Reversed approach to S-farnesylation and S-palmitoylation: Application to an efficient synthesis of the C-terminus of lipidated human N-Ras hexapeptide

Article abstract of DOI:10.1021/jo0482357

(Chemical Equation Presented) A general reversed approach is described to synthesize S-palmitoylated and S-farnesylated peptides via S_N2 displacement of bromide by reaction of a thiol group containing lipid as nucleophile with bromoalanine-cont

Full text of DOI:10.1021/jo0482357

The wide interest in Ras proteins is largely based on
the existence of various Ras oncogenes.4 It is estimated
that oncogenic Ras proteins are present in about 30% of
all human tumors.5 The Ras proteins have both farnesyl
thioether and palmitic acid thioester moieties. Protein
farnesylation is a stable and irreversible protein modi-
fication that plays a critical role in directing the modified
protein to the plasma membranes.2 Unlike farnesylation,
palmitoylation is a reversible reaction suggesting that it
may be particularly important for regulating protein
function.6 This palmitoylation is required for transforma-
tion of cells by H-Ras.
The synthesis of peptide conjugates such as the char-
acteristic C-terminal lipidated hexapeptide of the human
N-Ras protein is more complicated due to its pronounced
acid and base lability.7 The pioneering work of Waldmann
et al. enabled the synthesis of substantial amounts of
such types of peptide conjugates through multistep
solution-phase7 as well as solid-phase method.8 Recently,
van der Donk et al. also synthesized fluorescent farne-
sylated Ras peptide.9 In continuation of our work to
synthesize peptide conjugates, particularly thio glyco-
peptides,10 we became interested in developing a general
method to synthesize S-farnesylated and S-palmitoylated
peptides from bromoalanine (BrAla) containing peptides.
Herein, we report on this reversed approach to the
S-farnesylation and S-palmitoylation and exploitation of
this method to the synthesis of the C-terminus of human
N-Ras hexapeptide.
Attachment of lipid moieties such as the palmitoyl or
the farnesyl group to the cysteine is generally performed
by reacting the sulfhydryl group of cysteine with excess
palmitoyl chloride7 and excess farnesyl bromide under
basic conditions.11 The method reported in this paper is
opposite to these literature methods. Reaction of nucleo-
philic thiopalmitic acid or farnesyl mercaptan with
electrophilic bromoalanine containing peptide segments
under basic conditions should give the corresponding
S-palmitoyl and S-farnesyl peptides (Scheme 1). For the
present investigation, the required starting material
thiopalmitic acid 2 was prepared by the reaction of
N-succinimide ester 1 with H2S in the presence of Et3N
in dioxane (Scheme 2).
Reversed Approach to S-Farnesylation and
S-Palmitoylation: Application to an
Efficient Synthesis of the C-Terminus of
Lipidated Human N-Ras Hexapeptide
Kandasamy Pachamuthu, Xiangming Zhu, and
Richard R. Schmidt*
Fachbereich Chemie, Universita¨t Konstanz, Fach M 725,
D-78457 Konstanz, Germany
richard.schmidt@uni-konstanz.de
Received October 7, 2004
A general reversed approach is described to synthesize
S-palmitoylated and S-farnesylated peptides via SN2 dis-
placement of bromide by reaction of a thiol group containing
lipid as nucleophile with bromoalanine-containing peptides
as electrophile. By employing this approach, lipidated pep-
tides, including characteristic partial structures of human
Ras peptides, were synthesized in good yields. This method
gives access to farnesylated, palmitoylated, and doubly
lipidated peptides.
The biological activities of several cellular proteins
require association with the inner surface of the plasma
membrane, and this membrane localization is dependent
on the post-translational attachment of lipid residues
such as farnesyl, myristoyl, and palmitoyl moieties to the
carboxy terminus of the protein. Covalently modified
proteins (for instance, by lipid attachment) play impor-
tant roles in various biological processes,1 as signal
transduction and cell-cycle control. Among the different
types of covalently modified proteins, lipidation is par-
ticularly relevant.2 For example, the transmembrane G
proteins are S-palmitoylated, the heterotrimeric G pro-
teins are N-myristoylated, S-palmitoylated, and S-far-
nesylated, and most of the Ras proteins carry S-palmitoyl
and S-farnesyl groups. Further, membrane-associated
proteins such as the enzyme NOx synthetase3 and various
viral envelope proteins are S-palmitoylated. The lipid
groups are believed to be involved in protein-protein and
protein-lipid interactions, and they serve as anchors of
the proteins to different membranes.
(4) Clarke, S. Annu. Rev. Biochem. 1992, 61, 355.
(5) (a) Grand, R. J. A.; Owen, D. Biochem. J. 1991, 279, 609-631.
(b) Barbacid, M. Annu. Rev. Biochem. 1987, 56, 779.
(6) (a) Milligan, G.; Parenti, M.; Magee, A. I. Trends Biochem. Sci.
1995, 20, 181-187. (b) Mumby, S. M. Curr. Opin. Cell Biol. 1997, 9,
148-154.
(7) (a) Schelhaas, M.; Na¨gele, E.; Kuder, N.; Bader, B.; Kuhlmann,
J.; Wittinghofer, A.; Waldmann, H. Chem. Eur. J. 1999, 5, 1239-1252.
(b) Schelhaas, M.; Glomsda, S.; Ha¨nsler, M.; Jakubke, H.-D.; Wald-
mann, H. Angew. Chem. 1996, 108, 82-85; Angew. Chem., Int. Ed.
Engl. 1996, 35, 106-109.
(8) (a) Ludolph, B.; Waldmann, H. Chem. Eur. J. 2003, 9, 3683-
3691. (b) Kragol, G.; Lumbierres, M.; Palomo, J. M.; Waldmann, H.
Angew. Chem. 2004, 116, 5963-5966; Angew. Chem., Int. Ed. 2004,
43, 5839-5842.
(9) Zhu, Y.; van der Donk, W. A. Org. Lett. 2001, 3, 1189-1192.
(10) Zhu, X.; Schmidt, R. R. Tetrahedron Lett. 2003, 44, 6063-
6067.
(1) (a) Hancock, J. F.; Magee, A. I.; Childs, J. E.; Marshall, C. J.
Cell 1989, 57, 1167-1177. (b) Casey, P. J.; Solski, P. A.; Der, C. J.;
Buss, J. E. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 8323-8327. (c)
Schafer, W. R.; Trueblood, C. E.; Yang, C.-C.; Mayer, M. P.; Rosenberg,
S.; Poulter, C. D.; Kim, S.-H.; Rine, J. Science 1990, 1113-1139.
(2) Casey, P. J. Science 1995, 268, 221.
(11) (a) Brown, M. J.; Milano, P. D.; Lever, D. C.; Epstein, W. W.;
Poulter, C. D. J. Am. Chem. Soc. 1991, 113, 3176-3177. (b) Yang, C.-
C.; Marlowe, C. K.; Kania, R. J. Am. Chem. Soc. 1991, 113, 3177-
3178.
(3) Robinson, L. J.; Michel, T. Proc. Natl. Acad. Sci. U.S.A. 1995,
92, 11776.
10.1021/jo0482357 CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/02/2005
3720
J. Org. Chem. 2005, 70, 3720-3723

SCHEME 1. Reversed Approach To Synthesize
S-Palmitoyl and S-Farnesylated Cysteine and
Lipidated Peptides
SCHEME 2. Synthesis of Thiopalmitic Acid 2
SCHEME 3. Synthesis of Farnesyl Mercaptan 5
FIGURE 1. Structure of the C-terminus of human N-Ras
protein.
SCHEME 5. Retrosynthetic Analysis of the
C-Terminal Hexapeptide 22
SCHEME 4. Synthesis of S-Farnesylated Di- and
Tripeptides 7, 8, and 10
7 in 76% yield. Reaction of the same tripeptide with
farnesyl mercaptan 5 in the presence of Cs2CO3 as base
in DMF gave the corresponding S-farnesylated product
8 in 56% yield. In a similar manner, reaction of dipeptide
AlocSerBrAlaOMe 9 with farnesyl mercaptan 5 provided
the S-farnesylated product 10 in 70% yield.
A biologically relevant target compound, the S-farne-
sylated, S-palmitoylated C-terminus of human N-Ras
hexapeptide was chosen in order to demonstrate the
feasibility of the present reaction conditions (Figure 1).
The retrosynthetic pathway is depicted in Scheme 5. The
target compound 227a should be available from the
S-farnesylated bromoalanine containing hexapeptide 21
which was disconnected into intermediates 13 and 20.
N-Aloc-protected bromoalanine 11 was synthesized in
one pot by (i) heating of N-Aloc protected serine with 2.5
equiv of TMSCl in dry THF (ii) followed by the addition
of CBr4 and PPh3 at 0 °C (Scheme 6). The direct
conversion of the hydroxy group of serine to bromide,
Farnesyl mercaptan 5 was prepared by modifying the
reported method.12 We first reacted farnesyl bromide with
thioacetic acid in the presence of K2CO3 in DMF followed
by deacetylation in the presence of catalytic amounts of
NaOMe in methanol yielding farnesyl mercaptan 5,
which could be purified by column chromatography
(Scheme 3, 93% overall yield).
As a model substrate, a tripeptide AlocMetTyr-
BrAlaOMe 6 was synthesized by coupling dipeptide
AlocMetTyrOH with bromoalanine methyl ester
(BrAlaOMe)13,14 in the presence of EEDQ in dry dichlo-
romethane (Scheme 4). Reaction of thiopalmitic acid with
the tripeptide 6 in the presence of 1.1 equiv of NaOAc in
DMF afforded the corresponding S-palmitoylated product
(14) To a stirred solution of N-Boc BrAlaOMe (282 mg, 1 mmol) in
DCM (4 mL) was added TFA (1 mL) at 0 °C. The reaction mixture
was stirred at room temp. for 1 h. Evaporation of the solvent and
coevaporation with toluene yielded the corresponding TFA salt of
BrAlaOMe and this was used as such for further reaction.
(12) Gilbert, B. A.; Tan, E. W.; Perez-Sala, D.; Rando, R. R. J. Am.
Chem. Soc. 1992, 114, 3966-3973.
(13) Dhanalekshmi, S.; Christian, L.; Rolf, S. Helv. Chim. Acta 1996,
79, 288-94.
J. Org. Chem, Vol. 70, No. 9, 2005 3721

SCHEME 6. Synthesis of AlocBrAlaMetOH 13
coupled with dipeptide 13 in the presence of EEDQ in
dichloromethane at room temp. for 12 h yielding the
required S-farnesylated, bromoalanine containing hexa-
peptide 21 in 55% yield. EEDQ as coupling reagent gave
the best results throughout our studies in peptide seg-
ment connections. For instance, coupling of dipeptide 14
with proline tert-butyl ester worked well to give tripeptide
15 only when EEDQ was employed as a coupling agent
and coupling was not as successful when other coupling
reagents such as DCC, DIC, EDC, or PyBOP/DIPEA were
used. Finally, reaction of 21 with thiopalmitic acid in the
presence of NaOAc afforded the desired S-palmitoylated,
S-farnesylated hexapeptide 22 which had physical data
1
(optical rotation, H, and 13C NMR) in excellent agree-
SCHEME 7. Synthesis of
AlocCys(Pal)MetGlyLeuProCys(Far)OMe 22
ment with those previously reported.7a On incorporation
of bromoalanine into peptides, side reactions especially
with coupling reagents were not observed. Further,
intramolecular alkylation, for instance, of methionine
side chains or neighboring carbonyl groups with bro-
moalanine, was not noticed under the employed reaction
conditions.
Conclusion
In conclusion, an efficient reversed approach to the
synthesis of S-pamitoylated and S-farnesylated peptides
has been demonstrated where instead of alkylating a
peptide cysteine residue with a halogenated lipid, a thiol-
containing lipid was used to displace the bromide of a
bromoalanine containing peptide. This method was suc-
cessfully applied to the convergent synthesis of the
lipidated C-terminus of human N-Ras hexapeptide. Un-
der the required reaction conditions neither the acid nor
the base sensitivity of this molecule was affected, thus
pure product could be obtained in good yields. All starting
materials can be readily prepared; the synthesis of
N-Aloc-protected bromoalanine from N-Aloc-protected
serine could be performed in one-pot.
Experimental Section
trans,trans-Farnesyl Thioacetate (4). To a stirred solution
of farnesyl bromide 3 (568 mg, 2 mmol) and thioacetic acid (167
mg, 2.2 mmol) in DMF (10 mL) was added K2CO3 (276 mg, 2
mmol) at 0 °C. The reaction mixture was stirred at rt for 12 h.
The solution was extracted with ethyl acetate followed by
washing with 5% dilute HCl, water, and brine, and the organic
layer was dried over anhydrous MgSO4. After concentration of
the organic solvent, the crude material was purified by flash
column chromatography (9:1, petroleum ether/ethyl acetate) to
give the product 4 in 98% yield (555 mg) as a colorless liquid
which was immediately used in the next step.
trans,trans-Farnesyl Mercaptan (5). NaOMe (27 mg, 0.5
mmol) was added to the solution of 4 (555 mg, 1.98 mmol) in
dry methanol (15 mL), and the reaction mixture was allowed to
stir at rt for 6 h. The solvent was evaporated, the solution was
extracted with ethyl acetate and washed with 5% dilute HCl,
water, and brine, and the combined organic layer was dried over
anhydrous MgSO4. Evaporation of the solvent followed by
purification by flash column chromatography yielded the product
5 (449 mg, 95%) as a colorless liquid which was identical with
previously obtained material.12
without protecting the carboxyl group, was not successful.
Compound 12 was obtained by coupling of 11 with
commercially available methionine tert-butyl ester in the
presence of EEDQ in dry dichloromethane in 98% yield.
The tert-butyl ester was cleaved using 50% TFA in
dichloromethane to give the dipeptide 13.
For the synthesis of 20 (Scheme 7), N-Aloc-protected
dipeptide 14 was prepared by employing standard reac-
tion conditions.7a Coupling of 14 with commercially
available proline tert-butyl ester in the presence of EEDQ
in dry dichloromethane led to tripeptide 15 in 88% yield.
Cleavage of the tert-butyl group (f 16) and reaction with
the TFA salt of bromoalanine methyl ester13 (17) in the
presence of EEDQ in dry dichloromethane afforded
tetrapeptide 18 in 72% yield. Reaction of 18 with farnesyl
mercaptan C15H25-SH 5 in the presence of Cs2CO3 in
DMF gave the corresponding S-farnesylated product 19
in 75% yield. Cleavage of the N-allyloxy group using
catalytic amounts of Pd[PPh3]4 and piperidine as a
scavenger led to tripeptide 20. This compound was
N-Allyloxycarbonyl-L-methionyl-L-tyrosyl-(S-palmitoyl)-
L-cysteine Methyl Ester (AlocMetTyCys(Pal)OMe) (7). A
solution of 2 (39 mg, 0.14 mmol) in THF (5 mL) was added to a
stirred solution of 6 (40 mg, 0.07 mmol) and NaOAc (12 mg, 0.14
mmol) in DMF (5 mL) at 0 °C, and then the reaction mixture
was allowed to stir at rt for 12 h. The solution was extracted
3722 J. Org. Chem., Vol. 70, No. 9, 2005

with ethyl acetate followed by washing with 5% dilute HCl,
water, and brine. The combined organic layer was dried over
anhydrous MgSO4, and concentration of the solvent in vacuo
yielded the crude product, which was purified by flash column
chromatography (petroleum ether/ethyl acetate, 1.2:0.8) to yield
7 (40 mg, 76%) as an amorphous solid: Rf ) 0.6 (petroleum ether/
ethyl acetate, 1:1); [R]D ) -2.8 (c ) 1, CHCl3); 1H NMR (250
MHz, CDCl3) δ 0.88 (t, J ) 6.5 Hz, 3H, CH3), 1.25 (s, CH2, 24H,
12 × CH2), 1.58-1.70 (m, 2H, CH2), 1.86-2.04 (m, 2H, CH2),
2.07 (s, 3H, SCH3), 2.48-2.59 (m, 4H, 2 × CH2), 2.89-3.08 (m,
2H, CH2), 3.30 (t, J ) 6.2 Hz, 2H, CH2), 3.73 (s, 3H, OCH3),
4.25-4.34 (m, 1H, CH), 4.56 (br s, 2H, OCH2), 4.61-4.80 (m,
2H, CH), 5.20-5.34 (m, 2H, CHdCH2), 5.64-5.67 (m, 1H, NH),
5.82-6.03 (m, 1H, CH)CH2), 6.71 (d, J ) 8.2 Hz, 2H, aromatic),
under argon and in darkness for 1 h at rt. The solvent was
removed in vacuo, and the product was isolated by flash column
chromatography (silica gel, ethyl acetate/methanol, 9:1) to give
free amine 20 in 80% yield (177 mg) which was used immediately
for further reaction.
To a stirred solution of 12 (105 mg, 0.29 mmol) in dichlo-
romethane (4 mL) was added 1 mL of TFA at 0 °C. The solution
was stirred for 2 h at rt. Evaporation of the solvent and
coevaporation with toluene yielded the compound 13 which was
dissolved in dichloromethane (5 mL). To this solution were added
compound 20 (177 mg, 0.29 mmol) and EEDQ (108 mg, 0.44
mmol) in dichloromethane (10 mL) at 0 °C. The reaction mixture
was stirred at rt for 12 h. Removal of the solvent in vacuo and
purification of the crude material by flash column chromatog-
raphy (silica gel, ethyl acetate/methanol, 17:3) gave pentapeptide
21 (155 mg, 55%) as a waxy solid: Rf ) 0.6 (ethyl acetate/
methanol, 17:3); [R]D ) -84.1 (c ) 1.7, CHCl3); 1H NMR (250
MHz, CDCl3) δ 0.92-1.00 (m, 6H, 2 × CH3), 1.59, 1.65, 1.68 (s,
12H, 4 × CH3), 1.84-2.30 (m, 17H, 8 × CH2, CH), 2.08 (s, 3H,
SCH3), 2.50-3.30 (m, 6H, 3 × CH2), 3.56-3.80 (m, 4H, 2 × CH2),
3.72 (s, 3H, OCH3), 4.19 (br s, 2H, CH2), 4.58-5.46 (m, 12H,
OCH2, CHdCH2, 5 × CH, 3 × olefinic), 5.80-6.00 (1H, CH)CH2),
6.40 (br s, 1H, NH), 7.41, 7.80, 7.90, 8.10 (br s, 4H, NH); 13C
NMR (62.5 MHz, CDCl3) δ 15.3, 16.0, 16.1, 17.6, 21.9, 23.3, 24.7,
24.8, 25.6, 26.5, 26.7, 28.5, 29.5, 30.0, 31.9, 32.8, 33.9, 40.0, 42.2,
43.3, 47.5, 49.0, 52.0, 52.1, 52.3, 55.1, 59.7, 66.2, 118.0, 120.0,
123.8, 124.3, 131.2, 132.5, 135.3, 139.8, 156.1, 168.1, 168.7, 171.2
(2C), 171.3, 171.9. Anal. Calcd for C44H71BrN6O9S2 (970.39): C,
54.36; H, 7.36; N, 8.65. Found: C, 54.45; H, 7.48; N, 8.54.
N-Allyloxycarbonyl(S-palmitoyl)-L-cysteyl-L-meth-
ionylglycyl-L-leucyl-L-prolyl(S-farnesyl)-L-cysteine Methyl
Ester (AlocCys(Pol)MetGlyLeuProCys(Far)OMe) (22). A
solution of 5 (26 mg, 0.094 mmol) in THF (5 mL) was added at
0 °C to a stirred solution of 21 (45 mg, 0.047 mmol) and NaOAc
(8 mg, 0.094 mmol) in DMF (5 mL), and the reaction mixture
was allowed to stir at rt for 12 h. The solution was extracted
with ethyl acetate followed by washing with dilute HCl, water,
and brine. The combined organic layer was dried over anhydrous
MgSO4, and concentration of the solvent in vacuo yielded the
crude product, which was purified by flash column chromatog-
raphy (petroleum ether/ethyl acetate/methanol, 9.5:9.5:1) to
yield 22 (30 mg, 55%) as a waxy solid: Rf ) 0.4 (petroleum
ether/ethyl acetate/methanol, 9.5:9.5:1); [R]D ) -33.2 (c ) 0.7,
CHCl3) [lit.7a [R]D ) -34.0 (c ) 0.7, CHCl3)]; 1H NMR (250 MHz,
CDCl3) δ 0.84-0.98 (m, 9H, 3 × CH3), 1.24 (s, 24H, 12 × CH2),
1.56-1.76 (m, 14H), 1.95-2.35 (m, 17H), 2.09 (s, 3H, SCH3),
2.50-3.39 (m, 10H), 3.63-3.91 (m, 3H), 3.71 (s, 3H, OCH3),
4.27-4.34 (m, 1H, CH), 4.51-4.95 (m, 7H), 5.08-5.33 (m, 5H,
CHdCH2, 3 × olefinic(Far)), 5.80-6.04 (m, 2H, NH, CHdCH2),
7.26, 7.36, 7.81, 7.84, (br d, 4H, NH); .13C NMR (62.5 MHz,
CDCl3) δ 14.0, 15.1, 16.0, 16.1, 17.6, 21.8, 22.6, 23.3, 24.7, 24.9,
25.6, 25.62, 26.5, 26.7, 28.2, 28.9, 29.2, 29.3, 29.4, 29.6, 29.62,
29.65, 30.2, 30.8, 31.5, 31.9, 32.9, 39.7, 41.7, 43.4, 44.0, 47.3,
49.2, 52.0, 52.3, 52.5, 55.7, 60.0, 66.2, 117.9, 119.7, 123.8, 124.3,
131.2, 132.5, 135.3, 140.0, 156.3, 168.7, 169.8, 171.2, 171.24,
171.4, 172.2, 200.4.
6.85 (br s, 3H, NH, OH), 7.00 (d, J ) 8.2 Hz, 2H, aromatic); 13
C
NMR (62.5 MHz, CDCl3) δ 14.1, 15.2, 22.7, 25.5, 28.9, 29.2, 29.3,
29.4, 29.6, 29.65, 29.7, 30.1, 31.4, 31.9, 37.3, 44.0, 52.4, 52.7,
54.1, 54.4, 66.1, 68.2, 115.7, 118.1, 128.8, 130.4, 130.8, 132.5,
155.1, 170.0, 170.6, 171.1, 199.0; MS (MALDI) m/z 774.5 (M +
Na)+.
N-Allyloxycarbonyl-L-methionyl-L-tyrosyl-(S-farnesyl)-
L-cysteine Methyl Ester (AlocMetTyrCys(Far)OMe) (8).
Cs2CO3 (33 mg, 0.1 mmol) was added to a stirred solution of 6
(58 mg, 0.1 mmol) and farnesyl mercaptan 5 (26 mg, 0.11 mmol)
in DMF (5 mL) at 0 °C, and then the reaction mixture was
allowed to stir at rt for 1 h. The solution was extracted with
ethyl acetate followed by washing with 5% dilute HCl, saturated
NaHCO3, water, and brine. The combined organic layer was
dried over anhydrous MgSO4. The ethyl acetate layer was
concentrated and purified by flash column chromatography
(petroleum ether/ethyl acetate, 1.2:0.8) to give 8 (140 mg, 56%)
as colorless viscous oil: Rf ) 0.6 (petroleum ether/ethyl acetate,
1:1); [R]D ) -20.0 (c ) 1, CHCl3); 1H NMR (250 MHz, CDCl3) δ
1.60, 1.70 (s, 12H, CH3), 1.90-2.06 (m, 13H, SCH3, CH2), 2.44-
2.58 (m, 2H, CH2), 2.73-3.20 (m, 6H, CH2), 3.75 (s, 3H, OCH3),
4.30-4.35 (m, 1H, CH), 4.60 (br s, 2H, CH2), 4.70-4.90 (m, 2H,
CH), 5.05-5.40 (m, 5H, CHdCH2, olefinic(Far)), 5.70-6.00 (m,
2H, CHdCH2, NH), 6.70 (d, J ) 8.2 Hz, 2H, aromatic), 6.90-
7.04 (m, 3H, NH, OH), 7.00 (d, J ) 8.2 Hz, 2H, aromatic); MS
(MALDI) m/z 740.6 (M + Na)+. Anal. Calcd for C37H55N3O7S2
(717.35): C, 61.90; H, 7.72; N, 5.85. Found: C, 62.20; H, 7.83;
N, 5.72.
N-Allyloxycarbonylglycyl-L-leucyl-L-prolyl(S-farnesyl)-
L-cysteine Methyl Ester (AlocGlyLeuProCys(Far)OMe)
(19). To a stirred solution of 18 (60 mg, 0.12 mmol) and thio-
farnesol 5 (30 mg, 0.13 mmol) in DMF (5 mL) was added
Cs2CO3 (38 mg, 0.12 mmol) at 0 °C, and the reaction mixture
was allowed to stir at rt for 1 h. The solution was extracted with
ethyl acetate followed by washing with dilute HCl, saturated
NaHCO3, water, and brine. The combined organic layer was
dried over anhydrous MgSO4. The ethyl acetate layer was
concentrated and purified by flash column chromatography
(ethyl acetate/methanol, 9.9:0.1) to give 19 (61 mg, 75%) as a
colorless viscous oil: Rf ) 0.2 (ethyl acetate/methanol, 9.9:0.1);
[R]D ) -37.3 (c ) 1, CHCl3); 1H NMR (250 MHz, CDCl3) δ 0.86-
0.94 (m, 6H, 2 × CH3), 1.30-2.30 (m, 15H, 7 × CH2, CH), 1.56,
1.64 (s, 12H, 4 × CH3), 2.63-3.21 (m, 4H, 2 × CH2), 3.51-4.03
(m, 4H, 2 × CH2), 3.70 (s, 3H, OCH3), 4.56-4.70 (m, 4H, CH2, 2
× CH), 4.86-4.88 (m, 1H, CH), 5.03-5.30 (m, 5H, CHdCH2,
olefinic(Far)), 5.57 (br s, 1H, NH), 5.80-6.00 (m, 1H, CH)CH2),
7.38, 7.50 (br d, 2H, NH); MS (MALDI) m/z 713 (M + Na)+. Anal.
Calcd for C36H58N4O7S (690.40): C, 62.58; H, 8.46; N, 8.11.
Found: C, 62.73; H, 8.35; N, 7.89.
Acknowledgment. This work was supported by the
Deutsche Forschungsgemeinschaft and the Fonds der
Chemischen Industrie. K.P. is grateful for an Alexander
von Humboldt Fellowship.
Supporting Information Available: General experimen-
tal details, procedures for the synthesis of compounds 6, 9,
11, 12, 14, and 15 and physical data of these compounds, and
NMR spectra of all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
N-Allyloxycarbonyl-L-bromoalanyl-L-methionylglycyl-
L-leucyl-L-prolyl(S-farnesyl)-L-cysteine Methyl Ester
(AlocBrAlaMetGlyLeuProCys(Far)OMe) (21). Morpholine
(63 mg, 0.73 mmol) and tetrakis(trisphenylphosphine)palladium-
(0) (42 mg) were added to a solution of the tetrapeptide 19 (251
mg, 0.364 mmol) in THF (10 mL), and the mixture was stirred
JO0482357
J. Org. Chem, Vol. 70, No. 9, 2005 3723