Solid-phase synthesis of lipidated peptides

Article abstract of DOI:10.1002/chem.200500476

A new flexible and efficient methodology for the solid-phase synthesis of lipidated peptides has been developed. The approach is based on the use of previously synthesized building blocks and overcomes the limitations of previously reported methods, since long doubly lipidated peptides can be synthesized by using this route. Furthermore, it was thus possible to prepare a large number of N- and H-Ras peptides bearing a wide range of reporter and/or linking groups - efficient tools for the investigation of biological processes. In terms of efficiency and flexibility this solid-phase method is superior to the solution-phase synthesis. It gives pure peptides in multi-milligram amounts within a much shorter time and with superior overall yield.

Full text of DOI:10.1002/chem.200500476

FULL PAPER
DOI: 10.1002/chem.200500476
Solid-Phase Synthesis of Lipidated Peptides
Maria Lumbierres,[a] Jose M. Palomo,[a] Goran Kragol,[a] Sonja Roehrs,[b]
Oliver Müller,[b] and Herbert Waldmann*[a]
Abstract: A new flexible and efficient
methodology for the solid-phase syn-
thesis of lipidated peptides has been
developed. The approach is based on
the use of previously synthesized build-
ing blocks and overcomes the limita-
tions of previously reported methods,
since long doubly lipidated peptides
can be synthesized by using this route.
Furthermore,it was thus possible to
prepare a large number of N- and H-
Ras peptides bearing a wide range of
reporter and/or linking groups—effi-
cient tools for the investigation of bio-
logical processes. In terms of efficiency
and flexibility this solid-phase method
is superior to the solution-phase syn-
thesis. It gives pure peptides in multi-
milligram amounts within
a much
Keywords: building block strategy ·
lipidated peptides · lipoproteins ·
solid-phase synthesis
shorter time and with superior overall
yield.
Introduction
ester (Figure 1). Lipidation of these proteins is essential for
the biological function.[4] The lipid groups are believed to
serve as anchors of the proteins to the membranes and are
possibly involved in protein–protein and protein–lipid inter-
actions.
Tailor-made lipidated peptides representing the character-
istic functional parts of their parent proteins are efficient
tools for the investigation of biological processes.[5–6] There-
fore,the availability of a flexible solid-phase technology for
their synthesis is of great importance. To develop such a
general and flexible solid-phase method that would be appli-
The Ras proteins are plasma membrane bound lipoproteins
that serve as central molecular switches in biological signal
transduction. They cycle between an inactive GDP-bound
state and an active GTP-bound state.[1] In the active state,
the Ras proteins translate growth-promoting signals into
changes in gene expression. These proteins are involved in
the regulation of diverse cellular processes such as cell
growth and differentiation,the cell cycle,and apoptosis
[2]
.
The importance of the correct functioning of the Ras signal
transduction pathway is demonstrated by the fact that a
point of mutation in the ras oncogenes is found in approxi-
mately 30% of all human cancers.[3] The Ras proteins con-
tain both acid-labile farnesyl thioethers and base-sensitive
palmitic acid thioesters and terminate in a cysteine methyl
[a] Dr. M. Lumbierres,Dr. J. M. Palomo,Dr. G. Kragol,
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
Chemische Biologie (Germany)
Fax : (+49)231-133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
[b] S. Roehrs,Dr. O. Müller
Max-Planck-Institut für molekulare Physiologie
Abteilung Strukturelle Biologie
Otto-Hahn-Strasse 11,44227 Dortmund (Germany)
Figure 1. Schematic structure of the human N-Ras protein.
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cable to the synthesis of both acid- and base-labile lipidated
peptides several demands must be fulfilled (Figure 2). First,
the method requires the use of suitable orthogonally stable
protecting groups as well as a linker to the solid support
that can be cleaved under mild conditions[6] and which
allows release of the peptides as methyl ester,or—if re-
quired—equipped with a different functional group,for ex-
ample,a fluorophore at the C-terminus. Second,the ap-
proach should also allow an easy introduction of reporter
and/or linking groups needed for further biological investi-
gations.
At the outset of our work only one method for the solid-
phase synthesis of double lipidated peptides was available.
This employed on-resin lipidation of cysteines after cleavage
of suitable protecting groups as the key transformation.[7]
However,this method suffers from several drawbacks that
hinder its general applicability: 1) the use of a large excess
of (sometimes not readily available) lipidation reagents is
required,2) it is not suitable for the synthesis of longer pep-
tides (>10 amino acids),3) it is not readily automatable.
The major challenge to be met by a new more general and
flexible approach lies in the elongation of the peptide chain
after incorporation of the palmitoylated cysteine,since the
thioester rapidly undergoes an intramolecular S,N-acyl shift
once the amino group is deprotected (Figure 2).[8]
Herein we describe the development of a technology that
fulfils the demands and solves the problems mentioned
above. This approach employs the synthesis of lipidated
building blocks in solution,which substantially reduces the
amount of lipidation reagents required,and subsequent use
of these building blocks in the solid-phase synthesis of the
target peptides. The use of Fmoc-4-hydrazinobenzoic acid as
a linker allows direct access to the methyl esters or free
acids after cleavage under mild oxidative conditions.[9] Par-
ticularly remarkable is the establishment of coupling condi-
tions that minimize the S,N-acyl shift after incorporation of
the palmitoylated cysteine. We demonstrate the efficiency of
the new approach in the synthesis of a variety of C-terminal
N-Ras and H-Ras peptides that bear a wide range of fluo-
rescent and/or reporter and linking groups. Part of this work
has been published in a preliminary communication.[10]
Results and Discussion
Synthesis of the lipidated and labeled building blocks: The
building blocks required for the development of the new
solid-phase method were synthesized in high overall yields
as shown in Scheme 1 using in part transformations de-
scribed earlier.[11] Only one equivalent of lipidation reagent
(farnesyl or palmitoyl chloride) was required,which is nota-
bly advantageous in the case of the N-methylanthraniloyl
(Mant)-labeled cysteine 6,since this building block requires
a laborious multistep synthesis starting from geraniol.[12]
Thus a possible use of this reagent in fivefold excess as is re-
quired in the on-resin lipidation method[7] is clearly unde-
sired. Not only different lipidated cysteine building blocks
were synthesized (2, 4, 5,and 6),but also both a NBD (4-ni-
trobenz-2-oxa-1,3-diazole) fluorescent label (8) and a benzo-
phenone group (10) were attached to the side chain of
lysine.
Solid-phase synthesis of farnesylated and palmitoylated pep-
tides using the pre-lipidated building-blocks: The lipidated
cysteines 2 and 4 were used in the solid-phase synthesis of
the N- and H-Ras peptides 11 and 12 (Scheme 2). The solid-
phase synthesis was performed using the commercially avail-
able Fmoc-4-hydrazinobenzoic acid functionalized amino-
methyl polystyrene resin (Novabiochem) and standard
Fmoc chemistry. The Fmoc group was removed with 20%
piperidine in DMF. Standard couplings were performed with
five equivalents of amino acid/HBTU/HOBt and 10 equiva-
lents of diisopropylethylamine in DMF for 2 h. Cysteine
building blocks were coupled using HBTU/HOBt/trimethyl-
pyridine in CH2Cl2/DMF (1:1) to avoid racemization.[13] In
these cases extended coupling times were required due to
the bulkiness of the lipid residues. The doubly lipidated pep-
tides 11 and 12 were obtained after cleavage from the resin.
The hydrazide bond is oxidized by copper acetate. After the
nucleophilic attack of methanol,the peptide is released
from the resin as a methyl ester (Scheme 2). Filtration of
the crude reaction products through a silica gel cartridge
allows the peptides to be obtained in high yields (69% for
11 and 60% for 12) and with high purity (>90%,Figure 3).
Figure 2. Building block approach to the solid-phase synthesis of doubly lipidated peptides. FL: fluorescent label.
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Solid-Phase Synthesis of Lipidated Peptides
FULL PAPER
Scheme 1. Synthesis of building blocks 2, 4, 5, 6, 8,and 10. a) 5% TFA,3% TES,CH 2Cl2,1 h,RT; b) TMS-Cl (1.1 equiv),2 h,reflux,CH
Cl or HD-I (3 equiv),Et 3N (1.5 equiv) dropwise,3 h,CH 2Cl2,RT; c) Far-Cl (1 equiv),4 n NH3/methanol,3 h,0 8C,1 h,RT; d) Fmoc-OSu (1.1 equiv),
Et3N (1.1 equiv),CH 2Cl2,2 h,RT; e) GerMantCl (1 equiv),4 n NH3/methanol,3 h,0 8C,1 h,RT; f) NBDCl,THF/methanol,65 8C,2 h; g) 1.1 equiv
N,N-diisopropylethylamine,Far =farnisyl,Fmoc: 9-fluorenylmethyloxycar-
2Cl2,then Pal-
HATU,2 equiv DIPEA,DMF,10 min; h) Fmoc-Lys-OH,5 h,RT. DIPEA:
bonyl,HD-I: hexadecyl iodide,Pal =palmitoyl,TES: triethylsilane,TFA: trifluoroacetic acid,TMS: trimethylsilane,Su: succinimide,HATU:
benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate.
O-(7-aza-
Elongation of the peptide chain after incorporation of the
palmitoylated cysteine: Further elongation of the peptide
chain required finding new conditions to remove the Fmoc
protecting group from the N-terminal S-palmitoylated cys-
teine (Scheme 3),since the thioester is sensitive to nucleo-
philic attack (Figure 2) and therefore piperidine can not be
used. However,the main challenge at this point was to
avoid the quick S!N acyl shift once the amino group was
reduce the percentage of shifted peptide to 15% when a 7:1
mixture was used (entry 3,Table 1). The change of the base
from DIPEA to TMP did not improve the results (entry 4,
Table 1).
Furthermore,the coupling reagent also influenced the
extent of the undesired S,N-shift. Thus, the best results were
obtained using HATU (5 equiv) as a coupling reagent in
CH2Cl2/DMF (7:1) (entries 5–7,Table 1). The addition of
larger amounts of HATU (10 equiv) did not improve the
result (entry 6,Table 1). However,the addition of a fourfold
excess of base relative to the reagents gave the best results;
less than 5% of the undesired S,N-shifted peptide 17 was
formed (entry 7,Table 1). Taking these criteria into consid-
eration,we obtained the desired N-Ras peptide 15 in 98%
yield by using HATU (5 equiv) and DIPEA (20 equiv)
(Table 1). The development of the coupling conditions ini-
tially was performed by using peptide 15 in which the amino
acid sequence of the N-Ras C-terminus was incorporated. In
subsequent experiments these results were confirmed in the
synthesis of peptide 16,which corresponds to the C-terminus
of H-Ras (see below). Notably,in this case the bulky O-
trityl-protected Fmoc-serine was attached to the N-depro-
tected S-palmitoylated cysteine. This demonstrates that the
results obtained for the N-Ras sequence in which a glycine
free.[8]
Gratifying8ly-,d1i,azobicyclo[5.4.0]undec-7-ene
(DBU)—a non-nucleophilic hindered base—permitted a fast
removal of the Fmoc group and left the thioester intact. The
Fmoc group in 13 (Scheme 3) can be cleaved by double
treatment with a solution of 1% DBU in DMF each time
for 30 s.[14] Fast washing of 14 with DMF (ca. 20 s) and im-
mediate addition of a large excess (10 equiv) of preactivated
(10 min) amino acid minimize the S,N-acyl shift of the palm-
itoyl group. After substantial experimentation we discovered
that the polarity of the solvent plays a crucial role in this
step (Table 1). In the first attempts we performed the cou-
pling in DMF. The amino acid was activated by using
HBTU/HOBt with DIPEA as the base (entry 1,Table 1). In
this way we obtained almost exclusively the S,N-shifted N-
Ras peptide 17. By decreasing the polarity of the solvent
with different mixtures of CH2Cl2/DMF,it was possible to
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H. Waldmann et al.
Scheme 2. Synthesis of N- and H-Ras peptides 11 and 12 using the preli-
pidated cysteines. a) 1. 20% piperidine/DMF,2. Fmoc-Cys(Far)-OH
(4 equiv),HBTU/HOBt/TMP (4 equiv),CH 2Cl2/DMF (1:1),4 h; b) stan-
dard solid-phase synthesis: 1. 20% piperidine/DMF,2. Fmoc-AA-OH
(5 equiv),HBTU/HOBt (5 equiv)/DIPEA (10 equiv),DMF,2 h; c) 1.
20% piperidine/DMF,2. Fmoc-Cys(Pal)-OH (4 equiv),HBTU/HOBt/
Scheme 3. Elongation of the peptide chain after incorporation of the pal-
mitoylated cysteine. a) 1% DBU/DMF; b) coupling of Fmoc-AA-OH.
is introduced,are also valid for elongation of the peptide
chain with a sterically demanding bulky amino acid.
N-coupled peptides, 15 and 16,and SN, -shifted analogues,
17 and 18 could be clearly distinguished in RP-HPLC
TMP (4 equiv),CH 2Cl2/DMF (1:1),4 h; d) Cu(OAc) (0.5 equiv),pyri-
2
dine (30 equiv),acetic acid (50 equiv),methanol (215 equiv),CH
2Cl2,
oxygen,3 h. HBTU: N-[(1H-benzotriazol-1-yl)(dimethylamino)methy-
lene]-N-methylmethanaminium hexafluorophosphate N-oxide,HOBt: 1-
hydroxybenzotriazole,TMP: trimethylpyridine.
(Figure 4). These peptides were also identified by means of
1
their H NMR spectra,since the CH protons neighboring
2
the carbonyl group of the palmitoyl moiety display different
chemical shifts for the N-coupled (ca. 2.50 ppm) and the
S,N-shifted (ca. 2.15 ppm) peptides.
Applicability of the method to the synthesis of long lipidat-
ed peptides: Several farnesylated and palmitoylated pep-
tides (15–21) were synthesized to demonstrate the efficiency
of the method.(Figure 5) In all cases,pre-lipidated building
blocks were used for the solid-phase synthesis. After incor-
poration of the palmitoylated cysteine,1% DBU in DMF
was used for the Fmoc deprotection and the first coupling
was performed under the optimized conditions described
above (entry 7,Table 1) to avoid the SN, -shift of the palmi-
toyl group. Standard conditions (HBTU/HOBt/DIPEA in
DMF) were used for the following couplings. It is particular-
ly remarkable that with the new approach it was possible to
obtain C-terminal N-Ras decapeptide 19 and tetradecapep-
tide 20. Such long farnesylated and palmitoylated peptides
were not accessible with the previously reported solid-phase
method.[7] The superiority of the new method is also demon-
Figure 3. RP-HPLC trace of Fmoc-Cys(Pal)-Lys(Aloc)-Cys(Far)-OMe,
11.
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Solid-Phase Synthesis of Lipidated Peptides
FULL PAPER
Table 1. Study of conditions for minimizing S,N-acyl shift in the coupling of the next amino acid after palmitoylated cysteine in the synthesis of N-Ras
peptide 15.[a]
Entry
Coupling method
Base
Solvent
Yield
Yield
[CH2Cl2/DMF]
15 [%]
17 [%]
1
2
3
4
5
6
7
AA(10 equiv),HBTU (10 equiv),HOBt (10 equiv)
AA(10 equiv),HBTU (10 equiv),HOBt (10 equiv)
AA(10 equiv),HBTU (10 equiv),HOBt (10 equiv)
AA(10 equiv),HBTU (10 equiv),HOBt (10 equiv)
AA(5 equiv),HATU (5 equiv)
DIPEA(20 equiv)
DIPEA (20 equiv)
DIPEA (20 equiv)
TMP (10 equiv)
DIPEA (10 equiv)
DIPEA (20 equiv)
DIPEA (20 equiv)
0:1
<5
55
85
75
94
>95
45
15
25
6
1:1
7:1
7:1
7:1
7:1
7:1
AA(10 equiv),HATU (10 equiv)
AA(5 equiv),HATU (5 equiv)
95
98
5
2
[a] Abbreviations: AA: amino acid,that is Gly in the synthesis of the N-Ras sequence and Ser (Trt) for the H-Ras peptide,TMP: trimethylpyridine.
Figure 4. RP-HPLC trace of mixture 15/17 (95:5).
strated by means of the synthesis of the palmitoylated hexa-
decapeptide 21,which represents a lipidated part of the Wnt
protein,a signaling protein involved in numerous events in
animal development,such as proliferation and differentia-
tion in several tissues and structures during embryogene-
sis.[15]
Figure 5. Longer lipidated peptides synthesized by applying the optimized
conditions.
Wnt3a cells,served as the positive control. The conditioned
medium activated the Wnt pathway in the L cells,as shown
by the increase of the relative amount of b-catenin
(Figure 6). The b-catenin level did not increase after incuba-
tion with the peptide 21. There was no detectable b-catenin
in L cells incubated with the peptide up to a concentration
of 5 mm,which is approximately 1000 times higher than the
minimal concentration,in which the native protein showed
activity.[15a] The lack of activity of the peptide was not due
to the solvent. DMSO showed no negative or positive influ-
ence on the b-catenin level no matter whether the Wnt3a
protein was present or not. Thus,we conclude that the con-
served lipidated sequence incorporated into the peptide 21
is not sufficient to activate the Wnt pathway in L cells.
Analysis of the biological activity of Wnt peptide 21: The
Wnt proteins are a family of lipid-modified extracellular
proteins,[16] which are highly conserved in vertebrates and
non-vertebrates. The extracellular Wnt proteins activate the
Wnt pathway,which has an impact on cell proliferation,cell
[17]
transformation,and cell differentiation.
The lipid modifi-
cation is essential for the biological activity of the Wnt pro-
tein. The peptide 21 corresponds to the highly conserved se-
quence of the Wnt proteins. Given this sequence conserva-
tion it might be possible that the lipidated core peptide
motif is sufficient to activate the downstream signaling path-
way. To investigate this possibility,we measured the ability
of this peptide to activate the Wnt pathway. The relative
level of the intracellular proto-oncoprotein b-catenin served
as a parameter for the activity of the Wnt pathway. L cells,
which were incubated with a conditioned medium from L
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H. Waldmann et al.
peptides 25 and 26 carrying the benzophenone group (Fig-
ure 7b). This reporter group was placed in the side chain of
the lysine using the previously synthesized building block 10
(Scheme 1). The introduction of the biotin marker should
facilitate both the detection as well as the purification of the
cross-linked products.
Furthermore,we synthesized a wide range of N- and H-
Ras peptides fluorescently labeled with,for example,the
NBD group (Table 2). Several of these peptides bear the
NBD marker at the N-terminus,whereas in other cases an
Fmoc-Lys(NBD)-OH 8 building block was used for the syn-
thesis. Most of these peptides also incorporate the MIC
group which can be employed for ligation to proteins.[5a–b]
The presence of an N-terminal cysteine in 27 and 32 also
permits the coupling to proteins by way of expressed protein
ligation.[19] Moreover,the peptide 35 with the d-configura-
tion was synthesized for further stereochemical studies.
Finally,Mant and Bodipy-FL (4,-difluoro-4-borata-3a-
azonia-4a-aza-s-indacene)[20] were also used as fluorescent
labels (Table 2). Peptide 37 incorporates the GerMant-ana-
logue of the farnesyl group. The introduction of the fluores-
cent label into the lipid moiety leaves the C-terminus free
for possible ligation to proteins.
Figure 6. Analysis of the biological activity of peptide 21. The sizes of
marker fragments are indicated on the left in kilodaltons (kDa). Top:
Western blot analysis of b-catenin or of GAPD in total lysates of L cells
after incubation with conditioned media from L cells (L cell CM),condi-
tioned medium from L Wnt3a cells (Wnt3a CM),peptide 21 or DMSO at
the indicated concentrations. Bottom: Control experiment to analyze the
influence of the solvent DMSO on the b-catenin level in activated and
non-activated L cells. The b-catenin level was analyzed by Western blot
in total lysates of L cells after incubation with L cell CM or with Wnt3a
CM in the presence or absence of 1% DMSO.
We did not synthesize a previously labeled lysine building
block for the synthesis of the peptide 38,since Bodipy-FL is
not stable under typical Fmoc deprotection conditions (20%
piperidine). Fmoc-Lys(Aloc)-OH was used instead as a
building block for the peptide synthesis. After coupling of
Fmoc-Gly-OH,the Aloc group was removed,and the
Bodipy-FL was coupled to the lysine on the resin. Then,the
Fmoc group of Gly was cleaved with 1% DBU in DMF,and
the MIC group was attached to the N-terminus.
Introduction of fluorescent labels and/or reporter and link-
ing groups into the Ras peptides for further biological inves-
tigations: Once the efficiency of the new methodology in
the synthesis of long doubly lipidated peptides was demon-
strated,the scope of the new approach was explored. For
this purpose labeled peptides bearing reporter and/or link-
ing groups required for further biological investigations
were synthesized. To demonstrate the versatility of the new
method the labels were placed in different positions on the
lipidated peptide: 1) within the lipid residue,2) in the
amino acid side chain,3) at the C-terminus,and 4) at the N-
terminus.
The incorporation of a male-
imidocaproyl group (MIC) at
the N-terminus of the peptide
allows the ligation to proteins
through conjugate addition of
cysteine thiol groups.[5a,b] We
have synthesized several N-Ras
peptides carrying the maleimi-
do group with different combi-
nations of lipid patterns (22–
24),such as the farnesylated
and palmitoylated peptide 22 or
the double farnesylated com-
pound 24 (Figure 7a). Ras pro-
teins bearing a photoactivatable
benzophenone group are effi-
cient tools to study the interac-
tion and cross-linking with pos-
sible Ras-binding proteins.[18]
The interest in such proteins
led us to synthesize lipidated Figure 7. Peptides incorporating reporter and linking groups.
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Solid-Phase Synthesis of Lipidated Peptides
FULL PAPER
Table 2. Results of the syntheses of fluorescent labeled peptides by means of the new method.
Experimental Section
Entry
Compound
Peptide
Yield [%]
28
1H and 13C NMR data were recorded
on a 400 MHz spectrometer. Electro-
spray mass spectrometry (ESI-MS)
was performed in the positive mode
on an Agilent 1100 series instrument.
Analytical reverse-phase HPLC sepa-
ration was performed by using a C4
column with a flow rate of 1 mLminꢀ1.
A solvent system was used that con-
sisted of a isocratic elution with 80%
acetonitrile/water,0.1% formic acid
for 1 min followed by a linear gradient
to 100% acetonitrile for 7 min and
then again isocratic elution with 100%
acetonitrile for 4 min. FAB measure-
1
27
2
3
28
29
22
28
4
5
30
31
32
25
ments were taken with
a Jeol SX
102 A apparatus by using a 3-nitroben-
zyl alcohol (3-NBA) matrix. Optical
6
7
8
32
33
34
38
37
12
rotations were measured with
a
Perkin-Elmer 241 Polarimeter. Melt-
ing points were measured with a Büchi
B-540 apparatus. MALDI MS spectra
was recorded by using 2,5-dihydroxy-
benzoic acid (DHB) or a-cyano-4-hy-
droxy cinnamic acid (CHCA) as
matrix.
General procedure for the synthesis of
lipidated and labeled peptides: Com-
mercially available Fmoc-4-hydrazino-
benzoyl NovaGel resin was used for
all solid-phase reactions. The 50-mL
reactor flask had a frit at the bottom,
a stopcock,which permitted rapid fil-
tration and washing of the resin,and a
side arm,which allowed the supply of
nitrogen or argon. The yield and scale
of the solid-phase reactions are given
with respect to the amount of the first
amino acid coupled onto the resin.
The resin loading was determined by
measuring the Fmoc groups remaining
on the resin by the established UV
method. To this end,a small amount
9
35
36
40
22
10
11
12
37
38
31
30
Conclusion
of resin (~3 mg) was treated with 20% piperidine/DMF solution
(7.5 mL) for 30 min,and the UV absorption of the solution at 301 nm
(e=7800mꢀ1 cmꢀ1) was determined.
We have developed a flexible and efficient methodology for
the solid-phase synthesis of lipidated peptides based on the
use of pre-synthesized building blocks. This approach over-
comes the limitations of the previously reported method,
since it gives access to long doubly lipidated peptides and
furthermore it meets the demands for a widely applicable
potentially automatable methodology. Its efficiency is dem-
onstrated by the synthesis of a series of N- and H-Ras pep-
tides carrying not only different combinations of lipid pat-
terns,but also a wide range of reporter and/or linking
groups needed for further biological,biophysical,and bio-
chemical investigations. In terms of efficiency and flexibility
this solid-phase method is superior to the solution-phase
synthesis. It gives pure peptides in multimilligram amounts
within much shorter time and with superior overall yield.
All amino acids and NBD-labeled Lys or benzophenone-labeled Lys
were coupled by using HBTU/HOBt chemistry. Typically,the amino acid
(AA) (5 equiv) was treated for 2 min with HBTU (5 equiv),HOBt
(5 equiv),and DIPEA (10 equiv) in DMF. The solution was added to the
resin,which was then agitated for 2 h at room temperature. The resin
was washed after coupling and Fmoc-deprotection with DMF (5”2 min).
Every step was carried out under an argon atmosphere except the wash-
ings. The trityl group was cleaved by shaking the resin-bound peptides in
a
solution of 1% TFA and 2% triethylsilane in dichloromethane
(2.5 mL/0.1 g of resin) for 2 h.
Cysteine building blocks were coupled by using HBTU/HOBt/TMP in
CH2Cl2/DMF (1:1). After optimization of the coupling times for the lipi-
dated building blocks,the following conditions were used: Fmoc-Cys-
(Far)-OH was coupled by using four equivalents for 5 h,Fmoc-Cys(Pal)-
OH using four equivalents overnight,and Fmoc-Cys(GerMant)-OH
using 1.5 equivalents for 24 h. Standard Fmoc chemistry and piperidine
deprotection (20% in DMF 4”2 min) were used until palmitoylated Cys
was incorporated in the peptide. After incorporation of Fmoc-Cys(Pal)-
OH,1% DBU/DMF (1 mL/0.1 g of resin,2”30 s) was used for Fmoc de-
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H. Waldmann et al.
protection. To avoid an S,N-shift, HATU in CH2Cl2/DMF (7:1) was used
for the subsequent coupling of amino acids.
perature for 2 h under an argon atmosphere. The solvent was evaporated
under reduced pressure and the crude product was washed three times
with toluene and after each washing it was evaporated to dryness. The
solid residue was transferred to a sinter funnel and washed twice with
pentane to remove the released triphenylmethane. The remaining solid
(0.3 g) was dissolved in DMF and cooled to 08C,then triethylamine
(5 equiv) and a solution of hexadecyl iodide (3 equiv) in DMF was
added. The mixture was stirred at 08C and then at room temperature
overnight. The solvent was evaporated and the crude mixture was puri-
fied by flash chromatography on silica gel using a gradient of 0–20%
ethyl acetate in cyclohexane as the eluent. The eluent furnished a white
solid (0.14 g,30%). [ a]D20 =ꢀ5 (c=1.00 in CH2Cl2). 1H NMR (400 MHz,
Oxidative cleavage of peptides from the solid support: The resin was
treated with a solution of Cu(OAc)2 (0.5 equiv with respect to the initial
resin loading),pyridine (30 equiv),acetic acid (50 equiv),and nucleophile
(215 equiv)—methanol in dichloromethane or water in THF (5 mL/0.1 g
of resin)—for 3 h under an oxygen atmosphere. The resin was filtered,
and the solvent was evaporated under reduced pressure. The crude resi-
due was washed three times with toluene and after each washing it was
evaporated to dryness. The oily residue was purified by using a short
silica gel column (2 or 5 mL) with ethyl acetate and methanol (0–5% de-
pending on the peptide) as the eluent.
CDCl3): d=7.77 (d, J=7.4 Hz,2H),7.61 (m,2H),7.41 (t,
J=7.4 Hz,
Fmoc-Cys(Pal)-OH (2): Trifluoroacetic acid (2.5 mL) and triethylsilane
2H),7.32 (t, J=7.4 Hz,2H),4.65 (m,1H,Fmoc),4.42 (m,2H),4.25 (t,
(1.5 mL) were added to
a solution of Fmoc-Cys(Trt)-OH (1) (2 g,
J=7.0 Hz,1H),3.05 (m,2H),2.56 (m,2H),1.57 (t,
J=7.2 Hz,2H),1.26
3.4 mmol) in CH2Cl2 (50 mL),and the mixture was stirred at room tem-
perature for 2 h under an argon atmosphere. The solvent was evaporated
under reduced pressure,and the crude residue was washed three times
with toluene and after each washing it was evaporated to dryness. The
solid residue was transferred to a sinter funnel and washed twice with
pentane to remove the released triphenylmethane. The remaining solid
(1.2 g) was dissolved in CH2Cl2 (25 mL),trimethylsilyl chloride (0.48 mL,
3.7 mmol) was added,and the reaction mixture was heated to reflux for
2 h. After the mixture was cooled to room temperature,palmitoyl chlo-
ride (3.1 mL,10.2 mmol) was added,followed by dropwise addition of a
(m,28H),0.89 ppm (t, J=7.0 Hz,3H); 13C NMR (100 MHz,CDCl ): d=
3
174.9,156.6,143.7,141.3,127.7,127.1,125.1,120.0,67.4,53.6,47.1,34.2,
32.9,31.9,29.7,29.4,29.2,28.8,22.7,14.1 ppm; (FAB,3-NBA): calcd for
C34H49NO4S [M+Na]+ 590.3280,found 590.3300.
Fmoc-Cys(GerMant)-OH (6): A solution of cysteine hydrochloride mono-
hydrate (0.39 g,2.25 mmol) in MeOH (8 mL) was cooled to 0 8C,and a
7n solution of ammonia in MeOH (10 mL) was added slowly. After
[12]
5 min,GerMant-chloride
((E,E)-8-O-(2-N-methyl-aminobenzoyl)-3,7-
dimethyl-2,6-octandiene-1-chloride) (0.47 g, 1.5 mmol) was added and the
mixture was stirred at 08C for 3 h and then at room temperature for 1 h.
The solvent was evaporated,the solid residue was washed with pentane
(3”5 mL),and CH 2Cl2 (20 mL) was added. The resulting suspension was
cooled to 08C and triethylamine (0.31 mL,2.25 mmol) and Fmoc-succini-
mide (0.76 g,2.25 mmol) were added. The reaction mixture was stirred
overnight at room temperature and then concentrated in vacuum. The
oily residue was purified by flash chromatography on silica gel using a
gradient of 0–4% methanol in dicholoromethane as eluent to afford a
colorless oil (0.53 g,57%). Rf =0.7 (dichloromethane/methanol 9:1).
[a]2D0 =ꢀ1.0 (c=0.93 in CHCl3). 1H NMR (400 MHz,CDCl 3): d=7.93 (d,
J=8.0 Hz,1H),7.75 (d, J=7.5 Hz,2H),7.62–7.58 (m,2H),7.42–7.34 (m,
3H),7.28 (t, J=7.5 Hz,2H),6.66 (d, J=8.0 Hz,1H),6.59 (t, J=8.0 Hz,
1H),5.66 (d, J=6.9 Hz,1H),5.46 (t, J=6.9 Hz,1H),5.21 (t, J=7.5 Hz,
1H),4.63–4.58 (m,3H,distinguished singlet at 4.63 ppm),4.45–4.38 (m,
2H),4.22 (t, J=7.0 Hz,1H),3.25–3.13 (m,2H),3.04–2.88 (m,5H,distin-
guished singlet at 2.89 ppm),2.20–2.15 (m,2H),2.10–2.05 (m,2H),1.71
(s,3H),1.65 ppm (s,3H); 13C NMR (100 MHz,CDCl 3): d=175.1,168.6,
156.1,152.2,143.8,141.4,139.7,134.8,131.8,130.8,128.6,127.9,127.3,
125.3,120.3,120.2,114.6,111.1,110.2,70.1,67.7,53.8,47.5,39.3,33.6,
solution of triethylamine (0.78 mL,5.6 mmol) in CH Cl2 (15 mL) over 3 h
2
under an argon atmosphere. After the mixture had been stirred for one
hour,the solvent was evaporated,and the crude mixture was purified by
flash chromatography on silica gel using a gradient of 0–20% ethyl ace-
tate in cyclohexane. The eluent furnished a white solid (1.6 g,82%). Rf =
0.6 (ethyl acetate/cyclohexane (2:1),1% acetic acid). m.p. 85–86 8C.
1
[a]2D0 =ꢀ7.2 (c=1.69 in CH2Cl2). H NMR (400 MHz,CDCl ): d=7.76 (d,
3
J=7.5 Hz,2H),7.62–7.55 (m,2H),7.40 (t,
J=7.5 Hz,2H),7.29 (t, J=
7.5 Hz,2H),5.64 (d, J=7.8 Hz,1H),4.62 (m,1H),4.39 (d,
2H),4.24 (t, J=7.2 Hz,1H),3.50–3.34 (m,2H),2.59 (d,
1.70–1.60 (m,2H),1.23 (s,24H),0.89 ppm (t,
J=7.2 Hz,
J=7.6 Hz,2H),
J=6.9 Hz,3H); 13C NMR
(100 MHz,CDCl 3): d=199.5,174.5,156.2,143.8,141.4,127.9,127.3,
125.4,120.2,67.8,62.8,54.3,47.4,44.5,32.4,30.8,30.12,30.11,30.10,
30.08,30.03,29.9,29.8,29.7,29.4,26.0,23.1,14.6 ppm; MS (FAB,
3-NBA): calcd for C34H47NO5S [M+Na]+ 604.3073,found 604.3085.
Fmoc-Cys(Far)-OH (4): A solution of cysteine hydrochloride monohy-
drate (3) (1.0 g,5.7 mmol) in MeOH (11 mL) was cooled to 0 8C,and a
7n solution of ammonia in MeOH (15 mL) was slowly added. After
5 min farnesyl chloride (1.38 mL,5.7 mmol) was added. The reaction
mixture was stirred at 08C for 3 h and then at room temperature for 1 h,
the solvent was evaporated,the solid residue was washed with pentane
(3”10 mL),and CH 2Cl2 (50 mL) was added. The resulting suspension
was cooled to 08C and triethylamine (0.88 mL,6.28 mmol) and Fmoc-
succinimide (2.12 g,6.28 mmol) were added. The reaction mixture was
stirred overnight at room temperature and concentrated in vacuum. The
product was obtained by flash chromatography on silica gel using a gradi-
ent of 0–4% methanol in dicholoromethane as the eluent to give a pale
yellow oil that can be triturated with pentane to yield a sticky solid
(2.3 g,65%). Rf =0.7 (dichloromethane/methanol (9:1)). [a]2D0 =ꢀ4.3 (c=
30.4,30.0,26.4,16.6,14.6 ppm; MS (FAB,3-NBA): calcd for
C36H40N2O6S [M+H]+ 629.2685,found 629.2653,[ M+Na]+ 651.2505,
found 651.2496.
Fmoc-Lys(BP)-OH (10): Fmoc-Lys-OH (0.40 g,1.10 mmol) was added
was added to a solution of 4-benzoylbenzoic acid (0.25 g,1.10 mmol),
HATU (0.42 g,1.10 mmol),and DIPEA (0.20 mL,2.20 mmol) in DMF
(20 mL). The solution was stirred at room temperature for 5 h. After
evaporation of the solvent,the residue was dissolved in AcOEt and
washed with H2O,dried over MgSO 4,and concentrated in vacuum to
afford a white solid (0.48 g,75%). [ a]2D0 =ꢀ 2.1 (c=0.92 in CH2Cl2).
1H NMR (400 MHz,CDCl 3) d=7.85 (d, J=8.4 Hz,2H),7.74–7.69 (m,
6H),7.57–7.52 (m,3H),7.44 (t, J=7.4 Hz,2H),7.34 (t, J=7.4 Hz,2H),
7.24 (t, J=7.4 Hz,2H),4.40–4.30 (m,2H),44.17–4.09 (m,2H),3.46 (m,
1
0.94 in CHCl3). H NMR (400 MHz,CDCl 3): d=7.76 (d, J=7.4 Hz,2H),
7.61 (d, J=7.5 Hz,2H),7.40 (t, J=7.4 Hz,2H),7.31 (t, J=7.4 Hz,2H),
5.60 (d, J=8.0 Hz,1H),5.22 (t, J=7.4 Hz,1H),5.12–5.06 (m,2H),4.63
13C NMR (100 MHz,CDCl 3): d=196.5,
(m,1H),4.47–4.39 (m,2H),4.24 (t,
J=7.0 Hz,1H),3.27–3.15 (m,2H),
2H),1.95–1.47 ppm (m,6H);
3.04–2.90 (m,2H),2.14–1.93 (m,8H),1.68 (s,3H),1.65 (s,3H),
1.59 ppm (s,6H); 13C NMR (100 MHz,CDCl 3): d=175.7,156.1,143.8,
141.4,140.5,135.6,131.5,127.9,127.3,125.3,124.5,123.9,120.2,119.6,
67.7,53.9,47.5,40.1,40.0,33.5,30.5,27.2,26.9,26.2,18.2,16.6,16.5 pm;
MS (FAB,3-NBA): calcd for C 33H41NO4S [M+Na]+ 570.2654,found
570.2760.
167.6,156.6,150.5,143.8,141.5,140.3,137.1,133.2,130.3,128.7,127.3,
120.2,67.3,60.7,47.3,40.1,32.0,29.0,22.6,14.2 ppm; MS (FAB,3-NBA):
calcd for C35H33N2O6S [M+H]+ 577.2260,found 577.2365.
Fmoc-Cys(Pal)-Lys(Aloc)-Cys(Far)-OMe (11): Yield: 0.051 mmol; 69%.
[a]2D0 =ꢀ21.2 (c=0.20 in CH2Cl2). 1H NMR (400 MHz,CDCl 3): d=7.68
(d, J=7.6 Hz,2H,Fmoc),7.52 (d,
J=9.6 Hz,2H; Fmoc),7.32 (t, J=
Fmoc-d-Cys(Far)-OH: The synthesis was performed as described for the
l-enantiomer. [a]2D0 =+ 4.2 (c=0.94 in CHCl3).
7.4 Hz,2H,Fmoc),7.23 (t, J=7.4 Hz,2H; Fmoc),6.94 (s,1H; NH),6.82
(s,1H),5.83–5.75 (m,2H),5.19 (d, J=18 Hz,1H),5.15–4.97 (m,5H),
4.66 (m,1H),4.50–4.24 (m,6H,Fmoc,Aloc),4.14 (m,1H),3.67 (s,3H,
OMe),3.30–2.75 (m,8H),2.51 (t, J=7.4 Hz,2H,Pal),2.1–1.81 (m,8H,
Far),1.68–1.59 (m,2H,Pal),1.60 (s,3H,Far),1.58 (s,3H; CH 3 Far),1.52
Fmoc-Cys(HD)-OH (5): Trifluoroacetic acid (1 mL) and triethlysilane
(0.6 mL) were added to a solution of Fmoc-Cys(Trt)-OH (1) (0.5 g,
3.4 mmol) in CH2Cl2 (20 mL),and the mixture was stirred at room tem-
7412
www.chemeurj.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
Chem. Eur. J. 2005, 11,7405 – 7415

Solid-Phase Synthesis of Lipidated Peptides
FULL PAPER
(s,6H,Far),1.46–1.43 (m,2H),1.36–1.33 (m,2H),1.18 (s,24H,Pal),
0.81 ppm (t, J=7.0 Hz,3H,Pal); MS (ESI +): calcd for C63H94N4O9S2
[M+H]+: 1115.7; found: 1115.3.
MIC-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe
(22):
Yield:
1
0.022 mmol; 30%. [a]2D0 =ꢀ22.0 (c=0.42 in CH2Cl2). H NMR (400 MHz,
CDCl3): d=6.62 (s,2H,MIC),5.12 (t, J=7.2 Hz,1H,Far),5.02 (m,2H,
Far),4.52 (m,1H) 4.33 (t, J=7.0 Hz,1H),4.20 (t, J=7.0 Hz,1H),3.99
(m,2H),3.78–3.44 (m,7H,distinguished singlet at 3.68,OMe),3.12–3.23
Fmoc-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe
(12):
Yield:
1
0.044 mmol; 60%. [a]2D0 =ꢀ17.0 (c=0.40 in CH2Cl2). H NMR (400 MHz,
(m,3H),2.89–3.04 (m,2H),2.65–2.70 (m,1H),2.54 (t,
J=7.2 Hz,2H,
[D6]DMSO): d=7.69 (d, J=7.6 Hz,2H,Fmoc),7.53 (m,2H,Fmoc),7.33
Pal),2.46 (t, J=7.2 Hz,2H),2.19–1.95 (m,19H),1.66–1.53 (m,17H,dis-
tinguished singlets at 1.66,1.62 and 1.58,Far),1.35–1.15 (m,30H),0.92–
0.79 ppm (m,9H,Leu,Pal); MS (ESI): calcd for C 68H112N8O12S3 [M+H]+
1329.8; found 1329.6.
(t, J=7.4 Hz,2H,Fmoc),7.24 (t,
J=7.2 Hz,2H,Fmoc),5.10 (t,
J=
7.2 Hz,1H,Far),5.01 (m,2H,Far),4.70 (m,1H),4.49 (m,1H),4.30 (m,
2H),4.15 m (1H),36, 5–3.48 (m,5H,distinguished singlet at 3.60,OMe),
2.51 (t, J=7.3 Hz,2H,Pal),2.15–1.81 (m,17H),1.65–1.40 (m,14H,dis-
tinguished singlets at 1.60 and 1.52,Far),1.35–1.17 (m,30H),0.92–0.85
MIC-Gly-Cys(HD)-Met-Gly-Leu-Pro-Cys(Far)-OH
(23):
Yield:
0.012 mmol; 25%. [a]2D0 =ꢀ20.4 (c=0.30 in CH2Cl2). H NMR (400 MHz,
1
(m,4H),0.80 ppm (t,
J=7.0 Hz,6H,Leu,Pal); MS (ESI): calcd for
C71H108N6O10S3 [M+H]+ 1301.7; found 1301.4.
CDCl3): d=6.65 (s,2H,MIC),5.10 (t, J=7.2 Hz,1H,Far),5.00 (m,2H,
Far),4.50 (m,1H),4.30 (t,
J=7.0 Hz,1H),4.20 (t, J=7.0 Hz,1H),3.99
Fmoc-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe
(15):
Yield:
(m,2H),3.78–3.44 (m,4H),3.12–3.23 (m,3H),2.89–3.04 (m,2H),2.65–
2.70 (m,1H),2.45 (t, J=7.2 Hz,2H),2.19–1.95 (m,21H),1.64–1.53 (m,
17H,distinguished singlets at 1.64,1.61 and 1.57,Far),1.30–1.10 (m,
32H),0.92–0.79 ppm (m,9H,Leu,HD); MS (ESI): calcd for
C67H112N8O11S3 [M+H]+ 1300.8 found 1300.9.
1
0.031 mmol; 40%. [a]2D0 =ꢀ27.0 (c=0.22 in CH2Cl2). H NMR (400 MHz,
CDCl3): d=7.74 (d, J=7.3 Hz,2H,Fmoc),7.60 (m,2H,Fmoc),7.37 (t,
J=7.3 Hz,2H,Fmoc),72,8 (t,
J=7.3 Hz,2H,Fmoc),5.13 (t, J=7.2 Hz,
1H,Far),5.07 (m,2H,Far),4.82 (m,1H),4.65 (m,2H),4.51 (m,1H),
4.33 (t, J=7.0 Hz,1H),4.20 (t, J=7.0 Hz,1H),3.99 (m,2H),3.77–3.45
(m,5H,distinguished singlet at 3.68,OMe),3.12–3.23 (m,3H),2.89–3.04
MIC-Gly-Cys(Far)-Met-Gly-Leu-Pro-Cys(Far)-OMe
(24):
Yield:
0.048 mmol; 14%. [a]2D0 =ꢀ15.5 (c=1.28 in CH2Cl2). H NMR (400 MHz,
CDCl3): d=6.62 (s,2H,MIC),5.02–5.12 (m,6H,Far),4.7–4.2 (m,8H),
3.6–3.1 (m,7H),2.6–2.15 (m,10H),2.1–1.3 (m,27H),1.3–0.99 (m,24H,
1
(m,2H),2.65–2.70 (m,1H),2.52 (t,
J=7.2 Hz,2H,Pal),2.46 (t,
J=
7.2 Hz,2H),2.19–1.95 (m,17H),1.66–1.53 (m,17H,distinguished sin-
glets at 1.66,1.62 and 1.58,Far),1.35–1.15 (m,24H),0.95–0.75 ppm (m,
Far),0.6–0.95 ppm (6H,Leu); MS (ESI): calcd for
C
67H106N8O11S3
9H,Leu,Pal); MS (ESI): calcd for C 73H111N7O11S3 [M+H]+ 1358.8;
[M+H]+ 1295.7; found 1295.7.
found 1358.6.
MIC-Gly-Cys(Pal)-Met-Lys(BP)-Leu-Pro-Cys(Far)-OMe (25): Yield:
0.018 mmol; 25%. [a]2D0 =ꢀ25.0 (c=0.20 in MeOH). 1H NMR (400 MHz,
Fmoc-Ser(Trt)-Cys(Pal)-Lys(Aloc)-Cys(Far)-OMe
(16):
Yield:
1
0.041 mmol; 56%. [a]2D0 =ꢀ20.3 (c=0.21 in CH2Cl2). H NMR (400 MHz,
CDCl3): d=7.81 (d, J=8.4 Hz,2H,BP),7.74 (d,
J=7.3 Hz,2H,BP),
J=
J=7.2 Hz,1H,Far),5.02 (m,
CDCl3): d=7.69 (d, J=7.5 Hz,2H,Pal),7.57–7.52 (m,2H,Pal),7.37–
7,65 (d, J=7.3 Hz,2H,BP),7.55 (t,
8.4 Hz,2H,BP),6.60 (s,2H,MIC),5.13 (t,
J=7.4 Hz,1H,BP),74,6 (2H,
7.20 (m,19H,Pal,Trt),5.87–5.75 (m,2H),5.21 (d,
J=17.2 Hz,1H,Far)
5.15–4.93 (m,5H,Far),4.68–3.92 (m,10H),3.78–3.65 (m,5H,distin-
2H,Far),4.52 (m,1H),4.30 (t,
J=7.0 Hz,1H),4.20 (t, J=7.0 Hz,1H),
guished singlet at 3.68 ppm,OMe),3.35–2.72 (m,8H),2.52 (t,
2H,Pal),2.05–1.85 (m,10H),1.65–1.25 (m,18H,distinguished singlets
at 1.60,1.59,and 1.52,Far),1.18 and 1.16 (2”s,24H),0.81 ppm (t,
J=7.6 Hz,
3.96 (m,2H),3.75–3.44 (m,7H,distinguished singlet at 3.65,OMe),
3.12–3.20 (m,3H),2.89–3.04 (m,4H),2.65–2.70 (m,1H),2.51 (t,
7.3 Hz,2H,Pal),2.15–1.90 (m,25H),1.62–1.47 (m,17H,distinguished
singlets at 1.65,1.62 and 1.57,Far),1.35–1.15 (m,30H),0.92–0.79 ppm
J=
J=
6.9 Hz,3H,Pal); MS (ESI): calcd for C 85H113N5O11S2 [M+Na]+ 1466.8,
found 1446.8.
(m,9H,Leu,Pal); MS(ESI): calcd for C 86H129N9O14S3 [M+H]+ 1608.9;
Fmoc-Gly-Thr-Gln(Trt)-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe
(19): Yield: 0.022 mmol; 30%. [a]2D0 =+ 1.3 (c=0.74 in CH2Cl2).
1H NMR (400 MHz,CDCl 3): d(characteristic signals)=7.67 (m,Fmoc),
7.50 (m,Fmoc),7.33 (m,Fmoc),7.00–7.22 (m,Fmoc),5.01 (m,Far),
3.60–3.70 (m,distinguished singlet at 3.65,OMe),1.42–1.70 (m,distin-
found 1608.6.
Biotin-Aca-Gly-Cys(Pal)-Met-Lys(BP)-Leu-Pro-Cys(Far)-OMe
(26):
Yield: 0.015 mmol,20%. [ a]2D0 =ꢀ24.0 (c=0.25 in MeOH). 1H NMR
(400 MHz,CDCl 3): d(characteristic signals)=7.85 (d, J=8.2 Hz,2H,
BP),7.78 (d, J=8,2 Hz, 2H, BP), 7.72 (d, J=7.0 Hz,2H,BP),7.55 (t, J=
7.4 Hz,1H,BP),7.43 (t, J=7.8 Hz,2H,BP),5.29 (m,1H),5.14 (m,1H,
Far),5.02 (m,2H,Far),3.69–3.60 (m,7H,distinguished singlet at 3.69,
OMe),2.02–1.89 (m,14H),1.65–1.45 (m,12H,distinguished singlets at
1.61 and 1.53,Far),1.35–1.10 (m,30H,Pal),0.83–0.77 ppm (m,9H,Leu,
Pal); MS(ESI): calcd for C92H143N11O14S4 [M+H]+ 1754.0; found 1754.7.
guished singlets at 1.62 and 1.53,4 CH Far),0.90–14, 0 (m,12 CH
Pal),
2
3
0.75–0.85 ppm (m,CH
C
Pal,2 CH
Leu); MS (ESI): calcd for
3
3
103H143N11O16S3 [M+H]+ 1887.0; found 1887.3.
Fmoc-Ser-Ser-Asp(OtBu)-Asp(OtBu)-Gly-Thr(OtBu)-Gln(Trt)-Gly-Cys-
(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe (20): Yield: 0.010 mmol; 12%.
[a]2D0 =ꢀ20.4 (c=0.21 in CH2Cl2). 1H NMR (400 MHz,CDCl 3): d(charac-
teristic signals)=7.69 (m,Fmoc),7.54 (m,Fmoc),7.33 (m,Fmoc),7.04–
7.23 (m,Fmoc,Trt),5.01 (m,Far),3.60–3.70 (m,distinguished singlet at
Aloc-Cys(S-tBu)-Met-Ser-Cys(Pal)-Lys(NBD)-Cys(Far)-OMe
(27):
Yield: 0.020 mmol; 28%. [a]2D0 =ꢀ18.4 (c=0.30 in CH2Cl2). 1H NMR
(400 MHz,CDCl 3): d(characteristic signals)=8.46 (m,1H,NBD),6.15
3.66,OMe),1.42–1.70 (m,distinguished singlets at 1.60 and 1.53,4 CH
(m,1H,NBD),5.88 (m,1H,Aloc),3.72 (s,3H,OCH
7.1 Hz,2H,Pal),1.99 (s,3H),1.65 (s,3H,Far),1.57 (s,6H,Far),1.31 (s,
9H, tBu),1.22 (s,24H,Pal),0.85 ppm (t, J=7.1 Hz,3H,Pal); MS
69H112N10O14S5 [M+Na]+ 1487.7,found
3),2.55 (t, J=
3
Far),0.90–14,0 (m,12 CH
Pal, tBu),0.76–0.85 ppm (m,CH Pal,2 CH
3 3
2
Leu); MS (MALDI,DHB): calcd for C 129H187N15O26S3 [M+Na]+ 2481.3;
found 2481.8; [M+K]+ 2497.3; found 2497.9.
(MALDI,DHB): calcd for
1490.0.
C
NH2-Gly-Cys(Pal)-Gln-His-Gln-Phe-Arg-Gly-Arg-Arg-Trp-Asn-Cys-Thr-
NBD-Aca-Gly-Cys(SBut)-Met-Gly-Leu-Pro-Cys(Far)-OMe (28): Yield:
Thr-Val-OH (21): The Pbf,Boc,Trt,and
tBu groups were cleaved from
the amino acid residues on resin with 95% TFA and 5% water for 2 h.
The resin was treated with a solution of NBS (20 equiv) and pyridine
(20 equiv) in CH2Cl2 (3 mL) for 5 min,washed with DCM (3”5 mL),and
then with THF (3”5 mL). A solution of water (50 equiv) in THF (3 mL)
was added and the mixture was stirred for 4 h at room temperature. The
resin was filtered off and washed four times with THF and the solvent
0.013 mmol,22%.
[
a]2D0 =ꢀ 32.3 (c=0.6 in CH2Cl2). 1H NMR
(400 MHz.CDCl3): d=8.42 (d, J=8.2,1H,NBD),6.1 (m1, H,NBD),5.2–
4.95 (m,3H,Far),4.78–4.1 (m,8H),3.7–3.3 (m,9H),3.2–2.5 (m,6H),
2.31–2.15 (m,2H),2.1–1.6 (m,19H),1.6–1.3 (m,2H),1.3–0.99 (m,27H),
0.6–0.95 ppm (m,6H); MS (MALDI): calcd for C 58H91N11O12S4 [M+Na]
1285.67 found 1286.28.
was evaporated under reduced pressure. Yield: 0.023 mmol; 45%. [a]D20
=
MIC-Gly-Cys(Pal)-Met-Lys(NBD)-Leu-Pro-Cys(Far)-OMe (29): Yield:
1
1
0.020 mmol; 28%. [a]2D0 =ꢀ28.3 (c=0.60 in CH2Cl2). H NMR (400 MHz,
+3.9 (c=0.43 in MeOH). H NMR (400 MHz,CD 3CN): d=(characteris-
tic signals) 8.77 (m,2H,NH Trp,NH His),7.98–7.93 (m,19H,C
=NH
CDCl3): d=8.36 (d, J=8.4 Hz,1H,NBD),6.62 (s,2H,MIC),6.09 (m,
1H),5.12 (t, J=7.2 Hz,1H,Far),5.02 (m,2H,Far),4.52 (m,1H) 4.33 (t,
J=7.0 Hz,1H),4.20 (t, J=7.0 Hz,1H),3.99 (m,2H),3.78–3.44 (m,7H,
distinguished singlet at 3.68,OMe),3.12–3.23 (m,3H),2.89–3.04 (m,
4H),2.65–2.70 (m,1H),2.54 (t, J=7.2 Hz,2H,Pal),2.19–1.95 (m,25H),
Arg,NH),7.73–7.63 (m,6H,
m-Phe,Trp),7.34–7.27 (m,3H,
o-Phe, p-
Phe),7.00 (s,2H,His,Trp),2.67 (t,
J=7.0 Hz,2H,Pal),1.35–1.29 (m,
28H,Pal),1.21 (d, J=4.3 Hz,6H,Thr),0.96–0.89 ppm (m,9H,Pal,Val);
MS (ESI): calcd for C97H155N31O23S2 [M+2Na]2+ 1116.1; found 1115.7.
Chem. Eur. J. 2005, 11,7405 – 7415
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
7413

H. Waldmann et al.
1.66–1.53 (m,17H,distinguished singlets at 1.66,1.62 and 1.58,Far),
1.35–1.15 (m,30H),0.92–0.79 ppm (m,9H,Leu,Pal). MS (ESI): calcd
for C78H122N12O15S3 [M+H]+ 1563.8; found 1563.6.
6.95 (m,1H,Bodipy),6.82–6.87 (m,1H,Bodipy),6.61 (s,2H,MIC),5.28
(m,1H),5.02 (m1, H),3.70–3.59 (m,5H),2.49 (m,2H),2.18–2.16 (m,
2H),1.98–1.92 (m,6H),1.70–1.40 (m,12H,distinguished singlets at 1.60
and 1.53),1.35–1.10 (m,28H,Pal),0.83–0.77 ppm (m,12H,Leu,Pal);
MS (ESI): calcd for C86H134BF2N11O13S3 [M+H]+ 1674.94,found 1674.78;
[MꢀF+H]+ 1655.94,found 1655.14.
NBD-Aca-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe (30): Yield:
1
0.019 mmol; 32%. [a]2D0 =ꢀ18.4 (c=0.30 in CH2Cl2). H NMR (400 MHz,
CDCl3): d=8.1 (d, J=8.2,1H,NBD),6.5 (m,1H,NBD),5.4–5.2 (m,
3H,Far),4.8–4.1 (m,8H),3.7–3.3 (m,9H),3.3–2.7 (m,6H),2.6–2.4 (m,
4H),2.1–1.6 (m,19H,CH 2),1.7–13 (m,16H,Far),1.3–1.0 (m,30H),0.7–
Biological assay: The peptide 21 was solubilized in DMSO at 5 mm con-
centration. L-M(TK-) cells (L cells) from murine subcutaneous connec-
tive tissue and Wnt3a transfected L cells (L Wnt3a cells) were purchased
from ATCC and cultured in Dulbeccoꢁs modified Eagleꢁs medium with
10% fetal calf serum at 378C and 10% CO2.[21] L Wnt3a cells express
and secrete biologically active Wnt-3a.[22] Conditioned media from L cells
or from L Wnt3a cells were prepared as described.[16] Assays to test the
activity of the Wnt peptide were performed as described for the purified
protein.[15a] L cells at approximately 75% confluence were incubated for
4.5 h with conditioned media from L cells or from L Wnt3a cells. In par-
allel,L cells were incubated with different concentrations of the peptide
21 and of DMSO as control. Assays were performed with the peptide or
DMSO in a full medium containing 10% fetal calf serum. After incuba-
tion,the cells were washed with PBS and lysed in cell lysis buffer (25 m m
Tris/HCl,2 m m EDTA,10% glycerol,1% Triton,2 m m DTE,pH 7.8) by
incubation for 5 min on ice. Total lysates were equilibrated to equal pro-
tein concentrations with cell lysis buffer. Samples were electrophoresed
on 12.5% SDS polyacrylamide gels and electro-blotted on a PVDF mem-
brane (Amersham Biosciences). Membranes were blocked in TPBS
(PBS,0.1% Tween) with 5% powdered milk. The membrane was probed
with a monoclonal anti-b-catenin antibody (Transduction Laboratories)
at 1:1000 dilution or with a monoclonal anti-GAPDH antibody (abcam)
at 1:30000 dilution in TPBS for 1 h at room temperature. After the mem-
brane had been washed three times in TPBS,it was probed with a horse-
raddish peroxidase coupled goat-anti-mouse antibody (Amersham Biosci-
ences) as secondary antibody. Luminescence detection was performed
with the commercial ECL Plus Western blot detection system (Amer-
sham Biosciences).
0.95 (6H,CH
Leu,Pal) ppm. MS (ESI): calcd for C 70H113N11O13S3
3
[M+H]+ 1412.7 found 1412.9.
NBD-Aca-Gly-Ser-Met-Gly-Leu-Pro-Cys(Far)-Val-Val-Met-OH
(31):
Yield: 0.018 mmol,25%. [ a]2D0 =ꢀ27.3 (c=0.36 in CH2Cl2). 1H NMR
(400 MHz.CDCl3): d=8.42 (m,1H,NBD),6.9 (m1,H,NBD),5.3 (m,
3H,Far),4.0–3.2 (m,20H),3.0–2.5 (m,8H),2.4–2.2 (m,4H),2.3–1.5 (m,
23H),1.5–1.0 (m1,8H),0.5–0.96 ppm (m,18H); MS (ESI): calcd for
C68H108N14O16S3 [M+H]+ 1473.7,found 1473.3.
H-Cys(S-tBu)-Met-Lys(NBD)-Leu-Pro-Cys(Far)-OMe
(32):
(0.028 mmol,38%). [a]2D0 =ꢀ 15.4 (c=0.40 in CH2Cl2). 1H NMR
(400 MHz,CDCl ): d=8.40 (d, J=8.4 Hz,1H,NBD),6.09 (m,1H),5.13
3
(m,1H,Far),5.01 (m,2H,Far),4.55–4.65 (m,2H),3.66–3.72 (m,5H,
OMe),3.41–3.53 (m,2H),2.95–3.15 (m,2H),1.90–2.05 (m,17H),1.53–
1.61 (m,21H),1.19 (s,9H,Leu),0.81–0.87 ppm (m,6H); MS (ESI):
calcd for C54H86N10O10S4 [M+H]+ 1163.5; found 1163.4.
NBD-Aca-Gly-Cys(S-tBu)-Met-Gly-Leu-Pro-Cys(Far)-OH (33): Yield:
0.011 mmol,37%. [ a]2D0 =ꢀ6.2 (c=0.93 in CH2Cl2). 1H NMR (400 MHz,
CDCl3): d=8.2 (d, J=8.3,1H,NBD),6.08 (m,1H,NBD),5.4–5.2 (m,
3H,Far),4.7–4.1 (m,8H),3.7–3.38 (m,6H),3.1–2.5 (m,6H),2.15 (m,
2H),2.00–1.8 (m,19H),1.8–1.4 (m,2H),1.3–1.0 (m,27H),1.0–0.7 ppm
(m,6H,Leu); MS-MALDI (DBH): calcd for C 57H89N11O12S4 [M+Na]
1271.7; found 1271.9.
Ac-Met-Ser-Cys(SBut)-Lys(NBD)-Cys(Far)-OMe (34): Acetylation of
N-terminus: after Fmoc-cleavage from the Met residue,the N-terminus
was acetylated using a cocktail of five equivalents of acetic acid/HBTU/
HOBt/DIPEA in CH2Cl2/DMF (1:1) for 4 h. Yield: 0.009 mmol; 12%.
[a]2D0 =95.2 (c=0.70 in CH2Cl2). 1H NMR (400 MHz.CDCl3): d=8.42
(m1, H,NBD),6.10 (m,1H,NBD),5.30 (m1, H,Far),5.00 (m,2H,Far),
4.78–4.10 (m,4H),3.70–3.00 (m,10H,OMe),2.80 (m,2H),3.21–2.50
(m,6H),2.10–1.81 (m,15H),1.80–1.20 (m,6H) 1.20–1.02 ppm (m,21H,
Far); MS-(ESI): calcd for C48H75N9O11S4 [M+H]+ 1083.43; found
1083.50.
Acknowledgements
This research was supported by the Deutsche Forschungsgemeinschaft,
The Max-Planck-Gesellschaft,and the Fonds der Chemischen Industrie.
Jose M. Palomo would like to thank the European Molecular Biology
Organization (EMBO) for a long-term Fellowship and G. Kragol is grate-
ful to the Humboldt Foundation for a Research Fellowship.
NBD-Aca-Gly-d-Cys(Trt)-d-Met-Gly-d-Leu-d-Pro-d-Cys(Far)-OMe (35):
Yield: 0.035 mmol; 40%. [a]2D0 =+3.0 (c=0.71 in CH2Cl2). 1H NMR
(400 MHz.CDCl3): d=8.42 (m ,1H, NBD), 7.06–7.45 (m,15H, Trt), 6.9
(m,1H, NBD),5.2–4.95 (m, 3H, Far), 4.78–4.1 (m, 8H), 3.7–3.3 (m, 9H),
3.2–2.5 (m,6H),2.31–2.15 (m,2H),2.1–1.3 (m,21H,Far),1.3–0.99 (m,
18H,Far,Aca),0.6–0.95 ppm (m,6H,Leu); MS (ESI): calcd for
C73H97N11O12S3 [M+Na]+ 1438.65; found 1438.71.
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3
3
CHCA): calcd for C72H113N9O13S3 [M+Na]+ 1430.8,found 1434.2; calcd
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1
CDCl3): d=7.63–7.58 (m,1H,Bodipy),7.41–7.38 (m,1H,Bodipy),7.11–
7414
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Chem. Eur. J. 2005, 11,7405 – 7415

Solid-Phase Synthesis of Lipidated Peptides
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Published online: September 29,2005
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Chem. Eur. J. 2005, 11,7405 – 7415
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
7415