Synthesis and application in SPPS of a stable amino acid isostere of palmitoyl cysteine

Article abstract of DOI:10.1016/j.tet.2008.11.033

(S)-2-Amino-4-(2-pentadecyl-1,3-dioxolan-2-yl)-butanoic acid, 11, Pdiob, a synthetic analogue C-isostere of palmitoylated cysteine, has been prepared starting from tetrabenzyl glutamic acid. The less hindered benzyl carboxylate ester was transformed into

Full text of DOI:10.1016/j.tet.2008.11.033

Tetrahedron 65 (2009) 844–848
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Synthesis and application in SPPS of a stable amino acid isostere of palmitoyl
cysteine
,
Elena Cini a, Lucia Raffaella Lampariello b, Manuela Rodriquez c, Maurizio Taddei a
*
a
`
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy
b
c
`
Dipartimento di Chimica, Universita degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Salerno, Via Ponte don Melillo, 84084 Fisciano, Salerno, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
(S)-2-Amino-4-(2-pentadecyl-1,3-dioxolan-2-yl)-butanoic acid, 11, Pdiob, a synthetic analogue C-isostere
Received 5 September 2008
Received in revised form 30 October 2008
Accepted 13 November 2008
Available online 18 November 2008
of palmitoylated cysteine, has been prepared starting from tetrabenzyl glutamic acid. The less hindered
benzyl carboxylate ester was transformed into the corresponding b-ketophosphonate and subjected to
a Horner–Wadsworth–Emmons reaction followed by hydrogenation/hydrogenolysis. This product was
used for the preparation on solid phase and under microwave dielectric heating of a highly lipidated
peptide that can be considered as an acid stable analogue of the C-terminus of the H-Ras heptapeptides
180–186.
Keywords:
Amino acid
Microwaves
Ó 2008 Elsevier Ltd. All rights reserved.
Hydrogenation
Peptidomimetics
Solid-phase synthesis
1. Introduction
proteins to the plasma membrane inducing the proteolysis of the
three last amino acids followed by the carbomethylation of the new
Covalent lipid modified proteins play a strategic role in many
signal transduction processes involving cell membrane-anchorage
mechanism. The high hydrophobicity, required for a proper sub-
cellular localization of such proteins, allows favourable interactions
with membranes, controls the active conformation of proteins
within a membrane layer and is involved in the switch on/off of
effective signal transduction.1 Post-translational linkage of long
fatty acyl chains on target proteins occurs through a thioether-
ification and thioesterification processes on specific cysteine resi-
dues (S-palmitoylation and S-farnesylation)2 or amidation of lysine
residues (N-myristoylation).3 A variety of transmembrane protein
and heterodimeric G-proteins and many other important signalling
proteins are lipidated4,5 and, based on structural elements adjacent
to the palmitoylation site, they can be divided into N-terminal
myristoyl class and C-terminal farnesyl class, which includes N-, H-
and K-Ras proteins. As the Ras pathway controls cell growth and
proliferation, when deregulation happens, uncontrolled cellular
growth and cancer may occur. S-Farnesylation of Ras cysteines
provides an irreversible modification that drives the lipidated
C-terminal Cys. The Ras carboxyl-terminal CAAX box (C is cysteine,
A is an aliphatic amino acid and X is usually Met or Ser) is recog-
nized by another transferase enzyme (palmitoyl acyltransferase
(PAT) in H-Ras, for example), which brings about S-palmitoylation
of cysteine. The latter is a reversible process involved in the steering
of biological events like regulated membrane trapping mechanism,
suggesting its importance in regulating protein role.6
Although many attempts have been made to investigate the
interactions of these proteins with the enzyme, the demand of new
peptide analogues is still high to enhance our understanding of
these phenomena.7 Moreover, depending on the nature of the lipid
motif, several problems have been encountered in some synthetic
approaches. The solid-phase synthesis of characteristic lipidated
Ras peptides containing both farnesylated and palmitoylated cys-
teine may be complicated by the acid and base sensitivity of these
groups.8 Consequently, several methods that employ particular
protections or synthetic strategies different from standard solid-
phase peptide synthesis have been successfully developed.9 Most of
these syntheses follow an Fmoc protocol adapted in order to in-
troduce the palmitoylated amino acid (or carry out palmitolylation)
in one of the final steps. However, the instability of the final peptide
remains a potential issue with the risk of lipid loss during purifi-
cation work-up or during biological evaluation. In continuation of
* Corresponding author. Tel.: þ39 0577234280; fax: þ39 0577234275.
E-mail address: taddei.m@unisi.it (M. Taddei).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.11.033

E. Cini et al. / Tetrahedron 65 (2009) 844–848
845
our work in peptidomimetics and modified or not-coded
a
-amino
Na2CO3) or in the presence of Boc2O introduced with the aim to
protect the NH2 before imine formation. In this last case, only
compound 6 was obtained as a single cis diastereoisomers as
revealed by 1H NMR and NOE spectroscopic analysis.
acids,10 we considered that the C-isostere of palmitoylated cysteine
1 (Scheme 1) could behave as an effective and stable surrogate of
the original post-translational modified amino acid.
NH2
NH2
H2
C
H
2 90 psi, Pd(OH)2/C
HO
S
HO
HOOC
N
R
rt, (Boc2O)
4
O
O
O
O
90%
5 R = H
1
6 R = Boc
Palmitoyl cysteine
C-Isostere of
palmitoyl cysteine
NBn2
H2 90 psi,
Pd(OH)2/C
rt, Boc2O
NaBH4,
H2O, rt
BnOOC
Scheme 1. Comparison between palmitoyl cysteine and C-isostere 1.
4
OH
86%
75%
Moreover, due to the presence of the carbonyl group, 1 could be
considered as a transition state analogue of natural S-palmitoylated
cysteine for potential inhibition of acyl-protein thioesterase en-
zyme. The N-Fmoc protected derivative of 1 could be also suitably
employed in standard solid-phase synthesis of stable lipidated
peptides as the final C-terminus of N-Ras protein.
NHBoc
7
HOOC
X
8 X = CHOH
9 X = C=O
ox.
2. Results and discussion
Scheme 3. Attempts to obtain N-Fmoc C-isostere of palmitoyl cysteine directly from 4.
The carbonyl group of 4 was then reduced to the alcohol with
NaBH4 in H2O to give compound 7 in good yield. Further hydro-
genolysis (H2 90 psi, rt, MeOH, 24 h) in the presence of Boc2O gave
alcohol 8 on which oxidation to 9 was immediately attempted.
However, Dess–Martin periodinane, Swern oxidation or Cr(VI) re-
agents did not give acceptable results. As the carbonyl protection
could be useful also during peptide synthesis, 4 was transformed
into dioxolane 10 (Scheme 4) using ethylene glycol and TsOH (tol-
uene, Dean–Stark apparatus, 84%).
The synthesis of 1 was approached starting from L-glutamic acid
that was fully protected as the tetrabenzyl derivative 2 as recently
reported in the literature.11 This product was then treated with
dimethyl methylphosphonate in the presence of BuLi at ꢀ78 ꢁC in
THF. We hypothesized that the hindrance around the a-carboxylic
group should direct the nucleophile towards the other carboxylic
group to produce compound 3 (Scheme 2).
NBn2
BnBr, NaOH,
Na2CO3
(MeO)2POMe,
BuLi, -78 °C
L-Glu
BnOOC
COOBn
96%
65%
2
NBn2
NBn2
HOCH2CH2OH
TsOH
H2, 90 psi,
Pd(OH)2/C
90%
OMe
P
O
BnOOC
C13H27CHO,
DIPEA, LiCl
O
OMe
O
BnOOC
NBn2
4
84%
O
85%
3a
BnOOC
O
10
NH2
NBn2
FmocOSu,
Et3N
HOOC
O
MeO
MeO
O
NHFmoc
O
P
O
COOBn
4
95%
O
HOOC
3b
O
11 Pdiob
Scheme 2. Synthesis of 4.
12
NHR
First attempt carried out with (MeO)2POMe and BuLi in THF
gave a mixture of the two regioisomers 3a and 3b in a 1:1 ratio
together with some starting material. Changing the base (LDA,
KN(SiMe3)2, i-PrMgBr) did not improve the formation of the re-
quired isomer. The best result was achieved by slow addition of the
tetrabenzyl glutamic acid 2 to a solution of the lithium phospho-
nate in THF at ꢀ78 ꢁC and doing the aqueous work-up at this
temperature after 4 h of stirring. Thus 3a was obtained in 65%
isolated yields after flash chromatography that is required to sep-
arate it from 20% of 3b.
Horner–Wadsworth–Emmons reaction of 3a under anhydrous
conditions in MeCN using LiCl and DIPEA with tetradecanal12 gave
unsaturated ketone 4 in 85% yield. Compound 4 was then subjected
to H2 under Pd catalysis in order to get simultaneous removal of the
benzyl protections and reduction of the double bond. During this
step, 5-pentadecyl proline 5 was obtained in good yield (Scheme 3).
The free NH2 (or the intermediate NHBn group) reacted with the
carbonyl forming an imine that was further reduced to 5. Cycliza-
tion also occurred under neutral or alkaline conditions (MeOH/
FarnesylBr
NH3/MeOH
S
HOOC
L-Cys
96%
13 R = H
FmocOSu
Et3N
71%
14 R = Fmoc
Scheme 4. Synthesis of 12 and 14.
Amine 10 was deprotected (H2 90 psi, Pd(OH)2/C, rt, MeOH,
24 h) to give the free amino acid, (S)-2-amino-4-(2-pentadecyl-1,3-
dioxolan-2-yl)-butanoic acid, 11 (Pdiob). Introduction of the Fmoc
gave 12 in 72% overall yield.13 At the same time, the N-Fmoc far-
nesyl cysteine 14 was also prepared through a direct alkylation of
free cysteine with farnesyl bromide in NH3/MeOH14 followed by
standard introduction of the Fmoc protection with FmocOSu. Fol-
lowing this procedure, 14 was isolated in 68% overall yield.
With compounds 12 and 14 in hand, we approached the solid-
phase synthesis of an isosteric C-terminus (residues 180–186) of

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E. Cini et al. / Tetrahedron 65 (2009) 844–848
the lipidated human H-Ras peptide.15 H-Ras is a small protein that
is active when the binding to membranes is achieved by farnesy-
lation at Cys186 followed by reversible palmitoylation at Cys184
and Cys18.16 The synthetic availability of mimetic peptides, which
are palmitoylated as well as farnesylated, should be useful to better
characterize the activities of key enzymes such as farnesyl protein
transferase or PAT and their biochemical regulation.17 Moreover, it
has been demonstrated that inhibition of farnesyl protein trans-
ferase by Ras carboxyl-terminal CAAX box analogues results in
anticancer effect.18
The synthesis of 15 was attempted by following typical solid-
phase peptide chemistry Fmoc protocol using a 2-chlorotrityl
chloride-PS resin as the support and HBTU/HOBT as the coupling
agent. Unfortunately, after cleavage from the resin with AcOH,
peptide 15 was obtained in low yields as a mixture with several
other compounds as shown by HPLC/MS analysis. In order to in-
crease the efficacy of the synthesis, we tried to carry out the pro-
tocol under microwave dielectric heating.19 Using a microwave
oven dedicated to peptide synthesis (Liberty from CEM)20 Fmoc-
Cys(S-Farnesyl)-OH 14 was loaded on the resin in DMF with
a double coupling protocol at 23 W and 75 ꢁC. Fmoc deprotection
and HBTU/HOBT mediated coupling with Fmoc-Lys(Boc)-OH were
carried out in 5 min at 23 W and 75 ꢁC. Further couplings were
carried out with Fmoc-Pdiob-OH 12, Fmoc-Ser(O-t-Bu)-OH, Fmoc-
Met-OH and FmocGly-OH (staggered by piperidine/DMF depro-
tection) under the same reaction conditions.
This procedure yielded the highly lipidated peptide 18 on the
resin without any particular difficulty, as pointed out by the Kaiser
tests. The fully protected precursor 18 was then cleaved from the
resin using AcOH/TFE/DCM for 3 h. With this standard cleavage
protocol, we obtained a mixture of 15 together with other species
carrying some of the side protections employed during the syn-
thesis. In order to get a complete removal of the protection, the
residue obtained from the resin cleavage was treated with TFA/
DMSO/H2O 1:1:1 for 48 h. Alternatively, a more rapid approach was
realized reacting the product coming from the cleavage with TFA/
H2O 1:1 under microwave dielectric heating for 20 min at 80 ꢁC
(150 W of power). Pure compound 15 was obtained in 30% overall
yield (Scheme 5). The analytical data registered on the synthetic
sample confirmed the formation of the expected isostere of the H-
Ras lipid-anchor portion with the unprotected carbonyl groups
(Fig. 1). Moreover, the formation of a single compound at HPLC
analysis pointed out that Fmoc-amino acids 12 and 14 were
obtained without racemization.
3. Conclusions
In conclusion, we have developed the synthesis of a new kind of
a lipophilic amino acid that is a structural analogue to palmitoyl
cysteine and that can be used for the synthesis of highly lipidated
peptides without particular precautions. This synthetic approach
can be extended in principle to the synthesis of different other long
1) Piperidine / DMF, MW, 75 °C, 5 min.
2) FmocAaxOH, HBTU, HOBt, DIPEA, MW, 75 °C, 5 min
3) Repetition of steps 1 and 2
Cl
Fmoc-Cys(S-Farnesyl)-OH
Fmoc-Cys(S-Farnesyl)
17
16
AcOH/TFE/DCM (2:2:6,) 3 h
followed by TFA/H2O (1:1),
MW, 150W, 80 °C, 20 min
H-Gly-Pdiob-Met-Ser(Ot-Bu)-Pdiob-Lys(Boc)-Cys(S-Farnesyl)
NH2
18
O
OH
O
O
O
S
H
N
H
H
N
Gly-Cys-Met-Ser-Cys-Lys-Cys-OMe
H2N
N
N
H
N
H
OH
S
S
S
O
O
O
Palmitoyl
Palmitoyl
Farmesyl
SMe
O
H-Ras, residue 180-186,
lipid-anchor portion
15
Isostere of the C-
terminus of H-Ras
heptapeptide 180-186
Scheme 5. Solid-phase synthesis of the H-Ras heptapeptide isostere.
Figure 1. HPLC graph of compound 15 prepared under microwave dielectric heating (for HPLC conditions see Section 4).

E. Cini et al. / Tetrahedron 65 (2009) 844–848
847
chain chiral
a
-amino acids that can be considered as useful tools in
(petroleum ether/EtOAc 20:1) to give compound 10 as a colourless
oil (0.450 g, 84% yield). nmax (Neat) 3080, 2983, 2865, 1732, 1643. 1H
drug delivery.21
NMR (CDCl3, 400 MHz):
d
¼0.91 (t, J¼6.4, 3H, CH3), 1.24–1.57 (m,
4. Experimental
22H, 9-H, 10-H, 11-H, 12-H, 13-H, 14-H, 15-H, 16-H, 17-H, 18-H, 19-
H), 1.84–1.99 (m, 4H, 3-H, 8-H), 2.18–2.34 (m, 2H, 4-H), 3.37 (t,
J¼6.4, 1H, 2-H), 3.72 (AB system, J¼8, 4H, NCH2Ph), 3.78–3.93 (m,
4H, OCH2CH2O), 5.22 (AB system, J¼8, 2H, OCH2Ph), 5.27–5.53 (m,
3H, 2-H, 6-H, 7-H), 7.22–7.41 (m, 15H, Ar–H). 13C NMR (100 MHz,
4.1. (S)-2-Dibenzylamino-6-(dimethoxy-phosphoryl)-5-oxo-
hexanoic acid benzyl ester (3a)
To
a
solution of dimethyl methylphoshonate (0.191 g,
CDCl3):
d
¼14.1, 22.7, 22.5, 29.3, 29.4, 29.7, 32.0, 32.7, 33.5, 40.8, 54.4,
1.54 mmol) in dry THF (17 mL) cooled at ꢀ78 ꢁC, BuLi (0.62 mL of
a 2.5 M solution in hexane) was added dropwise. After 40 min,
a solution of tetrabenzyl glutamic acid (0.390 g, 7.7 mmol) in dry
THF (14 mL) was added. After stirring for 4 h at ꢀ78 ꢁC, a saturated
aqueous solution of NH4Cl (6 mL) was added followed by ethyl
acetate (30 mL). After warming to rt, the organic phase was sepa-
rated. The aqueous phase was extracted with EtOAc, all the organic
fractions collected and dried over Na2SO4. The solvent was removed
in vacuo and the crude mixture was purified by column chroma-
tography (petroleum ether/EtOAc 1:3) to give compound 3a as
colourless oil (0.26 g, 65% yield). nmax (Neat) 3012, 2990, 2980, 1745,
60.9, 64.9, 65.9, 111.1, 124.3, 127.1, 128.4, 128.8, 129.0, 134.4, 136.2,
139.6, 172.7. HRMS (ES) calcd for C43H59NO4Na (MþH)þ: 676.4342,
found: 676.4310.
4.4. (S)-2-Amino-4-(2-pentadecyl-[1,3]dioxolan-2-yl)-
butenoic acid (11)
To a solution of 10 (0.400 g, 0.61 mmol) in CH2Cl2/MeOH (1:9,
20 mL) in a bottle connected to a Parr apparatus for medium
pressure hydrogenation, Pd(OH)2 on C (20%) (17 mg) was added.
The bottle was filled with H2 at 6 atm and shaken at rt for 12 h. The
bottle was degassed, the catalyst filtered (attention: the residue Pd
may be pyrophoric) and washed several times with MeOH. The
solvent was removed in vacuo to give compound 11 as a white gel
(0.212 g, 90% yield). nmax (Nujol) 3067–2901, 1660. 1H NMR (CDCl3,
1710. 1H NMR (CDCl3, 400 MHz):
d
¼1.95–2.02 (m, 2H, H-3), 2.41–
2.78 (m, 2H, H-4), 2.79–2.97 (m, 2H, H-6), 3.29 (t, J¼6.8, 1H, H-2),
3.67 (AB system, J¼7.0, 4H, NCH2Ph), 3.68 (d, J¼2.8, 3H, OCH3), 3.71
(d, J¼2.8, 3H, OCH3), 5.19 (AB system, J¼7.9, 2H, OCH2Ph) 7.19–7.36
(m, 15H, Ar–H). 13C NMR (50 MHz, CDCl3):
d¼22.6, 40.0, 40.4, 42.5,
400 MHz):
d¼0.79 (t, J¼7.2, 3H, CH3),1.19–1.43 (m, 26H, 7-H, 8-H, 9-
52.8, 53.0, 54.4, 59.8, 66.1, 127.1 54.5, 59.8, 66.2, 127.1, 128.2, 128.3,
128.5, 128.6, 128.9, 135.9(2C), 139.21, 172.13, 200.7, 200.8. HRMS
(ES) calcd for C29H35NO6P (MþH)þ: 524.2202, found: 524.2180.
H, 10-H, 11-H, 12-H, 13-H, 14-H, 15-H, 16-H, 17-H, 18-H, 19-H), 1.46–
1.54 (m, 2H, 3-H), 1.63–1.81 (m, 2H, 6-H), 1.82–2.04 (m, 2H, 4-H),
3.41–3.69 (m, 1H, H-2), 3.81–3.97 (m, 4H, OCH2CH2O). 13C NMR
(100 MHz, CDCl3):
d¼13.9, 22.6, 23.7, 24.4, 29.6, 31.8, 32.1, 37.0, 64.9,
4.2. (S,E)-2-Dibenzylamino-5-oxo-6-icosenoic acid benzyl
ester (4)
110.9, 172.7. HRMS (ES) calcd for C22H44NO4 (MþH)þ: 386.3270,
found: 386.3264.
To a solution of phosphonate 3a (0.900 g,1.7 mmol) in dry MeCN
4.5. (S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-4-(2-
(18 mL), dry LiCl (72.0 mg, 1.7 mmol) was added followed by freshly
pentadecyl-[1,3]dioxolan-2-yl)-butanoic acid (12)
distilled DIPEA (180 mg, 239 mL, 1.4 mmol). After stirring for 2 h at
rt, tetradecanal (0.276 g, 1.3 mmol) in MeCN was added and the
mixture was stirred at rt for 72 h. A saturated aqueous solution of
NaCl was used for quenching and the organic layer was separated.
The aqueous phase was extracted with EtOAc, all the organic frac-
tions collected and dried over Na2SO4. The solvent was removed in
vacuo and the crude mixture was purified by column chromatog-
raphy (petroleum ether/EtOAc 5:1) to give compound 4 as colour-
less oil (0.673 g, 85% yield). nmax (Neat) 3113, 3081, 2982, 2975,1742,
To a solution of 11 (0.210 g, 0.54 mmol) in H2O (6 mL), Et3N
(203 mL, 147 mg, 1.46 mmol) was added followed by FmocOSu
(0.376 g, 0.70 mmol) in CH3CN (6 mL). The mixture was stirred at rt
for 30 min. HCl (0.5 M) was added until the mixture reached pH 3–
4. The organic layer was separated and the aqueous phase was
extracted with EtOAc, all the organic fractions collected and dried
over Na2SO4. The solvent was removed in vacuo. The crude mixture
was purified by column chromatography (CH2Cl2/MeOH 99:1) to
give compound 12 as a colourless oil (0.314 g, 95% yield). HPLC
analysis (Chiralpack IB, 0.46ꢂ15 cm, eleuent isocratic i-PrOH/hex-
ane 25:75, adsorbance 254 nm): tR¼16.43 min, MS/ES (MþH)þ 631,
>95%; tR¼17.93 min, MS/ES (MþH)þ 631, <5%. nmax (Neat) 360,
1710–1690. 1H NMR (CDCl3, 400 MHz):
d
¼0.91 (t, J¼6.6, 3H, CH3),
1.24–1.47 (m, 20H, 10-H, 11-H, 12-H, 13-H, 14-H, 15-H, 16-H, 17-H,
18-H, 19-H), 1.45 (m, 2H, 9-H), 2.07–2.19 (m, 4H, 8-H, 3-H), 2.41–
2.69 (m, 2H, 4-H), 3.39 (dd, J¼8.4, 6.2, 1H, 2-H), 3.73 (AB system,
J¼8, 4H, NCH2Ph), 5.23 (AB system, J¼8, 2H, OCH2Ph), 6.00 (d,
J¼15.6, 1H, H-7), 6.71 (dt, J¼15.6, 7.0, 1H, H-6), 7.22–7.43 (m, 15H,
3417, 3342, 1720, 1703, 1684. 1H NMR (CDCl3, 400 MHz):
d
¼0.88 (t,
J¼7.2, 3H, CH3), 1.15–1.48 (m, 26H, 7-H, 8-H, 9-H, 10-H, 11-H, 12-H,
13-H, 14-H, 15-H, 16-H, 17-H, 18-H, 19-H), 1.51–1.58 (m, 2H, 3-H),
1.59–2.43 (m, 4H, 4-H, 6-H), 3.90 (s, 4H, OCH2CH2O), 4.21 (t, J¼6.8,
1H, 2-H), 4.38–4.49 (m, 3H, OCOCH2CH, OCOCH2CH), 5.79 (d, J¼6.8,
1H, NH), 7.23–7.41 (m, 4H, Ar–H), 7.50–7.59 (m, 2H, Ar–H), 7.73 (d,
J¼7.2, 2H, Ar–H), 10.31 (br s, 1H, COOH). 13C NMR (100 MHz, CDCl3):
Ar–H). 13C NMR (100 MHz, CDCl3):
d
¼14.0, 20.8, 22.6, 23.3, 28.0,
29.1, 29.2, 29.3, 29.4, 29.5, 31.8, 32.3, 36.3, 54.4, 60.1, 65.9, 126.9,
128.1, 128.2, 128.4, 128.4, 128.7, 130.1, 135.9, 139.2, 147.2, 172.2,
199.3. HRMS (ES) calcd for C41H55NO3Na (MþNa)þ: 632.4080,
found: 632.4062.
d
¼14.2, 22.7, 23.9, 26.3, 29.4, 29.7, 30.0, 32.0, 32.5, 37.4, 47.1, 53.8,
4.3. (S,E)-2-Dibenzylamino-4-(2-pentadec-1-enyl-[1,3]-
dioxolan-2-yl)-butanoic acid benzyl ester (10)
65.0, 67.2, 111.3, 120.0, 125.1, 127.1, 127.7, 141.3, 143.7, 176. HRMS (ES)
calcd for C37H53NO6Na (MþNa)þ: 630.3771, found: 630.3762.
To a solution of 4 (0.500 g, 0.82 mmol) in benzene (10 mL),
4.6. L-Farnesyl cysteine (13)
ethylene glycol (112 mg, 100 mL, 1.80 mmol) and p-TsOH (6 mg,
0.03 mmol) were added and the reaction mixture was heated at
reflux for 12 h with a Dean–Stark apparatus for azeotropic water
removal. Water was added and the organic layer was separated. The
aqueous phase was extracted with Et2O, all the organic fractions
collected and dried over Na2SO4. The solvent was removed in vacuo
and the crude mixture was purified by column chromatography
To a solution of cysteine (0.194 g, 1.60 mmol) in MeOH (5.2 mL)
cooled to 0 ꢁC, a solution of ammonia in MeOH (6.8 mL of a 7 M
solution) was added followed by farnesyl bromide (0.228 g,
1.60 mmol). The mixture was stirred at 0 ꢁC for 3 h and at rt for 1 h.
The solvent was removed at reduced pressure and the residue was
partitioned between 1-butanol and water. The butanol layer was

848
E. Cini et al. / Tetrahedron 65 (2009) 844–848
dried over MgSO4 and the solvent was removed under reduced
pressure. The residue was dissolved in MeOH and washed with
hexane. The MeOH was removed under reduced pressure to give 13
as a white solid (0.500 g, 96%). Mp 145–146 ꢁC (lit.14 148–150 ꢁC).
nmax (KBr) 3428–2640, 1620–1542.1H NMR (CDCl3, 200 MHz):
solvent system (A/B) was as follows: 0.1% TFA in water (A) and
acetonitrile (B). The adsorbance was detected at 254 nm. The HPLC
analysis showed one main peak at tR¼10.573 min that was identi-
fied as pure 15 on the basis of HRMS (ES) calcd for
C73H133N7S2O10Na (MþNa)þ: 1354.9453, found: 1354.9274.
d
¼1.57 (s, 6H, CH3CCH3), 1.64 (s, 3H, CH3CCH2), 1.72 (d, 2H, J¼7.1, 3H,
CH3CCH2), 1.89–2.20 (m, 8H, CH2), 2.91–3.31 (m, 2H, 3-H), 3.49–
Acknowledgements
3.88 (m, 3H, H-2, 10-H), 4.71 (br s, 2H, NH2), 4.99–5.11 (m, 2H, 60-H,
100-H), 5.12–5.34 (m, 1H, 20-H). 13C NMR (75 MHz, CDCl3):
d¼16.2,
This work was financially supported by MIUR (Rome) within the
PRIN project 2006. The authors thank also Mr. Luigi Verrengia for
the skilful technical help.
16.3, 17.8, 26.0, 27.5, 27.8, 30.3, 33.6, 40.8, 40.9, 55.2, 121.0, 125.0,
125.3, 132.0, 136.2, 140.9, 172.5. ES-MS: m/z 324 (MꢀH)ꢀ.
4.7. L-Fmoc farnesyl cysteine (14)
References and notes
To a solution of 13 (100 mg, 0.31 mmol) in CH2Cl2 (1.2 mL),
1. Resh, M. D. Science STKE 2006, 359, 14–19.
2. Clarke, S. Annu. Rev. Biochem. 1992, 61, 355–386.
3. Towler, D.; Gordon, J. I.; Adams, S. P.; Glaser, L. Annu. Rev. Biochem.1988, 57, 69–73.
4. Mumby, S. M. Curr. Opin. Cell Biol. 1997, 9, 148–154.
5. Resh, M. D. Cell Signalling 1996, 8, 403–412.
6. (a) Milligan, G.; Parenti, M.; Magee, A. I. Trends Biochem. Sci. 1995, 20, 181–184;
(b) Veit, M.; Schmidt, M. F. G. Methods Mol. Biol. 1998, 88, 227–239.
7. Stradley, S. J.; Rizo, J.; Gierasch, L. M. Biochemistry 1993, 32, 12586–12591; (b)
Schroeder, H.; Leventis, R.; Rex, S.; Schelhaas, M.; Na¨gele, E.; Waldmann, H.;
Silvius, J. R. Biochemistry 1997, 36, 13102–13121; (c) Xia, Z. D.; Smith, C. D. J. Org.
Chem. 2001, 66, 5241–5244.
FmocOSu (114 mg, 0.34 mmol) was added followed by Et3N (34 mg,
47 mL, 0.34 mmol). The mixture was stirred at rt for 2 h. HCl (1 M)
was added until the mixture reached pH¼1. The organic layer was
separated and the aqueous phase was extracted with CH2Cl2, all the
organic fractions collected and dried over Na2SO4. The solvent was
removed in vacuo and the crude mixture was purified by column
chromatography (CHCl3/MeOH 9:1) to give compound 14 as col-
ourless oil (119 mg, 71%). nmax (Neat) 3391, 3367, 2925, 1738, 1691.
1H NMR (CDCl3, 200 MHz):
d
¼1.57 (s, 3H, CH3), 1.59 (s, 3H, CH3),
8. Lumbierres, M.; Palomo, J. M.; Kragol, G.; Waldmann, H. Tetrahedron Lett. 2006,
47, 2671–2674.
1.61 (s, 3H, CH3), 1.66 (s, 3H, CH3), 1.90–2.18 (m, 8H, CH2), 2.75–3.04
(m, 2H, 1-H), 3.21 (d, J¼6.9, 2H, 10-H), 4.19 (t, J¼6.2, 1H, CH–Fmoc),
4.40 (d, J¼6.2, 2H, CH2–Fmoc), 4.49–4.74 (m,1H, 2-H), 5.01–5.31 (m,
2H, 60-H, 100-H), 5.21 (t, J¼6.9, 1H, 20-H), 5.75 (br s, 1H, NH), 7.23–
7.41 (m, 4H, Ar–H), 7.50–7.63 (m, 2H, Ar–H), 7.73 (d, J¼7.2, 2H, Ar–
9. (a) Kuhn, K.; Owen, D. J.; Bader, B.; Wittinghofer, A.; Kuhlmann, J.; Waldmann,
H. J. Am. Chem. Soc. 2001, 123, 1023–1035; (b) Kragol, G.; Lumbierries, M.;
Palomo, J. M.; Waldmann, H. Angew. Chem., Int. Ed. 2004, 43, 5839–5842; (c)
Rijkers, D. T. S.; Kruijtzer, J. A. W.; Killian, J. A.; Liskamp, R. M. J. Tetrahedron Lett.
2005, 46, 3341–3345; (d) Lumbierries, M.; Palomo, J. M.; Kragol, G.; Roehrs, S.;
Mu¨ller, O.; Waldmann, H. Chem.dEur. J. 2005, 11, 7405–7415.
10. Lampariello, L. R.; Piras, D.; Rodriquez, M.; Taddei, M. J. Org. Chem. 2003, 68,
7893–7895; Rodriquez, M.; Bruno, I.; Cini, E.; Marchetti, M.; Taddei, M.; Gomez-
Paloma, L. J. Org. Chem. 2006, 71, 103–107; See also: Ciapetti, P.; Falorni, M.;
Taddei, M. Tetrahedron 1996, 52, 7379–7380.
H). 13C NMR (50 MHz, CDCl3):
d
¼15.9,16.1,17.6, 25.6, 26.4, 26.7, 30.1,
33.4, 39.5, 39.6, 47.1, 67.3, 119.5, 119.9, 123.7, 124.3, 125.1, 127.0,
127.6, 131.2, 135.3, 140.0, 141.2, 143.7, 143.8, 156.1, 172.5. MS (ES) m/z
546 (MꢀH)ꢀ. HRMS (ES) calcd for C33H41NO4SNa (MþNa)þ:
570.2654, found: 570.2664.
11. Rodriquez, M.; Taddei, M. Synthesis 2005, 493–496.
12. Tetradecanal was obtained by Swern oxidation of commercially available 1-
tetradecanol. Chandrasekhar, S.; Shameen, S. S.; Kiranmai, K.; Narsihmulu, Ch.
Tetrahderon Lett. 2007, 48, 2373–2375.
4.8. Peptide (15)
13. The optical integrity of 12 was determined by HPLC analysis using a column
Chiralpack IB 0.46ꢂ15 cm.
14. Brown, M. J.; Milano, P. D.; Lever, D. C.; Epstein, W. W.; Poulter, C. D. J. Am. Chem.
Soc. 1991, 113, 3176–3177.
The synthesis was carried out using an automatic microwave
peptide synthesizer. (a) Loading of the resin (2-ClTrt-Cl). 2-ClTrt-
Cl resin (25 mg) was loaded with a solution of Fmoc-Farnesyl-Cys
15. Brunsveld, L.; Kuhlmann, J.; Alexandrov, K.; Wittinghofer, A.; Goody, R. S.;
Waldmann, H. Angew. Chem., Int. Ed. 2006, 45, 6622–6646; (b) Rotblat, B.; Prior,
I. A.; Muncke, C.; Parton, R. G.; Kloog, Y.; Henis, Y. I.; Hancock, J. F. Mol. Cell. Biol.
2004, 24, 6799–6810; (c) Jaumot, M.; Yan, J.; Clyde-Smith, J.; Sluimer, J.; Han-
cock, J. F. J. Biol. Chem. 2002, 277, 272–278.
14 (102 mg, 0.19 mmol) and DIPEA (32 mL, 0.19 mmol) in dry DMF
(3 mL) with a double coupling protocol at 23 W and 75 ꢁC. (b) Fmoc
deprotection. Removal of the Fmoc protecting group was carried
out using 20% piperidine in DMF (3 mL) at 23 W for 3 min at 75 ꢁC.
(c) Peptide coupling conditions. Fmoc-amino acid (5 equiv), HOBt
(5 equiv), HBTU (5 equiv) and DIPEA (5 equiv) were stirred under
N2 in DMF (3 mL) for 5 min at 23 W and 75 ꢁC. (d) Cleavege. The
dried resin was treated for 3 h with AcOH/TFE/DCM (2:2:6, 3 mL),
and then the resin was filtered off and washed with neat cleavage
16. Gorfe, A. A.; Babakhani, A.; McCammon, J. A. J. Am. Chem. Soc. 2007, 129,
12280–12286.
17. Draper, J. M.; Xia, Z.; Smith, C. D. J. Lipid Res. 2007, 48, 1873–1884.
18. (a) Whyte, D. B.; Kirschmeier, P.; Hockenberry, T. N.; Nunez-Oliva, I.; James, L.;
Catino, J. J.; Bishop, W. R.; Pai, J.-K. J. Biol. Chem. 1997, 272, 14559–14569; (b)
Deck, P.; Pendzialek, D.; Biel, M.; Wagner, M.; Popkirova, B.; Ludolph, B.; Kragol,
G.; Kuhlman, J.; Giannis, A.; Waldmann, H. Angew. Chem., Int. Ed. 2005, 44,
4975–4979.
19. For some examples on MW assisted synthesis of peptides see: Chighine, A.;
Sechi, G.; Bradley, M. Drug Discov. Today 2007, 12, 459–464; Collins, J. M.; Cox,
Z.; Singh, S. K. Biopolymers 2007, 88, 641–642; Cezamar, M.; Craig, D. K. J. Pept.
Sci. 2008, 14, 683–689; Grieco, P.; Cai, M.; Liu, L.; Mayrov, A.; Chandler, K.;
Trivedi, K.; Lin, G.; Campiglia, P.; Novellino, E.; Hruby, V. J. J. Med. Chem. 2008,
51, 2701–2707.
20. For information and leading references see: http://www.cem.com/biosciences/
autopep.asp.
21. Yaghmur, A.; Laggner, P.; Zhang, S.; Rappolt, M. PLoS ONE 2007, 2. doi:10.1371/
journal.pone.0000479 e479; Toth, I.; Horvath, A.; Olive, C.; Wong, A.; Clair, T.;
Yarwood, P.; Good, M. J. Med. Chem. 2002, 45, 1387–1390 and references therein.
mixture (3ꢂ1 mL). A solution of TFA/H2O (1:1, 200
mL) was added
and the solution stirred under microwave heating (150 W and
80 ꢁC) for 20 min to complete the deprotection of amino acids side
chains. After addition of hexane (15 mL), the product was concen-
trated and lyophilized to give compound 15 (38 mg, 61%). The
product was analyzed by analytic LC–MS (EC 125/4.6 NUCLEODUR
100–5C18ec) using the following gradient: from 0% B to 15% B over
15 min, then 15% B for 5 min at flow rate of 1 mL/min. The binary