Acid-labile protecting groups for the synthesis of lipidated peptides

Article abstract of DOI:10.1002/1521-3765(20010316)7:6<1184::AID-CHEM1184>3.0.CO;2-5

Lipidated peptides and their neolipoprotein derivatives are efficient tools for the investigation of biological processes in molecular detail. These compounds are often acid- and base-labile, and their synthesis requires the use of a combination of blocki

Full text of DOI:10.1002/1521-3765(20010316)7:6<1184::AID-CHEM1184>3.0.CO;2-5

FULL PAPER
Acid-Labile Protecting Groups for the Synthesis of Lipidated Peptides
Dieter Kadereit,[a, b] Patrick Deck,[a] Ines Heinemann,[a] and Herbert Waldmann*[a]
Abstract: Lipidated peptides and their
neolipoprotein derivatives are efficient
tools for the investigation of biological
processes in molecular detail. These
compounds are often acid- and base-
labile, and their synthesis requires the
use of a combination of blocking groups
that can be removed under very mild
conditions. In this article we demon-
strate that the Boc urethane and differ-
ent trityl-type protecting groups can be
cleaved selectively under acidic condi-
tions that are mild enough to be com-
patible with the demands of lipopeptide
synthesis. Thus, the Boc group was
cleaved with TMS triflate in the pres-
ence of lutidine, and the methyltrityl
(Mtt) and the methoxytrityl (Mmt)
group were removed with 1% TFA in
dichloromethane in the presence of
triethylsilane as cation scavenger. Re-
moval of the phenylfluorenyl group was
achieved with up to 3% TFA in di-
chloromethane in the presence of trie-
thylsilane at 08C. These protecting-
group techniques were successfully ap-
plied in the synthesis of differently
lipidated H-Ras peptides.
Keywords: lipids
´ lipoproteins ´
peptides ´ protecting groups ´ Ras
proteins
the synthesis of lipidated peptides is paramount to the success
of this method. These peptide conjugates are both base-labile
(if they embody palmitic acid thioesters) and acid-sensitive (if
they embody prenyl groups, i.e. farnesyl- or geranylgeranyl
thioethers) ruling out the use of many established protecting
groups. While the introduction of enzyme- and noble-metal-
sensitive protecting groups[3] provided efficient solutions to
this synthetic problem additional degrees of freedom are
urgently required if longer peptides and peptides incorporat-
ing amino acids with reactive side-chain functionalities like
NH2 and SH groups have to be constructed.[4] This problem is
particularly apparent in the case of our target compound, the
maleimido-modified H-Ras C-terminus 1a (Scheme 1). Apart
from the maleimido moiety, the structure carries an acid-
labile farnesyl thioether, a base-labile thioester and a
nucleophilic NH2 side chain that requires protection during
peptide synthesis or is susceptible to an undesired S ! N acyl
Introduction
Lipidated proteins embodying palmitic acid thioesters and
farnesyl or geranylgeranyl thioethers play important roles in
the transduction of extracellular signals across the cell
membrane and in numerous intracellular processes like
vesicle formation and targeting.[1, 2] Synthetic lipidated pro-
teins carrying different lipid modifications may be employed
as invaluable tools for the study of such biological processes in
molecular detail. For example, we recently described the
synthesis of lipidated Ras proteins based on the combination
of the techniques of molecular biology and organic synthesis,
that is, the bacterial expression of a truncated non-lipidated
Ras protein and its coupling with different lipidated peptides
via a maleimido linker.[2] Biophysical and cell-biological
investigation of these neolipoproteins then gave insight into
the mechanisms by which Ras proteins are specifically
targeted to the plasma membrane, a process which is crucial
to signalling via Ras. The availability of efficient methods for
NH2
MIC
[a] Prof. Dr. H. Waldmann, Dr. D. Kadereit, Dipl.-Chem. P. Deck,
Dipl.-Chem., Dipl.-Biol. I. Heinemann
Max-Planck-Institut für molekulare Physiologie
Universität Dortmund
O
N-Met-Ser-Cys-Lys-Cys-OMe
N
5
O
S
R
S
O
Fachbereich 3, Organische Chemie
R=Pal: 1a
R=Hd: 1b
R=StBu: 1c
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
Fax : (‡49)231-133-2499
O
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Pal:
Hd:
S
[b] Dr. D. Kadereit
StBu:
Institut für Organische Chemie
Universität Karlsruhe
Richard-Willstätter-Allee 2, 76128 Karlsruhe (Germany)
Scheme 1. Differently modified target peptides corresponding to the
H-Ras C-terminus for the synthesis of protein ± peptide conjugates.
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Chem. Eur. J. 2001, 7, No. 6

1184±1193
shift. Therefore, for the NH2 functionality a protecting group
was required that is compatible with the demands of lipo-
peptide synthesis. The obvious Pd0-catalyzed removal of allyl-
type protecting groups, which is one of the most powerful
methods for lipopeptide synthesis, cannot be employed in the
presence of maleimido groups since under the conditions
required for the removal of this blocking group undesired side
reactions occur, in particular attack of the alkyl-accepting
nucleophile and/or the phosphine ligands on the a,b-unsatu-
rated double bond.
Treatment of dipeptide 4 as test substrate with SnCl4 led to
rapid cleavage of the Boc group as well as to side reactions in
the farnesyl moiety, which were clearly evident as judged by
NMR. In contrast, application of a TMSOTf/lutidine mixture
removed the Boc group cleanly without side reactions
(Scheme 2). Furthermore, a wide range of amines could be
EEDQ
Fmoc-Lys(Boc)-Cys(Far)-OMe
Fmoc-Lys(Boc)-OH H-Cys(Far)-OMe
+
76 %
2
3
4
The very pronounced base lability of the palmitic acid
thioesters (hydrolysis occurs at pH > 7) excludes the use of
base-labile protection groups, an assumption that was easily
confirmed by some preliminary experiments. Deprotection by
oxidation or reduction was considered to be equally problem-
atic owing to the presence of the thioether and the double
bonds of the prenyl groups. However, initial orientating
experiments revealed that the prenyl thioethers tolerated very
low concentrations of acids like TFA. Since the palmitic acid
thioesters are stable to acid treatment we investigated the use
of different acid-sensitive protecting groups in lipopeptide
synthesis. In this paper we report that the Boc urethane and
different trityl-type blocking groups can be employed suc-
cessfully for the synthesis of multifunctional lipopeptides. We
demonstrate their use in the construction of maleimido-
modified H-Ras peptides and the coupling of these peptide
conjugates to a truncated H-Ras protein.
NH2
TMSOTf, lutidine,
CH2Cl2
H
N
Fmoc
CO2Me
N
75 %
H
O
S
EEDQ:
N
O
5
O
O
Scheme 2. Cleavage of a Boc group in the presence of a farnesyl moiety.
EEDQ: 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.
used including volatile amines like EtMe2N. Evaporation of
the excess amine and solvent after completion of the reaction
avoids an aqueous workup step, which can be a tedious pro-
cedure when multiply lipid-modified peptides are involved.
As an alternative for lysine side-chain protection, the
4-methyltrityl group[9] (Mtt) was investigated. This group can
be removed by treatment with a mixture of acetic acid,
trifluoroethanol, and CH2Cl2 (1:2:7) or a solution of 1% TFA
in CH2Cl2.[9] Application of the latter protocol resulted in
cleavage of the Mtt group in dipeptide 7. However, as we were
concerned about concentrating the TFA during solvent
removal, an excess of EtMe2N was added prior to distillation.
After neutralization partial retritylation occurred, indicating
that the addition of a cation scavenger is necessary. Sub-
sequently, treatment of dipeptide 7 with the TFA solution in
the presence of EtSiH3 gave 5 in excellent yield (Scheme 3).
Results and Discussion
From the numerous acid-labile protecting groups available for
the one approach, we chose the Boc group since it is one of the
standard urethanes widely employed in peptide chemistry for
which numerous deprotection conditions are available. For
the other approach, trityl-type groups removable under mild
conditions were investigated as possible alternatives for side-
chain protection, since for this purpose urethane protecting
groups are not necessary and since the lability of the trityl core
can be fine-tuned by introducing appropriate substituents into
the aromatic ring systems.
Initial experiments evaluating the suitability of these
protecting groups were carried out with model dipeptides of
the type Fmoc-Lys(Xxx)-Cys(Far)-OMe, which were synthe-
sized from an appropriately protected amino acid derivative
and S-farnesylated cysteine methyl ester.[5]
From the numerous different procedures for Boc cleavage,
treatment with SnCl4 in dichloromethane[6] and amine-buf-
fered trimethylsilyl trifluoromethanesulfonate (TMSOTf)[7]
were chosen as promising methods. Both had previously been
successfully used in the presence of acid-labile functionalities.
The former method was used for Boc deprotection in the
presence of thioamides,[6] while the latter represents a
procedure to remove tert-butyloxycarbonyl urethanes in the
presence of tert-butyl esters or the acid-labile Rink linker.[8]
Additionally, this technique offers the advantage that the
reactivity can be adjusted by the choice and the relative
amount of amine base.
EEDQ
Fmoc-Lys(Mtt)-Cys(Far)-OMe
Fmoc-Lys(Mtt)-OH H-Cys(Far)-OMe
+
75%
6
3
7
NH2
1% TFA/CH2Cl2,
Et3SiH
H
N
Mtt:
Fmoc
CO2Me
N
95 %
H
O
S
5
Scheme 3. Removal of the methyltrityl group in the presence of a farnesyl
moiety.
Motivated by this success, we investigated the limitations of
TFA-mediated removal of protecting groups in the presence
of acid-labile prenyl groups. Test reactions demonstrated that
Chem. Eur. J. 2001, 7, No. 6
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0947-6539/01/0706-1185 $ 17.50+.50/0
1185

H. Waldmann et al.
FULL PAPER
peptides incorporating the farnesyl group can be treated with
up to 5 vol% of TFA in CH2Cl2 for one hour without side
reactions. This was demonstrated by the removal of the
phenylfluorenyl group (PhFl) from 9 (Scheme 4). The PhFl
To start with, the allyloxycarbonyl (Aloc) group in the
palmitoylated tripeptide 14a[11] was replaced by the maleimi-
do linker group in a two-step procedure. Subsequent acid-
mediated removal of the tert-butyl ester gave fully modified
building block 15a (Scheme 6). The other building blocks, 16
EDC, HOBt
PhFl-Glu(OMe)-OH + H-Cys(Far)-OMe
PhFl-Glu(OMe)-Cys(Far)-OMe
96 %
1. [Pd(PPh3)4], DMB
2. MIC-OH, EEDQ
3. TFA
3
8
9
CO2CH3
H
Aloc-Met-Ser-Cys(Pal)-OtBu
MIC-Met-Ser-Cys(Pal)-OH
60 %
14a
15a
3 % TFA/DCM, Et3SiH, 0 °C
95 %
N
CO2CH3
H2N
O
Et2NH
CH2Cl2
S
PhFl:
Fmoc-Lys(PG)-Cys(Far)-OMe
H-Lys(PG)-Cys(Far)-OMe
PG=Boc: 4
PG=Mtt: 7
PG=Boc: 16
PG=Mtt: 17
10
Me2N
EDC:
· HCl
N C NEt
15a, EEDQ
MIC-Met-Ser-Cys(Pal)-Lys(PG)-Cys(Far)-OMe
Scheme 4. Removal of the phenylfluorenyl group in the presence of a
farnesyl moiety. EDC: 1-ethyl-3(dimethylamino)propylcarbodiimide hy-
drochloride, HOBt: 1-hydroxybenzotriazole.
PG=Boc: 76 %
PG=Mtt: 58 %
PG=Boc: 18a
PG=Mtt: 19a
1 % TFA
TMSOTf
lutidine
Et3SiH
96 %
group is 6000 times more stable towards hydrolysis than the
parent trityl group.[10] Nevertheless, it can be cleaved under
mild conditions. Upon treatment of PhFl-protected glutamyl
peptide 9 with 3% TFA in methylene chloride in the presence
of Et3SiH as cation scavenger, dipeptide 10 was isolated in
95% yield.
PG=Boc
PG=Mtt
side reaction
in prenyl group
S
MIC-Met-Ser-Cys-Lys-Cys-OMe
S
O
1a
Scheme 6. Application of Boc and Mtt protecting-group strategies in the
synthesis of 1a. DMB: N,N'-dimethyl barbituric acid.
Trityl-type protecting groups with comparable acid lability
are known for other functionalities as well. For instance, the S-
methoxytrityl group (Mmt) represents the equivalent thiol
group protection, and can be removed by treatment with 1%
TFA in dichloromethane. This was demonstrated for dipep-
tide 12 as a test substrate. The free thiol 13 was isolated in
quantitative yield under similar conditions to those used for
N-Mtt removal (Scheme 5).
and 17, were accessible by careful removal of the Fmoc group
from Boc- and Mtt-protected dipeptides 4 and 7, respectively.
Dipeptides of this type carry the intrinsic risk of diketopiper-
azine formation by nucleophilic attack of the unmasked lysine
a-amino group on the cysteine methyl ester, thereby render-
ing isolation of the dipeptides 16 and 17 problematic. This side
reaction was not observed during Fmoc removal. However,
after subsequent block condensation the diketopiperazines
were isolated as the major by-products.
EDC/HOBt
Fmoc-Cys(Mmt)-Cys(Far)-OMe
Fmoc-Cys(Mmt)-OH H-Cys(Far)-OMe
+
89 %
11
3
12
As the final step, the protecting groups on the lysine side
chain in pentapeptides 18a and 19a had to be removed. In the
case of the Boc group the conditions that were successfully
applied for deprotection of dipeptide 4 permitted undesired
side reactions in the prenyl group. Variation of this procedure
did not result in an improvement. However, to our delight,
treatment of Mtt-protected pentapeptide 19a with 1% TFA
in dichloromethane and Et3SiH gave target peptide 1a in 96%
isolated yield (Scheme 6). No undesired side reactions were
observed at all.
1% TFA/CH2Cl2,
Et3SiH
HS
Fmoc
H
N
OMe
Mmt:
CO2Me
N
quant.
H
O
S
13
Scheme 5. Removal of the S-methoxytrityl group (Mmt) in the presence of
This result prompted us to select the Mtt protecting group
for the synthesis of the differently lipid-modified analogous
target peptides 1b and 1c. These analogues were synthesized
by means of the strategy developed for palmitoylated peptide
1a. The required tripeptide building blocks were synthesized
by condensation of dipeptide 20, which is accessible by N-hydr-
oxysuccinimide(HOSu)-mediated coupling of Fmoc-Met-OH
with serine,[12] with the already modified cysteine tert-butyl ester
21b or 21c. Fmoc removal, condensation with maleimidocap-
roic acid, and subsequent treatment with TFA delivered the
tripeptide building blocks 15b and 15c (Scheme 7).
a farnesyl moiety.
Having established two different types of acid-labile protect-
ing groups, namely the Boc urethane and a trityl-type blocking
function, for the synthesis of prenylated peptides, we tested
the Boc and Mtt groups in the synthesis of the palmitoylated,
farnesylated, and maleimido-modified target peptide 1a. For
this purpose a block condensation strategy was chosen
because it readily gives access to differently protected
pentapeptides and differently lipidated analogues thereof.
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Chem. Eur. J. 2001, 7, No. 6

Protecting Groups for Lipidated Peptides
1184±1193
residue was dissolved in ethyl acetate (50 mL). The solution was extracted
twice with 0.5n hydrochloric acid (50 mL) and saturated NaHCO3 solution
(50 mL), dried over MgSO4, and filtered. The solvent was evaporated in
vacuo and the product was isolated by flash chromatography (hexane/ethyl
acetate 2:1) to yield 4 (425 mg, 0.538 mmol, 76%) as a colorless solid. M.p.
EEDQ
Fmoc-Met-Ser-Cys(R)-OtBu
Fmoc-Met-Ser-OH + H-Cys(R)-OtBu
R=Hd: 64 %
R=StBu: 58 %
R=Hd: 21b
R=StBu: 21c
R=Hd: 22b
R=StBu: 22c
20
1. Et2NH
2. MIC-OH, EEDQ
3. TFA
578C; [a]2D0
ˆ
8.9 (c ˆ 1.0, CHCl3); 1H NMR (CDCl3, 500 MHz, TMS):
H-Lys(Mtt)-Cys(Far)-OMe 17
EEDQ
d ˆ 7.76 (d, J ˆ 7.5 Hz, 2H, Fmoc-CH), 7.60 (d, J ˆ 7.0 Hz, 2H, Fmoc-CH),
MIC-Met-Ser-Cys(R)-OH
7.40 (t, J ˆ 7.4 Hz, 2H, Fmoc-CH), 7.31 (t, J ˆ 7.4 Hz, 2H, Fmoc-CH), 6.71
R=Hd: 69 %
R=StBu: 58 %
R=Hd: 26 %
R=StBu: 28 %
R=Hd: 15b
R=StBu: 15c
ˆ
(b, 1H, NH), 5.50 (b, 1H, NH), 5.15 ± 5.19 (m, 1H, CH C Far), 5.04 ± 5.12
ˆ
(m, 2H, CH C Far), 4.72 ± 4.79 (m, 1H, a-CH Cys), 4.66 (b, 1H, NH),
1 % TFA
Et3SiH
4.38 ± 4.45 (m, 2H, Fmoc-CH2), 4.20 ± 4.26 (m, 2H, a-CH Lys, Fmoc-CH),
3.74 (s, 3H, COOCH3), 3.06 ± 3.20 (m, 4H, CH2 Far, e-CH2 Lys), 2.95 (dd,
J ˆ 13.9 Hz, J ˆ 4.8 Hz, 1H, b-CH2a Cys), 2.85 (dd, J ˆ 13.9 Hz, J ˆ 6.3 Hz,
1H, b-CH2b Cys), 1.93 ± 2.10 (m, 8H, CH2 Far), 1.80 ± 1.92 (m, 1H, CH2a
Lys), 1.38 ± 1.74 (m, 5H, CH2 Lys), 1.68 (s, 3H, CH3 Far), 1.62 (s, 6H, CH3
Far), 1.60 (s, 3H, CH3 Far), 1.43 (s, 9H, CH3 Boc); 13C NMR (CDCl3,
MIC-Met-Ser-Cys(R)-Lys(Mtt)-Cys(Far)-OMe
R=Hd: 97 %
R=StBu: 76 %
19b,c
R
S
ˆ
ˆ
ˆ
100 MHz, TMS): d ˆ 171.7 (C O), 171.1 (C O), 156.2 (C O), 143.9 (quart,
Fmoc), 143.7 (quart, Fmoc), 141.3 (quart, Fmoc), 140.1 (quart, Far), 135.3
(quart, Far), 131.3 (quart, Far), 127.7 (Fmoc-CH), 127.1 (Fmoc-CH), 125.1
(Fmoc-CH), 124.3 (Far-CH), 123.7 (Far-CH), 120.0 (Fmoc-CH), 119.4 (Far-
CH), 79.1 (quart, Boc), 67.1 (Fmoc-CH2), 54.6 (a-CH), 52.6 (COOCH3),
51.8 (a-CH), 47.1 (Fmoc-CH), 39.9 (CH2), 39.7 (CH2), 39.6 (CH2), 33.1
(CH2), 32.2 (CH2), 29.8 (CH2), 29.6 (CH2), 28.4 (Boc-CH3), 26.7 (CH2), 26.4
(CH2), 25.7 (Far-CH3), 22.3 (CH2), 17.7 (Far-CH3), 16.1 (Far-CH3), 16.0
MIC-Met-Ser-Cys-Lys-Cys-OMe
S
R=Hd: 1b
R=StBu: 1c
Scheme 7. Synthesis of the analogous peptides 1b and 1c by the Mtt
strategy.
‡
‡
(Far-CH3); MS (FAB, 3-NBA): m/z: 790.4 [M‡H] , 690.4 [M Boc‡H] .
In the case of both tripeptides 15b and 15c the subsequent
block condensation with dipeptide 17 gave the desired
pentapeptides in only low yield. It proved difficult to improve
this result as significant amounts of diketopiperazine were
formed as a side product during the condensation reaction,
and the separation of diketopiperazine and pentapeptide was
problematic. The final deprotection reaction under the
conditions described above once more proceeded smoothly
and without any undesired side reactions. The target peptides
1b and 1c were isolated in 97% and in 76% yield,
respectively.
N(a)-Fluorenylmethoxycarbonyl-N(e)-(4-tolyldiphenylmethyl)-(l)-lysyl-S-
farnesyl-(l)-cysteine methyl ester (Fmoc-Lys(Mtt)-Cys(Far)-OMe) (7):
EEDQ (142 mg, 0.575 mmol) was added to a solution of Fmoc-Lys(Mtt)-
OH[9] (6; 276 mg, 0.442 mmol) and H-Cys(Far)-OMe[5] (3; 150 mg,
0.442 mmol) in dichloromethane (5 mL). After 20 hours the solvent was
evaporated in vacuo and the residue was dissolved in ethyl acetate (30 mL).
The solution was extracted twice with 0.5n hydrochloric acid (20 mL) and
saturated NaHCO3 solution (20 mL), dried over MgSO4, and filtered. The
solvent was evaporated in vacuo and the product was isolated by flash
chromatography (hexane/ethyl acetate 3:1) to yield 6 (312 mg, 0.330 mmol,
1
75%) as a colorless oil. [a]D20
ˆ
10.1 (c ˆ 1.0, CHCl3); H NMR (CDCl3,
500 MHz, TMS): d ˆ 7.74 (d, J ˆ 7.1 Hz, 2H, Fmoc-CH), 7.57 (d, J ˆ 7.9 Hz,
2H, Fmoc-CH), 7.45 (d, J ˆ 7.6 Hz, 4H, Mtt-CH), 7.23 ± 7.38 (m, 10H,
Fmoc-CH, Mtt-CH), 7.15 (t, J ˆ 7.3 Hz, 2H, Mtt-CH), 7.06 (d, J ˆ 8.1 Hz,
2H, Mtt-CH), 6.59 (b, 1H, NH), 5.29 (d, J ˆ 7.7 Hz, 1H, NH), 5.14 ± 5.17
Conclusion
ˆ
ˆ
(m, 1H, CH C Far), 5.05 ± 5.10 (t, J ˆ 7.7 Hz, 2H, CH C Far), 4.72 ± 4.79
(m, 1H, a-CH Cys), 4.37 ± 4.43 (m, 2H, Fmoc-CH2), 4.16 ± 4.22 (m, 2H, a-
CH Lys, Fmoc-CH), 3.69 (s, 3H, COOCH3), 3.15 (dd, J ˆ 13.1 Hz, J ˆ
8.2 Hz, 1H; CH2a Far), 3.06 (dd, J ˆ 13.2 Hz, J ˆ 7.4 Hz, 1H, CH2b Far),
2.93 (dd, J ˆ 13.9 Hz, J ˆ 4.8 Hz, 1H, b-CH2a Cys), 2.85 (dd, J ˆ 13.9 Hz,
J ˆ 5.0 Hz, 1H, b-CH2b Cys), 2.29 (s, 3H, Mtt-CH3), 1.93 ± 2.12 (m, 10H,
CH2 Far, e-CH2 Lys), 1.78 ± 1.88 (m, 1H, CH2a Lys), 1.45 ± 1.74 (m, 3H, CH2
Lys), 1.67 (s, 3H, CH3 Far), 1.62 (s, 3H, CH3 Far), 1.59 (s, 3H, CH3 Far), 1.58
(s, 3H, CH3 Far), 1.41 ± 1.49 (m, 2H, CH2 Lys); 13C NMR (CDCl3, 125 MHz,
Acid-labile protecting groups can be cleaved in the presence
of acid-sensitive prenyl groups under mild conditions. In
particular, trityl-based groups, namely the N-methyltrityl
(Mtt), the N-phenylfluorenyl (PhFl), and the S-methoxytrityl
(Mmt) group, may be applied advantageously in the synthesis
of lipidated H-Ras peptides.
ˆ
ˆ
ˆ
TMS): d ˆ 171.6 (C O), 171.0 (C O), 156.0 (C O), 146.4 (quart, Mtt),
143.9 (quart, Fmoc), 143.7 (quart, Fmoc), 143.3 (quart, Mtt), 141.3 (quart,
Fmoc), 140.1 (quart, Far), 135.3 (quart, Far), 135.4 (quart, Mtt), 131.3
(quart, Far), 128.6 (Mtt, CH), 128.6 (Mtt, CH), 128.5 (Mtt, CH), 127.7
(Fmoc-CH), 127.1 (Fmoc-CH), 126.1 (Mtt, CH), 125.1 (Fmoc-CH), 124.3
(Far-CH), 123.7 (Far-CH), 120.0 (Fmoc-CH), 119.5 (Far-CH), 70.6 (quart,
Mtt), 67.1 (Fmoc-CH2), 54.8 (a-CH), 52.6 (COOCH3), 51.7 (a-CH), 47.2
(Fmoc-CH), 43.4 (CH2), 39.7 (CH2), 39.6 (CH2), 33.1 (CH2), 32.9 (CH2),
30.7 (CH2), 29.9 (CH2), 29.7 (CH2), 26.4 (CH2), 25.7 (Far-CH3), 23.2 (CH2),
20.9 (Mtt-CH3), 17.7 (Far-CH3), 16.1 (Far-CH3), 16.0 (Far-CH3); MS (FAB,
Experimental Section
General: 1H and 13C NMR spectra were recorded on Bruker AC-250,
Bruker AM-400, and Bruker DRX-500 spectrometers and were based on
tetramethylsilane (TMS) as internal standard. EI-MS and FAB-MS were
recorded on a Finnigan MAT 90 mass spectrometer with 3-nitrobenzyl
alcohol (3-NBA) as a matrix for FAB-MS. Combustion analysis was carried
out with a Heraeus CHN-Rapid analyser. Flash chromatography was
executed on columns packed with Baker silica gel (30 ± 60 mm). TLC was
performed on Kieselgel 60F254 aluminum sheets (Merck Darmstadt,
Germany). All reagents were obtained from Fluka, Aldrich, Sigma, or
Novabiochem. All solvents were dried and distilled by standard proce-
dures.
‡
‡
‡
3-NBA): m/z: 945.4 [M‡H] , 868.4 [M C6H5] , 854.4 [M C6H4CH3] ,
‡
257.1 [Mtt] .
N(a)-Fluorenylmethoxycarbonyl-(l)-lysyl-S-farnesyl-(l)-cysteine
methyl
ester (Fmoc-Lys-Cys(Far)-OMe) (5): By Boc deprotection: TMSOTf
(12.0 mL, 66.5 mmol) was added to a solution of Fmoc-Lys(Boc)-Cys(Far)-
OMe (4; 10.5 mg, 13.3 mmol) and 2,6-lutidine (15.4 mL, 133 mmol) in dry
dichloromethane (0.7 mL). After one hour, dichloromethane (20 mL) was
added, the solution was extracted twice with 0.5n hydrochloric acid
(20 mL) and saturated NaHCO3 solution (20 mL), dried over MgSO4, and
filtered. The solvent was evaporated in vacuo and the product was isolated
by flash chromatography to yield 5 (6.9 mg, 9.98 mmol, 75%) as a colorless
solid.
N(a)-Fluorenylmethoxycarbonyl-N(e)-tert-butyloxycarbonyl-(l)-lysyl-S-far-
nesyl-(l)-cysteine methyl ester (Fmoc-Lys(Boc)-Cys(Far)-OMe) (4):
1-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
(EEDQ;
227 mg,
0.917 mmol) was added to a solution of Fmoc-Lys(Boc)-OH (2; 331 mg,
0.705 mmol) and H-Cys(Far)-OMe[5] (3; 239 mg, 0.705 mmol) in dichloro-
methane (10 mL). After 16 h the solvent was evaporated in vacuo and the
Chem. Eur. J. 2001, 7, No. 6
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0706-1187 $ 17.50+.50/0
1187

H. Waldmann et al.
FULL PAPER
By Mtt removal: Et3SiH (14.1 mL, 88.8 mmol) was added to a solution of
Fmoc-Lys(Mtt)-Cys(Far)-OMe (7; 8.4 mg, 8.88 mmol) in a 1% solution of
TFA in dichloromethane (0.7 mL). Within five minutes the mixture
changed from yellow to colorless. After 60 minutes a solution of 1%
EtMe2N in dichloromethane (0.8 mL) was added, the solvent was
evaporated in vacuo, and the product was isolated by flash chromatography
(CHCl3/MeOH 8:1) to yield 5 (5.8 mg, 8.41 mmol, 95%) as a colorless solid.
(CH Far), 124.1 (CH Far), 119.9 (CH Far), 54.8 (a-CH), 52.9 (OCH3), 52.1
(OCH3), 51.8 (a-CH), 40.0 (CH2), 33.8 (CH2), 30.7 (CH2), 30.5 (CH2), 30.2
(CH2), 27.1 (CH2), 26.8 (CH2), 26.1 (CH2), 18.1 (CH3 Far), 16.5 (CH3 Far),
16.4 (CH3 Far); MS (FAB, 3-NBA): m/z calcd for C25H42N2O5S: 482.6775;
found: 482.6736.
N-Fluorenylmethoxycarbonyl-(S-4-methoxytrityl)-(l)-cysteyl-(S-farnesyl)-
(l)-cysteine methyl ester (Fmoc-Cys(Mmt)-Cys(Far)-OMe) (12): At 08C,
HOBt (48 mg, 0.35 mmol) and EDC (54 mg, 0.28 mmol) were added to a
solution of Fmoc-Cys(Mmt)-OH[14] (145 mg, 2.40 mmol) and 3 (80 mg,
2.4 mmol) in dichloromethane (20 mL). The solution was stirred at room
temperature for 15 h. Ethyl acetate was added and the solution was
extracted twice with 0.5n hydrochloric acid (15 mL) and with 0.5n
NaHCO3 solution (15 mL), and once with brine (15 mL), dried over
Na2SO4, and filtered. The solvent was evaporated in vacuo and the product
was isolated by flash chromatography (n-hexane/ethyl acetate 2:1) to yield
M.p. 578C; [a]2D0
ˆ
4.3 (c ˆ 0.3, CHCl3); 1H NMR (CDCl3/CD3OD 6:1,
500 MHz, TMS): d ˆ 7.77 (d, J ˆ 7.5 Hz, 2H, Fmoc-CH), 7.60 ± 7.64 (m, 2H,
Fmoc-CH), 7.40 (t, J ˆ 7.4 Hz, 2H, Fmoc-CH), 7.30 ± 7.6 (m, 2H, Fmoc-
ˆ
ˆ
CH), 5.19 (t, J ˆ 7.5 Hz, 1H, CH C Far), 5.08 (t, J ˆ 6.9 Hz, 2H, CH C
Far), 4.67 (dd, J ˆ 7.5 Hz, J ˆ 5.0 Hz, 1H, a-CH Cys), 4.37 ± 4.44 (m, 2H,
Fmoc-CH2), 4.23 (t, J ˆ 6.8 Hz, 2H, a-CH Lys, Fmoc-CH), 3.75 (s, 3H,
COOCH3), 3.20 (dd, J ˆ 12.9 Hz, J ˆ 8.4 Hz, 1H, CH2a Far), 3.10 (dd, J ˆ
12.7 Hz, J ˆ 7.3 Hz, 1H, CH2b Far), 2.90 ± 2.97 (m, 3H, b-CH2a Cys, e-CH2
Lys), 2.81 (dd, J ˆ 13.9 Hz, J ˆ 7.6 Hz, 1H, b-CH2b Cys), 1.93 ± 2.10 (m, 10H,
CH2 Far, CH2 Lys), 1.78 ± 1.85 (m, 1H, CH2a Lys), 1.55 ± 1.74 (m, 1H, CH2b
Lys), 1.68 (s, 3H, CH3 Far), 1.65 (s, 3H, CH3 Far), 1.60 (s, 3H, CH3 Far), 1.59
(s, 3H, CH3 Far), 1.31 ± 1.42 (m, 2H, CH2 Lys); 13C NMR (CDCl3/CD3OD
12 (197 mg, 89%) as a colorless oil. [a]D20
ˆ
3.2 (c ˆ 0.5); 1H NMR
(CDCl3, 500 MHz, TMS): d ˆ 7.74 (dd, J ˆ 9.8 Hz, J ˆ 7.7 Hz, 2H, Fmoc-
CH), 7.56 (b, 2H, Fmoc-CH), 7.42 (d, J ˆ 7.6 Hz, 4H, Mmt-CH), 7.25 ± 7.40
(m, 10H, Fmoc-CH, Mmt-CH), 7.20 (t, J ˆ 7.3 Hz, 2H, Mmt-CH), 6.80 (d,
J ˆ 8.9 Hz, 2H, Mmt-CH), 6.61 (d, J ˆ 7.8 Hz, 1H, NH), 5.14 (t, J ˆ 7.6 Hz,
ˆ
ˆ
ˆ
6:1, 125 MHz, TMS): d ˆ 172.5 (C O), 171.5 (C O), 156.8 (C O), 144.0
(quart, Fmoc), 143.8 (quart, Fmoc), 141.4 (quart, Fmoc), 140.2 (quart, Far),
135.5 (quart, Far), 131.5 (quart, Far), 127.9 (Fmoc-CH), 127.2 (Fmoc-CH),
125.2 (Fmoc-CH), 124.4 (Far-CH), 123.8 (Far-CH), 120.0 (Fmoc-CH), 119.6
(Far-CH), 67.2 (Fmoc-CH2), 54.2 (a-CH), 52.7 (COOCH3), 52.2 (a-CH),
47.2 (Fmoc-CH), 39.8 (CH2), 39.8 (CH2), 39.3 (CH2), 32.7 (CH2), 31.9
(CH2), 29.7 (CH2), 26.8 (CH2), 26.7 (CH2), 26.6 (CH2), 25.7 (Far-CH3), 22.0
(CH2), 17.7 (Far-CH3), 16.1 (Far-CH3), 16.0 (Far-CH3); MS (FAB, 3-NBA):
ˆ
ˆ
1H, CH C Far), 5.03 ± 5.09 (m, 2H, CH C Far), 4.35 ± 4.36 (m, 2H, Fmoc-
CH2), 4.21 (t, J ˆ 6.9 Hz, 1H, Fmoc-CH), 3.82 ± 3.86 (m, 2H, a-CH Cys),
3.76 (s, 3H, COOCH3), 3.69 (s, 3H, OCH3), 3.11 (dd, J ˆ 13.1 Hz, J ˆ
8.2 Hz, 1H, CH2a Far), 3.05 (dd, J ˆ 13.0 Hz, J ˆ 7.4 Hz, 1H, CH2b Far),
2.89 (dd, J ˆ 13.9 Hz, J ˆ 5.0 Hz, 1H, b-CH2a Cys), 2.80 (dd, J ˆ 13.9 Hz,
J ˆ 6.0 Hz, 1H, b-CH2b Cys), 2.72 (dd, J ˆ 13.3 Hz, J ˆ 7.7 Hz, 1H, b-CH2a
Cys), 2.65 (dd, J ˆ 13.3 Hz, J ˆ 5.3 Hz, 1H, b-CH2b Cys), 1.94 ± 2.06 (m, 8H,
CH2 Far), 1.67 (s, 3H, CH3 Far), 1.62 (s, 3H, CH3 Far), 1.59 (s, 3H, CH3 Far),
‡
m/z: 690.5 [M‡H] ; HRMS (FAB, 3-NBA) calcd for C40H55N3O5S (689.95):
690.3941; found: 690.3954.
1.57 (s, 3H, CH3 Far); 13C NMR (CDCl3, 125 MHz, TMS): d ˆ 170.7 (C O),
ˆ
ˆ
ˆ
170.0 (C O), 158.3 (C O), 155.9 (quart, Mmt), 144.7 (quart, Mmt), 143.8
(quart, Fmoc), 143.7 (quart, Fmoc), 141.3 (quart, Fmoc), 140.0 (quart, Far),
136.4 (quart, Mmt), 135.3 (quart, Far), 131.3 (quart, Far), 130.8 (Mmt, CH),
129.5 (Mmt, CH), 128.1 (Mmt, CH), 127.7 (Fmoc-CH), 127.1 (Fmoc-CH),
126.9 (Mmt, CH), 125.1 (Fmoc-CH), 124.3 (Far-CH), 123.7 (Far-CH), 120.0
(Fmoc-CH), 119.5 (Far-CH), 113.4 (Mmt, CH), 67.2 (Fmoc-CH2), 66.9
(quart, Mmt), 55.2 (COOCH3), 53.9 (OCH3), 52.5 (a-CH), 52.0 (a-CH),
47.1 (Fmoc-CH), 39.7 (CH2), 39.6 (CH2), 33.8 (CH2), 33.2 (CH2), 29.9
(CH2), 26.7 (CH2), 26.4 (CH2), 25.7 (CH3), 17.7 (CH3), 16.1 (CH3), 16.0
N-[9-(9-Phenylfluorenyl)]-a-(l)-(g-methylglutamyl)-S-farnesyl-(l)-cys-
teine methyl ester (PhFl-Glu(OMe)-Cys(Far)-OMe) (9): At 08C, 1-ethyl-
3(dimethylamino)propylcarbodiimide hydrochloride (EDC; 173 mg,
0.884 mmol) was added to
a
solution of PhFl-Glu(OMe)-OH[13] (8,
300 mg, 0.736 mmol), H-Cys(Far)-OMe (3, 250 mg, 0.736 mmol) and
1-hydroxybenzotriazole (HOBt; 230 mg, 1.47 mmol) in dichloromethane
(40 mL). After 20 h the solution was extracted twice with 0.5n hydrochloric
acid (10 mL) and 10% Na2CO3 solution (10 mL), dried over Na2SO4, and
filtered. The solvent was evaporated in vacuo and the product was isolated
‡
(CH3); HRMS (FAB, 3-NBA): m/z calcd for C57H64NaN2S2O6 ([M ‡ Na] ):
by flash chromatography (hexane/ethyl acetate 3:1) to yield 9 (518 mg,
1
0.7 mmol, 96%) as a colorless oil. [a]D20
ˆ
8.9 (c ˆ 1.0, CHCl3); H NMR
959.4139; found: 959.4104.
(CDCl3, 400 MHz, TMS): d ˆ 7.69 ± 7.09 (m, 14H, NH, PhFl), 5.19 (m, 1H,
N-Fluorenylmethoxycarbonyl-(l)-cysteyl-(S-farnesyl)-(l)-cysteine methyl
ester (Fmoc-Cys-Cys(Far)-OMe) (13): At room temperature, 1% trifluoro-
acetic acid in dichloromethane (1.5 mL) was added to a solution of 12
(30 mg, 32 mmol) in dichloromethane (0.2 mL). Et3SiH (51 mL, 0.32 mmol)
was added to the orange solution. After 30 min toluene (4 mL) was added
and the solvents were evaporated in vacuo. The addition of toluene and
evaporation was repeated twice. The product was isolated by flash
ˆ
ˆ
CH C Far), 5.10 (m, 2H, CH C Far), 4.48 (m, 1H, a-CH Cys), 3.75 (s, 3H,
OCH3 Cys), 3.59 (s, 3H, OCH3 Glu), 3.09 (m, 3H, CH2 Far, NH), 2.75 (dd,
J ˆ 14 Hz, J ˆ 5 Hz, 1H, b-CH2a Cys), 2.68 (dd, J ˆ 14 Hz, J ˆ 5 Hz, 1H, b-
CH2b Cys), 2.48 (m, 1H, a-CH Glu), 2.40 ± 2.25 (m, 2H, g-CH2 Glu), 2.13 ±
1.96 (m, 8H, CH2 Far), 1.75 (m, 2H, b-CH2 Glu), 1.68 (s, 3H, CH3 Far), 1.66
(s, 3H, CH3 Far), 1.60 (s, 6H, CH3 Far); 13C NMR (CDCl3, 100 MHz, TMS):
ˆ
ˆ
d ˆ 174.4 (C O), 171.7 (C O), 141.1 (quart, PhFl), 140.8 (quart, PhFl),
140.4 (quart, Far), 135.8 (quart, Far), 131.7 (quart, Far), 128.9 (quart, PhFl),
128.4 (CH PhFl), 127.8 (CH PhFl), 127.7 (CH PhFl), 126.9 (CH PhFl), 125.3
(CH PhFl), 124.7 (CH Far), 124.1 (CH Far), 120.4 (CH PhFl), 120.3 (CH
PhFl), 119.9 (CH Far), 73.4 (quart, PhFl), 56.6 (a-CH), 52.9 (OCH3), 52.0
(OCH3), 51.6 (a-CH), 40.1 (CH2), 34.1 (CH2), 30.6 (CH2), 30.3 (CH2), 30.1
(CH2), 27.1 (CH2), 26.8 (CH2), 26.1 (CH2), 18.1 (CH3 Far), 16.5 (CH3 Far),
16.4 (CH3 Far); MS (FAB, 3-NBA): m/z calcd for C44H54N2O5S: 722.9760;
found: 722.9719.
chromatography (n-hexane/ethyl acetate 2:1) to yield 13 quantitatively as
1
a colorless solid. M.p. 67 ± 698C; [a]D20
ˆ
8.5 (c ˆ 0.7); H NMR (CDCl3,
500 MHz, TMS): d ˆ 7.77 (d, J ˆ 7.5 Hz, 2H, Fmoc-CH), 7.60 (d, J ˆ 6.7 Hz,
2H, Fmoc-CH), 7.40 (t, J ˆ 7.5 Hz, 2H, Fmoc-CH), 7.31 ± 7.34 (m, 2H,
ˆ
Fmoc-CH), 6.91 (b, 1H, NH), 5.75 (b, 1H, NH), 5.17 ± 5.20 (m, 1H, CH C
ˆ
Far), 5.07 ± 5.10 (m, 2H, CH C Far), 4.74 ± 4.78 (m, 1H, a-CH Cys), 4.45 ±
4.47 (m, 3H, a-CH Cys, Fmoc-CH2), 4.24 (t, J ˆ 6.8 Hz, 1H, Fmoc-CH),
3.76 (s, 3H, COOCH3), 3.19 (dd, J ˆ 13.1 Hz, J ˆ 8.2 Hz, 1H, CH2a Far),
3.06 ± 3.15 (m, 1H, b-CH2a Cys), 3.11 (dd, J ˆ 13.1 Hz, J ˆ 7.4 Hz, 1H, CH2b
Far), 2.99 (dd, J ˆ 13.9 Hz, J ˆ 4.7 Hz, 1H, b-CH2a Cys), 2.85 (dd, J ˆ
13.4 Hz, J ˆ 6.3 Hz, 1H, b-CH2b Cys), 2.69 ± 2.80 (m, 1H, b-CH2b Cys),
1.95 ± 2.11 (m, 8H, CH2 Far), 1.68 (s, 3H, CH3 Far), 1.65 (s, 3H, CH3 Far),
1.60 (s, 3H, CH3 Far), 1.59 (s, 3H, CH3 Far); 13C NMR (CDCl3, 125 MHz,
a-(l)-(g-Methylglutamyl)-S-farnesyl-(l)-cysteine methyl ester (H-Glu-
(OMe)-Cys(Far)-OMe) (10): At 08C, Et3SiH (440 mL, 2.77 mmol) was
added to a solution of 9 (200 mg, 0.277 mmol) in a 3% solution of TFA in
dichloromethane. Within five minutes the mixture turned from yellow to
colorless. After 3 h at 08C Et2MeN (1 mL) was added, the solvent was
evaporated in vacuo, and the product was isolated by flash chromatography
(CHCl3/MeOH 30:1) to yield 10 (127 mg, 0.263 mmol, 95%) as a yellowish
ˆ
ˆ
ˆ
TMS): d ˆ 170.9 (C O), 169.5 (C O), 155.9 (C O), 155.9, 143.8 (quart,
Fmoc), 143.6 (quart, Fmoc), 141.4 (quart, Fmoc), 140.3 (quart, Far), 135.4
(quart, Far), 131.3 (quart, Far), 127.8 (Fmoc-CH), 127.1 (Fmoc-CH), 125.0
(Fmoc-CH), 124.3 (Far-CH), 123.7 (Far-CH), 120.1 (Fmoc-CH), 119.3 (Far-
CH), 67.3 (Fmoc-CH2), 55.9 (COOCH3), 52.8 (a-CH), 51.9 (a-CH), 47.2
(Fmoc-CH), 39.7 (CH2), 39.6 (CH2), 39.3 (CH2), 34.3 (CH2), 33.1 (CH2),
29.8 (CH2), 27.2 (CH2), 26.7 (CH2), 26.4 (CH2), 25.7 (CH3), 17.7 (CH3), 16.1
(CH3), 16.0 (CH3); HRMS (FAB, 3-NBA): m/z calcd for C37H49N2S2O5:
665.3118; found: 665.3083.
oil. [a]2D0
ˆ
12.7 (c ˆ 1.0, CHCl3); 1H NMR (CDCl3, 400 MHz, TMS): d ˆ
ˆ
ˆ
5.21 (m, 1H, CH C Far), 5.09 (m, 2H, CH C Far), 4.75 (m, 1H, a-CH Cys),
3.76 (s, 3H, OCH3 Cys), 3.68 (s, 3H, OCH3 Glu), 3.48 (m, 1H, NH), 3.24 ±
3.11 (m, 3H, CH2 Far, a-CH Glu), 2.95 (dd, J ˆ 14 Hz, J ˆ 5 Hz, 1H, b-CH2a
Cys), 2.88 (dd, J ˆ 14 Hz, J ˆ 5 Hz, 1H, b-CH2b Cys), 2.53 ± 2.46 (m, 2H, g-
CH2 Glu), 2.15 ± 1.88 (m, 10H, CH2 Far, b-CH2 Glu), 1.68 (s, 6H, CH3 Far),
1.60 (s, 6H, CH3 Far); 13C NMR (CDCl3, 100 MHz, TMS): d ˆ 174.2 (C O),
(l)-Methionyl-(l)-seryl-S-palmitoyl-(l)-cysteine tert-butyl ester (H-Met-
Ser-Cys(Pal)-OtBu): PhSiH3 (34 mL, 0.279 mmol) and [Pd(PPh3)4] (16 mg,
ˆ
ˆ
171.7 (C O), 140.4 (quart, Far), 135.8 (quart, Far), 131.7 (quart, Far), 124.7
1188
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0706-1188 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 6

Protecting Groups for Lipidated Peptides
1184±1193
0.0139 mmol) were added under an argon atmosphere to a solution of
Aloc-Met-Ser-Cys(Pal)-OtBu[11] (14a; 100 mg, 0.139 mmol) in THF
(1 mL). After two hours the solvent was evaporated in vacuo and
H-Met-Ser-Cys(Pal)-OtBu was isolated by size-exclusion chromatography
(Sephadex LH-20, CHCl3/MeOH 1:1) as a colorless, waxy solid (50 mg,
(CH2), 25.4 (CH2), 22.9 (CH2), 15.3 (SCH3 Met), 14.2 (CH3 Pal); MS (FAB,
‡
‡
3-NBA): m/z: 793.4 [M‡Na] , 771.4 [M‡H] ; HRMS (FAB, 3-NBA) calcd
for C37H63N4O9S2: 771.4036; found 771.4068.
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-palmitoyl-(l)-cysteyl-N(e)
-
tert-butoxycarbonyl-(l)-lysyl-S-farnesyl-(l)-cysteine methyl ester (MIC-
Met-Ser-Cys(Pal)-Lys(Boc)-Cys(Far)-OMe) (18a): Et2NH (0.1 mL) was
added to a solution of 7 (28.8 mg, 36.5 mmol) in dichloromethane (0.4 mL).
After 2 h the solvent was evaporated in vacuo, the residue was dissolved in
dichloromethane (2 mL), and 15a (23.4 mg, 30.4 mmol) and EEDQ (9.8 mg,
36.5 mmol) were added. After 18 h the solvent was evaporated in vacuo and
the product was isolated by flash chromatography (dichloromethane/
ethanol 30:1, then 20:1) to yield 18a (30.3 mg, 22.9 mmol, 76%) as a
0.0789 mmol, 57%). [a]D20
ˆ
20.9 (c ˆ 1.0, CHCl3); 1H NMR (500 MHz,
CDCl3, TMS): d ˆ 8.06 (d, J ˆ 7.7 Hz, 1H, NH), 7.27 (d, J ˆ 7.3 Hz, 1H,
NH), 4.66 ± 4.70 (m, 1H, a-CH), 4.47 ± 4.50 (m, 1H, a-CH), 4.07 (dd, J ˆ
11.5 Hz, J ˆ 5.3 Hz, 1H, b-CH2a Ser), 3.60 (dd, J ˆ 11.5 Hz, J ˆ 4.7 Hz, 1H,
b-CH2b Ser), 3.59 (dd, J ˆ 8.1 Hz, J ˆ 4.7 Hz, 1H, a-CH Met), 3.47 (dd, J ˆ
14.1 Hz, J ˆ 4.1 Hz, 1H, b-CH2a Cys), 3.27 (dd, J ˆ 14.1 Hz, J ˆ 6.1 Hz, 1H,
b-CH2b Cys), 2.60 ± 2.65 (m, 2H, g-CH2 Met), 2.56 (t, J ˆ 7.6 Hz, 2H, CH2
Pal), 2.43 (b, 2H, NH2), 2.11 ± 2.19 (m, 1H, b-CH2a Met), 2.11 (s, 3H, SCH3
Met), 1.77 ± 1.84 (m, 1H, b-CH2b Met), 1.58 ± 1.65 (m, 2H, CH2 Pal), 1.46 (s,
9H, CO2C(CH3)3), 1.25 (s, 24H, CH3(CH2)12(CH2)2COS), 0.87 (t, J ˆ
6.9 Hz, 3H, w-CH3 Pal); 13C NMR (125 MHz, CDCl3, TMS): d ˆ 199.4,
colorless, waxy solid. M.p. 1198C; [a]D20
ˆ
24.6 (c ˆ 1.3, CHCl3); 1H NMR
(CDCl3, 500 MHz): d ˆ 7.63 (b, 1H, NH), 7.45 (b, 1H, NH), 7.29 (b, 1H,
NH), 7.15 (b, 1H, NH), 6.98 (d, J ˆ 7.3 Hz, 1H, NH), 6.69 (s, 2H, MIC-CH),
ˆ
ˆ
5.17 ± 5.27 (m, 1H, CH C Far), 5.07 ± 5.15 (m, 2H, CH C Far), 4.74 ± 4.86
(m, 1H, a-CH), 4.63 ± 4.72 (m, 2H, a-CH), 4.44 ± 4.51 (m, 1H, a-CH),
4.05 ± 4.10 (m, 1H, a-CH), 3.95 ± 4.03 (m, 1H, b-CH2a Ser), 3.78 ± 3.88 (m,
1H, b-CH2b Ser), 3.76 (s, 3H, COOCH3), 3.50 (t, J ˆ 7.2 Hz, 2H, MIC-
NCH2), 3.05 ± 3.45 (m, 6H, 2 CH2 Far, 2e-CH2 Lys, b-CH2 CysPal), 2.94 ± 3.04
(m, 1H, b-CH2a CysFar), 2.85 (m, 1H, b-CH2 CysFar), 2.49 ± 2.67 (m, 4H, g-
CH2 Met, CH2 Pal), 2.21 ± 2.27 (m, 2H, MIC), 1.88 ± 2.19 (m, 14H, b-CH2
Met, SCH3 Met, CH2a Lys, CH2 Far), 1.35 ± 1.72 (m, 11H, CH2 Pal, CH2
MIC, CH2 Lys), 1.68 (s, 3H, CH3 Far), 1.62 (s, 6H, CH3 Far), 1.60 (s, 3H,
CH3 Far), 1.45 (s, 9H, CH3 Boc), 1.22 ± 1.34 (m, 26H,
CH3(CH2)12(CH2)2COS, CH2 MIC), 0.88 (t, 3H, J ˆ 6.9 Hz, w-CH3 Pal);
ˆ
175.3, 170.7, 168.8 (4 C O), 83.3 (CO2C(CH3)3), 62.8 (b-CH2 Ser), 54.2,
54.0, 53.0 (3a-CH), 44.1 (b-CH2 Cys), 33.9, 31.9, 30.6, 30.2, 29.7, 29.7, 29.6,
29.4, 29.4, 29.2, 29.0 (11CH2), 27.9 (CO2C(CH3)3), 25.6, 22.7 (2 CH2), 15.3
‡
(SCH3 Met), 14.1 (CH3 Pal); MS (FAB, 3-NBA): m/z: 634.4 [M‡H] ;
elemental analysis calcd (%) for C31H59N3O6S2: C 58.55, H 8.84, N 5.85;
found:C 58.49, H 8.61, N 5.54.
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-palmitoyl-(l)-cysteine
tert-
butyl ester (MIC-Met-Ser-Cys(Pal)-OtBu): EEDQ (29.3 mg, 118 mmol)
was added to a solution of MIC-OH (18.3 mg, 86.8 mmol) and H-Met-Ser-
Cys(Pal)-OtBu (50 mg, 78.9 mmol) in dichloromethane (1 mL). After
18 hours the solvent was evaporated in vacuo and the product was isolated
by flash chromatography (dichloromethane/ethanol 30:1, then 20:1) as a
13C NMR (CDCl3, 125 MHz): d ˆ 173.4 (C O), 171.7 (C O), 170.8 (C O),
ˆ
ˆ
ˆ
ˆ
ˆ
ˆ
ˆ
169.4 (C O), 167.5 (C O), 166.9 (C O), 156.3 (C O), 140.0 (quart, Far),
135.3 (quart, Far), 134.1 (2CH MIC), 131.3 (quart, Far), 124.3 (Far-CH),
123.8 (Far-CH), 119.5 (Far-CH), 79.1 (quart, Boc), 64.2 (b-CH2 Ser), 54.9
(a-CH), 53.8 (a-CH), 53.4 (a-CH), 52.9 (a-CH), 52.6 (COOCH3), 44.1
(CH2), 40.4 (CH2), 39.7 (CH2), 39.6 (CH2), 37.7 (CH2), 36.0 (CH2), 35.5
(CH2), 32.7 (CH2), 31.9 (CH2), 29.7 (CH2), 29.7 (CH2), 29.6 (CH2), 29.5
(CH2), 29.4 (CH2), 29.1 (CH2), 29.0 (CH2), 28.4 (Boc-CH3), 26.8 (CH2), 26.6
(CH2), 26.4 (CH2), 25.7 (Far-CH3), 25.7 (CH2), 22.6 (CH2), 17.7 (Far-CH3),
16.2 (Far-CH3), 16.0 (Far-CH3) 15.4 (SCH3 Met), 14.1 (CH3 Pal); MS (FAB,
colorless, waxy solid (41.5 mg, 50.2 mmol, 64%). [a]D20
ˆ
3.6 (c ˆ 1.0,
CHCl3); 1H NMR (CDCl3, 500 MHz, TMS): d ˆ 7.37 ± 7.39 (m, 2H, NH),
6.78 (d, J ˆ 7.9 Hz, 1H, NH), 6.69 (s, 2H, MIC-CH), 4.63 ± 4.74 (m, 2H, a-
CH Cys, a-CH Met), 4.57 ± 4.62 (m, 1H, a-CH Ser), 3.99 (dd, J ˆ 11.4 Hz,
J ˆ 4.2 Hz, 1H, b-CH2a Ser), 3.69 (dd, J ˆ 11.5 Hz, J ˆ 5.6 Hz, 1H, b-CH2b
Ser), 3.49 ± 3.53 (m, 2H, MIC-NCH2), 3.46 (dd, J ˆ 14.0 Hz, J ˆ 4.4 Hz, 1H,
b-CH2a Cys), 3.32 (dd, J ˆ 14.0 Hz, J ˆ 6.1 Hz, 1H, b-CH2b Cys), 2.52 ± 2.61
(m, 4H, g-CH2 Met, CH2 Pal), 2.23 (t, J ˆ 7.5 Hz, 2H, MIC), 2.08 ± 2.15 (m,
1H, b-CH2a Met), 2.11 (s, 3H, SCH3 Met), 1.95 ± 2.03 (m, 1H, b-CH2b Met),
1.56 ± 1.69 (m, 6H, CH2 Pal, CH2 MIC), 1.46 (s, 9H, CO2C(CH3)3), 1.24 ±
‡
‡
‡
3-NBA): m/z: 1342.9 [M‡Na] , 1322.0 [M‡H] , 1220.8 [M Boc‡H] ;
‡
HRMS (FAB, 3-NBA) calcd for C62H106N7O11S3 ([M Boc‡H] ):
1.36 (m, 26H, CH3(CH2)12(CH2)2COS, CH2 MIC), 0.88 (t, J ˆ 6.9 Hz, 3H,
1220.711; found: 1220.719.
13
ˆ
w-CH3 Pal); C NMR (CDCl3, 125 MHz, TMS): d ˆ 199.0 (C O), 173.2
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-palmitoyl-(l)-cysteyl-N(e)-(4-
tolyldiphenylmethyl)-(l)-lysyl-S-farnesyl-(l)-cysteine methyl ester (MIC-
Met-Ser-Cys(Pal)-Lys(Mtt)-Cys(Far)-OMe) (19a): Et2NH (0.2 mL) was
added to a solution of 7 (50.0 mg, 55.1 mmol) in dichloromethane (0.8 mL).
After 4 h the solvent was evaporated in vacuo, the residue was dissolved in
dichloromethane (2 mL), and 15a (32.7 mg, 42.4 mmol) and EEDQ
(13.6 mg, 55.1 mmol) were added. After 16 h the solvent was evaporated
in vacuo and the product was isolated by flash chromatography (dichloro-
methane/ethanol 30:1, then 20:1) to yield 19a (36.5 mg, 24.7 mmol, 58%) as
ˆ
ˆ
ˆ
ˆ
ˆ
(C O), 171.9 (C O), 170.9 (2 C O MIC), 170.1 (C O), 168.9 (C O), 134.1
(2 CH MIC), 83.3 (CO2C(CH3)3), 62.8 (b-CH2 Ser), 54.6 (a-CH), 53.1 (a-
CH), 52.3 (a-CH), 44.1 (b-CH2 Cys), 37.6 (CH2), 36.1 (CH2), 33.6 (CH2),
31.9 (CH2), 30.3 (CH2), 30.1 (CH2), 29.7 (CH2), 29.7 (CH2), 29.6 (CH2), 29.4
(CH2), 29.4 (CH2), 29.2 (CH2), 29.0 (CH2), 28.2 (CH2), 27.9 (CO2C(CH3)3),
26.3 (CH2), 26.2 (CH2), 25.6 (CH2), 25.0 (CH2), 22.7 (CH2), 15.3 (SCH3
‡
‡
Met), 14.1 (CH3 Pal); MS (FAB, 3-NBA): m/z: 849 [M‡Na] , 827 [M‡H] ,
‡
771 [M tBu‡H] ; HRMS (FAB, 3-NBA): calcd for C41H71N4O9S2:
827.4662; found: 827.4622.
a
colorless, waxy solid. M.p. 1838C; [a]D20
ˆ
23.8 (c ˆ 1.6, CHCl3);
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-palmitoyl-(l)-cysteine (MIC-
Met-Ser-Cys(Pal)-OH) (15a): TFA (1 mL) was added to a solution of MIC-
Met-Ser-Cys(Pal)-OtBu (35.7 mg, 43.2 mmol) in dichloromethane (1 mL).
After 3 h toluene (2 mL) was added and the solvents were evaporated in
vacuo. The addition of toluene and evaporation was repeated twice to yield
1H NMR (CDCl3, 500 MHz, TMS): d ˆ 7.65 (b, 1H, NH), 7.52 (b, 1H,
NH), 7.49 (d, J ˆ 7.6 Hz, 4H, Mtt-CH), 7.41 (d, J ˆ 7.2 Hz, 2H, Mtt-CH),
7.34 ± 7.38 (m, 6H, Mtt-CH), 7.21 (d, J ˆ 8.1 Hz, 2H, Mtt-CH), 6.69 (s, 2H,
ˆ
ˆ
MIC-CH), 5.16 ± 5.20 (m, 1H, CH C Far), 5.07 ± 5.11 (m, 2H, CH C Far),
4.57 ± 4.63 (m, 1H, a-CH), 4.47 ± 4.53 (m, 1H, a-CH), 4.38 ± 4.44 (m, 2H, a-
CH), 4.15 ± 4.23 (m, 1H, a-CH), 3.88 ± 3.95 (m, 1H, b-CH2a Ser), 3.75 ± 3.80
(m, 1H, b-CH2b Ser), 3.68 (s, 3H, COOCH3), 3.48 (t, J ˆ 7.2 Hz, 2H, MIC-
NCH2), 3.39 ± 3.45 (m, 1H, b-CH2a CysPal), 3.13 ± 3.25 (m, 2H, 1 CH2a Far, b-
CH2 CysPal), 3.07 ± 3.12 (m, 1H, 1 CH2b Far), 2.81 ± 3.01 (m, 4H, b-CH2
CysFar , b-CH2 Lys), 2.49 ± 2.62 (m, 4H, g-CH2 Met, CH2 Pal), 2.35 (s, 3H,
Mtt-CH3), 2.19 ± 2.28 (m, 2H, MIC), 1.92 ± 2.15 (m, 14H, b-CH2 Met, SCH3
Met, CH2a Lys, CH2 Far), 1.35 ± 1.72 (m, 11H, CH2 Pal, CH2 MIC, CH2 Lys),
1.68 (s, 3H, CH3 Far), 1.65 (s, 3H, CH3 Far), 1.60 (s, 6H, CH3 Far), 1.19 ± 1.38
(m, 26H, CH3(CH2)12(CH2)2COS, CH2 MIC), 0.88 (t, J ˆ 6.7 Hz, 3H, w-
15a quantitatively as a colorless solid. M.p. 1618C; [a]D20
ˆ
8.5 (c ˆ 1.1,
1
CHCl3/CH3OH 2:1); H NMR (CDCl3/CD3OD 6:1, 500 MHz, TMS): d ˆ
6.75 (s, 2H, MIC-CH), 4.65 (dd, J ˆ 7.4 Hz, J ˆ 4.6 Hz, 1H, a-CH Cys), 4.53
(dd, J ˆ 8.3 Hz, J ˆ 5.6 Hz, 1H, a-CH Met), 4.44 (t, J ˆ 5.1 Hz, 1H, a-CH
Ser), 3.87 (dd, J ˆ 11.4 Hz, J ˆ 4.8 Hz, 1H, b-CH2a Ser), 3.73 (dd, J ˆ
11.4 Hz, J ˆ 5.3 Hz, 1H, b-CH2b Ser), 3.50 ± 3.54 (m, 3H, MIC-NCH2, b-
CH2a Cys), 3.27 (dd, J ˆ 14.0 Hz, J ˆ 7.5 Hz, 1 H, b-CH2b Cys), 2.52 ± 2.61
(m, 4H, g-CH2 Met, CH2 Pal), 2.25 (t, J ˆ 7.5 Hz, 2H, MIC), 2.09 ± 2.17 (m,
1H, b-CH2 Met), 2.12 (s, 3H, SCH3 Met), 1.92 ± 1.99 (m, 1H, b-CH2a Met),
1.57 ± 1.70 (m, 6H, CH2 Pal, 4CH2 MIC), 1.24 ± 1.36 (m, 26H,
CH3 Pal); 13C NMR (CDCl3, 125 MHz, TMS): d ˆ 173.3 (C O), 171.8
ˆ
ˆ
ˆ
ˆ
CH3(CH2)12(CH2)2COS, CH2 MIC), 0.88 (t, J ˆ 6.9 Hz, 3H, w-CH3 Pal);
(C O), 170.7 (C O), 168.9 (C O), 146.5 (quart, Mtt), 143.2 (quart, Mtt),
140.0 (quart, Far), 135.5 (quart, Mtt), 135.3 (quart, Far), 134.0 (2CH MIC),
131.3 (quart, Far), 128.6 (Mtt, 3 CH), 127.1 (Mtt, CH), 126.1 (Mtt, CH),
124.3 (Far-CH), 123.8 (Far-CH), 119.5 (Far-CH), 70.6 (quart, Mtt), 54.7 (a-
CH), 53.8 (a-CH), 53.2 (a-CH), 52.5 (COOCH3), 44.0 (CH2), 40.4 (CH2),
43.6 (CH2), 39.7 (CH2), 39.6 (CH2), 37.7 (CH2), 36.0 (CH2), 35.7 (CH2), 32.8
(CH2), 31.9 (CH2), 30.2 (CH2), 29.7 (CH2), 29.7 (CH2), 29.4 (CH2), 29.1
13
ˆ
C NMR (CDCl3/CD3OD 6:1, 125 MHz, TMS): d ˆ 199.9 (C O), 174.8
ˆ
ˆ
ˆ
ˆ
ˆ
(C O), 172.6 (C O), 172.1 (C O), 171.6 (2C O MIC), 170.8 (C O), 134.5
(2CH MIC), 62.3 (b-CH2 Ser), 55.3 (a-CH), 52.9 (a-CH), 52.8 (a-CH), 44.3
(b-CH2 Cys), 37.9 (CH2), 36.1 (CH2), 34.1 (CH2), 32.2 (CH2), 31.6 (CH2),
30.4 (CH2), 30.3 (CH2), 30.0 (CH2), 29.9 (CH2), 29.9 (CH2), 29.7 (CH2), 29.6
(CH2), 29.5 (CH2), 29.3 (CH2), 28.5 (CH2), 26.6 (CH2), 26.5 (CH2), 25.8
Chem. Eur. J. 2001, 7, No. 6
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0706-1189 $ 17.50+.50/0
1189

H. Waldmann et al.
FULL PAPER
[15]
(CH2), 28.4 (CH2), 27.9 (CH2), 26.8 (CH2), 26.6 (CH2), 26.4 (CH2), 25.7 (Far-
CH3), 25.6 (CH2), 25.2 (CH2), 22.7 (CH2), 20.9 (Mtt-CH3), 17.7 (Far-CH3),
16.2 (Far-CH3), 16.0 (Far-CH3), 15.3 (SCH3 Met), 14.1 (CH3 Pal); MS (FAB,
solution of (H-Cys-OtBu)2
(500 mg, 2.27 mmol) and Et3N (630 mL,
4.54 mmol) in dichloromethane (10 mL). After 100 min, dichloromethane
(40 mL) was added and the solution was extracted three times with water
(50 mL), dried over MgSO4, and filtered. The solvent was evaporated in
vacuo, the residue was dissolved in dry DMF (10 mL), and hexadecyl
bromide (2.77 mL, 9.08 mmol) and Et3N (630 mL, 4.54 mmol) were added
under an argon atmosphere. After two days, water (50 mL) was added and
the solution was extracted three times with diethyl ether (50 mL). The
combined organic layers were dried over MgSO4 and filtered, the solvent
was evaporated in vacuo, and the product was isolated by flash chroma-
tography (hexane/ethyl acetate 2:1) to yield 21b (723 mg, 1.80 mmol, 40%)
‡
‡
3-NBA): m/z: 1474.5 [M H] , 1399.6 [M C6H5] , 1385.5 [M
‡
‡
‡
C6H4CH3] , 1220.8 [M Mtt‡H] , 257.3 [Mtt] .
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-palmitoyl-(l)-cysteyl-(l)-ly-
syl-S-farnesyl-(l)-cysteine methyl ester (MIC-Met-Ser-Cys(Pal)-Lys-Cys-
(Far)-OMe) (1a): Et3SiH (16.5 mL, 104 mmol) was added to a solution of
19a (15.3 mg, 10.4 mmol) in a 1% solution of TFA in dichloromethane
(0.5 mL). After 70 min a solution of 1% EtMe2N in dichloromethane
(0.6 mL) was added, the solvent was evaporated in vacuo, and the product
was isolated by size-exclusion chromatography (Sephadex LH-20, CHCl3/
MeOH 1:1) to yield 1a (12.1 mg, 9.91 mmol, 96%) as a colorless, waxy solid.
as
a
colorless oil. [a]D20
ˆ
3.5 (c ˆ 2.0, CHCl3); 1H NMR (CDCl3,
500 MHz, TMS): d ˆ 3.52 (dd, J ˆ 7.3 Hz, J ˆ 4.7 Hz, 1H, a-CH Cys), 2.89
(dd, J ˆ 13.3 Hz, J ˆ 4.6 Hz, 1H, b-CH2a Cys), 2.74 (dd, J ˆ 13.3 Hz, J ˆ
7.3 Hz, 1H, b-CH2b Cys), 2.54 (t, J ˆ 7.4 Hz, 2H, CH2 Hd), 2.00 (s, 2H,
NH2), 1.53 ± 1.61 (m, 2H, CH2 Hd), 1.48 (s, 9H, CO2C(CH3)3), 1.31 ± 1.39
(m, 2H, CH2 Hd), 1.25 (s, 24H, CH3(CH2)12(CH2)3S), 0.88 (t, J ˆ 6.8 Hz,
M.p. 1608C; [a]2D0
ˆ
24.0 (c ˆ 0.6, CHCl3); 1H NMR (CDCl3/CD3OD 4:1,
500 MHz, TMS): d ˆ 7.99 (d, J ˆ 6.8 Hz, 1H, NH), 7.89 ± 7.93 (m, 2H, NH),
7.85 (d, J ˆ 6.7 Hz, 1H, NH), 7.70 (d, J ˆ 8.0 Hz, 1H, NH), 6.73 (s, 2H, MIC-
ˆ
ˆ
CH), 5.17 ± 5.23 (m, 1H, CH C Far), 5.05 ± 5.14 (m, 2H, CH C Far), 4.62 ±
4.67 (m, 1H, a-CH), 4.38 ± 4.50 (m, 3H, a-CH), 4.31 ± 4.36 (m, 1H, a-CH),
3.77 ± 3.81 (m, 2H, b-CH2 Ser), 3.76 (s, 3H, COOCH3), 3.51 (t, J ˆ 7.2 Hz,
2H, 2MIC-NCH2), 3.35 ± 3.41 (m, 1H, b-CH2a CysPal), 3.19 ± 3.28 (m, 2H,
CH2a Far, b-CH2b CysPal), 3.09 ± 3.16 (m, 1H, CH2b Far), 2.92 ± 3.00 (m, 3H,
b-CH2a CysFar , e-CH2 Lys), 2.78 ± 2.84 (m, 1H, b-CH2a CysFar), 2.51 ± 2.62 (m,
4H, g-CH2 Met, CH2 Pal), 2.24 ± 2.32 (m, 2H, MIC), 1.88 ± 2.14 (m, 14H, b-
CH2 Met, SCH3 Met, CH2a Lys, CH2 Far), 1.52 ± 1.76 (m, 9H, CH2 Pal, CH2
MIC, CH2 Lys), 1.68 (s, 6H, CH3 Far), 1.60 (s, 6H, CH3 Far), 1.38 ± 1.52 (m,
2H, CH2 Lys), 1.21 ± 1.38 (m, 26H, CH3(CH2)12(CH2)2COS, CH2 MIC), 0.88
(t, J ˆ 6.9 Hz, 3H, w-CH3 Pal); 13C NMR (CDCl3/CD3OD 4:1, 125 MHz,
3H, w-CH3 Hd); 13C NMR (CDCl3, 125 MHz, TMS): d ˆ 173.2 (C O), 81.6
ˆ
(CO2C(CH3)3), 54.7 (a-CH Cys), 37.4 (CH2), 32.8 (CH2), 31.9 (CH2), 29.7
(CH2), 29.7 (CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 29.2 (CH2), 28.8
(CH2), 28.0 (CO2C(CH3)3), 22.7 (CH2), 14.1 (CH3 Hd); MS (FAB, 3-NBA):
‡
‡
m/z: 402.4 [M‡H] , 346.3 [M tBu‡H] .
N-Fluorenylmethoxycarbonyl-(l)-methionyl-(l)-seryl-S-hexadecyl-(l)-cys-
teine tert-butyl ester (Fmoc-Met-Ser-Cys(Hd)-OtBu) (22b): EEDQ
(382 mg, 1.55 mmol) was added to a solution of 20 (600 mg, 1.31 mmol)
and H-Cys(Hd)-OtBu (21b; 478 mg, 1.19 mmol) in DMF (7 mL). After
3 days, ethyl acetate (50 mL) was added and the solution was extracted
twice with 0.5n hydrochloric acid (50 mL), saturated NaHCO3 solution
(50 mL), and brine (50 mL), dried over MgSO4, and filtered. The solvent
was evaporated in vacuo and the product was isolated by flash chromatog-
ˆ
ˆ
ˆ
ˆ
TMS): d ˆ 200.6 (C O), 175.2 (C O), 173.0 (C O), 172.0 (C O), 171.4
ˆ
ˆ
ˆ
(C O), 171.3 (C O), 170.8 (C O), 140.3 (quart, Far), 135.5 (quart, Far),
134.3 (2CH MIC), 131.5 (quart, Far), 124.5 (Far-CH), 123.9 (Far-CH), 119.7
(Far-CH), 61.8 (b-CH2 Ser), 55.8 (a-CH), 54.6 (a-CH), 53.1 (a-CH), 52.8
(a-CH), 52.7 (COOCH3), 52.3 (a-CH), 44.2 (CH2), 39.8 (CH2), 39.6 (CH2),
37.8 (CH2), 35.9 (CH2), 32.7 (CH2), 32.1 (CH2), 31.0 (CH2), 30.9 (CH2), 30.4
(CH2), 29.8 (CH2), 29.8 (CH2), 29.7 (CH2), 29.6 (CH2), 29.5 (CH2), 29.4
(CH2), 29.2 (CH2), 28.4 (CH2), 26.9 (CH2), 26.6 (CH2), 26.4 (CH2), 26.4
(CH2), 25.7 (Far-CH3), 25.7 (CH2), 25.2 (CH2), 22.8 (CH2), 22.1 (CH2), 17.7
(Far-CH3), 16.2 (Far-CH3), 16.1 (Far-CH3), 15.3 (SCH3 Met), 14.1 (CH3
raphy (hexane/ethyl acetate 1:1) to yield 22b (639 mg, 0.759 mmol, 64%)
as a colorless solid. M.p. 1148C; [a]D20
1
ˆ
12.3 (c ˆ 1.5, CHCl3); H NMR
(CDCl3, 500 MHz, TMS): d ˆ 7.76 (d, J ˆ 7.5 Hz, 2H, Fmoc-CH), 7.59 (dd,
J ˆ 7.4 Hz, J ˆ 3.8 Hz, 2H, Fmoc-CH), 7.40 (t, J ˆ 7.4 Hz, 2H, Fmoc-CH),
7.32 (t, J ˆ 7.4 Hz, 2H, Fmoc-CH), 7.03 ± 7.09 (m, 2H, NH), 5.50 (d, J ˆ
7.4 Hz, 1H, NH), 4.61 ± 4.69 (m, 1H, a-CH), 4.48 ± 4.54 (m, 1H, a-CH),
4.38 ± 4.45 (m, 3H, Fmoc-CH2, a-CH), 4.22 (t, J ˆ 6.9 Hz, 1H, Fmoc-CH),
4.05 ± 4.12 (m, 1H, b-CH2a Ser), 3.62 ± 3.71 (m, 1H, b-CH2b Ser), 2.98 (dd,
J ˆ 13.8 Hz, J ˆ 4.5 Hz, 1H, b-CH2a Cys), 2.91 (dd, J ˆ 13.6 Hz, J ˆ 6.1 Hz,
1H, b-CH2b Cys), 2.52 ± 2.61 (m, 2H, g-CH2 Met), 2.50 (t, J ˆ 7.4 Hz, 2H,
CH2 Hd), 2.11 (s, 3H, SCH3 Met), 2.08 ± 2.16 (m, 1H, b-CH2a Met), 1.95 ±
2.03 (m, 1H, b-CH2b Met), 1.49 ± 1.58 (m, 2H, CH2 Hd), 1.47 (s, 9H,
‡
Pal); MS (FAB, 3-NBA): m/z: 1220.5 [M‡H] .
N-Fluorenylmethoxycarbonyl-(l)-methionyl-(l)-serine
(Fmoc-Met-Ser-
OH) (20): DCC (1.00 g, 4.85 mmol) was added at 08C to a solution of
Fmoc-Met-OH (1.50 g, 4.04 mmol) and N-hydroxysuccinimide (0.47 g,
4.04 mmol) in dry THF (5 mL). After 2.5 h the solution was filtered and
the solvent was evaporated in vacuo. The residue was dissolved in dioxane
(10 mL) and added at 08C to a solution of serine (0.64 g, 6.06 mmol) and
NaOH (0.24 g, 6.06 mmol) in water (10 mL). After the mixture had spent
two days at ambient temperature, water (50 mL) was added and the
solution was extracted twice with ethyl acetate (50 mL). By addition of
hydrochloric acid the pH of the aqueous layer was adjusted to 3 and the
solution was again extracted with ethyl acetate (3 Â 50 mL). The combined
organic layers were dried over MgSO4 and filtered, and the solvent was
evaporated in vacuo to yield 20 (1.52 mg, 3.32 mmol, 82%) as a colorless
solid, which was used without further purification. [a]2D0 ˆ ‡10.6 (c ˆ 1.0,
chloroform/methanol 2:1); 1H NMR (CDCl3/CD3OD 6:1, 500 MHz, TMS):
d ˆ 7.75 (d, J ˆ 7.5 Hz, 2H, CH Fmoc), 7.61 (t, J ˆ 6.5 Hz, 2H, CH Fmoc),
7.39 (t, J ˆ 7.4 Hz, 2H, CH Fmoc), 7.30 (t, J ˆ 7.5 Hz, 2H, CH Fmoc), 4.55 (t,
J ˆ 3.5 Hz, 1H, a-CH Ser), 4.43 (dd, J ˆ 10.5 Hz, J ˆ 7.2 Hz, 1H, a-CH
Met), 4.34 ± 4.39 (m, 2H, CH2 Fmoc), 4.21 (t, J ˆ 6.9 Hz, 1H, CH Fmoc),
3.98 (dd, J ˆ 11.5 Hz, J ˆ 3.9 Hz, 1H, b-CH2a Ser), 3.86 (dd, J ˆ 11.5 Hz, J ˆ
3.4 Hz, 1H, b-CH2b Ser), 2.51 ± 2.60 (m, 2H, g-CH2 Met), 2.05 ± 2.14 (m,
CO2C(CH3)3), 1.22 ± 1.38 (m, 26H, CH3(CH2)13(CH2)2S), 0.88 (t, J ˆ 6.9 Hz,
13
ˆ
3H, w-CH3 Hd); C NMR (CDCl3, 125 MHz, TMS): d ˆ 171.8 (C O),
ˆ
ˆ
ˆ
170.2 (C O), 169.7 (C O), 156.2 (C O), 143.8 (quart, Fmoc), 143.7 (quart,
Fmoc), 141.3 (quart, Fmoc), 127.7 (Fmoc-CH), 127.1 (Fmoc-CH), 125.1
(Fmoc-CH), 120.0 (Fmoc-CH), 83.1 (CO2C(CH3)3), 67.2 (Fmoc-CH2), 62.9
(b-CH2 Ser), 54.5 (a-CH), 54.0 (a-CH), 52.6 (a-CH), 47.1 (Fmoc-CH), 34.0
(CH2), 32.7 (CH2), 32.0 (CH2), 31.9 (CH2), 30.1 (CH2), 29.7 (CH2), 29.7
(CH2), 29.6 (CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 28.8
(CH2), 28.0 (CO2C(CH3)3), 22.7 (CH2), 15.3 (SCH3 Met), 14.1 (CH3 Hd);
‡
‡
MS (FAB, 3-NBA): m/z: 864.5 [M‡Na] , 842.7 [M‡H] , 786.4 [M
‡
tBu‡H] ; elemental analysis calcd (%) for C46H71N3O7S2: C 65.60, H
8.50, N 4.99; found: C 65.55, H 8.36, N 4.97.
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-hexadecyl-(l)-cysteine tert-
butyl ester (MIC-Met-Ser-Cys(Hd)-OtBu): Et2NH (4 mL) was added to a
solution of 22b (203 mg, 0.242 mmol) in dichloromethane (4 mL). After
1.5 h the solvents were evaporated in vacuo, the residue was dissolved in
dichloromethane (2 mL), and MIC-OH (56.1 mg, 0.226 mmol) and EEDQ
(77.6 mg, 0.314 mmol) were added. After 15 h the solvent was evaporated
in vacuo and the residue was dissolved in ethyl acetate (40 mL). The
solution was extracted twice with 0.5n hydrochloric acid (40 mL), saturated
NaHCO3 solution (40 mL), and brine (40 mL), dried over MgSO4, and
filtered. The solvent was evaporated in vacuo and the product was isolated
by flash chromatography (hexane/ethyl acetate 1:3) to yield the product
1H, b-CH2a Met), 2.09 (s, 3H, Met-CH3), 1.86 ± 1.98 (m, 1H, b-CH2b Met);
13
ˆ
C NMR (CDCl3/CD3OD 6:1, 125 MHz, TMS): d ˆ 172.5 (C O), 172.3
ˆ
ˆ
(C O), 157.0 (C O), 144.1 (quart, arom, Fmoc), 143.9 (quart, arom, Fmoc),
141.5 (quart, arom, Fmoc), 128.0 (arom, Fmoc-CH), 127.3 (arom, Fmoc-
CH), 125.3 (arom, Fmoc-CH), 120.2 (arom, Fmoc-CH), 67.3 (Fmoc-CH2),
62.3 (b-CH2 Ser), 55.0 (a-CH), 54.2 (a-CH), 47.3 (Fmoc-CH), 32.1 (g-CH2
Met), 30.2 (b-CH2 Met), 15.2 (SCH3 Met); MS (FAB, 3-NBA): m/z: 481.2
[M‡Na] , 459.2 [M‡H] ; HRMS (FAB, 3-NBA) calcd for C23H27N2O6S:
459.1590; found 459.1564, elemental analysis calcd (%) for C23H26N2O6S: C
60.25, H 5.72, N 6.11; found:C 60.09, H 5.82, N 5.63.
(135 mg, 0.166 mmol, 69%) as a colorless solid. M.p. 888C; [a]D20
ˆ
12.0
‡
‡
(c ˆ 1.0, CHCl3); 1H NMR (CDCl3, 500 MHz, TMS): d ˆ 7.12 (d, J ˆ 7.7 Hz,
2H, NH), 6.69 (s, 2H, MIC-CH), 6.40 (d, J ˆ 7.5 Hz, 1H, NH), 4.60 ± 4.68
(m, 2H, 2a-CH), 4.49 ± 4.54 (m, 1H, a-CH), 4.54 ± 4.58 (m, 1H, a-CH), 4.06
(dd, J ˆ 11.4 Hz, J ˆ 3.6 Hz, 1H, b-CH2a Ser), 3.69 (dd, J ˆ 11.5 Hz, J ˆ
5.5 Hz, 1H, b-CH2b Ser), 3.51 (t, J ˆ 7.2 Hz, 2H, MIC-NCH2), 2.99 (dd, J ˆ
S-Hexadecyl-(l)-cysteine tert-butyl ester (H-Cys(Hd)-OtBu) (21): Under
an argon atmosphere, dithiothreitol (1.4 g, 9.08 mmol) was added to a
1190
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0706-1190 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 6

Protecting Groups for Lipidated Peptides
1184±1193
13.7 Hz, J ˆ 4.7 Hz, 1H, b-CH2a Cys), 2.91 (dd, J ˆ 13.6 Hz, J ˆ 6.2 Hz, 1H,
b-CH2b Cys), 2.53 ± 2.61 (m, 2H, g-CH2 Met), 2.52 (t, J ˆ 7.4 Hz, 2H, CH2
Hd), 2.22 (t, J ˆ 7.5 Hz, 2H, MIC), 2.08 ± 2.14 (m, 1H, b-CH2a Met), 2.12 (s,
3H, Met-CH3), 1.96 ± 2.04 (m, 1H, b-CH2b Met), 1.50 ± 1.70 (m, 6H, CH2
MIC, CH2 Hd), 1.48 (s, 9H, CO2C(CH3)3), 1.22 ± 1.37 (m, 28H,
(CH2), 30.6 (CH2), 30.3 (CH2), 29.8 (CH2), 29.7 (CH2), 29.7 (CH2), 29.4
(CH2), 29.0 (CH2), 28.3 (CH2), 26.8 (CH2), 26.6 (CH2), 26.4 (CH2), 26.3
(CH2), 25.7 (Far-CH3), 25.0 (CH2), 22.8 (CH2), 20.9 (Mtt-CH3), 17.7 (Far-
CH3), 16.2 (Far-CH3), 16.0 (Far-CH3), 15.4 (SCH3 Met), 14.1 (CH3 Hd); MS
‡
‡
‡
(FAB, 3-NBA): m/z: 1484.6 [M‡Na] , 1462.4 [M‡H] , 1460.4 [M H] ,
‡
‡
‡
CH3(CH2)13(CH2)2S, CH2 MIC), 0.88 (t, J ˆ 6.9 Hz, 3H, w-CH3 Hd);
1384.5 [M C6H5 H] , 1371.5 [M C6H4CH3] , 1206.6 [M Mtt‡H] ,
13
‡
ˆ
ˆ
C NMR (CDCl3, 125 MHz, TMS): d ˆ 173.1 (C O), 171.5 (C O), 170.9
257.2 [Mtt] .
ˆ
ˆ
ˆ
(2C O MIC), 170.1 (C O), 169.7 (C O), 134.1 (2CH MIC), 83.1
(CO2C(CH3)3), 62.7 (b-CH2 Ser), 54.5 (a-CH), 52.5 (a-CH), 52.5 (a-CH),
37.6 (CH2), 36.2 (CH2), 34.0 (CH2), 32.6 (CH2), 31.9 (CH2), 31.3 (CH2), 30.3
(CH2), 29.7 (CH2), 29.7 (CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 29.2
(CH2), 28.8 (CH2), 28.2 (CH2), 28.0 (CO2C(CH3)3), 26.3 (CH2), 24.9 (CH2),
22.7 (CH2), 15.3 (SCH3 Met), 14.1 (CH3 Hd); MS (FAB, 3-NBA): m/z: 835.5
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-hexadecyl-(l)-cysteyl-(l)-ly-
syl-S-farnesyl-(l)-cysteine methyl ester (MIC-Met-Ser-Cys(Hd)-Lys-Cys-
(Far)-OMe) (1b): Et3SiH (16.2 mL, 102 mmol) was added to a solution of
19b (14.9 mg, 10.2 mmol) in a 1% solution of TFA in dichloromethane
(0.8 mL). After 50 min a solution of 1% EtMe2N in dichloromethane
(0.9 mL) was added, the solvent was evaporated in vacuo, and the product
was isolated by size-exclusion chromatography (Sephadex LH-20, CHCl3/
‡
‡
‡
[M‡Na] , 813.5 [M‡H] , 757.4 [M tBu‡H] ; HRMS (FAB) calcd for
‡
C41H72N4NaO8S2 ([M‡Na] ): 835.4689; found: 835.4727.
MeOH 1:1) to yield 1b (11.9 mg, 9.86 mmol, 97%) as a colorless, waxy solid.
M.p. 1938C; [a]2D0
ˆ
39.0 (c ˆ 0.3, CHCl3/MeOH 2:1); H NMR (CDCl3/
1
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-hexadecyl-(l)-cysteine (MIC-
Met-Ser-Cys(Hd)-OH) (15b): TFA (0.25 mL) was added to a solution of
MIC-Met-Ser-Cys(Hd)-OtBu (61.0 mg, 75.0 mmol) in dichloromethane
(0.25 mL). After 3 h, toluene (4 mL) was added and the solvent was
evaporated in vacuo. Addition of toluene and evaporation were repeated
twice to yield 15b in quantitative yield as a colorless solid. M.p. 1418C;
CD3OD 6:1, 500 MHz, TMS): d ˆ 6.74 (s, 2H, MIC-CH), 5.20 (t, J ˆ 7.3 Hz,
ˆ
ˆ
1H, CH C Far), 5.06 ± 5.13 (m, 2H, CH C Far), 4.63 (dd, J ˆ 8.2 Hz, J ˆ
5.0 Hz, 1H, a-CH), 4.42 ± 4.48 (m, 3H, 3a-CH), 4.38 (t, J ˆ 4.6 Hz, 1H, a-
CH), 3.92 (dd, J ˆ 11.5 Hz, J ˆ 4.4 Hz, 1H, b-CH2a Ser), 3.79 (dd, J ˆ
11.5 Hz, J ˆ 4.9 Hz, 1H, b-CH2b Ser), 3.75 (s, 3H, COOCH3), 3.52 (t, J ˆ
7.2 Hz, 2H, MIC-NCH2), 3.24 (dd, J ˆ 13.1 Hz, J ˆ 8.3 Hz, 1H, CH2a Far),
3.12 (dd, J ˆ 13.2 Hz, J ˆ 7.2 Hz, 1H, CH2b Far), 3.00 (dd, J ˆ 13.6 Hz, J ˆ
6.5 Hz, 1H, b-CH2a CysHd), 2.93 ± 2.99 (m, 3H, b-CH2a CysFar , e-CH2 Lys),
2.89 (dd, J ˆ 13.6 Hz, J ˆ 7.9 Hz, 1H, b-CH2b CysHd), 2.80 (dd, J ˆ 13.9 Hz,
J ˆ 8.2 Hz, 1H, b-CH2b CysFar), 2.51 ± 2.61 (m, 4H, g-CH2 Met, CH2 Hd),
2.29 (t, 2H, J ˆ 7.0 Hz, MIC), 1.94 ± 2.15 (m, 14H, b-CH2 Met, SCH3 Met,
CH2a Lys, CH2 Far), 1.54 ± 1.78 (m, 9H, CH2 Hd, CH2 MIC, CH2 Lys), 1.68
(s, 6H, CH3 Far), 1.61 (s, 6H, CH3 Far), 1.41 ± 1.50 (m, 2H, CH2 Lys), 1.21 ±
1.39 (m, 28H, CH3(CH2)13(CH2)2S, CH2 MIC), 0.88 (t, J ˆ 7.0 Hz, 3H, w-
CH3 hexadecyl); 13C NMR (CDCl3/CD3OD 6:1, 125 MHz, TMS): d ˆ 175.3
[a]2D0
ˆ
9.6 (c ˆ 1.0, CHCl3/MeOH 2:1); 1H NMR (CDCl3/CD3OD 6:1,
500 MHz, TMS): d ˆ 6.72 (s, 2H, MIC-CH), 4.48 ± 4.70 (m, 3H, 3 a-CH),
3.88 ± 3.93 (m, 1H, b-CH2a Ser), 3.73 ± 3.78 (m, 1H, b-CH2b Ser), 3.52 (t, J ˆ
7.2 Hz, 2H, MIC-NCH2), 3.04 (dd, J ˆ 13.8 Hz, J ˆ 4.4 Hz, 1H, b-CH2a
Cys), 2.95 (dd, J ˆ 13.9 Hz, J ˆ 6.8 Hz, 1H, b-CH2b Cys), 2.49 ± 2.58 (m, 4H,
g-CH2 Met, CH2 Hd), 2.24 (t, J ˆ 7.3 Hz, 2H, MIC), 2.06 ± 2.16 (m, 1H, b-
CH2a Met), 2.11 (s, 3H, Met-CH3), 1.90 ± 1.98 (m, 1H, b-CH2b Met), 1.53 ±
1.69 (m, 6H, CH2 MIC, CH2 Hd), 1.22 ± 1.41 (m, 28H, CH3(CH2)13(CH2)2S,
CH2 MIC), 0.88 (t, J ˆ 6.8 Hz, 3H, w-CH3 Hd); 13C NMR (CDCl3/CD3OD
ˆ
ˆ
ˆ
6:1, 125 MHz, TMS): d ˆ 174.4 (C O), 172.7 (C O), 172.4 (C O), 171.3
ˆ
ˆ
ˆ
ˆ
ˆ
ˆ
ˆ
ˆ
(2C O MIC), 170.6 (C O), 134.3 (2CH MIC), 62.4 (b-CH2 Ser), 55.0 (a-
CH), 52.6 (a-CH), 52.3 (a-CH), 37.8 (CH2), 36.0 (CH2), 33.7 (CH2), 32.6
(CH2), 32.0 (CH2), 31.6 (CH2), 30.3 (CH2), 29.8 (CH2), 29.8 (CH2), 29.7
(CH2), 29.7 (CH2), 29.5 (CH2), 29.4 (CH2), 29.0 (CH2), 28.3 (CH2), 26.4
(CH2), 25.1 (CH2), 22.8 (CH2), 15.3 (SCH3 Met), 14.2 (CH3 Hd); MS (FAB,
(C O), 173.0 (C O), 172.3 (C O), 171.7 (C O), 171.4 (C O), 171.4 (C O),
140.3 (quart, Far), 135.6 (quart, Far), 134.4 (2CH MIC), 131.5 (quart, Far),
124.5 (Far-CH), 124.0 (Far-CH), 119.8 (Far-CH), 62.0 (b-CH2 Ser), 55.8 (a-
CH), 53.9 (a-CH), 53.3 (a-CH), 52.9 (a-CH), 52.7 (COOCH3), 52.4 (a-
CH), 39.9 (CH2), 39.7 (CH2), 37.8 (CH2), 36.0 (CH2), 33.0 (CH2), 32.8
(CH2), 32.5 (CH2), 32.1 (CH2), 31.0 (CH2), 30.7 (CH2), 30.5 (CH2), 29.9
(CH2), 29.9 (CH2), 29.8 (CH2), 29.7 (CH2), 29.6 (CH2), 29.5 (CH2), 29.1
(CH2), 28.4 (CH2), 26.9 (CH2), 26.7 (CH2), 26.5 (CH2), 26.5 (CH2), 25.8
(Far-CH3), 25.3 (CH2), 22.9 (CH2), 22.2 (CH2), 17.8 (Far-CH3), 16.2 (Far-
CH3), 16.1 (Far-CH3), 15.3 (SCH3 Met), 14.2 (CH3 Hd); MS (FAB, 3-NBA):
‡
‡
3-NBA): m/z: 779.5 [M‡Na] , 757.4 [M‡H] ; HRMS (FAB) calcd for
‡
C37H64N4NaO8S2 ([M‡Na] ): 779.4063; found: 779.3996.
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-hexadecyl-(l)-cysteyl-N(e)-(4-
tolyldiphenylmethyl)-(l)-lysyl-S-farnesyl-(l)-cysteine methyl ester (MIC-
Met-Ser-Cys(Hd)-Lys(Mtt)-Cys(Far)-OMe) (19b): Et2NH (0.4 mL) was
added to a solution of 7 (90.0 mg, 95.1 mmol) in dichloromethane (1.6 mL).
After 4 h the solvent was evaporated in vacuo, the residue was dissolved in
a mixture of dichloromethane (5 mL) and trifluoroethanol (0.1 mL), and
15b (55.9 mg, 73.8 mmol) and EEDQ (23.7 mg, 95.9 mmol) were added.
After 15 h the solvent was evaporated in vacuo and the product was
isolated by flash chromatography (dichloromethane/ethanol 30:1) to yield
19b (28.3 mg, 19.3 mmol, 26%) as a colorless, waxy solid. M.p. 1758C;
‡
m/z: 1206.5 [M‡H] .
N-Fluorenylmethoxycarbonyl-(l)-methionyl-(l)-seryl-S-tert-butylthio-(l)-
cysteine tert-butyl ester (Fmoc-Met-Ser-Cys(tBu)-OtBu) (22c): EEDQ
(488 mg, 1.97 mmol) was added to a solution of Fmoc-Met-Ser-OH (20;
835 mg, 1.82 mmol) and H-Cys(tBu)-OtBu (21c; 403 mg, 1.82 mmol) in
DMF (8 mL). After 15 h ethyl acetate (50 mL) was added and the solution
was extracted twice with 0.5n hydrochloric acid (50 mL), saturated
NaHCO3 solution (50 mL), and brine (50 mL), dried over MgSO4, and
filtered. The solvent was evaporated in vacuo and the product was isolated
by flash chromatography (hexane/ethyl acetate 2:1) to yield 22c (618 mg,
[a]2D0
ˆ
21.2 (c ˆ 1.0, CHCl3); 1H NMR (CDCl3/CD3OD 6:1, 500 MHz,
TMS): d ˆ 7.42 (d, J ˆ 7.6 Hz, 4H, Mtt-CH), 7.22 ± 7.33 (m, 2H, Mtt-CH),
7.24 ± 7.34 (m, 6H, Mtt-CH), 7.13 ± 7.20 (m, 2H, Mtt-CH), 7.05 ± 7.09 (m, 2H,
Mtt-CH), 6.71 (s, 1H, MIC-CH), 6.70 (s, 1H, MIC-CH), 5.15 ± 5.23 (m, 1H,
0.875 mmol, 58%) as a colorless solid. M.p. 1338C; [a]D20
ˆ
49.8 (c ˆ 1.1,
CH C Far), 5.05 ± 5.12 (m, 2H, CH C Far), 4.34 ± 4.64 (m, 5H, 5a-CH),
3.88 ± 3.95 (m, 1H, b-CH2a Ser), 3.69 ± 3.78 (m, 1H, b-CH2b Ser), 3.69 (s, 3H,
COOCH3), 3.47 ± 3.55 (m, 2H, MIC-NCH2), 3.21 (dd, J ˆ 13.1 Hz, J ˆ
8.2 Hz, 1H, CH2a Far), 3.09 (dd, J ˆ 13.1 Hz, J ˆ 7.4 Hz, 1H, CH2b Far),
2.90 ± 2.98 (m, 4H, b-CH2 CysHd, b-CH2a CysFar), 2.79 (dd, J ˆ 13.8 Hz, J ˆ
7.8 Hz, 1H, b-CH2b CysFar), 2.45 ± 2.58 (m, 6H, g-CH2 Met, CH2 Hd, e-CH2
Lys), 2.30 (b, 3H, Mtt-CH3), 2.18 ± 2.26 (m, 2H, MIC), 1.82 ± 2.15 (m, 14H,
b-CH2 Met, SCH3 Met, CH2a Lys, CH2 Far), 1.48 ± 1.69 (m, 11H, CH2 MIC,
CH2 Lys, CH2 Hd), 1.68 (s, 3H, CH3 Far), 1.67 (s, 3H, CH3 Far), 1.60 (s, 6H,
CH3 Far), 1.19 ± 1.39 (m, 28H, CH3(CH2)13(CH2)2S, CH2 MIC), 0.88 (t, J ˆ
6.8 Hz, 3H, w-CH3 Hd); 13C NMR (CDCl3/CD3OD 6:1, 125 MHz, TMS):
CHCl3); 1H NMR (CDCl3, 500 MHz, TMS): d ˆ 7.75 (d, J ˆ 7.5 Hz, 2H,
Fmoc-CH), 7.58 (dd, J ˆ 7.4 Hz, J ˆ 3.7 Hz, 2H, Fmoc-CH), 7.39 (t, J ˆ
7.5 Hz, 2H, Fmoc-CH), 7.27 ± 7.34 (m, 3H, J ˆ 7.4 Hz, 2 Fmoc-CH, 1 NH),
7.24 (d, J ˆ 6.9 Hz, 1H, NH), 5.50 (d, J ˆ 7.7 Hz, 1H, NH), 4.72 ± 4.79 (m,
1H, a-CH), 4.57 ± 4.63 (m, 1H, a-CH), 4.35 ± 4.47 (m, 3H, a-CH, Fmoc-
CH2), 4.21 (t, J ˆ 6.9 Hz, 1H, Fmoc-CH), 4.06 (dd, J ˆ 11.3 Hz, J ˆ 3.0 Hz,
1H, b-CH2a Ser), 3.70 (dd, J ˆ 11.0 Hz, J ˆ 5.1 Hz, 1H, b-CH2b Ser), 3.22
(dd, J ˆ 13.6 Hz, J ˆ 4.1 Hz, 1H, b-CH2a Cys), 3.10 (dd, J ˆ 13.6 Hz, J ˆ
6.1 Hz, 1H, b-CH2b Cys), 2.52 ± 2.65 (m, 2H, g-CH2 Met), 2.07 ± 2.18 (m,
1H, b-CH2a Met), including 2.10 (s, 3H, Met-CH3), 1.95 ± 2.05 (m, 1H, b-
CH2b Met), 1.47 (s, 9H, CO2C(CH3)3), 1.30 (s, 9H, SC(CH3)3); 13C NMR
ˆ
ˆ
ˆ
ˆ
ˆ
ˆ
ˆ
ˆ
ˆ
d ˆ 174.3 (C O), 172.2 (C O), 172.1 (C O), 171.2 (2C O MIC), 170.6
(CDCl3, 125 MHz, TMS): d ˆ 171.6 (C O), 170.2 (C O), 169.2 (C O),
ˆ
ˆ
ˆ
ˆ
(C O), 176.9 (C O), 169.4 (C O), 146.4 (quart, Mtt), 143.3 (quart, Mtt),
140.1 (quart, Far), 135.8 (quart, Mtt), 135.4 (quart, Far), 134.2 (2CH MIC),
131.4 (quart, Far), 128.7 (Mtt, 3CH), 127.8 (Mtt, CH), 126.2 (Mtt, CH),
124.4 (Far-CH), 123.9 (Far-CH), 119.6 (Far-CH), 70.7 (quart, Mtt), 62.1 (b-
CH2 Ser), 54.9 (a-CH), 53.6 (a-CH), 53.0 (a-CH), 52.8 (a-CH), 52.5
(COOCH3), 52.1 (a-CH), 43.6 (CH2), 39.8 (CH2), 37.7 (CH2), 35.9 (CH2),
33.9 (CH2), 33.5 (CH2), 32.8 (CH2), 32.6 (CH2), 32.4 (CH2), 32.0 (CH2), 31.3
156.2 (C O), 143.9 (quart, arom, Fmoc), 143.7 (quart, arom, Fmoc), 141.3
(quart, arom, Fmoc), 127.7 (arom, Fmoc-CH), 127.1 (arom, Fmoc-CH),
125.1 (arom, Fmoc-CH), 120.0 (arom, Fmoc-CH), 83.3 (CO2C(CH3)3), 67.2
(Fmoc-CH2), 62.7 (b-CH2 Ser), 54.3 (a-CH), 54.0 (a-CH), 53.1 (a-CH), 48.4
(SC(CH3)3), 47.1 (Fmoc-CH), 42.2 (b-CH2 Cys), 32.0 (CH2), 30.1 (CH2),
29.8 (SC(CH3)3), 27.9 (CO2C(CH3)3), 15.3 (SCH3 Met); MS (FAB, 3-NBA):
‡
‡
‡
‡
m/z: 737.1 [M‡K] , 728.2 [M‡Na] , 706.3 [M‡H] , 650.2 [M tBu‡H] ,
Chem. Eur. J. 2001, 7, No. 6
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0706-1191 $ 17.50+.50/0
1191

H. Waldmann et al.
FULL PAPER
‡
593.1 [M 2tBu‡H] ; elemental analysis calcd (%) for C34H47N3O7S3: C
CH2b Ser, COOCH3), 3.44 ± 3.52 (m, 2H, MIC-NCH2), 3.05 ± 3.22 (m, 4H,
b-CH2 CysStBu, CH2 Far), 2.94 (dd, J ˆ 14.8 Hz, J ˆ 4.9 Hz, 1H, b-CH2a
CysFar), 2.84 (dd, J ˆ 14.1 Hz, J ˆ 7.2 Hz, 1H, b-CH2b CysFar), 2.48 ± 2.62 (m,
4H, g-CH2 Met, e-CH2 Lys), 2.29 (b, 3H, Mtt-CH3), 2.19 ± 2.28 (m, 2H,
MIC), 1.82 ± 2.15 (m, 14H, b-CH2 Met, SCH3 Met, CH2a Lys, CH2 Far),
1.45 ± 1.72 (m, 9H, CH2 MIC, CH2 Lys), 1.67 (s, 3H, CH3 Far), 1.65 (s, 3H,
CH3 Far), 1.59 (s, 6H, CH3 Far), 1.24 ± 1.41 (m, 11H, SC(CH3)3, CH2 MIC);
57.85, H 6.71, N 5.95; found: C 58.13, H 6.77, N 6.11.
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-tert-butylthio-(l)-cysteine
tert-butyl ester (MIC-Met-Ser-Cys(tBu)-OtBu): Et2NH (3 mL) was added
to a solution of 22c (100 mg, 0.142 mmol) in dichloromethane (3 mL).
After 5 h the solvent was evaporated in vacuo, the residue was dissolved in
dichloromethane (2 mL), and MIC-OH (32.9 mg, 0.156 mmol) and EEDQ
(45.5 mg, 0.184 mmol) were added. After 18 h dichloromethane (40 mL)
was added, the solution was extracted twice with 0.5n hydrochloric acid
(40 mL), saturated NaHCO3 solution (40 mL), and brine (40 mL), dried
over MgSO4, and filtered. The solvent was evaporated in vacuo and the
product was isolated by flash chromatography (dichloromethane/ethanol
30:1, then 20:1) to yield the product (55.8 mg, 82.4 mmol, 58%) as a
13C NMR (CDCl3, 125 MHz, TMS): d ˆ 173.4 (C O), 171.1 (C O), 170.9
ˆ
ˆ
ˆ
ˆ
(C O), 170.5 (C O), 146.5 (quart, Mtt), 143.2 (quart, Mtt), 140.1 (quart,
Far), 135.5 (quart, Mtt), 135.3 (quart, Far), 134.1 (2CH MIC), 131.3 (quart,
Far), 128.6 (Mtt, 3 CH), 127.8 (Mtt, CH), 126.2 (Mtt, CH), 124.3 (Far-CH),
123.8 (Far-CH), 119.5 (Far-CH), 70.6 (quart, Mtt), 62.3 (b-CH2 Ser), 54.3
(a-CH), 53.5 (a-CH), 52.8 (a-CH), 52.5 (COOCH3), 51.8 (a-CH), 48.6
(CH2), 48.4 (SC(CH3)3), 41.0 (CH2), 39.7 (CH2), 37.6 (CH2), 36.2 (CH2), 36.1
(CH2), 32.8 (CH2), 31.0 (CH2), 30.9 (CH2), 30.3 (CH2), 29.8 (SC(CH3)3),
29.7 (CH2), 28.3 (CH2), 28.2 (CH2), 26.7 (CH2), 26.5 (CH2), 26.4 (CH2), 26.3
(CH2), 25.7 (Far-CH3), 24.9 (CH2), 21.0 (Mtt-CH3), 17.7 (Far-CH3), 16.2
(Far-CH3), 16.0 (Far-CH3), 15.4 (SCH3 Met); MS (FAB, 3-NBA): m/z:
colorless solid. M.p. 988C; [a]D20
ˆ
41.7 (c ˆ 0.8, CHCl3); 1H NMR
(CDCl3, 500 MHz, TMS): d ˆ 7.29 (d, J ˆ 7.8 Hz, 1H, NH), 7.23 (d, J ˆ
7.2 Hz, 1H, NH), 6.69 (s, 2H, MIC-CH), 6.52 (d, J ˆ 7.3 Hz, 1H, NH), 4.72 ±
4.77 (m, 1H, a-CH), 4.62 ± 4.68 (m, 1H, a-CH), 4.54 ± 4.58 (m, 1H, a-CH),
4.06 (dd, J ˆ 11.5 Hz, J ˆ 3.9 Hz, 1H, b-CH2a Ser), 3.71 (dd, J ˆ 11.5 Hz, J ˆ
5.4 Hz, 1H, b-CH2b Ser), 3.51 (t, J ˆ 7.2 Hz, 2H, MIC-NCH2), 3.23 (dd, J ˆ
13.7 Hz, J ˆ 4.4 Hz, 1H, b-CH2a Cys), 3.12 (dd, J ˆ 13.6 Hz, J ˆ 6.2 Hz, 1H,
b-CH2b Cys), 2.53 ± 2.65 (m, 2H, g-CH2 Met), 2.23 (t, J ˆ 7.5 Hz, 2H, MIC),
2.09 ± 2.18 (m, 1H, b-CH2a Met), 2.12 (s, 3H, Met-CH3), 1.96 ± 2.05 (m, 1H,
b-CH2b Met), 1.64 ± 1.70 (m, 2H, CH2 MIC), 1.57 ± 1.63 (m, 2H, CH2 MIC),
‡
‡
‡
1348.5 [M‡Na] , 1326.5 [M‡H] , 1248.4 [M C6H5] , 1334.5 [M
‡
‡
‡
C6H4CH3] , 1070.5 [M Mtt‡H] , 257.1 [Mtt] .
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-tert-butylthio-(l)-cysteyl-(l)-
lysyl-S-farnesyl-(l)-cysteine methyl ester (MIC-Met-Ser-Cys(tBu)-Lys-Cys-
(Far)-OMe) (1c): Et3SiH (11.4 mL, 71.6 mmol) was added to a solution of
19c (9.5 mg, 7.2 mmol) in a 1% solution of TFA in dichloromethane
(0.8 mL). After 60 min a solution of 1% EtMe2N in dichloromethane
(0.9 mL) was added, the solvent was evaporated in vacuo and the product
was isolated by size-exclusion chromatography (Sephadex LH-20, CHCl3/
1.49 (s, 9H, CO2C(CH3)3), 1.28 ± 1.36 (m, 2H, CH2 MIC), 1.32 (s, 9H,
13
ˆ
SC(CH3)3), C NMR (CDCl3, 125 MHz, TMS): d ˆ 173.2 (C O), 171.7
ˆ
ˆ
ˆ
ˆ
(C O), 170.9 (2 C O MIC), 170.1 (C O), 169.2 (C O), 134.1 (2CH MIC),
83.3 (CO2C(CH3)3), 62.6 (b-CH2 Ser), 54.5 (a-CH), 53.1 (a-CH), 52.5 (a-
CH), 48.4 (SC(CH3)3), 42.2 (b-CH2 Cys), 37.6 (CH2), 36.1 (CH2), 31.5
(CH2), 30.3 (CH2), 29.8 (SC(CH3)3), 29.7 (CH2), 28.2 (CH2), 28.0
(CO2C(CH3)3), 26.3 (CH2), 24.9 (CH2), 15.3 (SCH3 Met); MS (FAB,
MeOH 1:1) to yield 1c (5.8 mg, 5.4 mmol, 76%) as a colorless, waxy solid.
1
M.p. 1558C; [a]2D0
ˆ
59.0 (c ˆ 0.2, CHCl3/MeOH 2:1); H NMR (CDCl3/
CD3OD 6:1, 500 MHz, TMS): d ˆ 6.74 (s, 2H, MIC-CH), 5.21 (t, J ˆ 7.3 Hz,
‡
‡
‡
ˆ
ˆ
1H, CH C Far), 5.08 ± 5.14 (m, 2H, CH C Far), 4.63 (dd, J ˆ 8.1 Hz, J ˆ
5.0 Hz, 1H, a-CH), 4.60 (dd, J ˆ 8.5 Hz, J ˆ 5.7 Hz, 1H, a-CH), 4.42 ± 4.56
(m, 2H, 2 a-CH), 4.38 (t, J ˆ 4.7 Hz, 1H, a-CH), 3.92 (dd, J ˆ 11.4 Hz, J ˆ
4.7 Hz, 1H, b-CH2a Ser), 3.81 (dd, J ˆ 11.4 Hz, J ˆ 5.0 Hz, 1H, b-CH2b Ser),
3.76 (s, 3H, COOCH3), 3.52 (t, J ˆ 7.2 Hz, 2H, 2 MIC-NCH2), 3.18 ± 3.26
(m, 2H, CH2a Far, b-CH2a CysStBu), 3.12 (dd, J ˆ 13.5 Hz, J ˆ 7.5 Hz, 1H,
CH2b Far), 3.08 (dd, J ˆ 13.6 Hz, J ˆ 8.5 Hz, 1H, b-CH2b CysStBu), 2.93 ± 2.99
(m, 3H, b-CH2a CysFar , e-CH2 Lys), 2.80 (dd, J ˆ 13.9 Hz, J ˆ 8.2 Hz, 1H, b-
CH2b CysFar), 2.52 ± 2.61 (m, 2H, g-CH2 Met), 2.30 (td, J ˆ 7.5 Hz, J ˆ
2.3 Hz, 2H, MIC), 1.92 ± 2.16 (m, 14H, b-CH2 Met, SCH3 Met, CH2a Lys,
CH2 Far), 1.59 ± 1.79 (m, 7H, CH2 MIC, CH2 Lys), 1.68 (s, 6H, CH3 Far),
1.61 (s, 6H, CH3 Far), 1.41 ± 1.50 (m, 2H, CH2 Lys), 1.30 ± 1.39 (m, 11H,
SC(CH3)3, CH2 MIC); 13C NMR (CDCl3/CD3OD 6:1, 125 MHz, TMS): d ˆ
3-NBA): m/z: 699.3 [M‡Na] , 677.3 [M‡H] , 621.3 [M tBu‡H] , 564.2
‡
[M 2tBu] ; HRMS (FAB) calcd for C29H49N4O8S3: 677.2713; found:
677.2728.
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-tert-butylthio-(l)-cysteine
(MIC-Met-Ser-Cys(tBu)-OH) (15c): TFA (0.25 mL) was added to
a
solution of MIC-Met-Ser-Cys(tBu)-OtBu (15.1 mg, 22.3 mmol) in dichloro-
methane (0.25 mL). After 3 h, toluene (1 mL) was added and the solvent
was evaporated in vacuo. Addition of toluene and evaporation were
repeated twice to yield 15c in quantitative yield as a colorless solid. M.p.
1238C; [a]2D0
ˆ
48.6 (c ˆ 0.7, CHCl3/MeOH 2:1); 1H NMR (CDCl3/
CD3OD 6:1, 500 MHz, TMS): d ˆ 7.78 (d, J ˆ 7.6 Hz, 1H, NH), 7.71 (d,
J ˆ 7.6 Hz, 1H, NH), 7.45 (d, J ˆ 7.7 Hz, 1H, NH), 6.73 (s, 2H, MIC-CH),
4.73 ± 4.79 (m, 1H, a-CH), 4.47 ± 4.55 (m, 2H, 2a-CH), 3.89 (dd, J ˆ
11.5 Hz, J ˆ 4.8 Hz, 1H, b-CH2a Ser), 3.75 (dd, J ˆ 11.5 Hz, J ˆ 5.3 Hz,
1H, b-CH2b Ser), 3.52 (t, J ˆ 7.2 Hz, 2H, MIC-NCH2), 3.26 (dd, J ˆ 13.7 Hz,
J ˆ 4.4 Hz, 1H, b-CH2a Cys), 3.11 (dd, J ˆ 13.7 Hz, J ˆ 7.6 Hz, 1H, b-CH2b
Cys), 2.52 ± 2.58 (m, 2H, g-CH2 Met), 2.24 (t, J ˆ 7.5 Hz, 2H, MIC), 2.07 ±
2.16 (m, 1H, b-CH2a Met), 2.12 (s, 3H, Met-CH3), 1.90 ± 1.99 (m, 1H, b-
CH2b Met), 1.56 ± 1.69 (m, 4H, CH2 MIC), 1.26 ± 1.35 (m, 2H, CH2 MIC),
1.33 (s, 9H, SC(CH3)3); 13C NMR (CDCl3/CD3OD 6:1, 125 MHz, TMS):
ˆ
ˆ
ˆ
ˆ
ˆ
175.4 (C O), 173.1 (C O), 172.2 (C O), 171.5 (C O), 171.4 (C O MIC),
171.3 (C O), 140.3 (quart, Far), 135.6 (quart, Far), 134.4 (2 CH MIC), 131.5
ˆ
(quart, Far), 124.5 (Far-CH), 124.0 (Far-CH), 119.8 (Far-CH), 61.9 (b-CH2
Ser), 55.9 (a-CH), 54.0 (a-CH), 53.4 (a-CH), 53.0 (a-CH), 52.7 (COOCH3),
52.4 (a-CH), 40.7 (CH2), 39.9 (CH2), 39.7 (CH2), 37.8 (CH2), 36.0 (CH2),
32.7 (CH2), 31.0 (CH2), 30.8 (CH2), 30.5 (CH2), 30.0 (SC(CH3)3), 29.7
(CH2), 28.5 (CH2), 26.9 (CH2), 26.7 (CH2), 26.5 (CH2), 25.8 (Far-CH3), 25.3
(CH2), 22.2 (CH2), 17.8 (Far-CH3), 16.2 (Far-CH3), 16.1 (Far-CH3), 15.4
ˆ
ˆ
ˆ
ˆ
d ˆ 174.5 (C O), 172.4 (C O), 171.3 (2C O MIC), 170.6 (C O), 134.3
(2CH MIC), 62.2 (b-CH2 Ser), 55.0 (a-CH), 52.7 (a-CH), 52.3 (a-CH), 48.3
(SC(CH3)3), 41.7 (b-CH2 Cys), 37.8 (CH2), 36.0 (CH2), 31.5 (CH2), 30.3
(CH2), 29.9 (SC(CH3)3), 28.4 (CH2), 26.4 (CH2), 25.2 (CH2), 15.3 (SCH3
‡
(SCH3 Met); MS (FAB, 3-NBA): m/z: 1070.5 [M‡H] ; HRMS (FAB) calcd
for C50H83N7O10S4: 1070.516; found: 1070.519.
‡
‡
Met); MS (FAB, 3-NBA): m/z: 659.3 [M‡K] , 643.2 [M‡Na] , 621.2
Acknowledgements
‡
[M‡H] ; HRMS (FAB) calcd for C25H41N4O8S3: 621.2087; found: 621.2094.
Maleimidocaproyl-(l)-methionyl-(l)-seryl-S-tert-butylthio-(l)-cysteyl-N(e)
-
This work was supported by the Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie.
(4-tolyldiphenylmethyl)-(l)-lysyl-S-farnesyl-(l)-cysteine methyl ester
(MIC-Met-Ser-Cys(tBu)-Lys(Mtt)-Cys(Far)-OMe) (19c): Et2NH (0.4 mL)
was added to a solution of 7 (85.6 mg, 90.5 mmol) in dichloromethane
(1.6 mL). After 4 h the solvent was evaporated in vacuo, the residue was
dissolved in a mixture of dichloromethane (2 mL) and trifluoroethanol
(0.1 mL), and 15c (47.1 mg, 69.6 mmol) and EEDQ (24.2 mg, 90.5 mmol)
were added. After 13 h the solvent was evaporated in vacuo and the
product was isolated by flash chromatography (dichloromethane/ethanol
30:1) to yield 19c (25.9 mg, 19.5 mmol, 28%) as a colorless, waxy solid. M.p.
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1558C; [a]2D0
ˆ
47.2 (c ˆ 0.5, CHCl3); 1H NMR (CDCl3, 500 MHz, TMS):
d ˆ 7.43 (d, J ˆ 7.8 Hz, 4H, Mtt-CH), 7.30 (d, J ˆ 8.2 Hz, 2H, Mtt-CH),
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ˆ
ˆ
5.16 ± 5.23 (m, 1H, CH C Far), 5.03 ± 5.11 (m, 2H, CH C Far), 4.40 ± 4.75
(m, 5H, 5 a-CH), 3.91 ± 3.99 (m, 1H, b-CH2a Ser), 3.60 ± 3.80 (m, 4H, b-
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1192
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Received: September 15, 2000 [F2731]
Chem. Eur. J. 2001, 7, No. 6
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