, Pro-Hg, Met-Hb), 1.89 (s, 3H, Ac), 1.93 (s, 3H, Met-CH3),
2.40–2.59 (m, 4H, Pro-Hb, Met- Hg), 2.63–2.70 (m, 2H, Cys
Cys(Pal)-Hba), 2.86–2.91 (m, 2H, Cys(Far)-Hb), 2.95–3.20 (m, 4H, Cys-
(Far)-CH2, Cys(Pal)-Hb), 3.51 (m, 1H, Pro-Hda), 3.65 (s, 3H, OCH3),
3.57–3.69 (m, 1H, Pro-Hdb), 3.75 (t, 2H, J=3.84 Hz, Gly-Ha), 4.45–4.68
(m, 5H, Cys(Far)-Ha, Met-Ha, Pro-Ha, Cys(Pal) Ha, Leu-Ha), 5.02 (m,
2H, Far), 5.13 (m, 1H, Far), 7.28–7.32 (m, 2H, Gly-NH, Leu-NH), 7.40–
7.42 (m, 1H, Cys-(Far)-NH), 7.51 (m, 2H, Gly-NH, Cys(Pal)-NH),
7.70 ppm (m, 1H, Met-NH); LC-MS: m/z: calcd for C60H103N7O10S3:
ACHTUNGTRENUN(NG Far)-Hba,
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
1178.70 [M+H]+;found 1178.19 [M+H]+, 1194.34 [M+NH3]+; tR
ACHTUNGTERNUNNG =
11.48 min; ES MS: m/z: calcd for C60H103N7O10S3: 1178.700013 [M+H]+;
found 1178.70107 [M+H]+.
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Acknowledgements
This research was supported by the Max Planck Society, the Fonds der
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Received: June 10, 2010
Published online: July 20, 2010
Chem. Eur. J. 2010, 16, 9585 – 9591
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9591