Inactivation of protein farnesyltransferase by active-site-targeted dicarbonyl compounds

Article abstract of DOI:10.1002/1521-4184(200106)334:6<194::AID-ARDP194>3.0.CO;2-M

Upon farnesylation by protein farnesyltransferase (FTase), key proteins become compartmentalized in cells. For example, cell membrane localization is essential for the mitogenic role of mutant Ras protein, which acts as a switch for cancer cell proliferation. We report that α-dicarbonyl compounds derived from the isoprenoid skeleton or other hydrophobic groups potently obstruct farnesylation of a Ras model peptide by human recombinant FTase in vitro. A geranyl-derived isoprenoid diketone, 5,9-dimethyl-8-decene-2,3-dione, at 17 μM caused a 62% reduction in FTase activity after 30 minutes. A farnesyl-derived isoprenoid diketone, 5,9,13-trimethyl-8,12-tetradecadiene-2,3-dione, at 93 μM caused a 94% reduction after 30 minutes. Other dicarbonyl compounds found to be effective against FTase in vitro were (±)-6-(camphorquinone-10-sulfonamido)-hexanoic acid, 4,4′-biphenyldiglyoxaldehyde, dehydroascorbic acid 6-palmitate, 2-oxododecanal, and phenylglyoxal. Higher concentrations of the α-dicarbonyl compound resulted in more rapid and more extensive inactivation. These findings demonstrate that α-dicarbonyl compounds targeted to FTase interfere with protein farnesylation in vitro and may lead to derivatives that have utility as chemotherapeutic agents.

Full text of DOI:10.1002/1521-4184(200106)334:6<194::AID-ARDP194>3.0.CO;2-M

194
Rose and co-workers
Inactivation of Protein Farnesyltransferase by Active-Site-Targeted
Dicarbonyl Compounds
Karl J. Okolotowicz, Wei-Jen Lee, Rosemarie F. Hartman, Ann Y. Kim, Steven R. Ottersberg, Dale E. Robinson, Jr.,
Scott R. Lefler, and Seth D. Rose
Department of Chemistry and Biochemistry, PO Box 871604, Arizona State University, Tempe, AZ 85287-1604, USA
Key Words: Farnesyltransferase inactivation; Ras; α-dicarbonyl compounds; arginine
Subcellular localization is essential for the mitogenic role
of Ras, which acts as a switch for cell division. Cycling
between the inactive form (GDP-bound Ras) and the active
form (GTP-bound Ras) is part of the normal mechanism of
cell growth regulation. Mutant forms of Ras protein, how-
ever, have lost GTPase activity and trigger unrestrained cell
division. Approximately one third of all human cancers ex-
Summary
Upon farnesylation by protein farnesyltransferase (FTase), key
proteins become compartmentalized in cells. For example, cell
membrane localization is essential for the mitogenic role of mutant
Ras protein, which acts as a switch for cancer cell proliferation.
We report that α-dicarbonyl compounds derived from the iso-
prenoid skeleton or other hydrophobic groups potently obstruct
farnesylation of a Ras model peptide by human recombinant FTase
in vitro. A geranyl-derived isoprenoid diketone, 5,9-dimethyl-8-
decene-2,3-dione, at 17 µM caused a 62% reduction in FTase
activity after 30 minutes. A farnesyl-derived isoprenoid diketone,
5,9,13-trimethyl-8,12-tetradecadiene-2,3-dione, at 93 µM caused
a 94% reduction after 30 minutes. Other dicarbonyl compounds
found to be effective against FTase in vitro were (±)-6-(camphor-
quinone-10-sulfonamido)-hexanoic acid, 4,4′-biphenyldiglyoxal-
dehyde, dehydroascorbic acid 6-palmitate, 2-oxododecanal, and
phenylglyoxal. Higher concentrations of the α-dicarbonyl com-
pound resulted in more rapid and more extensive inactivation.
These findings demonstrate that α-dicarbonyl compounds targeted
to FTase interfere with protein farnesylation in vitro and may lead
to derivatives that have utility as chemotherapeutic agents.
hibit a mutant ras genotype, with substantial variations in the
[11]
incidence, depending on tumor type
.
It has been found in recent years that some cancer cell lines
with mutant ras genotypes are insensitive to FTase inhibitors,
and some cancer cell lines without mutant ras genotypes are
[12]
sensitive to FTase inhibitors . A separate prenylation path-
way in cells involves the 20-carbon geranylgeranyl unit.
Recent studies demonstrate the importance of the ratio of
farnesylation and geranylgeranylation of the protein RhoB in
tumorigenesis, presumably as a result of variation in the
compartmentalization of farnesylated and geranylgerany-
[13,14]
lated RhoB
. Recognition of Ras and other protein sub-
strates of FTase is based on a structural motif consisting of a
C-terminal CAAX sequence, in which C represents cysteine,
A represents an aliphatic amino acid and X is preferentially
serine (H-Ras), methionine (N-Ras, K-Ras4a, and K-Ras4b),
cysteine, alanine, or glutamine. The related posttranslational
geranylgeranylation, in which the 20-carbon geranylgeranyl
chain is attached to the cysteine of CAAX by geranylgeranyl-
transferase (GGTase), occurs preferentially when X is
Introduction
Posttranslational modification of Ras and other proteins is
carried out by protein farnesyltransferase (FTase) , which
attaches the 15-carbon farnesyl group of farnesyl pyrophos-
phate to the sulfhydryl group of a cysteine residue near the
C-terminus of the protein. Because farnesylation is required
for proper cell compartmentalization of the proteins (e.g., Ras
becomes localized at the lipid bilayer of the cytosolic surface
1)
[15]
leucine . FTase inhibition results in sole geranylgeranyla-
tion of RhoB, with accompanying reversion of cancer cells to
normal phenotypes in early stages of transformed cells and
cytotoxicity in late stages of transformed cells. This, coupled
with low toxicity toward normal cells, makes FTase inhibi-
tion an attractive anticancer strategy.
of the cell membrane), inhibition of FTase has become a
prominant anticancer drug development strategy
Nucleophilic attack by the cysteine sulfhydryl group of
CAAX, activated by an enzyme-bound zinc ion, on FPP itself
(S 2) or an FPP-derived carbocation (S 1) results in Ras
[1–10]
.
———
N
N
[16–
Correspondence: Prof. Seth D. Rose, Department of Chemistry and Bio-
chemistry, PO Box 871604, Arizona State University, Tempe, AZ 85287-
1604, USA.
E-mail: srose@asu.edu
Fax: +1 480 727 6457
farnesylation, with departure of pyrophosphate from FPP
19]
[20–23]
. Details of FTase X-ray crystal structures
reveal that
291β
a key arginine (Arg
), known from mutagenesis studies to
be involved with FPP binding, is positioned to allow hydro-
gen bonding to the FPP oxygen atoms. A second arginine
1) The abbreviations used are: DMSO, dimethyl sulfoxide; Ds-GCVLS, the
dansyl-labeled pentapeptide N,N-dimethylaminonaphthalenesulfonyl-Gly-
Cys-Val-Leu-Ser; DTT, dithiothreitol; Fmoc, 9-fluorenylmethoxycarbonyl;
FTase, human recombinant protein farnesyltransferase; FPP, farnesyl pyro-
phosphate; GGPP, geranylgeranyl pyrophosphate; GGTase, protein geranyl-
geranyltransferase; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethane-
sulfonic acid; PGO, phenylglyoxal monohydrate; PCC, pyridinium chloro-
chromate; rt, room temperature; TFAA, trifluoroacetic anhydride; THF,
tetrahydrofuran; Tris, tris(hydroxymethyl)aminomethane.
residue (Arg
) at the bottom of the barrel-shaped active
202β
site is involved in peptide binding, via hydrogen bonding as
well as hydrophobic contact between the third isoprene unit
of FPP and the trimethylene group of the arginine side chain.
Active-site arginine residues are known to be subject to
chemical modification by phenylglyoxal and other simple
α-dicarbonyl compounds, with concomitant loss of enzyme
[24–26]
[27,28]
[29]
activity
. We
and others
have found that
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0365-6233/01/0606/0194 $17.50 +.50/0

Inactivation of Protein Farnesyltransferase
195
as the guanidinium group of arginine. To develop a new coup
de main against cancer, we have undertaken a strategy of
development of irreversible inhibitors, or inactivators, of
FTase, i.e., compounds that covalently react with critical
active site residues to block substrate binding or destroy
catalytic activity of active site residues. These new additions
to the armamentarium of farnesylation regulators consist of
α-dicarbonyl compounds tailored to the FTase active site.
Their structures are shown in Figure 1.
One of the two isoprenoid α-dicarbonyl compounds is
5,9-dimethyl-8-decene-2,3-dione (1), a geranyl-derived α-
diketone. The other isoprenoid is 5,9,13-trimethyl-8,12-
tetradecadiene-2,3-dione (2), a farnesyl-derived α-diketone.
A third compound tailored to the active site has a dicarbonyl
functionality tethered to a carboxylate (3), which is designed
to mimic the anionic carboxylate terminus of the CAAX
O
C
O
C
CH3
1
2
O
O
C
C
CH3
O
O
H
N
SO2
HO
O
3
recognition sequence, which participates in electrostatic
202β
Figure 1. Structures of compound 1, 2, and 3.
bonding to active-site Arg
of FTase.
The above compounds, as well as 4,4′-biphenyldiglyoxal-
phenylglyoxal inactivated FTase, a not unreasonable result in dehyde (4), dehydroascorbic acid 6-palmitate (5), 2-oxodo-
view of the fact that catalysis of farnesylation by FTase might decanal (6), and phenylglyoxal (7) (structures shown in
be expected to be facilitated by salt-bridge formation (i.e., Table 1), were preincubated with human recombinant FTase,
electrostatic stabilization) between the pyrophosphate leav- and enzyme activity was then assayed in vitro. The results of
ing group of FPP and a cationic amino acid side chain such these studies are described in this report.
Table 1. FTase inactivation (% of activity lost) following 30-min incubation with FTase.
Compound
Structure
FTase inactivation
O
C
O
C
82% at 68 µM
62% at 17 µM
(±)-1
CH3
O
O
C
94% at 93 µM
49% at 46 µM
(±)-2
C
CH3
O
78% at 93 µM
74% at 46 µM
O
H
N
(±)-3
SO2
HO
O
O
O
C
O
O
C
4
89% at 93 µM
78% at 141 µM
C
C
H
H
O
C
O
(CH2)14 CH3
OH
5
O
O
HO
OH
HO OH
O
O
6
7
78% at 5 mM
77% at 5 mM
C
C
H
O
C
O
C
H
Arch. Pharm. Pharm. Med. Chem. 334, 194–202 (2001)

196
Rose and co-workers
100
100
10
1
10
0
60
120
0
60
120
Incubation period (min)
Incubation period (min)
Figure 2. Kinetics of inactivation of FTase by compound 1. The rate of
inactivation of FTase is dependent on concentration of 1: solid squares, 1
absent; solid diamonds, 17 µM 1; open triangles, 68 µM 1; open circles,
170 µM 1. Each point is the average of duplicate determinations.
Figure 4. Kinetics of inactivation of FTase by compound 5. The rate of
inactivation of FTase is dependent on concentration of 5: solid squares, 5
absent; solid diamonds, 7 µM 5; open triangles, 28 µM 5; open circles,
141 µM 5. Each point is the average of duplicate determinations.
100
10
1
100
10
1
0.1
0.01
0.1
0
60
120
Incubation period (min)
0
30
60
90
120
150
180
Incubation period (min)
Figure 3. Kinetics of inactivation of FTase by compound 4. The rate of
inactivation of FTase is dependent on concentration of 4: solid squares, 4
absent; solid diamonds, 23 µM 4; open triangles, 93 µM 4. Each point is the
average of triplicate determinations.
Figure 5. Kinetics of inactivation of FTase by PGO. The rate of inactivation
of FTase is dependent on PGO concentration. Loss of enzyme activity is
faster at higher concentrations of PGO: solid squares, PGO absent; solid
diamonds, 5 mM PGO; opentriangles, 10mM PGO. At 20mM PGO, enzyme
activity was lost within 2 min of incubation (data not shown). Assays were
carried out after removal of PGO with a high molecular weight cutoff
microconcentrator. Each point is the average of duplicate determinations.
found for compound 2 (data not shown). The data for com-
pounds 4 and 5 are shown in Figures 3 and 4, respectively.
The data for PGO (7) are shown in Figure 5. Similar results
were found for compound 6 (data not shown). All of these
compounds have a hydrophobic portion that would be ex-
pected to be compatible with the highly hydrophobic FPP
binding site of FTase.
As shown in Table 1, the isoprenoid diketones 1 and 2 were
highly potent at impairing FTase after a 30-min incubation,
causing an 82% reduction in its activity in the case of 1 at 68
µM and a 94% reduction in the case of 2 at 93 µM. The
Results
Inactivation of FTase in vitro was monitored by a fluores-
cence assay, in which activity of FTase was determined from
the initial slope of a plot of the fluorescence increase vs. time.
FTase activity was measured after preincubation of FTase
with a dicarbonyl compound and in the absence of dicarbonyl
compound as a control.
The results for compounds 1 through 7 are summarized in
Table 1. The isoprenoid dicarbonyl compound 1 reduced
FTase activity in a concentration- and time-dependent man-
ner, as shown by the data in Figure 2. Similar results were bifunctional compound 3 was also very effective, causing a
Arch. Pharm. Pharm. Med. Chem. 334, 194–202 (2001)

Inactivation of Protein Farnesyltransferase
197
Inactivation of FTase by PGO was hampered by FPP, as
shown in Figure 6, which suggests an overlap of PGO and
FPP binding sites.
90
80
70
60
50
40
30
20
10
0
Compounds without the α-dicarbonyl functionality were
virtually ineffective at inactivating FTase, as expected. The
camphor derivative 8b (Figure 7), an analogue of 3, showed
an 8% reduction in FTase activity at 250 µM after 30 min.
The biphenylderivative 9 (Figure 7), an analogue of 4, caused
no decrease in FTase activity at 23 µM after 30 min, at which
concentration the dicarbonyl compound 4 induced a decrease
of 33% activity after 30 min.
Irreversibility (i.e., covalent modification of FTase) in the
case of PGO was evidenced by the fact that PGO was re-
moved by microconcentration of the protein and buffer re-
placement prior to the assay for activity. If the binding were
reversible, activity would have been restored upon removal
of the excess PGO by microconcentration.
0
5
10
FPP concentration ( M)
15
20
µ
Figure 6. Farnesyl pyrophosphate protection of FTase from inactivation by
PGO. Points are determined by 100 × (FTase activity after incubation with
FPP and PGO)/(FTase activity after incubation with FPP without PGO).
Assays were carried out after removal of FPP and PGO with a high molecular
weight cutoff microconcentrator. Each point is the average of duplicate
determinations.
Discussion
FTase was potently inactivated by several of the α-dicar-
bonyl compounds described in this study, as shown in Table
1. Enzyme inactivation does not offer an IC (concentration
50
required for 50% inhibition) for test substances because en-
zyme activity is lost in a time-dependent process, unlike
Michaelis-Menten kinetics. A measure of the effectiveness of
a test substance to act as an inactivator is the level of enzyme
activity observed after a specified preincubation period of
FTase with the test substance at a specified concentration.
The results reported in Table 1 reflect the extent of inactiva-
tion of FTase after a 30-min preincubation with the indicated
α-dicarbonyl compounds. The geranyl-derived dicarbonyl
compound 1 caused an 82% reduction in FTase activity at
68 µM, whereas the farnesyl-derived compound 2 caused a
94% reduction in activity at 93 µM.
74% reduction in FTase activity after 30 min at 46 µM.
Another bifunctional compound, 4,4′-biphenyldiglyoxalde-
hyde (4), was also very effective, with an 89% reduction in
FTase activity occurring after a 30-min incubation at 93 µM.
The dehydroascorbic acid derivative 5 caused a 78% reduc-
tion at 141 µM after 30 min. The simple aliphatic and aro-
matic compounds, 2-oxododecanal (6) and phenylglyoxal
(7), required a much higher concentration (5 mM) for a 78%
and a 77% reduction in enzyme activity, respectively, after a
30-min preincubation period. At 35 mM 6, enzyme activity
was lost within 2 min.
The fact that compounds 1 and 2 did not differ greatly in
effectiveness suggests that an optimal fit to the active site has
not yet been achieved in the cases of these isoprenoids. A
linker between the isoprenoid group (e.g. farnesyl group) and
the dicarbonyl functionality might be necessary to allow the
O
molecule to “seat” in the farnesyl binding pocket and for the
291β
-
dicarbonyl functionality to reach Arg
. Molecular model-
O3S
O
ing studies are in progress to address this possibility.
In contrast, the simple dicarbonyl compound 2-oxodode-
canal (6) required a concentration of 5 mM to reduce FTase
activity by 78% in 30 min, in spite of the fact that aldehydes
are much more electrophilic than ketones (e.g., 1, 2, and 3).
This difference may in part be attributable to the presence of
the less reactive aldehyde hydrate in solution, but it must also
in part be due to a less favorable fit of the simple poly-
methylene chain of 6 to the FTase active site.
8a
O
H
N
SO2
HO
O
8b
Also remarkable is the observation that (±)-6-(camphor-
quinone-10-sulfonamido)-hexanoic acid (ionized 3, Fig-
ure 1) brought about a 74% reduction in enzyme activity at
O
C
O
C
46 µM. This molecule was designed with the idea that the
291β
CH3
CH3
dicarbonyl functionality would react with Arg
, which
normally interacts with the pyrophosphate group of FPP, and
202β
the carboxylate terminus would bind to Arg
, which nor-
9
mally binds the C terminus of the substrate protein by hydro-
gen. The simpler (±)-camphorquinonesulfonate (8a,
Figure 7), which has a negative charge a short distance away
Figure 7. Structures of compounds 8a, 8b, and 9.
Arch. Pharm. Pharm. Med. Chem. 334, 194–202 (2001)

198
Rose and co-workers
from the dicarbonyl functionality, showed no inactivation
after 30 min at 100 µM. This demonstrates the importance of
This work has shown that FTase is sensitive to dicarbonyl
compounds targeted to the active site by structural similarities
spacer length in bifunctional agents tailored to the FTase to the substrate FPP. Further development of highly tailored
active site and encourages the design of further bifunctional inactivators, which can efficiently function at lower concen-
agents containing a dicarbonyl group and a charged group on trations and in the presence ofFPP, is in progress. Inactivation
a suitably designed spacer. Also, an analogue of 3 lacking a of FTase by dicarbonyl compounds thus potentially offers a
second carbonyl group (8b, Figure 7), showed only an 8% means of selective obstruction of protein prenylation by
FTase for probing FTase mechanism and eventually for pos-
sible cancer chemotherapy.
reduction in enzyme activity at 250 µM, highlighting the
importance of the α-dicarbonyl functionality.
Dehydroascorbic acid 6-palmitate (5) showed significant
activity against FTase (78% inactivation at 141 µM). Al-
though predominantly the dihydrate in aqueous solution, as
shown in Table 1, this substance presumably reacts with
FTase via a transient diketone (or successively formed
monoketones) produced by dehydration of the gem-diol
groups. The polymethylene chain of the palmitoyl group may
not offer optimal targeting to the FTase active site, so synthe-
sis of the 6-farnesoyl ester is in progress.
Acknowledgements
We thank Professor P. Casey and Dr. John F. Moomaw at Duke University
for the generous gift of human FTase, and Dr. Dan Brune at ASU for
assistance with the preparation of the dansyl peptide. We also gratefully
acknowledge support from the Arizona Disease Control Research Founda-
tion (Contract nos. 9723 and 10021).
Phenylglyoxal showed low reactivity toward FTase, requir-
ing 5 mM concentration to achieve 77% inactivation in
30 min. Previously, the stepwise formation of a 2:1 PGO-en-
zyme adduct has been seen kinetically as a biphasic time
course of inactivation of porcine liver prenyltransferase. This
was attributed to rapid formation of a 1:1 covalent adduct,
resulting in reduced activity of the enzyme, followed by
Experimental
All reagents were from Sigma-Aldrich and used without further purifica-
tion, except as noted. PGO was dissolved in boiling water that contained
charcoal, and the mixture was gravity filtered through Celite while hot. Upon
cooling, the solution was filtered by suction to obtain the PGO, and the PGO
was repeatedly recrystallized from water until its 1H NMR spectrum and
melting point indicated the absence of impurities, such as polymeric mate-
rial[21]. All PGO solutions were freshly prepared before use in the studies
described below.
slower conversion of the enzyme into a less active form
[26]
containing a 2:1 covalent adduct.
Some indication of
All 1H NMR spectra were recorded at 300 MHz in CDCl3 (except as noted)
and chemical shifts (δ) are reported in ppm relative to internal (CH3)4Si (or
indirectly to residual CHCl3 at δ 7.26).
similar behavior by human FTase is seen in Figure 5, espe-
cially at low PGO concentration, although this was not ob-
[29]
served with yeast FTase.
Incubation of FTase with FPP
during incubation with PGO decreased the ability of PGO to
Preparation of Ds-GCVLS
inactivate FTase, as shown in Figure 6. This was also ob-
[29]
served in a study of yeast FTase.
FPP can compete with
Ds-GCVLS was prepared by a method analogous to a published proce-
dure[31]. The synthesis was performed by use of a Millipore 9050 Plus
PepSynthesizer that employed standard Fmoc solid phase peptide synthesis
methodology. PS-PEG resin and Fmoc protected amino acids were obtained
from Millipore. Peptide purification was performed by RP-HPLC on an
Alltech Macrosphere RP300 C8 column. Elution was accomplished with a
linear gradient from aqueous 10 mM trifluoroacetic acid to 10 mM tri-
fluoroacetic acid in acetonitrile. Lyophilization yielded a pale yellow solid.
Stock solutions were prepared in 50 mM Tris containing 5 mM DTT and
0.2% n-octyl-β-D-glucopyranoside, pH 7.7. Concentrations of Ds-GCVLS
were determined by use of the extinction coefficient of the dansyl group at
340 nm (ε = 4250 M–1cm–1). Stock solutions were stored at –20 °C.
PGO for access to the active site, thereby decreasing the rate
of covalent modification and inactivation of FTase. Once
PGO has entered the active site and has covalently reacted
with the enzyme, however, subsequent displacement of PGO
by FPP would be unlikely to occur.
The analogue 4,4′-biphenyldiglyoxaldehyde (4) was con-
siderably more reactive than PGO, effecting 89% inactivation
in 30 min at 93 µM. This may in part be due to the greater
hydrophobicity of two phenyl rings than one, as well as the
possibility of reaction of 4 with two active site arginine
residues of FTase. The simple analogue lacking adjacent
carbonyl groups, 4,4′-diacetylbiphenyl (9, Figure 7) was in-
active against FTase, as expected.
It is likely that arginine side-chain modification by com-
pounds 1–7 interferes with the catalytic function of one or
more active-site arginines, the binding of FPP, the binding of
the substrate pentapeptide, or more than one of these factors.
The observation that only low concentrations of these com-
pounds, especially 1–5, are required for rapid inactivation of
FTase Assay
FTase was stored at –70 °C. The fluoresence assay conditions were
adapted from a published method[31]. The FTase assay consisted of the
measurement of the rate of FTase-catalyzed farnesylation of the fluorescently
labeled pentapeptide Ds-GCVLS. Upon farnesylation of the cysteine, the
fluoresence emission of the dansyl group is enhanced due to the proximity
of the hydrophobic isoprenoid group. Enzyme activity (approximately 15–
60 nM FTase) was monitored at saturating substrate concentrations (10 µM
farnesyl pyrophosphate, ammonium salt, prepared by successive dilutions of
a commercial 2.3 mM solution in methanol:10 mM aqueous NH4OH (7:3)
into assay buffer, and 1.0 µM Ds-GCVLS) by measurement of the increase
in fluorescence emission as FTase farnesylated Ds-GCVLS for a period of
5–10 min. The assay buffer was 50 mM NaHEPES, 5 mM DTT, 5 mM
MgCl2, 10 µM ZnCl2, and 0.2% n-octyl-β-D-glucopyranoside, pH 7.5. Fluo-
rescence emission was measured with a JASCO FP-77 spectrofluorimeter
fitted with a temperature-controlled cuvette holder connected to a 30 °C
constant temperature bath. All fluoresence assays were conducted in a 4-mm
× 4-mm quartz cuvette with excitation at 340 nm and emission monitored at
505 nm. Each determination was carried out at least in duplicate.
the enzyme is also consistent with the idea that modification
[30]
of active site rather than remote arginines has occurred
.
The twin menaces of resistance and toxicity plague current
cancer chemotherapy. Both result from exposure of cells to
the therapeutic agent. If a single dose of an irreversible
inhibitor were to be sufficient to eliminate FTase activity until
further endogenous biosynthesis produced additional FTase,
exposure of both normal and cancer cells would be more
limited, relative to a reversible inhibitor.
Arch. Pharm. Pharm. Med. Chem. 334, 194–202 (2001)

Inactivation of Protein Farnesyltransferase
199
O
O
a
OH
H
H
15
16
17
b
OH
NO2
c
18
d
OAc
NO2
e
NO2
20
19
f
OH
g
O
NO2
O
22
21
h
O
O
2
Scheme 1. a. PCC/CH2Cl2, rt, 24 h. b. NiCl2•6H2O/Al/THF, rt, 6 h. c. Nitroethane/Am-
berlyst A-21, rt, 24 h. d. Ac2O/cat. H2SO4, –10 °C, 15 min. e. t-BuOK/t-BuOH, rt, 1 h.
f. H2O2/OH–/MeOH/dioxane. g. Dowex-50(H+)/H2O/dioxane, reflux, 2 h.
h. DMSO/TFAA/Et3N, –77 °C, 1.25 h.
For assays, compounds 1, 2, 4, and 6 were dissolved in DMSO, and the
stock solutions were diluted with assay buffer without DTT to a final
concentration of up to 5% DMSO in the FTase preincubation solution.
Compound 5 was dissolved in DMSO, and this solution was diluted into
5 mM sodium acetate (pH 3.9) to a final concentration of 2% DMSO. The
resulting stock solution was kept cold and protected from light, and it was
used within several days of preparation. Compounds 3 and 7 were dissolved
in assay buffer.
The microconcentration step was omitted with compounds 1, 2, 3, and 5,
as there was no interfering fluorescence caused by these compounds.
Enzyme Kinetic Data Analysis
Inactivation kinetics differ from conventional inhibition kinetics. Inacti-
vation kinetics typically consist of a prior, reversible binding of the inactiva-
tor (X) to the enzyme to form a noncovalent complex (E•X), followed by
covalent reaction of X in the active site with E[32]. This results in the
conversion of the active form of the enzyme (E) into impaired and/or
completely inactive form(s) of the enzyme (E–X), as shown in the equation
below:
FTase was incubated at 30 °C in the presence of inactivator at various
concentrations in assay buffer without DTT. Periodically, a 70-µL aliquot of
the preincubation mixture was added to a cuvette containing FPP, assay
buffer (i.e., containing DTT), and Ds-GCVLS to give a final volume of
100 µL and a final concentration of 15 nM FTase. Control experiments were
performed with assay mixtures containing all components (including
DMSO) except the inactivator.
kinact
Kr
(1)
E + X
E–X
E·X
Because of interfering fluorescence from the inactivator in the cases of 4,
6, and 7, the aliquot of preincubation mixture was first subjected to solvent
exchange by size exclusion membrane concentration to remove the inactiva-
tor. The solvent exchange utilized Microcon (Millipore Co.) 30 kD molecular
weight cutoff microconcentrators. This also served to stop further inactiva-
tion of the enzyme by the inactivator. A 100-µL aliquot of FTase-inactivator
reaction mixture was pipetted into a Microcon concentrator, which was
centrifuged at 12,000 RPM for 8 min at 4 °C. The concentrate was resus-
pended in 100 µL of assay buffer (i.e., containing DTT), and centrifugation
was repeated. The final concentrate was resuspended in 90 µL of assay
buffer, and the resulting solution was recovered by inversion of the concen-
trator and centrifugation at 3500 RPM for 3 min at 4 °C. Finally, FPP and
Dn-GCVLS were added, and the solution was assayed for FTase activity. A
control was handled similarly, except that inactivator was omitted from the
incubation mixture.
The preequilibrium is governed by Kr = [E•X]/[E][X], and kinact is the
first-order rate constant for covalent bond formation within the E•X com-
plex. Under the typical conditions of X being present in excess over [E]0
(initial enzyme concentration), [X] is essentially constant, and the above
scheme obeys the following rate equation:
[E]t/[E]0 = exp(–k[X]t)
(2)
Thus, the ratio [E]t/[E]0 (i.e., the fraction of activity remaining at incuba-
tion time t) decreases at longer incubation time, and the decrease is more
rapid at higher concentrations of inactivator X. Data from the FTase assays
described above were plotted according to the logarithmic form of equa-
tion (2).
Arch. Pharm. Pharm. Med. Chem. 334, 194–202 (2001)

200
Rose and co-workers
FPP Protection of FTase against Inactivation by PGO
was dried over anhydrous MgSO4, and the solvent was removed by rotary
evaporation to yield 1.40 g of a mixture of stereoisomers of the nitroacetate
19. 1H NMR δ (ppm) 5.4 (br m, CHOCOCH3), 5.2 (t, CH3C(CH3)=CH
and CH2C(CH3)=CH), 4.4 (m, CHNO2), 2.1 (s, CH3CO2), 2.0 (m,
FTase was treated with FPP at various concentrations in assay buffer
without DTT for 2 min at 30 °C prior to addition of PGO at a final
concentration of 10 mM and incubation for 30 min at 30 °C. The control was
incubated with FPP at the corresponding concentration for the full length of
time, but without PGO. The mixtures were subsequently subjected to micro-
concentration and assay, as described above.
C=CHCH2CH2C(CH3)=CHCH2),
1.6–1.7
(all
s,
CH3C(CH3)=CHCH2CH2CCH3), 1.5 (d, CH3CHNO2), 1.2–1.5 (m,
CH2CH(CH3)CH2), 0.9 (d, CH2CH(CH3)CH2).
Synthetic Strategy
5,9,13-Trimethyl-2-nitro-2,8,12-tetradecatriene (20)
The isoprenoid dicarbonyl compounds 1 and 2 were synthesized according
to the method outlined in Scheme 1 for compound 2, based on procedures in
the literature for analogous compounds (step a,[33] b,[34] c,[35] d,[36] e,[36] f,[37]
g,[38], and h[39]) and described in detail below, beginning with the preparation
of 16. Compound 3 was prepared from (±)-camphorsulfonic acid by forma-
tion of the sulfonyl chloride, conversion to the sulfonamide, and oxidation
to the camphorquinone by analogy to a literature procedure[40] for camphor-
quinonesulfonamides of α-amino acids, as described in detail below. Com-
A solution of 1.30 g of 19 in 25 mL of t-butyl alcohol was added to a
solution of 30 mL of t-butyl alcohol containing 0.53 g of potassium t-butox-
ide. The reaction mixture was stirred at room temperature for 1 h, whereupon
it was added to 150 mL of ether and 20 mL of H2O. The organic layer was
separated and dried over MgSO4, and the solvent was removed by rotary
evaporation to afford 0.40 g of racemic 20, predominantly the (E)-isomer.
1H NMR δ (ppm) 7.18 (t, CH=CNO2), 5.2 (t, CH3C(CH3)=CH and
CH2C(CH3)=CH),
2.19
(s,
C=C(CH3)NO2),
2.2–2.3 (m,
2.0
(m,
pound 4 was prepared by a literature procedure[41]
.
C=CHCH2CH2C(CH3)=CHCH2),
CH2C=CNO2),
(CH3C=(CH3), 1.6–1.7 (all s, CH3C(CH3)=CHCH2CH2CCH3), 1.2–1.5 (m,
CH2CH(CH3)CH2), 0.95 (d, CH2CH(CH3)CH2).
Farnesal (16)
To a stirred solution of 15 g of PCC in 250 mL of dichloromethane was
added 10 g of (E,E)-farnesol (15). The reaction mixture was stirred at room
temperature overnight and filtered through a 55-cm (height) by 5-cm (diame-
ter) column of silica gel. The eluate was dried over MgSO4, and the solvent
was removed by rotary evaporation to give a greenish-yellow oil. The crude
farnesal was purified by column chromatography (silica gel/chloroform) to
yield 7.25 g of product 16, predominantly the (E,E)-isomer. 1H NMR δ (ppm)
10.0 (d, CHO), 5.9 (d, CH2C(CH3)=CHCO), 5.1 (t, CH3C(CH3)=CH
and CH2C(CH3)=CH), 2.3 (m, CH2C(CH3)=CH-CO), 2.5 (m,
CH2C(CH3)=CHCH2CH2), 2.2 (s, CH3C=CH-CO), 2.0 (m,
CH3C(CH3)=CHCH2CH2), 1.6–1.7 (all s, CH3C(CH3)=CHCH2CH2CCH3).
2,3-Epoxy-5,9,13-trimethyl-2-nitro-8,12-tetradecadiene (21)
To a solution of 600 mg of 20 in 9 mL of a 70/30 mixture of methanol–di-
oxane in an ice bath, 0.7 mL of 15% H2O2 was added. The reaction mixture
was allowed to stir for 10 min, whereupon 0.5 mL of 2 N KOH was added,
and the reaction mixture was stirred on ice for 30 min. The ice bath was
removed, and the reaction mixture was warmed to room temperature over
1.5 h. The reaction mixture was acidified to pH 2 by addition of 5% HCl and
was then extracted with ether. The ether extracts were washed with H2O and
saturated aqueous sodium bicarbonate and dried over anhydrous MgSO4, and
the solvent was removed by rotary evaporation to afford 200 mg of a mixture
of stereoisomers of crude epoxide 21. 1H NMR δ (ppm) 5.2 (t,
CH3C(CH3)=CH and CH2C(CH3)=CH), 3.4 (m, CHCNO2), 2.0 (m,
C=CHCH2CH2C(CH3)=CHCH2), 1.95 (s, CHC(CH3)NO2), 1.6–1.7 (all s,
CH3C(CH3)=CHCH2CH2CCH3), 1.2–1.5 (m, CH2CH(CH3)CH2), 0.9 (d,
CH2CH(CH3)CH2).
Dihydrofarnesal (17)
To a stirred solution of 0.53 g of 16 in 150 mL of dry THF in a round-bot-
tom flask equipped with a water-cooled condenser was added a solid mixture
of 31.04 g of NiCl2•6H2O and 4.27 g of Al powder (caution: exothermic).
The reaction mixture was stirred at room temperature for 6 h. The solid mass
was removed by filtration, and the solvent was dried over MgSO4 and
removed by rotary evaporation, which afforded 0.50 g of racemic 17,
predominantly the (E)-isomer. 1H NMR δ (ppm) 9.8 (t, CHO), 5.2 (t,
CH3C(CH3)=CH and CH2C(CH3)=CH), 2.3 (m, CH2C(CH3)=CH), 2.2–2.4
(qd, CH2CHO), 2.0 (m, C=CHCH2CH2C(CH3)=CHCH2), 1.6–1.7 (all s,
CH3C(CH3)=CHCH2CH2CCH3), 1.2–1.5 (m, CH2CH(CH3)CH2CO), 0.9
(d, CH2C(CH3)=CH).
3-Hydroxy-5,9,13-trimethyltetradeca-8,12-dien-2-one (22)
To 200 mg of 21 in 50/50 water–dioxane was added 100 mg of Dowex-50
(H+), and the mixture was stirred under reflux for 2 h. The reaction mixture
was cooled to room temperature, and the Dowex-50 was removed by filtra-
tion. To the filtrate was added two volumes of saturated aqueous NaCl, and
the solution was extracted with ether. The organic layer was dried over
anhydrous MgSO4, and the solvent was evaporated to yield 125 mg of a
mixture of stereoisomers of the hydroxy ketone 22. 1H NMR δ (ppm) 5.2 (t,
CH3C(CH3)=CH and CH2C(CH3)=CH), 4.2 (br d, CHOH), 2.1 (s,
CH3C=O), 2.0 (m, C=CHCH2CH2C(CH3)=CHCH2), 1.6–1.7 (all s,
CH3C(CH3)=CHCH2CH2CCH3), 1.2–1.5 (m, CH2CH(CH3)CH2), 0.9 (d,
CH2CH(CH3)CH2).
5,9,13-Trimethyl-2-nitrotetradeca-8,12-dien-3-ol (18)
To2.93gof 17 wasadded5 mLofnitroethanewithstirring. Approximately
5–6 g of Amberlyst A-21 was added, and the mixture was stirred at room
temperature overnight. The mixture was filtered and the ion exchange
resin was rinsed four times with 25 mL of dichloromethane. The filtrate was
dried over anhydrous MgSO4, and the solvent was removed by rotary
evaporation, which afforded 3.53 g (90%) of a mixture of stereoisomers of
the desired nitroalcohol 18. 1H NMR δ (ppm) 5.2 (t, CH3C(CH3)=CH
and CH2C(CH3)=CH), 4.4 (m, CHNO2), 2.2 (br s, CHOH),
5,9,13-Trimethyltetradeca-8,12-diene-2,3-dione (2)
To 50 mg of the hydroxy ketone 22 dissolved in 1 mL of dichloromethane
in a –78 °C bath was added 31 µL of DMSO and 45 µL of trifluoroacetic
anhydride. The reaction mixture was stirred under an N2 atmosphere for
30 min, after which time 153 µL of triethylamine was added. The reaction
mixture was warmed to room temperature over a 40-min period. The dichlo-
romethane solution was extracted with saturated aqueous NaCl, the solution
was dried over anhydrous MgSO4, and the solvent was removed by rotary
evaporation. The crude oil was purified by preparative TLC (silica gel/chlo-
roform), which afforded 10 mg of pure racemic diketone 2, predomi-
nantly the (E)-isomer. 1H NMR δ (ppm) 5.2 (t, CH3C(CH3)=CH and
CH2C(CH3)=CH), 2.5–2.7 (qd, CH2C=O), 2.3 (s, CH3C=O), 2.0
2.0 (m, C=CHCH2CH2C(CH3)=CHCH2),
1.6–1.7
(all
s,
CH3C(CH3)=CHCH2CH2CCH3), 1.5 (d, CH3CHNO2), 1.2–1.5 (m,
CH2CH(CH3)CH2), 0.9 (d, CH2CH(CH3)CH2).
5,9,13-Trimethyl-2-nitrotetradeca-8,12-dien-3-yl Acetate (19)
A flask containing 1.15 g of acetic anhydride was cooled to –10 °C with a
salt-ice bath, and 3.1 g of 18 was added with magnetic stirring under an N2
atmosphere. After 10–15 min, 25µL of concentrated H2SO4 was added. The
reaction mixture was allowed to stir for an additional 10 min, after which
it was diluted with 40 mL of ether and 10 mL of H2O. The mixture was
extracted three times with 15 mL of saturated aqueous sodium bicarbon-
ate and once with 10 mL of saturated aqueous NaCl. The ether layer
(m, C=CHCH2CH2C(CH3)=CHCH2),
1.6–1.7
(all
s,
CH3C(CH3)=CHCH2CH2CCH3), 1.2–1.5 (m, CH2CH(CH3)CH2), 0.9 (d,
CH2CH(CH3)CH2).
Arch. Pharm. Pharm. Med. Chem. 334, 194–202 (2001)

Inactivation of Protein Farnesyltransferase
201
5,9-Dimethyl-2-nitrodec-8-en-3-ol (10)
5,9-Dimethyldec-8-en-2,3-dione (1)
Nitroethane (7.5 g) was added to a stirred solution of 13.82 g of citronellal
in dichloromethane. Approximately 12–14 g of Amberlyst A-21 ion ex-
change resin was added, and the mixture was stirred at room temperature
overnight. After filtration, the resin was rinsed 4 times with 75 mL of
dichloromethane. The filtrate was dried over anhydrous MgSO4 and the
solvent was removed by rotary evaporation, affording 20.1 g (95%) of a
To a stirred solution of 1.51 g of PCC in 50 mL of dichloromethane was
added 950 mg of 14. The reaction mixture was stirred at room temperature
for 8 h, whereupon it was diluted with ether. The mixture was passed through
a 15-cm (height) by 2-cm (diameter) column of silica gel, which was
subsequently washed with ether. The effluents were combined and the
solvent was removed by rotary evaporation. The product was purified by
column chromatography (silica gel/chloroform) to yield 150 mg of racemic
1. 1H NMR δ (ppm) 5.2 (t, C=CH), 2.6–2.8 (qd, CH2C=O), 2.2 (s, CH3C=O),
2.0 (br m, C=CHCH2), 1.6 and 1.7 (both s, CH3C(CH3)=), 1.2–1.4 (m,
CH2CH(CH3)CH2), 0.95 (d, CH2CH(CH3)CH2).
1
mixture of stereoisomers of the desired nitroalcohol 10. H NMR δ (ppm)
5.2 (t, C=CH), 4.4 (m, CH3CHNO2), 2.5 (broad s, CHOH), 2.0 (br m,
=CHCH2), 1.60 and 1.65 (both s, CH3C(CH3)=), 1.5 (d, CH3CHNO2),
1.2–1.5 (m, CH2CH(CH3)CH2), 0.95 (d, CH2CH(CH3)CH2).
(±)-Camphor-10-sulfonyl Chloride
5,9-Dimethyl-2-nitrodec-8-en-3-yl Acetate (11)
Thionyl chloride (14.5 mL) was added to cold dimethylformamide (14.5
mL) and the solution was mixed for 10 min. (±)-Camphor-10-sulfonic acid
(3.63 g) was added to the reaction mixture, which was then stirred for 2.5 h
at 0 °C and 2 h at room temperature protected from atmospheric moisture.
The mixture was poured into crushed ice, whereupon a light yellow precipi-
tate formed. Ether was added to dissolve the yellow precipitate, and the
organic layer was dried over anhydrous MgSO4. The solvent was removed
by rotary evaporation, and light yellow crystals of (±)-camphor-10-sulfonyl
chloride were obtained (mp 85.5–87 °C, 3.32 g, 85%; lit.[42] 85 °C).
A flask containing 1.3 g of acetic anhydride was cooled to –10 °C with a
salt-ice bath, and 2.34 g of 10 was added with magnetic stirring under an N2
atmosphere. After 10–15 min, 2–5 drops of concentrated H2SO4 were added.
The reaction mixture was allowed to stir for an additional 10 min, after which
it was treated with 40 mL of ether and 10 mL of H2O. The mixture was
extracted three times with 15 mL of saturated aqueous sodium bicarbonate
and once with 10 mL of saturated aqueous NaCl. The ether layer was dried
over anhydrous MgSO4, and the solvent was removed by rotary evaporation
to yield 2.41 g (88%) of a mixture of stereoisomers of the nitroacetate 11. 1H
NMR δ (ppm) 5.4 (broad m, CH2CH(OAc)), 5.2 (t, C=CH), 4.65 (m,
CH3CHNO2), 2.1 (s, CH3CO2), 2.0 (br m, =CHCH2), 1.60 and 1.65 (both s,
CH3C(CH3)=), 1.5 (d, CH3CHNO2), 1.2–1.5 (m, CH2CH(CH3)CH2), 0.95
(d, CH2CH(CH3)CH2).
(±)-6-(Camphor-10-sulfonamido)-hexanoic Acid (8b)
To 33 mL of warm water was added 0.83 g of 6-aminocaproic acid, and
the solution was stirred for 5 min. Magnesium oxide (2.50 g) and dioxane
(21 mL) were added to the warm solution, and the mixture was stirred for
15 min. (±)-Camphor-10-sulfonyl chloride (3.32 g) in four portions was
added to the mixture over 24 h. The mixture was stirred for an additional 16
h, whereupon the MgO was removed by filtration. The filtrate was acidified
with 5% HCl and extracted with ether. The organic layer was dried over
anhydrous MgSO4, and the ether was evaporated to obtain (±)-6-(camphor-
10-sulfonamido)-hexanoic acid 8b as a light yellow oil (1.42 g, 65%). 1H
NMR δ (ppm) 5.18 (m, NH), 3.39 (d, CHHSO2), 3.16 (m, NHCH2), 2.90 (d,
CHHSO2), 2.40 (m, CH2CO2H), 2.10 (m, bicyclo-CCH2CH2CH,
bicyclo-CH2CH and bicyclo-CHCH2CO), 1.67 (m, NHCH2CH2 and
CH2CH2CO2H), 1.45 (m, CH2CH2CH2CH2CH2 and bicyclo-
CCH2CH2CH), 1.02 (s, CH3), 0.91 (s, CH3). IR , ν (cm–1): 3285 (NH), 2943
(CH), 1729 (C=O), 1325 (SO2), 1140 (SO2).
5,9-Dimethyl-2-nitro-2,8-decadiene (12)
A solution of 0.84 g of 11 in 10 mL of t-butyl alcohol was added to a
solution of 5 mL of t-butyl alcohol containing 0.38 g of potassium t-butoxide.
The reaction mixture was stirred at room temperature for 1 h, whereupon it
was added to 150 mL of ether and 20 mL of H2O. The organic layer was
separated and dried over anhydrous MgSO4, and the solvent was removed
by rotary evaporation to afford 0.60 g (94%) of racemic (E)- and (Z)-12. 1H
NMR δ (ppm) 7.18 (t, CH=CNO2), 5.2 (t, (CH3)2C=CH), 2.2–2.3 (m,
CH2C=CNO2), 2.19 (s, CH3CNO2), 2.0 (br m, (CH3)2C=CHCH2), 1.60 and
1.65 (both s, CH3C(CH3)=), 1.2–1.5 (m, CH2CH(CH3)CH2), 0.95 (d,
CH2CH(CH3)CH2).
(±)-6-(Camphorquinone-10-sulfonamido)-hexanoic Acid (3)
2,3-Epoxy-5,9-dimethyl-2-nitro-8-decene (13)
Selenium dioxide (0.48 g) (caution: highly toxic, corrosive) was dissolved
in a warm solution of dioxane–water (95:5, 4.3 mL). The solution was stirred
for 15 min, or until dissolved. (±)-6-(Camphor-10-sulfonamido)-hexanoic
acid 8b (1.00 g) was then added to the warm solution. The reaction mixture
was gently refluxed for 120 h with protection from both light and atmospheric
moisture. The resulting elemental selenium was removed by filtration
through Celite. Upon removal of the solvent by rotary evaporation, a dark
red-brown residue remained. This residue contained the desired product 3
and traces of selenium and dioxane (1.39 g). 1H NMR (acetone-d6) δ (ppm)
3.22 (d, CHHSO2), 3.18 (m, NHCH2), 2.90 (d, CHHSO2), 2.25 (m,
CCH2CH2, CH2CHCO, and CH2CO2H), 1.84 (m, NHCH2CH2 and
CH2CH2CO2H), 1.60 (m, CH2CH2CH2CH2CH2 and bicyclo-CH2CH2CH),
1.23 (s, CH3), 0.91 (s, CH3). IR (KBr), ν (cm–1): 3284.7 (NH), 2944 (CH),
1732 (C=O), 1326 (SO2), 1144 (SO2).
To a solution of 3.0 g of 12 in 60 mL of a 70/30 mixture of methanol–di-
oxane in an ice bath, 6.3 mL of 15% H2O2 was added. The reaction mixture
was allowed to stir for 10 min, whereupon 3.5 mL of 2 N NaOH was added,
and the reaction mixture was stirred on ice for 30 min. The ice bath was
removed, and the reaction mixture was warmed to room temperature over
1.5 h. The reaction mixture was acidified to pH 2 by addition of 5% HCl and
was then extracted with ether. The ether extracts were washed with H2O and
saturated aqueous sodium bicarbonate and dried overanhydrous MgSO4. The
solvent was removed by rotary evaporation to afford 2.99 g (94%) of a
mixture of stereoisomers of crude epoxide 13. 1H NMR δ (ppm) 5.2 (t,
C=CH), 3.45 (dd, CHO), 2.0 (br m, C=CHCH2), 1.9 (s, CH3CNO2), 1.4–1.5
(m, CH2CH(CH3)CH2), 1.2–1.3 (m, CH2CH(CH3)CH2), 1.60 and 1.65 (both
s, CH3C(CH3)=), 0.95 (d, CH2CH(CH3)CH2).
L-Dehydroascorbic Acid 6-Palmitate (5)
3-Hydroxy-5,9-dimethyldec-8-en-2-one (14)
This substance was prepared by the oxidation of L-ascorbic acid 6-palmi-
tate in 95% ethanol by iodine. A solution of 0.358 g of ascorbic acid
6-palmitate in 50 mL of 95% ethanol containing 1 g of NaHCO3 was treated
with 0.219 g I2 dissolved in 25 mL of 95% ethanol. The iodine was
decolorized by oxidation of the ascorbate. After approximately 30 min at
room temperature, the solvent was removed by rotary evaporation, and the
residue was dissolved in ether that was pretreated with alumina to remove
peroxides. (Caution: destroy any peroxides bound to the alumina before
disposal.) The ether solution was washed three times with water and then
with saturated aqueous sodium chloride. The organic layer was dried over
To 1.0 g of 13 in water–dioxane (50/50) was added 0.5 g of Dowex-50
(H+), and the mixture was stirred under reflux for 2 h. The reaction mixture
was cooled to room temperature, and the Dowex-50 was removed by filtra-
tion. To the filtrate was added two vols of saturated aqueous NaCl, and the
solution was extracted with ether. The organic layer was dried over anhy-
drous MgSO4, and the solvent was evaporated to yield 0.61 g (70%) of the
hydroxy ketone 14. 1H NMR δ (ppm) 5.2 (t, C=CH), 4.2 (dd, CHOH), 2.2
(s, CH3C=O), 2.0 (br m, C=CHCH2), 1.6 and 1.7 (both s, CH3C(CH3)=),
1.2–1.4 (m, CH2CH(CH3)CH2), 0.95 (d, CH2CH(CH3)CH2).
Arch. Pharm. Pharm. Med. Chem. 334, 194–202 (2001)

202
Rose and co-workers
anhydrous MgSO4, and the solvent was removed by rotary evaporation to
afford 5 as a white solid. The product’s identity was verified by comparison
of the 13C NMR signals to published[43] values.
[19] V. A. Weller, M. D. Distefano, J. Am. Chem. Soc. 1998, 120, 7975–
7976.
[20] H.-W. Park, S. R. Boduluri, J. F. Moomaw, P. J. Casey, L. S. Beese,
Science 1997, 275, 1800–1804.
Preparation of 2-Oxododecanal (6)
[21] P. Dunten, U. Kammlott, R. Crowther, D. Weber, R. Palermo, J. Birk-
A mixture of 2.41 g (21.7 mmole) of selenium dioxide (caution: highly
toxic corrosive) in 100 mL of 95% ethanol was stirred and heated to 40 °C
until all of the selenium dioxide had dissolved. Over a 5-min period, 4.00 g
(21.7 mmol) of dodecanal was added, and the reaction mixture was heated
at reflux for 14 h. After filtration to remove the precipitated selenium, the
solvent was removed in vacuo, and the crude product was subjected to silica
gel column chromatography. After elution of the residual aldehyde starting
material with 10% ethyl acetate in hexane, the product was eluted with ethyl
acetate. The fractions containing the product were combined, and the solvent
was evaporated in vacuo. The resulting solid, probably the aldehyde hydrate,
melted on heating and the substance was distilled at reduced pressure to yield
1.01 g (24%) of a bright yellow liquid, 2-oxododecanal 6 (bp 83–84 °C at
4 mm Hg). 1H NMR δ (ppm), 9.21 (s, H–C=O), 2.70 (t, O=CCH2CH2), 1.61
(m, O=CCH2CH2), 1.25–1.37 (m, (CH2)7CH3), 0.87 (t, CH3).
toft, Biochemistry 1998, 37, 7907–7912.
[22] S. B. Long, P. T. Casey, L. S. Beese, Biochemistry 1998, 37, 9612–
9618.
[23] C. L. Strickland, W. T. Windsor, R. Syto, L. Wang, R. Bond, Z. Wu, J.
Schwartz, H. V. Le, L. S. Beese, P. C. Weber, Biochemistry 1998, 37,
16601–16611.
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Lefler, A. Y. Kim, W.-J. Lee, D. E. Robinson, communication pre-
sented at Signal Transduction 2000, Luxembourg, January 26–29,
2000.
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Received: December 14, 2000 [FP536]
Arch. Pharm. Pharm. Med. Chem. 334, 194–202 (2001)