Design and synthesis of a transferable farnesyl pyrophosphate analogue to Ras by protein farnesyltransferase

Article abstract of DOI:10.1021/jo991735t

The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its membrane localization and is required for both its biological activity and ability to induce malignant transformation. We describe the design and synthesis of a farnesyl pyrophosphate (FPP) analogue, 8-anilinogeranyl pyrophosphate 3 (AGPP), in which the ω-terminal isoprene unit of the farnesyl group has been replaced with an aniline functionality. The key steps in the synthesis are the reductive amination of the α,β-unsaturated aldehyde 5 to form the lipid analogue 6, and the subsequent conversion of the allylic alcohol 7 to the chloride 8 via Ph₃PCl₂ followed by displacement with [(n-Bu)₄N]₃-HP₂O₇ to give AGPP (3). AGPP is a substrate for protein farnesyltransferase (FTase) and is transferred to Ras by FTase with the same kinetics as the natural substrate, FPP. AGPP is highly selective, showing little inhibitory activity against either geranylgeranyl-protein transferase type I (GGTase I) (K(i) = 0.06 μM, IC₅₀ = 20 μM) or squalene synthase (IC₅₀ = 1000 μM). AGPP is the first efficiently transferable analogue of FPP to be modified at the ω-terminus that provides a platform from which additional analogues can be made to probe the biological function of protein farnesylation. AGPP is the first example of a class of compounds that are alternate substrates for protein isoprenylation that are not inhibitors of squalene synthase.

Full text of DOI:10.1021/jo991735t

J . Org. Chem. 2000, 65, 3027-3033
3027
Design a n d Syn th esis of a Tr a n sfer a ble F a r n esyl P yr op h osp h a te
An a logu e to Ra s by P r otein F a r n esyltr a n sfer a se
Kareem A. H. Chehade,†,§ Douglas A. Andres,†,§ Hiromi Morimoto,| and
H. Peter Spielmann*,†,‡,§
Department of Biochemistry, Department of Chemistry, and Kentucky Center for Structural Biology,
University of Kentucky, Lexington, Kentucky 40536-0084 and the National Tritium Labeling Facility,
Lawrence Berkeley National Laboratory, Berkeley, California 94720
Received November 5, 1999
The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its
membrane localization and is required for both its biological activity and ability to induce malignant
transformation. We describe the design and synthesis of a farnesyl pyrophosphate (FPP) analogue,
8-anilinogeranyl pyrophosphate 3 (AGPP), in which the ω-terminal isoprene unit of the farnesyl
group has been replaced with an aniline functionality. The key steps in the synthesis are the
reductive amination of the R,â-unsaturated aldehyde 5 to form the lipid analogue 6, and the
subsequent conversion of the allylic alcohol 7 to the chloride 8 via Ph3PCl2 followed by displacement
with [(n-Bu)4N]3HP2O7 to give AGPP (3). AGPP is a substrate for protein farnesyltransferase (FTase)
and is transferred to Ras by FTase with the same kinetics as the natural substrate, FPP. AGPP is
highly selective, showing little inhibitory activity against either geranylgeranyl-protein transferase
type I (GGTase I) (Ki ) 0.06 µM, IC50 ) 20 µM) or squalene synthase (IC50 ) 1000 µM). AGPP is
the first efficiently transferable analogue of FPP to be modified at the ω-terminus that provides a
platform from which additional analogues can be made to probe the biological function of protein
farnesylation. AGPP is the first example of a class of compounds that are alternate substrates for
protein isoprenylation that are not inhibitors of squalene synthase.
In tr od u ction
membrane localization and/or protein-protein recogni-
tion. These downstream events are potentially interesting
alternate targets for interfering with Ras function.
Synthetic modifications of enzymatic substrates are
useful in studying the interactions between small mol-
ecules and proteins because they allows critical struc-
tures involved in ligand binding and catalysis to be
identified. Analogues of FPP that are alternative trans-
ferable substrates for FTase have been utilized to study
farnesylation and subsequent downstream processing
events.4f Simple FPP analogues stripped of most iso-
prenoid features such as methyl groups and unsaturation
are transferred to Ras by FTase.4f Although the structure
of these lipids attached to the Ras farnesylation site can
affect further processing, the precise structure of these
analogues appears to have little effect on the ability of
Ras to activate effector pathways once the modified Ras
has become membrane-localized.4f Other FPP analogues
that have been modified at the farnesyl C3 position of
FPP with vinyl or cyclopropyl substituents as well as the
Z,E and E,Z isomers of FPP are also alternative sub-
strates for FTase.7 While 3-vinyl FPP and its farnesol
Mutated forms of cellular Ras genes are among the
most common genetic abnormalities in human cancer,
occurring in 30% of all neoplasms.1 Ras proteins are
synthesized as cytosolic precursors which localize to the
inner leaflet of the plasma membrane only after undergo-
ing a series of well-defined posttranslational modifica-
tions.2 The first and obligatory step in this processing is
the transfer of the 15-carbon isoprene farnesyl from
farnesylpyrophosphate (FPP, 1) to a cysteine located four
residues from the Ras C-terminus by protein farnesyl-
transferase (FTase).2a,3 While farnesylation is essential
for Ras to become membrane associated and induce
cellular transformation,2b-d it is unknown whether the
prenyl group functions as a hydrophobic membrane
anchor,4 or by targeted protein-protein recognition.5
FTase inhibitors (FTIs) have been the subject of intense
research because they prevent Ras membrane localiza-
tion and function.6 However, inhibition of protein pre-
nylation by FTIs precludes the study of events down-
stream of farnesylation which may be important to
* To whom correspondence should be addressed: Tel: 606-257-4790.
Fax: 606-323-1037. e-mail: hps@pop.uky.edu.
(4) (a) Hancock, J . F.; Magee, A. I.; Childs, J . E.; Marshall, C. J .
Cell 1989, 57, 1167. (b) Hancock, J . F.; Cadwallader, K.; Paterson, H.;
Marshall, C. J . EMBO J . 1991, 10, 4033. (c) Hancock, J . F.; Cadwal-
lader, K.; Marshall, C. J . EMBO J . 1991, 10, 641. (d) Leevers, S. J .;
Paterson, H. F.; Marshall, C. J . Nature 1994, 369, 411. (e) Stokoe, D.;
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† Department of Biochemistry, University of Kentucky.
‡ Department of Chemistry, University of Kentucky.
§ Kentucky Center for Structural Biology, University of Kentucky.
| National Tritium Labeling Facility.
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10.1021/jo991735t CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/14/2000

3028 J . Org. Chem., Vol. 65, No. 10, 2000
Chehade et al.
analogue act as alternative substrates for FTase in
H-Ras-F cells and transformed NIH3T3 fibroblasts,8
respectively, it is unknown whether the above FPP
isomers support full function of Ras in vivo.
Studies aimed at understanding the biological role of
protein isoprenylation are limited by the lack of FPP
analogues containing heteroatoms and more widely
varied structures that are transferable to Ras by FTase.
There are a number of challenges to the design of
structurally diverse, transferable, isoprenoid analogues.
The ability of FTase to transfer lipid analogues to target
proteins is exquisitely sensitive to the structure of the
ω-terminal group of the prenyl chain.4f,9 Incorporation of
photoreactive functionality and heteroatoms on the ω-ter-
minus of FPP analogues that would allow further study
of downstream processing events, only generated FTIs.9
Recently, Waldmann and co-workers have successfully
synthesized ω-arylated photoprobes that act as alterna-
tive substrates for the prenyltransferase geranylgera-
nyltransferase II.10 Inspection of the four crystal struc-
tures of FTase with and without bound substrates11
indicates that there is sufficient room for modest modi-
fication at the ω-terminus of FPP. Although the synthesis
of ω-arylated farnesol analogues has been achieved via
a carbon-carbon linkage,12 we reasoned that a transfer-
able analogue would result from introduction of a het-
eroatom linkage between a geranyl pyrophosphate moiety
and an aniline ring.
F igu r e 1. Structure of farnesyl pyrophosphate 1, gera-
nylgeranyl pyrophosphate 2, and transferable analogue 8-a-
nilinogeranyl pyrophosphate 3 (AGPP).
over-oxidation to the R,â-unsaturated carboxylic acid, or
difficulty in product isolation. We found that a two-step
oxidation of 4 with SeO2 and tert-butyl hydroperoxide,
followed by MnO2, resulted in the selective conversion
of the ω-methyl group to the all trans aldehyde 5 in 73%
yield.
The key step in the synthesis of AGPP 3 was formation
of acetate 6 by reductive amination of aniline with
aldehyde 5 using NaBH(OAc)3.15 We required a very mild
reductive amination in this scheme, since we envision
incorporating anilines with sensitive functionality in the
future. We selected NaBH(OAc)3 because it is a very mild
reducing agent and gives consistently higher yields with
fewer side products than conventional reductive amina-
tion reagents such as NaCNBH3/MeOH, BH3-pyridine,
and catalytic hydrogenation.15d Only one example of
NaBH(OAc)3 reductive amination employing an R,â-
unsaturated aldehyde, cinnamaldehyde, has been
described.15d,16 A slight excess of NaBH(OAc)3 in 1,2-
dichloroethane at 25 °C resulted in the allylic aniline 6
in 85% yield. Integration of the 1H NMR spectrum of
amine 6 indicated that a mixture of cis/ trans isomers
about the 6,7 double bond was formed in a 7:93 ratio.
The acetate 6 was hydrolyzed to alcohol 7 with K2CO3/
MeOH/H2O in 95% yield. The geometry of the cis/ trans
isomers was confirmed by NOE experiments performed
on alcohol 7 by irradiating the resonances of the C8
methylene protons at 3.65 ppm (the desired E (trans)
isomer) and 3.71 ppm (Z (cis) isomer) (see Supporting
Information). Attempts to separate the isomers by col-
umn chromatography, silica-HPLC, or reverse-phase
HPLC were unsuccessful.
We report the design, synthesis and functional char-
acterization of 8-anilinogeranyl pyrophosphate (3) (AGPP),
which is recognized and transferred to Ras by FTase with
in vitro kinetic properties that are indistinguishable from
that of FPP.
Resu lts a n d Discu ssion
Design a n d Syn th esis. We conceptually composed
8-anilinogeranyl pyrophosphate (AGPP) 3 by replacing
the terminal isoprene of FPP 1 with an aniline function-
ality (Figure 1). We chose aniline because it is a conve-
nient platform for the incorporation of a large number
of highly functionalized synthons into the 8-anilinogera-
nyl skeleton. The synthesis of AGPP was accomplished
in five steps from commercially available geranyl acetate
4 as illustrated in Scheme 1. Previous attempts to
generate the R,â-unsaturated aldehyde 5 using only
SeO2,13 or SeO2 and tert-butyl hydroperoxide14 resulted
in poor yields due to numerous side reactions including
(8) Gibbs, B. S.; Zahn, T. J .; Mu, Y. Q.; Sebolt-Leopold, J . S.; Gibbs,
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The presence of the amine in our farnesol analogue
precluded the implementation of standard methods to
activate the lipid alcohol 7 for pyrophosphorylation (Ph3P,
CCl4;17 SOCl2;18 NCS, Me2S19). Attempts at employing
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Transferable Farnesyl Pyrophosphate Analogue
J . Org. Chem., Vol. 65, No. 10, 2000 3029
Sch em e 1a
a
Reaction conditions: (a) tert-butyl hydroperoxide, SeO2, salicylic acid, CH2Cl2; MnO2, CH2Cl2; (b) aniline, NaBH(OAc)3, HOAc,
ClCH2CH2Cl; (c) K2CO3, H2O, MeOH; (d) (Ph)3PCl2, Hu¨nig’s base, CH3CN; (e) [(n-Bu)4N]3HP2O7, CH3CN; (f) L-cysteine, NH3, MeOH.
these procedures resulted in low or no yield of allylic
chloride 8 due to amine oxidation, polymerization, or
difficulty in product isolation. Sandri and Viala found
mild conditions for converting homoallylic alcohols into
their corresponding bromides using Ph3PBr2.20 Utilizing
Ph3PCl2 in MeCN, we were able to convert allylic alcohol
7 into the chloride 8 in 75% yield. The chloride 8 is
unstable and decomposes upon short-term storage at -20
°C, apparently into polymeric materials. Consequently,
alcohol 7 was converted to pyrophosphate 3 without
isolating the intermediate allylic chloride 8. Other previ-
ously reported farnesyl chloride analogues were not
F igu r e 2. Differential inhibition of farnesyltransferase and
isolated prior to displacement reactions due to their
instability.7b Displacement of the allylic chloride 8 with
tris(tetra-n-butylammonium) hydrogen pyrophosphate21
followed by ion exchange chromatography and reverse-
phase HPLC gave AGPP (3) in 61% overall yield from
alcohol 7. AGPP decomposes upon prolonged exposure
to room light.
FTase transfers the lipid portion of FPP to a cysteine
residue on Ras forming a thioether linkage. To verify that
AGPP was appropriately transferred to Ras by FTase,
we required an authentic sample of the product of FTase
catalyzed transfer of the anilinogeranyl chain to cysteine.
We modified the procedure of Brown et al.22 to synthesize
cysteine adduct 9 from alcohol 7 via chloride 8 in 65%
yield.
CAAX geranylgeranyltransferase by AGPP. The assay mix-
tures contained (in a final volume of 25 µL) various components
as described in Experimental Procedures. After incubation for
15 min at 37 °C, the amount of [3H]prenyl group transferred
to the appropriate protein substrate [Ras-CVLS for FTase
(filled circles) and Ras-CVLL for CAAX GGTase (open tri-
angles)] was measured by precipitation with ethanol-HCl. The
assays contained 10 ng of recombinant rat FTase, 5 µM Ras,
and 0.6 µM [3H]FPP (33000 dpm/pmol) or 100 ng of recombi-
nant rat CAAX GGTase, 5 µM Ras-CVLL, and 1 µM [3H]GGPP
(33000 dpm/pmol) and the indicated concentration of unlabeled
AGPP. The 100% of control values were 1.1 and 5.5 pmol of
[3H]farnesyl or [3H]geranylgeranyl transferred per tube. Each
value is the average of duplicate incubations and is represen-
tative of two separate experiments.
Biologica l Eva lu a tion of AGP P : In h ibition Stu d -
ies. AGPP inhibited FTase-catalyzed farnesylation of Ras
in a concentration dependent manner when measured in
the presence of increasing concentrations of AGPP and
at a fixed concentration of [3H]FPP (Figure 2, filled
circles). The Ki (which is equivalent to the Km for an
alternative substrate competitive inhibitor) for AGPP was
calculated using the Km value for FPP (0.04 µM)23 along
with velocity measurements from Figure 2. These data
were fit to eq 1 to obtain a Ki ) Km ) 0.03 µM (see
Experimental Procedures) that is essentially identical to
that of FPP. The relative affinity of an inhibitor toward
an enzyme can be expressed as the IC50 value, since this
value is directly related to the Ki of the inhibitor. The
IC50 value for AGPP (0.5 µM) calculated from eq 2 was
comparable to the IC50 concentration reported by Moores
et al.24 for FPP (0.34 µM).
(18) Young, W. G.; Caserio, F. F.; Brandon, D. D. J . Am. Chem. Soc.
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R.; Stremler, K. E.; Muehlbacher, M.; Poulter, C. D. J . Org. Chem.
1986, 51, 4768.
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Casey, P. J . Biochemistry 1995, 34, 6857. (b) Pompliano, D. L.; Rands,
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Biochemistry 1992, 31, 3800.

3030 J . Org. Chem., Vol. 65, No. 10, 2000
Chehade et al.
Geranylgeranyltransferase-I (GGTase I) catalyzes the
transfer of a geranylgeranyl group from GGPP to proteins
having a C-terminal CAAX sequence where X is leucine.2a
Both CAAX-based tetrapeptides and other FPP analogues
have been shown to be selective inhibitors of FTase
relative to GGTase I.25 To evaluate the selectivity of
AGPP for these enzymes, we examined its inhibitory
activity against recombinant GGTase I, as measured by
incorporation of radioactivity from [3H]geranylgeranylpy-
rophosphate into Ras-CVLL. As expected for an FPP
analogue,26 AGPP inhibits GGTase I activity much less
efficiently than it inhibits FTase (Figure 2, open tri-
angles). A Ki ) 0.06 µM for GGTase I was estimated for
AGPP when the literature Km value for GGPP (0.003
µM)27 was used with eq 1. The IC50 value calculated for
GGTase I inhibition by AGPP (20 µM) compared favor-
ably to the reported IC50 value for FPP (16 µM).24
Therefore, AGPP is similar to FPP in that it is a 40-fold
better inhibitor of FTase than of GGTase I.
[3H]AGP P Is a Su bstr a te for F a r n esyltr a n sfer a se
In Vitr o. The observed inhibition kinetics could be a
manifestation of the ability of AGPP to act as an
alternative substrate for FTase. Even though the data
implied that the structure of AGPP was recognized by
FTase, previously reported ω-terminal modified FPP
analogues are not transferred very efficiently by FTase.9
To evaluate the potential of AGPP to serve as a FTase
substrate, [3H]-AGPP was prepared by a similar route
as described above, incorporating tritium with a specific
activity of 17 Ci/mmol (synthesis to be reported else-
where). [3H]-AGPP was an alternative substrate for
FTase when incubated with Ras-CVLS as a cosubstrate.
Figures 3A and 3C show that FTase incorporated radio-
activity from [3H]AGPP into Ras in a time and concen-
tration dependent fashion at 37 °C. The incorporated
radioactivity was detected as a band of the expected
molecular weight of farnesylated Ras on SDS-polyacry-
lamide gels (Figure 3B). The saturation curve from
Figure 3C was almost identical to that obtained with [3H]-
FPP,3a suggesting that FTase has a similar affinity for
AGPP and FPP. Either nonradiolabeled AGPP or FPP,
but not alcohol 7 or farnesol, were found to inhibit the
time and concentration dependent transfer of [3H]AGPP
to Ras (data not shown). Figure 3D shows that [3H]AGPP
and [3H]FPP are transferred with comparable affinities
by FTase. These sets of data suggest that the cis/ trans
isomer mixture of AGPP does not interfere with the
FTase catalyzed transfer of lipid to Ras. We applied
initial velocity measurements from Figure 3D and a Km
) 0.63 µM23b for Ras-CVLS to eq 3 to calculate the kinetic
parameters kcat and kcat/Km for AGPP and FPP. These
values are tabulated in Table 1. The kinetic values we
obtained for FPP were identical to those reported in the
literature.23b
F igu r e 3. Transfer of anilinogeranyl from [3H]AGPP to Ras
by farnesyltransferase. (A) Each reaction contained 1 µM [3H]-
AGPP (37400 dpm/pmol) and 10 ng of recombinant FTase in
the absence (open triangles) or presence (filled circles) of 5 µM
Ras. Duplicate samples were incubated for the indicated times
at 37 °C, and ethanol-HCl precipitated radioactivity was
quantified. (B) The autoradiograph shows the migration of
[3H]-AG modified Ras on a 12.5% SDS-polyacrylamide gel of
an aliquot from the reaction carried out for 1 h in the presence
(+) or absence (-) of Ras. The gel was treated with Amplify
solution (Amersham), dried, and exposed to Kodak X-OMAT
AR film for 2 days at -70 °C. The positions of molecular weight
markers placed in an adjacent lane is indicated. (C) The AGPP
substrate saturation curve for FTase was determined by
varying the amount of [3H]AGPP in the standard reaction
described in A. Assays were carried out in duplicate for 30
min at 37 °C, and the ethanol-HCl precipitable radioactivity
measured. The data are representative of three independent
experiments. (D) FTase (10 ng) was incubated in a final volume
of 25 µL in the presence of 1.0 µM [3H]AGPP (open triangles)
or [3H]FPP (filled circles) and 5 µM Ras. After samples were
incubated for the indicated times at 37 °C, the amount of [3H]-
prenyl transferred to Ras was determined. Each value is the
average of duplicate incubations and is representative of five
separate experiments.
Ta ble 1. Stea d y-Sta te Kin etic P a r a m eter s
substrate
Km (µM)
kcat (s-1
)
kcat/Km (M-1 s-1
)
FPP
AGPP
0.04
0.03
0.09
0.07
2 × 106
2 × 106
products to the authentic AG-cysteine standard 9. The
modified Ras was digested with Pronase E and the
butanol-soluble products were analyzed by reverse phase
chromatography. The chromatographic mobility of the
major radioactive product of this digestion was consistent
with authentic AG-cysteine 9.
AGP P Does Not In h ibit Squ a len e Syn th a se Ac-
tivity. Squalene synthase is involved in cholesterol
biosynthesis and has an important role as a secondary
regulatory protein in that pathway. Squalene synthase
catalyzes the reductive head-to-head coupling of two FPP
molecules to give squalene.28 FPP analogues have been
found through design and screening of chemical libraries
that are potent inhibitors of FTase but are not inhibitors
of squalene synthase.29 However, other FTIs, particularly
those competitive for FPP binding, are inhibitors of
Verification of appropriate transfer of the anilinogera-
nyl group to cysteine was accomplished by hydrolyzing
the [3H]AGPP-modified Ras and comparing the digestion
(24) Moores, S. L.; Schaber, M. D.; Mosser, S. D.; Rands, E.; O’Hara,
M. B.; Garsky, V. M.; Marshall, M. S.; Pompliano, D. L.; Gibbs, J . B.
J . Biol. Chem. 1991, 266, 14603.
(25) For a recent review, see: Ayral-Kaloustian, S.; Skotnicki, J . S.
In Annual Reports in Medicinal Chemistry; Bristol, J . A., Ed.; Academic
Press: San Diego, 1996; Vol. 31, pp 171-180.
(26) Armstrong, S. A.; Hannah, V. C.; Goldstein, J . L.; Brown, M.
S. J . Biol. Chem. 1995, 270, 7864.
(27) Zhang, F. L.; Moomaw, J . F.; Casey, P. J . J . Biol. Chem. 1994,
269, 23465.
(28) Watson, N. S.; Procopiou, P. A. Prog. Med. Chem. 1996, 33, 331.

Transferable Farnesyl Pyrophosphate Analogue
J . Org. Chem., Vol. 65, No. 10, 2000 3031
1
internal peak: 7.27 ppm for H, 77.4 ppm for 13C), C6D6 (C6D6
1
internal peak: 7.37 ppm for H), DMSO-d6 (DMSO-d6 internal
peak: 39.5 ppm for 13C). Mass spectra were obtained from the
University of Kentucky Mass Spectra Facility. Combustion
analyses were performed by Atlantic Microlabs, Inc., Norcross,
GA.
(E,E)-3,7-Dim eth yl-1-a cetoxy-2,6-octa d ien -8-a l (5). Ger-
anyl acetate was oxidized as described previously14 with some
modification. A 40 mL (0.36 mol) amount of 90% tert-butyl
hydroperoxide was poured into a stirred suspension of 1.11 g
(0.010 mol) of SeO2, 1.4 g (0.010 mol) of salicylic acid, and 75
mL of CH2Cl2 in a 250 mL round-bottom flask. The resulting
solution was allowed to stir to homogeneity at room temper-
ature before being placed at 0 °C. After 10 min, 21.5 mL (0.10
mol) of geranyl acetate 4 was introduced. The mixture was
stirred for 5 h at 0 °C, and then stirred for 24 h at room
temperature. The solution was diluted with 100 mL of toluene,
and solvents were removed under reduced pressure. The
residue was then dissolved in toluene, washed with 5%
NaHCO3 to remove H2SeO3, saturated CuSO4, saturated
aqueous Na2S203 (2×), water, and brine, dried over MgSO4,
and filtered. The solvent was removed under reduced pressure
to afford a crude oil, which was dissolved in 100 mL of dry
CH2Cl2 and placed at 0 °C. A 40.95 g (0.47 mol) amount of
activated MnO2 was added, and the suspension was stirred
for 5 h at 0 °C, and then stirred overnight at room tempera-
ture. The mixture was filtered over a short bed of Celite and
washed thoroughly with dry CH2Cl2. Solvent was evaporated
under reduced pressure to yield a crude, pale yellow oil which
was purified by flash chromatography on silica gel (hexane
(1000 mL), 5% EtOAc in hexane) affording 6.04 g of unreacted
4 and 10.68 g (73% based on consumed 4) of 5 as a colorless
oil; TLC: (Rf 0.38 aldehyde 5, Rf 0.19 alcohol); 1H NMR (C6D6,
500 MHz) δ 9.47 (s, 1H), 5.98 (t, 1H, J ) 6.9 Hz), 5.48 (t, 1H,
J ) 6.9 Hz), 4.75 (d, 2H, J ) 6.8 Hz), 2.11 (q, 2H, J ) 7.3 Hz),
1.92 (t, 2H, J ) 7.3 Hz), 1.91 (s, 3H), 1.79 (s, 6H), 1.59 (s, 3H);
13C NMR (50.3 MHz) δ 195.47, 171.39, 153.70, 140.73, 140.08,
120.01, 61.48, 38.13, 27.33, 21.37, 16.77, 9.62.
8-An ilin e-3,7-d im eth yl-1-a cetoxy-2,6-octa d ien e (6). The
reductive amination procedure of Abdel-Magid et al.15d was
modified as follows. Into a 250 mL three-neck flask was
introduced 150 mL of 1,2-dichloroethane, followed by 7.88 g
(37.48 mmol) of 5, 3.76 mL (41.22 mmol) of fresh aniline, and
2.58 mL (44.89 mmol) of glacial acetic acid. After stirring for
0.5 min at room temperature, 11.12 g (52.47 mmol) of NaBH-
(OAc)3 was added to the above solution. The solution was
stirred for 5 h at room temperature, and then quenched by
pouring into a separatory funnel containing 200 mL of 5%
NaHCO3. The product was extracted with ether (3 × 75 mL),
dried (MgSO4), and concentrated to give a pale yellow, viscous
oil. Purification by flash chromatography (10% EtOAc in
hexane) yielded 9.15 g (85%) of a light-straw colored to
colorless oil; TLC: (Rf 0.46 6); 1H NMR (200 MHz) δ 7.18 (m,
2H), 6.69 (m, 1H), 6.62 (m, 2H), 5.38 (m, 2H), 4.60 (d, 2H, J )
7.2 Hz), 3.82 (s, 1H), 3.65 (s, 2H), 2.33-2.02 (m, 4H), 2.08 (s,
3H), 1.72 (s, 3H), 1.69 (s, 3H); 13C NMR (50.3 MHz) δ 171.42,
148.84, 142.12, 133.13, 129.43, 125.71, 118.93, 117.44, 113.13,
61.68, 52.03, 39.50, 26.21, 21.37, 16.74, 15.00. Anal. Calcd for
F igu r e 4. Inhibition of squalene synthase by AGPP. The
initial rates of squalene synthase activity were determined
from bovine brain microsomal preparations in the presence of
the indicated concentration of unlabeled AGPP (filled squares)
or unlabeled FPP (open triangles). Each value is the average
of duplicate incubations and is representative of three separate
experiments.
squalene synthase.28 An important objective in the
development of our alternative FPP substrates for FTase
was the generation of molecules that do not interfere with
other FPP utilizing enzymes. AGPP was found to be a
far less potent in vitro inhibitor of squalene synthase
activity than FPP, with an IC50 ) 1000 µM (Figure 4).
We tested for the possibility that AGPP might act as a
substrate to form a homo-coupled product and the pos-
sibility that it might form hetero-coupled products with
FPP. No organic soluble products resulting from squalene
synthase-catalyzed coupling reactions of [3H]AGPP were
detected. Apparently, the structural differences between
AGPP and FPP do not allow AGPP to interfere with
squalene synthase activity. AGPP is the first example of
a class of compounds that are alternate substrates for
protein isoprenylation that are not inhibitors of squalene
synthase.
Con clu sion . AGPP is the prototype of a class of
alternative substrates for FTase that possess a modified
ω-terminus. The reductive amination of aniline with an
aliphatic R,â-unsaturated aldehyde and NaBH(OAc)3
provides a robust and versatile method to access a new
class of amino-functionalized isoprenyl analogues. An
alternative route to the synthesis of allylic chlorides from
their corresponding alcohols using dichlorotriphenylphos-
phorane has also been demonstrated.
Exp er im en ta l P r oced u r es
Gen er a l Ch em ica l P r oced u r es. Melting points are uncor-
rected. All reactions were conducted under dry argon, and all
reactions and chromatography procedures were conducted
under diminished light. Analytical TLC was performed on
precoated (0.25 mm) silica gel 60F-254 (Merck) plates and
developed with 30% ethyl acetate in hexane, except where
noted otherwise. Preparative TLC was performed on precoated
silica gel plates (E. Merck #13793-7). Visualization was
achieved either by UV irradiation, anisaldehyde-sulfuric acid
spray followed by heating, or subjecting the plates to a 5%
ethanolic phosphomolybdic acid solution followed by heating.
Flash chromatography was performed on Merck silica gel 60
(230-400 mesh ASTM). NMR spectra were obtained in CDCl3
(unless otherwise noted) at 200 MHz or at 500 MHz. Chemical
shifts for the following deuterated solvents are reported in ppm
downfield using the indicated reference peaks: CDCl3 (CDCl3
C
18H25NO2: C, 75.22; H, 8.77; N, 4.87. Found: C, 75.38; H,
8.63; N, 4.93.
8-An ilin e-3,7-d im eth yl-2,6-octa d ien -1-ol (7). To a 500
mL three-neck flask were added acetate 6 (8.00 g, 27.8 mmol)
and 250 mL of methanol. Potassium carbonate (11.53 g, 83.4
mmol) dissolved in 20 mL of water was added, and the reaction
mixture was stirred at room temperature overnight. The
mixture was then concentrated to ca. 100 mL on a rotary
evaporator. Water was added (100 mL), and the mixture was
extracted with ether (4 × 100 mL). The combined extracts were
washed with brine (100 mL), dried (MgSO4), and concentrated
to yield 6.48 g (95%) of 7 as a viscous, light-straw colored to
colorless oil; TLC: (Rf 0.20 7); 1H NMR (200 MHz) δ 7.18 (m,
2H), 6.71 (m, 1H), 6.61 (m, 2H), 5.40 (m, 2H), 4.13 (d, 2H, J )
6.9 Hz), 3.65 (s, 2H), 2.30-1.98 (m, 4H), 1.69 (s, 6H); 13C NMR
(50.3 MHz) δ 148.84, 139.45, 132.94, 129.45, 125.83, 124.09,
117.46, 113.18, 59.67, 52.01, 39.51, 26.24, 16.55, 15.04. Puri-
(29) Aoyama, T.; Satoh, T.; Yonemoto, M.; Shibata, J .; Nonoshita,
K.; Arai, S.; Kawakami, K.; Iwasawa, Y.; Sano, H.; Tanaka, K.;
Monden, Y.; Kodera, T.; Arakawa, H.; Suzuki-Takahashi, I.; Kamei,
T.; Tomimoto, K. J . Med. Chem. 1998, 41, 143.

3032 J . Org. Chem., Vol. 65, No. 10, 2000
Chehade et al.
fication by flash chromatography (30% EtOAc in hexane)
yielded the analytical sample of 7. Anal. Calcd for C16H23NO:
C, 78.32; H, 9.45; N, 5.71. Found: C, 78.13; H, 9.46; N, 5.88.
8-An ilin e-1-ch lor o-3,7-d im eth yl-2,6-octa d ien e (8). The
allylic chloride was synthesized as described for homoallylic
alcohols by Sandri and Viala20 with some modification. Into a
100-mL round-bottom flask were added alcohol 7 (1.24 g, 5.05
mmol), N,N-diisopropylethylamine (1.41 mL, 8.09 mmol), and
30 mL of dry acetonitrile. The solution was stirred at 0 °C for
10 min. Solid dichlorotriphenylphosphorane (2.45 g, 7.58
mmol) was then added evenly to the reaction mixture over a
7 min period. After the final addition of Ph3PCl2, the reaction
was allowed to stir at 0 °C for an additional 40 min. The
mixture was then loaded directly onto a silica gel column and
purified by flash chromatography (5% EtOAc in hexane) to
yield 1.00 g (75%) of pure chloride 8 as a colorless oil (which
rapidly turns brown upon exposure to light); TLC: (Rf 0.62
8); 1H NMR (200 MHz) δ 7.19 (m, 2H), 6.96 (m, 3 H), 5.31 (m,
2H), 4.02 (d, 2H, J ) 7.6 Hz), 3.68 (s, 2H), 2.18-1.86 (m, 4H),
1.69 (s, 3H), 1.63 (s, 3H); 13C NMR (50.3 MHz) δ 145.47, 141.27,
133.67, 131.60, 129.38, 122.19, 120.86, 117.68, 55.25, 41.25,
38.89, 26.05, 16.20, 15.35.
collected, concentrated, and lyophilized yielding 9 (0.46 g
overall, 65% from alcohol 7) as a white solid, mp 151-153 °C
(dec); 1H NMR (DMSO-d6, 500 MHz) δ 7.61 (bs, 1H), 7.02 (m,
2H), 6.53 (m, 2H), 6.47 (m, 1H), 5.82 (bs, 1H), 5.33 (t, 1H, J )
5.9 Hz), 5.17 (t, 1H, J ) 7.8 Hz), 3.52 (s, 2H), 3.32 (bs, 3H),
3.28 (dd, 1H, J ) 3.4 Hz), 3.15 (ddd, 1H, J ) 8.3 Hz), 2.99 (dd,
1H, J ) 3.4 Hz), 2.64 (dd, 1H, J ) 9.3 Hz), 2.10 (q, 2H, J )
7.3 Hz), 1.99 (t, 2H, J ) 7.3 Hz), 1.63 (s, 3H), 1.59 (s, 3H); 13
C
NMR (DMSO-d6, 125.7 MHz) δ 168.31, 149.01, 138.12, 132.66,
128.61, 123.94, 120.37, 115.21, 112.00, 53.51, 50.25, 38.91,
32.48, 28.53, 25.64, 15.84, 14.34. Anal. Calcd for C19H28N2O2S‚
0.5H2O: C, 63.83; H, 8.18; N, 7.84; S, 8.97. Found: C, 64.02;
H, 7.92; N, 7.74; S, 8.89. LRMS (MALDI): (MH+) 349.20.
Gen er a l Biologica l Ma ter ia ls a n d Meth od s. [1-3H]-
Farnesyl pyrophosphate ([3H]FPP, 15 Ci/mmol) and [1-3H]-
geranylgeranyl pyrophosphate ([3H]GGPP, 15 Ci/mmol) were
obtained from American Radiochemical Co. Recombinant Ras-
CVLS and its derivative Ras-CVLL were produced in bacteria
as described previously.30 Farnesyl cysteine (F-Cys) was
synthesized as described by Kamiya et al.31 and purified by
preparative TLC. All other solvents and chemicals were
reagent grade and purchased from standard commercial
sources.
8-An ilin e-3,7-d im eth yl-2,6-octa d ien e P yr op h osp h a te
(3). To a 50-mL glass centrifuge tube was added 7.45 g (7.6
mmol) of tris(tetrabutylammonium) hydrogen pyrophosphate21
and 12 mL of dry acetonitrile. After vortexing, the milky white
solution was then centrifuged at 2000 rpm for 10 min. The
clear supernatant was decanted into a flame-dried, 100-mL,
round-bottomed flask charged with 1.00 g (3.8 mmol) of
chloride 8. The solution was allowed to stir at room temper-
ature for 3 h. Solvent was removed, and the pale white residue
was dissolved in 3 mL of ion exchange buffer (ion exchange
buffer was generated by dissolving ammonium bicarbonate (2.0
g, 25.3 mmol) in 1.0 L of 2% (v/v) isopropyl alcohol/water). The
resulting milky white solution was loaded onto a preequili-
brated 2 × 30-cm column of Dowex AG 50W-X8 (100-200
P r od u ction of Recom bin a n t CAAX GGTa se-1 a n d
F Ta se in Sf9 Cells. Recombinant FTase was prepared by
coinfection of fall army worm ovarian (Sf9) cells with recom-
binant baculoviruses encoding the R- and â-subunits of rat
FTase and purified as previously described.30 The FTase
R-subunit had been modified to contain a 6xHistidine affinity
tag at its N-terminus to allow rapid purification using Ni2+
-
Sepharose affinity chromatography as described.32 A cDNA
clone that encodes the GGTase-1 â-subunit was kindly pro-
vided by Dr. Guy J ames (University of Texas, Southwestern
Medical Center, Dallas). Recombinant baculoviruses encoding
the GGTase-1 â-subunit were generated by cotransfection of
Sf9 cells with pVL-GGTB and linear BacPAK6 viral DNA
(Clontech) and plaque purified as described.32
+
mesh) cation-exchange resin (NH4 form). The flask was
washed with buffer (2 × 5 mL), and both washes were loaded
onto the column before elution with 190 mL (two column
volumes) of ion exchange buffer. The cloudy white eluant was
collected in a 600-mL freeze-drying flask, frozen, and lyoph-
ilized to yield 3.17 g of an off-white solid. A portion of the crude
AGPP was dissolved in 25 mM NH4HCO3 to a final concentra-
tion of ca. 5 mM AGPP. The colorless solution was loaded onto
an analytical Vydac C4 (214TP54) column and eluted under
the following gradient at a flow rate of 1 mL/min: 0-11 min
100% A, 11-12 min 80% A, 12-24 min 80% A, 24-27 min 5%
A, 27-31 min 5% A, 31-36 min 100% A, 36-45 min 100% A.
Solvents: A ) water; B ) 0.01% (v/v) TFA in 2-propanol. The
desired peak (tR ≈ 18.8 min) was immediately collected in a
vial containing 25 mM NH4HCO3, frozen, and lyophilized
yielding 3 as a white solid (1.05 g overall, 61%); 1H NMR (D2O,
200 MHz) δ 7.26 (m, 2H), 6.86 (m, 3H), 5.42 (m, 2H), 4.47 (t,
2H, J ) 6.7 Hz), 3.69 (s, 2H), 2.25-2.01 (m, 4H), 1.69 (s, 3H),
1.64 (s, 3H); 13C NMR (D2O, 50.3 MHz) δ 145.70, 135.39,
132.46, 129.78, 122.92, 122.76, 122.19, 118.34, 65.65 (d, J )
5.3 Hz), 54.35, 41.40, 28.18, 18.41, 16.63; 31P NMR (D2O, 202.4
MHz) δ -6.87 (1P, d, J ) 22 Hz), -10.84 (1P, dt, J ) 22 Hz).
LRMS (FAB+, H+ glycerol matrix): (M+ + H+) 406.2. LRMS
(FAB-, H+ glycerol matrix): (M+ - H+) 404.1.
8-An ilin e-3,7-d im eth yl-2,6-octa d ien e-S-L-cystein e (9).
The cysteine adduct was synthesized as described by Brown
et al.22 with some modification. The alcohol 7 (0.50 g, 2.04
mmol) was first converted into chloride 8 as described above.
Crude chloride 8 was then added to a 25-mL pear shaped flask,
followed by 0.32 g (2.67 mmol) of L-cysteine and 10 mL of a 2
N NH3 solution in MeOH. The solution began to form a
precipitate after 15 min, and stirring was continued for 1 d.
The reaction mixture was then filtered and the solid washed
with 10 mL of MeOH:H2O (9:1 v/v). Aliquots of the filtrate were
loaded onto a preparative Vydac C18 (218TP1010) column and
eluted under the following gradient at a flow rate of 5 mL/
min: 0-5 min 90% A, 5-20 min 5% A, 20-23 min 5% A, 23-
27 min 90% A, 27-31 min 90% A. Solvents: A ) 25 mM
NH4HCO3; B ) CH3CN. The desired peak (tR ≈ 15.7 min) was
Assa y for F a r n esyltr a n sfer a se Activity. Farnesyltrans-
ferase activity was assayed by measuring the amount of [3H]-
farnesyl or [3H]anilinogeranyl transferred from [3H]FPP or
[3H]AGPP to recombinant Ras, as described previously.33
Unless otherwise stated, each reaction mixture contained the
following components in a final volume of 25 µL: 50 mM Tris
(pH 7.4), 20 mM KCl, 0.2% octyl â-glucopyranoside, 1 mM
dithiothreitol, 10-20 ng recombinant FTase, 5 µM Ras, 3 mM
MgCl2, 50 µM ZnCl2, and 0.6 µM [3H]FPP (33000 dpm/pmol;
American Radiochemical Co.) or 0.1-5 µM [3H]AGPP (37400
dpm/pmol). Following incubation for the indicated times at 37
°C, the amount of [3H]prenyl transferred was measured by
ethanol-HCl precipitation and filtration on glass fiber filters
with modification as previously described.30 A blank value was
determined in parallel incubation mixtures containing no
enzyme. This blank value was subtracted from each reaction
before calculating pmol [3H]prenyl transferred.
Assa y for CAAX Ger a n ylger a n yltr a n sfer a se-1 Activity.
CAAX geranylgeranyltransferase activity was assayed by
measuring the amount of [3H]geranylgeranyl transferred from
[3H]GGPP to recombinant Ras CVLL, as described previ-
ously.26 Unless otherwise stated, each reaction contained the
following components in a final volume of 25 µL: 50 mM Tris-
HCl (pH 7.4), 5 mM dithiothreitol, 20 µM Zwittergent (3-14),
1 µM [3H]GGPP (33000 dpm/pmol; American Radiochemical
Co.), 5 µM Ras CVLL, 5 µM ZnCl2, 5 mM MgCl2, 100 ng
recombinant CAAX GGTase-1, and the indicated amount of
unlabeled AGPP. Following incubation at 37 °C for 15 min,
the amount of [3H]geranylgeranyl transferred was measured
(30) Crick, D. C.; Suders, J .; Kluthe, C. M.; Andres, D. A.; Waechter,
C. J . J . Neurochem. 1995, 65, 1365.
(31) Kamiya, Y.; Sakurai, A.; Tamura, S.; Takahashi, N.; Tsuchiya,
E.; Abe, K.; Fukui, S. Agric. Biol. Chem. 1979, 43, 363.
(32) Andres, D. A.; Seabra, M. C.; Brown, M. S.; Armstrong, S. A.;
Smeland, T. E.; Cremers, F. P.; Goldstein, J . L. Cell 1993, 73, 1091.
(33) Andres, D. A.; Goldstein, J . L.; Ho, Y. K.; Brown, M. S. J . Biol.
Chem. 1993, 268, 1383.

Transferable Farnesyl Pyrophosphate Analogue
J . Org. Chem., Vol. 65, No. 10, 2000 3033
by ethanol-HCl precipitation followed by filtration on glass
fiber filters. A blank value was determined in parallel incuba-
tion mixtures containing no enzyme and was subtracted from
each reaction.
IC50 value)37
[I]0.5 ) (1 + S/Km)Ki
(2)
where [I]0.5 is the IC50 value for AGPP. Equation 3 describes
the dependence of the steady-state velocity on substrate
concentration for a two-substrate random-equilibrium mech-
anism for FTase with FPP or AGPP and Ras-CVLS to form
the prenylated product Ras-C(isoprenoid)-VLS23a
Digestion of [3H]AGP P -Mod ified Ra s. An aliquot from
a standard FTase reaction carried out at 37 °C for 30 min in
the presence of [3H]AGPP and Ras was subjected to Pronase
E digestion as previously described.34 The labeled products,
extracted with n-butanol, were analyzed on C18 reverse-phase
TLC plates developed in CH3CN:H2O:HOAc (75:25:1 v/v/v).
Radioactive zones were located with a Bioscan Imaging System
200-IBM.
ν(SR + RR + âS + Râ)
kcat
)
(3)
ETSR
Assa y for Squ a len e Syn th a se Activity. Initial rates of
squalene synthase activity present in bovine brain microsome
fractions were assayed as previously described30 with minor
modifications. Typical reaction mixtures contained crude
bovine brain microsomes (0.2 mg membrane protein), 55 mM
HEPES (pH 7.4), 5.5 mM MgCl2, 11 mM KF, 1 mM NADPH,
and 0.05 mM [3H]FPP (105 cpm/pmol) in a total volume of 100
µL. Unlabeled AGPP and FPP were added at the indicated
concentrations. Assay mixtures were incubated for 30 min at
37 °C, and the reactions terminated by the addition of 50 µL
of 40% (w/v) KOH and then 100 µL of 95% ethanol. The
resulting mixtures were incubated at 60 °C for 2 h and then
cooled to room temperature. Authentic carrier squalene (20
µg) was added to each reaction tube, which were then extracted
with petroleum ether (3×). The lipid extracts were pooled,
washed with water (3×), and evaporated under a stream of
N2. The lipid residue was redissolved in 100 µL of CHCl3/CH3-
OH (2:1 v/v) and an aliquot (30 µL) was taken to determine
the amount of labeled lipid formed by scintillation spectrom-
etry. The remaining sample was analyzed on Silica gel G 60
TLC plates (Merck) by developing with hexane.35 In all
analyses the squalene standard was located by anisaldehyde
spray reagent,36 and radioactive zones were located with a
Bioscan Imaging System 200-IBM.
where ν is the velocity of product formation, S is the concen-
tration of the isoprenyl substrate FPP or AGPP, R is the
concentration of the protein acceptor Ras-CVLS, R is the Km
of FPP or AGPP, â is the Km for Ras-CVLS, and ET is the total
enzyme concentration.
Ack n ow led gm en t. This work was supported in part
by the National Institutes of Health grant (EY11231 to
D.A.A.), the Kentucky Medical Center Research Fund
(831) (to H.P.S.), and Kentucky-NSF EPSCoR Program
(EPS-9452895) (to H.P.S.), and the NTLF is supported
by the Biomedical Technology Area, National Center for
Research Resources, U.S. National Institutes of Health,
under Grant P41 RR01237, through DOE Contract DE-
AC03-76SF00098 with the University of California
(H.M.). We thank Dr. Phillip G. Williams at the Na-
tional Tritium Labeling Facility (Lawrence Berkeley
National Laboratory), and Drs. Guy J ames and Scott
Armstrong at the University of Texas, Southwestern
Medical Center, Dallas, for help with prenyltransferase
enzyme production. We also thank Dr. Dean Crick at
Colorado State University, Fort Collins, for his helpful
suggestions and assistance in prenyl group analysis
using Pronase digestion, Dr. J eff Rush for the brain
membranes used in the squalene synthase assays, and
Dr. Louis Hersh for his advice with the enzyme kinetics.
To evaluate the potential of AGPP as an alternative
substrate for squalene synthase, the above assay was repeated
as described with some minor modification. Where indicated,
0.05 mM [3H]FPP was substituted with 0.05 mM [3H]AGPP
(304 cpm/pmol), and no unlabeled isoprenyl analogues were
added. After incubation, the reactions were extracted, concen-
trated, and analyzed by TLC for possible products resulting
from any [3H]AGPP coupling reactions.
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C, 31P NMR,
and MS spectra for compound 3; 1H and 13C NMR spectra for
compound 6; 1H, 13C NMR, and NOE spectra for compound 7;
1
1H and 13C NMR spectra for compound 8; H, 13C NMR, and
Kin etic An a lysis. Equation 1 describes the expression for
the relative velocity or fractional activity in the presence and
absence of a competitive inhibitor37
MS spectra for compound 9. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O991735T
y ) 1 - [I/(I + Ki(1 + S/Km))]
(1)
(34) Crick, D. C.; Waechter, C. J .; Andres, D. A. Biochem. Biophys.
Res. Commun. 1994, 205, 955.
(35) Honda, A.; Salen, G.; Nguyen, L. B.; Tint, G. S.; Batta, A. K.;
Shefer, S. J . Lipid Res. 1998, 39, 44.
(36) Dunphy, P. J .; Kerr, J . D.; Pennock, J . F.; Whittle, K. J .; Feeney,
J . Biochim. Biophys. Acta 1967, 136, 136.
(37) Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid
Equilibrium and Steady-State Enzyme Systems, Wiley Classics Library
ed.; J ohn Wiley and Sons: New York, 1993; pp 100-118.
where y is the fractional inhibition of enzyme activity, I is the
concentration of the inhibitor AGPP, S is the concentration of
the substrate FPP or GGPP, and Km and Ki are the respective
Michaelis constants for the substrate and inhibitor. Equation
2 describes the expression for the concentration of inhibitor
that yields 50% inhibition of the relative enzyme activity (the