A R T I C L E S
Vo¨lkert et al.
General Procedure for Coupling to Cysteine Methyl Ester
(Preparation of Compounds 7). To a solution of 1.1 equiv of
N-chlorosuccinimide in CH2Cl2 (2-5 mL/mmol) was added dropwise
at - 40 °C dimethyl sulfide (2 equiv). After being warmed to 0 °C for
5 min, the mixture was cooled to -40 °C, and a solution of 5 in CH2-
Cl2 was added dropwise. The resulting mixture was allowed to warm
to 0 °C over 1 h and stirred at 0 °C for 1 h. The mixture was diluted
with cyclohexane, washed twice with ice-cold brine, dried over MgSO4,
and concentrated in vacuo. The residue was used for the alkylation
step without further purification.
thoroughly with CH2Cl2. 9b was cleaved from the resin with 10% TFA
in CH2Cl2, and the solvent was removed by evaporation in vacuo under
repeated addition of toluene for azeotropic removal of TFA. Purification
by flash chromatography (SiO2; EtOAc/MeOH, 10:1) yields 375.8 mg
of 9b (70% starting from proline-loaded resin) as a colorless oil. The
20
assignment of the proton signals was achieved by H-H COSY. [R]D
) -43.5° (c ) 2.67, MeOH). 1H NMR (400 MHz, CDCl3): δ ) 0.73-
0.98 (m, 6H, δ-Leu), 1.12-1.37 (m, 11H, CH2 MIC, t-Bu), 1.39-1.73
(m, 7H, 2 CH2 MIC, â-Leu, γ-Leu), 1.98-2.19 (m, 7H, â′-Met, â′-
Pro, γ-Pro, CH3 Met), 2.20-2.41 (m, 4H, R-CH2 MIC, â-Met, â-Pro),
2.47-2.74 (m, 2H, γ-Met), 2.98-3.19 (m, 2H, â-Cys), 3.42-3.57 (m,
2H, N-CH2 MIC), 3.58-3.69 (m, 1H, δ′-Pro), 3.77-4.04 (m, 5H, 2
R-Gly, δ-Pro), 4.09-4.23 (m, 1H, R-Pro), 4.24-4.56 (m, 2H, R-Met,
R-Cys), 4.62-4.79 (m, 1H, R-Leu), 6.69 (s, 2H, HCdCH). HPLC-
ESI-MS: tR ) 11.11 min, purity >90%; m/z 858.2 [M + H]+, 880.3
[M + Na]+. HR-FAB-MS: m/z calcd for C37H59N7O10S3 857.3536,
found 857.3464.
The residue was dissolved in MeOH or THF and added at 0 °C to
a solution of l-cysteine methyl ester hydrochloride (1.1 equiv) in 2 N
NH3/MeOH (5-10 mL/mmol). After 1 h, the reaction mixture was
allowed to warm to room temperature for 1 h. After dilution with CH2-
Cl2 or ether, the mixture was washed with water. After thorough back-
extraction the combined organic layers were dried over MgSO4 and
concentrated in vacuo. The crude product was purified by flash column
chromatography on silica gel. Following this procedure, 13 compounds
7xxx were prepared. Their characterization is given in the Supporting
Information.
Fragment Couplings of the BP-Labeled Cysteines 7 with the
Biotinylated Peptide 9a. For fragment coupling, 1.0 equiv of the
biotinylated hexapeptide 9a was stirred with 1.0 equiv of the prenylated
cysteine 7, 1.3 equiv of EDC, and 1.5 equiv of HOBt in CH2Cl2 with
5% (v/v) 2,2,2-trifluoroethanol at room temperature for 16-24 h. The
reaction mixture was diluted with EtOAc, washed successively with
0.5 N aqueous HCl, saturated NaHCO3, and brine, dried over MgSO4,
and concentrated in vacuo. The crude product was purified by flash
chromatography.
Synthesis of Biot-Aca-GC(S-t-Bu)MGLPC(8-(4-benzoylphenyl-
oxy)geranyl)-OMe (10bpt). The reaction was performed on a scale
of 62.7 µmol. Purification using EtOAc/MeOH, 5:1 (v/v), yields 10bpt
(40 mg, 43%) as a pale brown oil. [R]D20 ) -38.5° (c ) 1.13, CHCl3).
1H NMR (500 MHz, CDCl3): δ ) 0.89-0.98 (m, 6H), 1.20-1.75 (m,
17H), 1.28 (s, 9H), 1.68 (s, 3H), 1.74 (s, 3H), 2.01-2.40 (m, 14H),
2.50-2.68 (m, 4H), 2.72-3.23 (m, 10H), 3.50-3.70 (m, 2H), 3.73 (s,
3H), 3.80-4.02 (m, 2H), 4.20-4.36 (m, 2H), 4.43 (s, 2H), 4.52-4.63
(m, 2H), 4.64-4.80 (m, 3H), 5.17 (t, 1H, J ) 7.2 Hz), 5.50 (t, 1H, J
) 7.3 Hz), 7.14 (m, 1H), 7.30-7.40 (m, 3H), 7.40-7.52 (m, 2H), 7.52-
7.60 (m, 1H), 7.76-7.82 (d, 2H, J ) 8.2 Hz). HPLC-ESI-MS: tR )
26.01 min, purity >90%; m/z 1453.4 [M + H]+, 1476.4 [M + Na]+.
MALDI-MS (DHB, calibrated): m/z calcd for C70H104N10O13S5Na+
1475.629, found 1475.626.
Synthesis of Biot-Aca-GC(S-t-Bu)MGLPC(12-(4-benzoylphenyl-
oxy)farnesyl)-OMe (10cpt). The reaction was performed on a scale
of 35.6 µmol. Purification using EtOAc/MeOH, 9:1 (v/v), yields 10cpt
(22.9 mg, 42%) as a pale brown oil. [R]D20 ) -11.2° (c ) 1.17, MeOH).
1H NMR (400 MHz, MeOD): δ ) 0.73-1.00 (m, 6H), 1.29 (s, 9H),
1.19-1.79 (m, 17H), 1.62 (s, 3H), 1.65 (s, 3H), 1.73 (s, 3H), 1.93-
2.36 (m, 14 H), 2.07 (s, 3H), 2.46-2.78 (m, 4H), 2.84-3.04 (m, 3H),
3.06-3.22 (m, 4H), 3.40-4.14 (m, 6H), 3.71 (s, 3H), 4.18-4.32 (m,
1H), 4.42-4.73 (m, 8H), 5.11 (t, 1H, J ) 7.2 Hz), 5.19 (t, 1H, J ) 7.6
Hz), 5.57 (t, 1H, J ) 7.2 Hz), 7.04 (d, 2H, J ) 8.8 Hz), 7.52 (t, 2H,
J ) 7.6 Hz), 7.62 (t, 1H, J ) 7.6 Hz), 7.71 (d, 2H, J ) 7.2 Hz), 7.79
(d, 2H, J ) 8.8 Hz). HPLC-ESI-MS: tR ) 25.75 min, purity >90%;
m/z 1521.5 [M + H]+, 1543.6 [M + Na]+. MALDI-MS (DHB): m/z
1543.65 [M + Na]+, 1560.73 [M + K]+. FAB-MS (NBA): m/z 1543.1
(M+ + Na).
Fragment Couplings of the BP-Labeled Cysteines 7 with the
MIC-Modified Peptide 9b. For fragment coupling, 1.0 equiv of the
MIC hexapeptide 9b was stirred with 1.0 equiv of the prenylated
cysteine 7, 1.3 equiv of EDC, and 1.5 equiv of HOBt in CH2Cl2 at
room temperature for 16-24 h. The reaction mixture was diluted with
EtOAc, washed successively with 0.5 N aqueous HCl, saturated
NaHCO3, and brine, dried over MgSO4, and concentrated in vacuo.
The crude product was purified by flash chromatography.
Solid-Phase Synthesis of H-GC(S-t-Bu)MGLP 8. 2-Chlorotrityl
polystyrene resin (loading 1.08 mmol/g) was loaded by shaking with
1.2 equiv of Fmoc-proline and 4.8 equiv of DIPEA in 5 mL of dry
CH2Cl2 for 2 h. Unreacted linker groups were blocked by subsequent
treatment with MeOH and DIPEA in CH2Cl2 for 0.5 h. The loading of
the resin was determined to be 0.25 mmol/g. After Fmoc cleavage with
20% piperidine in DMF, the peptide 8 was synthesized using the
following protocol: The Fmoc-amino acids (4 equiv) were coupled to
the resin with 3.6 equiv of HBTU, 4.8 equiv of HOBt, and 8 equiv of
DIPEA in DMF for 90 min after 5 min of preactivation. For capping,
the resin was treated twice with 10% acetic anhydride in pyridine for
5 min. Deprotection was accomplished through repeated washing with
a solution of 20% piperidine in DMF for 10 min. Between all steps
the resin was washed thoroughly with DMF. For further elaboration
of the N-terminus the Fmoc group was removed by the standard
cleavage procedure. The quality of the product was assured by cleavage
of a test sample with 10% TFA in CH2Cl2 and analysis by HPLC-
ESI-MS: tR ) 16.57 min, purity 90%; m/z (M) calcd for C42H58N6O9S
886.34, found 885.2 [M - H]-.
Synthesis of Biot-Aca-GC(S-t-Bu)MGLP 9a. For the biotinylation
Biot-Aca-OH (4 equiv) was activated for 10 min with 3.6 equiv of
HBTU, 4.8 equiv of HOBt, and 16 equiv of DIPEA in DMF and
subsequently shaken for 2 h with the immobilized hexapeptide 8. After
the reaction the resin was washed thoroughly with CH2Cl2. 9a was
cleaved from the resin with 10% TFA in CH2Cl2, and the solvent was
removed by evaporation in vacuo under repeated addition of toluene
for azeotropic removal of TFA, yielding 393.6 mg of 9a (87% starting
from Pro-loaded resin) as a colorless oil. Further purification was not
required. The assignment of the proton signals was achieved by H-H
COSY. [R]D ) -27.0° (c ) 1.08, MeOH). 1H NMR (500 MHz,
20
MeOD): δ ) 0.96 (dd, 6H, J ) 10.0, 6.5 Hz, δ-Leu), 1.26 (s, 9H,
t-Bu), 1.21-1.73 (m, 15H, 6 CH2 chain, â-Leu, γ-Leu), 2.00 (s, 3H,
CH3 Met), 1.85-2.05 (m, 4H, γ-Pro, γ-Met), 2.07-2.20 (m, 6H, 2
CH2CdO Biot-Aca, â-Pro), 2.40-2.58 (m, 2H, δ-Pro), 2.60-2.65 (m,
2H, S-CH2 Biot), 2.82-2.87 (m, 1H, S-CH Biot), 2.93 (dd, 1H, J )
7.7, 13.6 Hz, â-Cys), 3.04-3.14 (m, 3H, N-CH2 Aca, â′-Cys), 3.65-
3.90 (m, 4H, 2 R-Gly), 4.20-4.24 (m, 2H, N-CH Biot, R-Pro), 4.32-
4.36 (m, 1H, R-Met), 4.39-4.43 (m, 1H, N-CH Biot), 4.53 (dd, 1H, J
) 7.7, 5.7 Hz, R-Cys), 4.61 (dd, 1H, J ) 10.3, 4.3 Hz, R-Leu). HPLC-
ESI-MS: tR ) 13.79 min, purity 85%; m/z 1004.4 [M + H]+, 1027.4
[M + Na]+. HR-FAB-MS: m/z calcd for C43H74N9O10S4 1004.444,
found 1004.446.
Synthesis of MIC-GC(S-t-Bu)MGLP 9b. MIC-OH (4 equiv) was
activated for 10 min with 3.6 equiv of HBTU, 4.8 equiv of HOBt, and
8 equiv of DIPEA in DMF and subsequently shaken for 90 min with
the immobilized hexapeptide 8. After the reaction the resin was washed
Synthesis of MIC-GC(S-t-Bu)MGLPC(4-(4-benzoylphenyloxy)-
prenyl)-OMe (11bpt). The reaction was performed on a scale of 51
µmol. Purification using EtOAc/MeOH, 10:1 (v/v), yields 11bpt (36
9
12756 J. AM. CHEM. SOC. VOL. 125, NO. 42, 2003