Photoactive analogs of farnesyl pyrophosphate containing benzoylbenzoate esters: Synthesis and application to photoaffinity labeling of yeast protein farnesyltransferase

Article abstract of DOI:10.1021/jo9602736

Farnesyl pyrophosphate (FPP) is involved in a large number of cellular processes including the prenylation of transforming mutants of Ras proteins implicated in cancer. Photoactive analogs could provide useful information about enzyme active sites that bind farnesyl pyrophosphate; however, the availability of such compounds is extremely limited. Molecules that incorporate benzophenone moieties are attractive photoaffinity labeling reagents because of their useful photochemical properties. Here, the syntheses of two compounds, 3a and 3b, containing para- and meta-substituted benzoylbenzoates are described. Compounds 3a and 3b are competitive inhibitors (with respect to FPP) of yeast protein farnesyltransferase (PFTase) with K(i) values of 910 and 380 nM, respectively. Both compounds inactivate PFTase upon photolysis, resulting in as much as 44% inactivation of enzyme activity. Photolysis of PFTase in the presence of [³²P]3a or of [³²P]3b results in preferential labeling of the β subunit, suggesting that this subunit is involved in prenyl group recognition. These compounds should be valuable tools for studying enzymes that utilize FPP as a substrate.

Full text of DOI:10.1021/jo9602736

7738
J . Org. Chem. 1996, 61, 7738-7745
P h otoa ctive An a logs of F a r n esyl P yr op h osp h a te Con ta in in g
Ben zoylben zoa te Ester s: Syn th esis a n d Ap p lica tion to
P h otoa ffin ity La belin g of Yea st P r otein F a r n esyltr a n sfer a se
Igor Gaon, Tammy C. Turek, Valerie A. Weller, Rebecca L. Edelstein, Satinder K. Singh, and
Mark D. Distefano*
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455
Received February 9, 1996 (Revised Manuscript Received J uly 25, 1996X)
Farnesyl pyrophosphate (FPP) is involved in a large number of cellular processes including the
prenylation of transforming mutants of Ras proteins implicated in cancer. Photoactive analogs
could provide useful information about enzyme active sites that bind farnesyl pyrophosphate;
however, the availability of such compounds is extremely limited. Molecules that incorporate
benzophenone moieties are attractive photoaffinity labeling reagents because of their useful
photochemical properties. Here, the syntheses of two compounds, 3a and 3b, containing para-
and meta-substituted benzoylbenzoates are described. Compounds 3a and 3b are competitive
inhibitors (with respect to FPP) of yeast protein farnesyltransferase (PFTase) with Ki values of
910 and 380 nM, respectively. Both compounds inactivate PFTase upon photolysis, resulting in
as much as 44% inactivation of enzyme activity. Photolysis of PFTase in the presence of [32P]3a or
of [32P]3b results in preferential labeling of the â subunit, suggesting that this subunit is involved
in prenyl group recognition. These compounds should be valuable tools for studying enzymes that
utilize FPP as a substrate.
In tr od u ction
Chemical modification techniques are useful in study-
ing the interactions between small molecules and pro-
teins because they allow critical residues involved in
ligand binding and catalysis to be identified. Photoaf-
finity labeling experiments utilize functional groups
incorporated into ligand or substrate structures that can
be activated by light; upon irradiation, they form reactive
intermediates that undergo bond insertion reactions with
a macromolecule.1 Since the intermediates produced by
photolysis are highly reactive, these species typically
react with residues that are in close proximity. This lack
of chemical specificity is useful because many different
amino acids can be labeled, and hence identified, using
these reagents.2
Farnesyl pyrophosphate (FPP, 1) and its C20 homo-
logue, geranylgeranyl pyrophosphate (2), shown in Figure
1, are isoprenoids that are involved in a number of
cellular processes including cholesterol biosynthesis,
glycoprotein synthesis, vitamin and cofactor synthesis,
and protein prenylation.3 Recently, the enzyme protein
farnesyltransferase (PFTase), which utilizes FPP as a
substrate, has attracted considerable attention as a
possible target for the design of chemotherapeutic agents.4
Photoaffinity labeling reagents could provide important
information concerning the active site of this enzyme;
however, the availability of such compounds is extremely
limited; very few photoactive FPP analogs have been
prepared.5 In 1988, Baba and Allen introduced a sub-
strate analog that incorporated a photoactive diazoester
moiety into a derivative of geraniol.6 While the resulting
F igu r e 1. Structures of FPP, GGPP, and photoaffinity label-
ing analogs.
molecule was nearly superimposible with FPP, the
pendant diazotrifluoropropionate cross-linking group pos-
sessed a number of less desirable features including low
cross-linking efficiency and the requirement for prolonged
short wavelength UV irradiation for photoactivation.2,7
Molecules that incorporate benzophenone moieties are
attractive alternatives to diazo- and azide-containing
compounds for photoaffinity labeling experiments.8 Ben-
zophenone-based cross-linking agents function via diradi-
cal intermediates that are not quenched by solvent
(5) (a) Das, N. P.; Allen, C. M. Biochem. Biophys. Res. Commun.
1991, 181, 729-735. (b) Omer, C. A.; Kral, A. M.; Diehl, R. E.;
Prendergast, G. C.; Powers, S.; Allen, C. M.; Gibbs, J . B.; Kohl, N. E.
Biochemistry 1993, 32, 5167-5176. (c) Yokoyama, K.; McGeady, P.;
Gelb, M. H. Biochemistry 1995, 34, 1344-1354. (d) Bukhtiyarov, Y.
E.; Omer, C. A.; Allen, C. M. J . Biol. Chem. 1995, 270, 19035-19040.
(6) (a) Baba, T.; Allen, C. M. Biochemistry 1984, 23, 1312-1322.
(b) Baba, T.; Muth, J .; Allen, C. M. J . Biol. Chem. 1985, 260, 10467-
10473.
X Abstract published in Advance ACS Abstracts, October 1, 1996.
(1) Chowdhry, V.; Westheimer, F. H. Ann. Rev. Biochem. 1979, 48,
293-325.
(2) Burnner, J . Ann. Rev. Biochem. 1993, 62, 483-514. (b) Fleming,
S. A. Tetrahedron 1995, 51, 12479-12520.
(3) (a) Goldstein, J .; Brown, M. S. Nature 1990, 343, 425-430. (b)
Sinensky, M.; Lutz, R. J . BioEssays 1992, 14, 25-31.
(4) (a) Buss, J . E.; Marsters, J . C. Chem. Biol. 1995, 2, 787-791.
(b) Gibbs, J . B.; Oliff, A.; Kohl, N. E. Cell 1994, 77, 175-178. (c)
Tamanoi, F. TIBS 1993, 18, 349-353.
(7) Chowdhry, V.; Vaughan, R.; Westheimer, F. H. Proc. Natl. Acad.
Sci. U.S.A. 1976, 73, 1406-1408.
(8) Dorman, G.; Prestwich, G. D. Biochemistry 1994, 33, 5661-5673.
S0022-3263(96)00273-3 CCC: $12.00 © 1996 American Chemical Society

Photoactive Analogs of Farnesyl Pyrophosphate
J . Org. Chem., Vol. 61, No. 22, 1996 7739
Sch em e 1a
a
Reaction conditions: (a) (ClCH2CO)2O, pyridine, DMAP, DMF; (b) tert-butyl hydroperoxide, H2SeO3, salicylic acid, CH2Cl2; (c)
p-benzoylbenzoyl chloride (6a ) or m-benzoylbenzoyl chloride (6b), pyridine; (d) NH3, MeOH, H2O; (e) N-chlorosuccinamide, Me2S, CH2Cl2;
(f) [(n-Bu)4N]3HP2O7, CH3CN.
groups including tert-butyldimethylsilyl and tert-butyl-
diphenylsilyl were also employed, but proved to be less
stable later in the syntheses. The resulting chloroacetate
was then oxidized with tert-butyl hydroperoxide and
catalytic H2SeO3 to yield 5.6 The E-stereoselectivity for
the hydroxylation reaction was confirmed by the disap-
pearance of the C-8 methyl group in the 13C NMR
spectrum of 5.10 To attach photoactive benzophenone
moieties to the C10 isoprenoid unit, 5 was acylated with
4-benzoylbenzoyl chloride and 3-benzoylbenzoyl chloride
to yield 6a and 6b, respectively. The chloroacetate
protecting groups of 6a and 6b were then selectively
F igu r e 2. Superposition of FPP and GPP (bold) on 3b (dashed
hydrolyzed in the presence of the benzoylbenzoates with
lines). Double bonds are not shown for clarity.
a NH3/MeOH/H2O mixture producing the free alcohols
7a and 7b, which are stable compounds that can be
stored at -20 °C indefinitely. Finally, 7a and 7b were
molecules and are hence highly efficient. Furthermore,
they are activated by irradiation at longer wavelengths
converted to their corresponding pyrophosphates by
where protein damage is less likely. Taking advantage
chlorination with N-chlorosuccinamide and dimethyl
of these features, Coleman and co-workers recently used
sulfide followed by displacement of the allylic chlorides
peptides incorporating benzophenones to study the prenyl
with tris(tetra-n-butylammonium) hydrogen pyrophos-
group acceptor binding site in PFTase.9 In this paper,
the syntheses of two FPP analogs, 3a and 3b, are
presented together with inhibition kinetics and photoin-
activation experiments with PFTase. The results clearly
indicate that these compounds are useful photoaffinity
labeling analogs for studying PFTase and other enzymes
that employ FPP as a substrate.
phate as described by Poulter and co-workers.11 Diphos-
phates 3a and 3b were purified by reversed-phase
chromatography and characterized by 1H NMR, 31P NMR,
and FAB mass spectrometry.
P r od u ct Stu d ies. To determine whether compounds
3a and 3b were substrates or inhibitors for PFTase, a
continuous fluorescence assay using the peptide N-
dansyl-GCVIA (9) was first employed. This assay, de-
Resu lts a n d Discu ssion
veloped by Pompliano et al. for the human PFTase and
adapted by Poulter and co-workers for the yeast PFTase,
monitors the time-dependent increase in the fluorescence
Design a n d Syn th esis. The design of 3a and 3b as
analogs for FPP originated from our observation of
of the dansyl group due to the local increase in hydro-
significant overlap in a comparison of the structures of
phobicity as the adjacent cysteine residue is isoprenyl-
FPP and benzophenone. This complementarity is il-
lustrated in Figure 2 where FPP is superimposed on 3b.
Also shown is a superposition of GGPP and 3b; com-
ated;12 under the assay conditions an approximately 10-
fold increase in the dansyl group fluorescence occurs upon
farnesylation of 9 to 10. In contrast, incubation of 3a or
pounds 3a and 3b may be useful analogs of this longer
3b with PFTase and 9 resulted in no discernible change
isoprenoid. The syntheses of compounds 3a and 3b were
in fluorescence suggesting that these compounds are not
each accomplished in six steps starting from commer-
cially available geraniol as illustrated in Scheme 1. The
hydroxyl group of geraniol was first protected by esteri-
fication with chloroacetic anhydride; other protecting
(10) Umbreit, M. A.; Sharpless, K. B. J . Am. Chem. Soc. 1977, 99,
5526-5527.
(11) Davisson, V. J .; Woodside, A. B.; Poulter, C. D. Methods
Enzymol. 1985, 110, 130-144.
(12) (a) Bond, P. D.; Dolence, J . M.; Poulter, C. D. Methods Enzymol.
1995, 250, 30-43. (b) Pompliano, D. L.; Gomez, R. P.; Anthony, N. J .
J . Am. Chem. Soc. 1992, 114, 7945-7946.
(9) Ying, W.; Sepp-Lorenzino, L.; Cai, K.; Aloise, P.; Coleman, P. S.
J . Biol. Chem. 1994, 269, 470-477.

7740 J . Org. Chem., Vol. 61, No. 22, 1996
Gaon et al.
F igu r e 3. Structures of expected products of enzymatic
prenylation of 9 by 3a and 3b.
enzyme substrates. To study this question in greater
detail, the expected products of enzymatic prenylation
of 9 with 3a and 3b were prepared by chemical synthesis
using the method of Brown et al.13 and purified by
reversed-phase HPLC to yield 11a and 11b shown in
Figure 3. Interestingly, the fluorescence intensities for
11a and 11b are 4-6-fold less than those for 9, which
suggests that the benzoylbenzoates present in 11a and
11b quench the dansyl group fluorescence and that a
time dependent decrease in the fluorescence should occur
if 11a and 11b are PFTase substrates; as noted above,
no such effect was observed. The possibility that 3a and
3b were substrates was also investigated by HPLC
analysis using authentic 10, 11a , and 11b as standards.
A large-scale enzyme reaction using 1 and 9 as substrates
resulted in the conversion of 9 to 10 in quantitative yield
(data not shown). In contrast, HPLC analysis of a
reaction mixture containing 9 and 3a did not result in
the formation of significant amounts of 11a (Figure 4A).
A similar result was obtained with 3b (Figure 4B).
HPLC analysis with fluorescence detection of the same
reaction mixtures did allow the detection of small amounts
of products in both reactions (Figure 5A,B). In both
cases, the yields of products formed were less than 1%.
Since these large-scale reactions were performed with 18-
fold greater amounts of enzyme and were allowed to
proceed for 6-fold longer times than would be necessary
to quantitatively convert a reaction containing 1 and 9
to 10, it can be estimated that 3a and 3b react 10 000-
fold slower than the natural substrate, 1, and are hence
not efficient substrates for PFTase.
F igu r e 4. HPLC analysis of reactions containing PFTase, 9,
and 3a or 3b. Panel A: chromatogram A is from a 10 mL
reaction mixture containing 9 and 3a . Chromatogram B is from
a sample of chemically synthesized 11a . The amount of 11a
injected is equal to the amount of product that would be formed
in the enzymatic reaction assuming 100% conversion. Panel
B: chromatogram A is from
a 10 mL reaction mixture
containing 9 and 3b. Chromatogram B is from a sample of
chemically synthesized 11b. The amount of 11b injected is
equal to the amount of product that would be formed in the
enzymatic reaction assuming 100% conversion.
with competitive inhibition with respect to the substrate,
FPP. The rate data were further analyzed by the method
of Eadie-Hoftsee to determine Ki values for each inhibitor.
Compound 3a yields a value of 910 nM, while 3b gives a
value of 380 nM. Comparison of these data with the KD
value of 75 nM obtained for FPP by Dolence et al.
indicates that 3a and 3b bind effectively to PFTase; the
presence of the benzophenone moieties in 3a and 3b
results in a 5-12-fold decrease in binding affinity for
PFTase when compared to the natural ligand, 1.14
P h otolysis Kin etics. Compounds 3a and 3b were
tested for their ability to inactivate PFTase upon UV
irradiation. These experiments were performed by ir-
radiating mixtures of the inhibitors and the enzyme,
withdrawing aliquots at regular intervals, and assaying
the resulting samples for residual activity. The results
of these experiments are summarized in Figure 7A (3a )
and B (3b). Irradiation of PFTase alone for up to 12 h
resulted in no decrease in enzyme activity. In contrast,
irradiation for 2 h in the presence of 3a or 3b at
saturating concentrations led to a 12% decrease in
enzyme activity with 3a and a 9% decrease with 3b. This
inactivation could be partially reversed by the addition
In h ibition Kin etics. To evaluate their potential as
enzyme inhibitors, the rate of PFTase-catalyzed farne-
sylation of N-dansyl-GCVIA was measured in the pres-
ence of fixed concentrations of 3a and 3b at various
concentrations of FPP. Double reciprocal plots of these
data for 3a (Figure 6A) and 3b (Figure 6B) both give
patterns of lines that intersect on the 1/v axis, consistent
(13) Brown, M. J .; Milano, P. D.; Lever, D. C.; Epstein, W. W.;
Poulter, C. D. J . Am. Chem. Soc. 1991, 113, 3176-3177.
(14) Dolence, J . M.; Cassidy, P. B.; Mathis, J . R.; Poulter, C. D.
Biochemistry 1995, 34, 16687-16694.

Photoactive Analogs of Farnesyl Pyrophosphate
J . Org. Chem., Vol. 61, No. 22, 1996 7741
F igu r e 5. HPLC analysis employing fluorescence detection
of reactions containing PFTase, 9, and 3a or 3b. Panel A:
chromatogram A is from a 10 mL reaction mixture containing
9 and 3a . Chromatogram B is from a sample of chemically
synthesized 11a . The amount of 11a injected is equal to the
amount of product that would be formed in the enzymatic
reaction assuming 0.5% conversion. Panel B: chromatogram
A is from a 10 mL reaction mixture containing 9 and 3b.
Chromatogram B is from a sample of chemically synthesized
11b. The amount of 11b injected is equal to the amount of
product that would be formed in the enzymatic reaction
assuming 0.5% conversion.
F igu r e 7. Time course for the photoinactivation of PFTase
by 3a and 3b. Irradiation of PFTase alone (E), in the presence
of 3a (E + I), or in the presence of 3a and FPP (E + I + S).
Panel A: inhibition by 3a . Panel B: inhibition by 3b.
observed with 3a and 4% inactivation with 3b. Greater
levels of inactivation were achieved with longer incuba-
tion times. Photolysis of PFTase in the presence of 3a
resulted in 21% inactivation after 6 h and 44% inactiva-
tion after 12 h. Similar results were obtained with 3b.
It should be noted that no inactivation occurred when
PFTase was incubated with 3a or 3b in the dark and
that inactivation ceased when irradiation of reaction
mixtures was stopped. These experiments indicate that
3a and 3b are true photoaffinity labeling reagents and
that no long-lived reactive intermediates are involved in
the inactivation process.
P h otolysis Rea ction s w ith [32P ]3a a n d [32P ]3b. To
determine the sites of cross-linking of PFTase with 3a
and 3b, the radiolabeled analogs [32P]3a and [32P]3b were
prepared and purified using a modification of the proce-
dure developed by Bukhtiyarov.5d Photolysis of PFTase
in the presence of [32P]3a resulted in preferential labeling
of the â subunit (Figure 8A, lane 3). Similar results were
obtained with [32P]3b (Figure 8B, lane 3). Addition of
substrate, 1, to photolysis reactions containing[32P]3a or
[32P]3b resulted in substantial protection from labeling
(Figure 8A, lane 4, and Figure 8B, lane 4). To quantitate
the relative labeling efficiencies for each reaction, phos-
phorimaging analysis was employed (Figure 9). Using
[32P]3a , the â subunit was labeled 3.7-fold more than the
R subunit. With [32P]3b, the â subunit was labeled 4.2-
fold more than the R subunit. Inclusion of substrate 1
in photolysis reactions containing PFTase and [32P]3a
resulted in a 4.5-fold decrease in â subunit labeling and
a 3.1-fold decrease in R subunit labeling. Similar experi-
F igu r e 6. Double reciprocal plots showing the competitive
inhibition of PFTase by 3a and 3b. The concentrations of 3a
(or 3b) are as follows: (O) 0 µM, (b) 1.0 µM, (0) 1.7 µM, (9)
2.0 µM, (4) 2.7 µM, (2) 3.4 µM. Inset: replot of slopes from
double reciprocal plot versus [3a ] (or [3b]). Panel A: inhibition
by 3a . Panel B: inhibition by 3b.
of substrate, FPP; in the case of reactions containing 3a
or 3b and FPP (100 µM), only 3% inactivation was

7742 J . Org. Chem., Vol. 61, No. 22, 1996
Gaon et al.
yeast PFTase is consistant with results obtained with
human PFTase using other photoprobes.5 Collectively,
these results implicate the â subunit in prenyl group
recognition and suggest that benzophenone-based pho-
toprobes may allow specific amino acid residues in the
prenyl group binding site to be identified. These experi-
ments are currently in progress.
Sta bility Stu d ies. Although the 32P-labeled com-
pounds employed here did allow the prenyl group binding
subunit to be identified, these allylic pyrophosphates and
their cross-linked products are not stable to the condi-
tions normally used to separate cross-linked peptides. For
peptide mapping, the radiolabel must be incorporated at
a position that is stable to the purification conditions;
consequently, the stability of the ester linkage in 7b was
evaluated. Incubation of 7b in CD3CN/D2O (9:1) contain-
ing 0.2% CF3CO2D for 2 weeks at 37 °C resulted in no
significant cleavage of the benzoylbenzoate ester linkage
1
as determined by H NMR. Since these conditions are
more severe than those typically used in the HPLC
analysis of tryptic digests of proteins that have been
subjected to photocross-linking, these results suggest that
radioactive labels can be incorporated into either the
benzophenone or the isoprenoid portions of 3a and 3b
without risking the loss of label during peptide analysis.
Con clu sion s
Compounds 3a and 3b are analogs of FPP that are
competitive inhibitors of PFTase with Ki values in the
submicromolar range. Upon photolysis, they inactivate
PFTase in a time-dependent manner, resulting in up to
44% inactivation of enzyme activity. Photolysis of PFTase
in the presence of [32P]3a or of [32P]3b resulted in
preferential labeling of the â subunit, suggesting that this
subunit is involved in prenyl group recognition. These
compounds should be valuable tools for studying enzymes
that utilize FPP as a substrate.
F igu r e 8. Analysis of photolabeling of PFTase by [32P]3a and
[32P]3b by SDS-polyacrylamide gel electrophoresis. Lanes 1
and 3 contain samples of PFTase irradiated in the presence
of [32P]3a (or [32P]3b), Lanes 2 and 4 contain samples of
PFTase irradiated in the presence of [32P]3a (or [32P]3b) and
substrate 1. Lanes 1 and 2 show the silver-stained proteins
and lanes 3 and 4 show the radiolabeled proteins. Panel A:
labeling with [32P]3a . Panel B: labeling with [32P]3b. The
lighter intensity of the R subunit compared to the â subunit
is an artifact of silver staining. In gels stained with coomassie
blue, both bands appear with equal intensities.
Exp er im en ta l Section
Gen er a l P r oced u r es. All reactions were conducted under
dry nitrogen and stirred magnetically. Reaction temperatures
refer to external bath temperatures. Analytical TLC was
performed on precoated (0.25 mm) silica gel 60F-254 plates
purchased from E. Merck. Visualization was done under UV
irradiation or by subjecting the plates to ethanolic phospho-
molybdic acid solutions followed by heating. Flash chroma-
tography silica gel (60-120 mesh) was obtained from E. M.
Science. CH2Cl2, CH3CN, and pyridine were distilled from
CaH2, and Et2O was distilled from sodium/benzophenone ketyl.
DMF was dried with 3 Å molecular sieves. Melting points
were recorded in open capillaries and are uncorrected. NMR
J values are given in Hz. Combustion analyses were per-
formed by M-H-W Laboratories, Phoenix, AZ. UV spectra were
obtained using a Hewlett-Packard 8452A spectrophotometer,
and fluorescence measurements were performed with a Perkin-
Elmer LS 50B luminescence spectrometer. HPLC analysis
was carried out using a Beckman Model 127/166 instrument
equiped with a diode array UV detector and a Beckman 6300A
fluorescence detector (305-395 nm excitation filter and 480-
520 nm emission filter). Preparative HPLC separations were
performed with a Rainin Dynamax Microsorb C18 column (2.14
F igu r e 9. Phosphorimaging analysis of photolabeling of R and
â subunits of PFTase by [32P]3a and [32P]3b. Column 3a:
PFTase irradiated in the presence of [32P]3a . Column 3a/1:
PFTase irradiated in the presence of [32P]3a and 1. Column
3b: PFTase irradiated in the presence of [32P]3b. Column 3b/
1: PFTase irradiated in the presence of [32P]3b and 1.
× 25 cm with
a 5 cm guard column), while analytical
separations employed a Phenomenex Luna C18 column (5 µm,
4.6 × 250 mm). Phosphorimaging analysis was performed
with a Moleular Dynamics 445 SI Phosphorimager. Cell line
YL1/2 (ECACC # 92092402) was obtained from the European
Collection of Animal Cell Cultures. Fetal bovine serum was
obtained from Hyclone, and DMEM/F-12 media and Protein-
Free Hybridoma Media were obtained from Gibco/BRL. Gam-
ments with [32P]3b yielded a 6.3-fold decrease in â
subunit labeling and a 4.0-fold decrease in R subunit
labeling. The preferential labeling of the â subunit of

Photoactive Analogs of Farnesyl Pyrophosphate
J . Org. Chem., Vol. 61, No. 22, 1996 7743
maBind Plus Sepharose resin, DE-52 resin, and Dowex 50W-
X8 resin were obtained from Pharmacia, Whatman, and
BioRad, respectively. Sep-Pak columns were obtained from
Waters. Asp-Phe and N-dansylglycine were purchased from
Sigma. [32P]H3PO4 (specific activity 8500-9120 Ci/mmol) was
obtained from DuPont NEN. E. coli DH5R/pGP114 was a
generous gift from Dr. C. D. Poulter, Department of Chemistry,
University of Utah.
6.8), 2.25-2.08 (4H, m), 1.73 (3H, s), 1.66 (3H, s); 13C NMR
(52.3 MHz, CDCl3) δ 196.3, 166.0, 141.6, 138.9, 137.2, 133.8,
133.2, 130.4, 130.0, 129.8, 129.7, 128.7, 124.3, 71.3, 59.6, 39.1,
26.2, 16.5, 14.3; HR-EI MS calcd for C24H26O4 [M]+ 378.1824,
found 378.1816. Anal. Calcd for C24H26O4: C, 76.21; H, 6.87.
Found: C, 76.13; H, 7.05.
(E,E)-8-O-(4-Ben zoylben zoyl)-3,7-d im eth yl-2,6-octa d i-
en e 1-Ch lor id e (8a ). N-Chlorosuccinamide (0.147 g 1.1
mmol) was dissolved in CH2Cl2 (4.5 mL) and cooled to -30
°C. Dimethyl sulfide (74 mg, 1.2 mmol) was added dropwise,
and the solution was then cooled to -40 °C. Compound 7a
(0.378 g, 1.0 mmol) dissolved in 1 mL of CH2Cl2 was slowly
added, and the reaction mixture was allowed to warm to 0 °C
over 1 h. After being stirred at 0 °C for an additional 15 min,
the mixture was poured into a separatory funnel containing
cold saturated NaCl solution (2.5 mL) and pentane (2.0 mL).
After extraction, the pentane layer was removed and retained,
and the aqueous layer was extracted two additional times with
pentane (2 mL). The organic fractions were then combined,
washed twice with cold saturated aqueous NaCl (10 mL each
time), dried over Na2SO4, filtered, and evaporated to yield the
allylic chloride as a pale yellow oil (190 mg, 48%): Rf 0.71,
(silica gel, toluene/EtOAc, 10:1, v/v); 1H NMR (200 MHz,
CDCl3) δ 8.15 (2H, d, J ) 8.0), 7.83 (2H, d, J ) 8.0), 7.79 (2H,
d, J ) 8.2), 7.64-7.45 (3H, m), 5.56-5.41 (2H, m), 4.86 (1H,
s), 4.07 (2H, d, J ) 7.8), 2.17-2.15 (4H, m), 1.7 (6H, s); 13C
NMR (52.3 MHz, CDCl3) δ 196.3, 165.8, 142.1, 141.5, 137.0,
133.6, 133.0, 130.6, 130.2, 129.9, 129.6, 129.1, 128.6, 121.0,
3,7-Dim eth yl-1-(ch lor oa cetoxy)-2,6-octa n d ien -8-ol (5).
To a solution of geraniol, 4 (1.54 g, 10 mmol), in DMF (10 mL)
was added chloroacetic anhydride (2.57 g, 15 mmol), pyridine
(2 mL), and DMAP (0.122 g, 1 mmol), and the resulting
solution was stirred at rt for 30 min. The solvents were
removed in vacuo, and the resulting oily residue was dissolved
in Et2O, washed with 1 M NaHCO3, dried with Na2SO4, and
filtered. The organic layer was then concentrated and purified
by flash chromatography on silica gel (hexanes/toluene, 2:1,
v/v), which afforded geranyl chloroacetate (2.26 g, 98%) as a
1
colorless oil: Rf 0.1 (silica gel, hexanes/toluene (2:1, v/v); H
NMR (200 MHz, CDCl3) δ 5.32 (1H, t), 5.04 (1H, t), 4.67 (2H,
d, J ) 7.2), 4.02 (2H, s), 2.13-1.97 (4H, m), 1.69 (3H, s), 1.65
(3H, s), l.57 (3H, s); 13C NMR (52.3 MHz, CDCl3) δ 167.5, 143.8,
132.1, 123.9, 117.7, 63.2, 41.2, 39.8, 26.5, 25.9, 17.9, 16.7; HR-
CI MS calcd for C12H23ClNO2 [M + NH4]+ 248.1417, found
248.1411.
Geranyl chloracetate (2.30 g, 10 mmol) and tert-butyl
hydroperoxide (4.0 mL, 36 mmol) were stirred in the presence
of H2SeO3 (26 mg, 0.2 mmol) and salicylic acid (140 mg, 1
mmol) in CH2Cl2 (10 mL) for 20 h at rt. The CH2Cl2 was
removed under reduced pressure, and the tert-butyl-hydro-
peroxide was removed by repeated (3×) addition of toluene
and evaporation. The residue was dissolved in Et2O, washed
with 1 M NaHCO3 to remove H2SeO3, dried with Na2SO4, and
filtered. The organic layer was concentrated and the crude
product purified by flash chromatography (toluene/EtOAc, 10:
1, v/v) to yield 5 as a colorless oil (1.11 g, 48%): Rf 0.26 (silica
71.0, 41.0, 38.9, 26.0, 16.2, 14.2; HR-EI MS calcd for C24H25
ClO3 [M] 396.1493, found 396.1489.
-
+
(E,E)-8-O-(4-Ben zoylben zoyl)-3,7-d im eth yl-2,6-octa d i-
en e1-Dip h osp h a te (3a ). The allylic chloride 8a (41 mg, 0.103
mmol) was pyrophosphorylated with [(n-Bu)4N]3HP2O7 (196
mg, 0.218 mmol) in anhydrous CH3CN (0.492 mL) for 3 h at
rt. The product was converted to the NH4+ salt using an ion-
exchange column (Dowex 50W-X8, NH4+ form) in 25 mM NH4-
HCO3/2-propanol (49:1, v/v), and the salt was obtained by
lyophilization. The final product was purified employing a C18
reversed-phase column (Sep-pak cartridge) in a 25 mM NH4-
HCO3/CH3CN solvent system. After lyophilization, 3a (30 mg,
50% yield) was obtained as a white powder: 1H NMR (300
MHz, D2O, adjusted to pH 8 with ND4OD) δ 8.03 (2H, d, J )
8.4), 7.73 (2H, d, J ) 8.4), 7.68 (1H, d, J ) 8.7), 7.58 (2H, d, J
) 7.2), 7.43 (2H, t, J ) 7.5), 5.50 (1H, t, J ) 5.7), 5.33 (1H, t,
J ) 7.5), 4.73 (2H, s), 4.31 (2H t, , J ) 7.5), 2.10 (2H, m), 1.99
(2H, m), 1.61 (3H, s), 1.57 (3H, s); 31P NMR (121.4 MHz, D2O,
adjusted to pH 8 with ND4OD) δ -6.84 (1P, d, J ) 23), -10.87
1
gel, toluene/EtOAc, 10:1, v/v); H NMR (200 MHz, CDCl3) δ
5.32 (2H, t, J ) 6.9), 4,67 (2H, d, J ) 7.2), 4.03 (2H, s), 3.95
(2H, s), 2.18-2.01 (4H, m), 1.69 (3H, s), l.63 (3H, s); 13C NMR
(52.3 MHz, CDCl3) δ 167.6, 143.3, 135.6, 125.1, 117.9, 68.8,
63.2, 41.1, 39.2, 25.8, 16.6, 13.8; HR-CI MS calcd for C12H23
-
ClNO3 [M + NH4]+ 264.1366, found 264.1363. Anal. Calcd
for C12H19O3Cl: C, 58.45; H, 7.70. Found: C, 58.26, H, 7.59.
(E,E)-8-O-(4-Ben zoylb en zoyl)-1-(ch lor oa cet oxy)-3,7-
d im eth yl-2,6-octa d ien e (6a ). Compound 5 (492 mg, 2.0
mmol) was acylated with 4-benzoylbenzoyl chloride (700 mg,
2.8 mmol) in pyridine (2.5 mL) at rt for 6 h. The reaction
mixture was then filtered, and the pyridine was removed under
reduced pressure. Toluene was added to the resulting residue,
and the mixture was filtered to remove excess 4-benzoylben-
zoyl chloride. The solution was concentrated, dissolved in
toluene/EtOAc (10:1, v/v), and purified by flash chromatogra-
phy using the same solvent. Evaporation of the solvent gave
6a as a light yellow oil (349 mg, 38%): Rf 0.58 (silica gel,
(1P, d, J ) 23); UV (H2O), λmax ) 262 nm, ꢀ ) 18 600 M-1cm-1
;
FAB MS calcd for C24H27O10P2 [M - H]- 537.0, found 537.1,
calcd for C24H26O10P2Na [M - 2H + Na]- 559.0, found 559.0;
HR-FAB MS calcd for C24H28O10P2Na [M + Na]+ 561.1048,
found 561.1088.
(E,E)-8-O-(3-Ben zoylb en zoyl)-1-(ch lor oa cet oxy)-3,7-
d im eth yl-2,6-octa d ien e (6b). Compound 6b was prepared
and purified using the procedures described for the synthesis
of 6a . After chromatography, 6b was obtained as a clear oil
1
toluene/EtOAc, 10:1, v/v); H NMR (200 MHz, CDCl3) δ 8.11
(2H, d, J ) 8.2), 7.78 (2H, d, J ) 8.2), 7.74 (2H, d, J ) 6.9),
7.56 - 7.39 (3H, m), 5.49 (1H, t, J ) 6.00), 5.32 (1H, t, J )
7.2), 4.67 (2H, d, J ) 7.2), 4.63 (2H, s), 4.00 (2H, s), 2.19-2.07
(4H, m), 1.70 (3H, s), 1.68 (3H, s); 13C NMR (52.3 MHz, CDCl3)
δ 196.0, 167.4, 165.8, 143.0, 141.5, 137.2, 133.7, 133.1, 130.7,
130.3, 130.0, 129.7, 129.2, 128.7, 118.2, 71.1, 63.1, 41.2, 39.0,
26.1, 16.7, 14.3; HR-EI MS calcd for C26H27ClO5 [M]+ 454.1547,
found 454.1561. Anal. Calcd for C26H27ClO5: C, 68.67; H,
5.94. Found: C, 68.78; H, 5.79.
1
(38%): Rf 0.50 (silica gel, toluene/EtOAc, 10:1, v/v); H NMR
(200 MHz, CDCl3) δ 8.43 (1H, s), 8.25 (1H, d, J ) 8.0), 7.97
(1H, d, J ) 8.0), 7.79 (1H, s), 7.75 (1H, s), 7.60-7.25 (4H, m),
5.49 (1H, t), 5.34 (1H, t), 4.69 (2H, d, J ) 6.0), 4.65 (2H, s),
4.03 (2H, s), 2.18-2.07 (4H, m), 1.78 (6H, s); 13C NMR (52.3
MHz, CDCl3) δ 195.8, 167.4, 165.7, 143.0, 138.1, 134.1, 133.3,
132.9, 131.1, 130.2, 129.1, 128.7, 128.6, 128.4, 128.2, 127.4,
118.1, 70.9, 63.0, 41.1, 39.0, 26.0, 16.7, 14.2; HR-EI MS calcd
for C26H27ClO5 [M]+ 454.1547, found 454.1558. Anal. Calcd
for C26H27ClO5: C, 68.67; H, 5.94. Found: C, 68.49; H, 5.77.
(E,E)-8-O-(3-Ben zoylben zoyl)-3,7-dim eth yl-2,6-octadien -
1-ol (7b). The procedures described for the preparation of 7a
were used for the synthesis and purification of 7b. After
purification by flash chromatography, 7b was isolated as a
white crystalline solid (43%): mp 37-38 °C; Rf 0.13 (silica gel,
(E,E)-8-O-(4-Ben zoylben zoyl)-3,7-dim eth yl-2,6-octadien -
1-ol (7a ). Compound 6a (0.454 g, 1.0 mmol) was hydrolyzed
with 4.5 mL of 0.1 M NH4OH in aqueous methanol (90%
MeOH, v/v) at rt for 1 h. The methanol was removed under
reduced pressure, dissolved in Et2O, and dried over Na2SO4.
The crude product was then concentrated in vacuo, dissolved
in toluene, and purified by flash chromatography by elution
with toluene/EtOAc (10:1, v/v) to yield 7a as a pale yellow oil
(158 mg, 42%): Rf 0.16 (silica gel, toluene/EtOAc, 10:1, v/v);
1H NMR (200 MHz, CDCl3) δ 8.14 (2H, d, J ) 8.20), 7.82 (2H,
d, J ) 8.28), 7.79 (2H, d, J ) 8.2), 7.76-7.25 (3H, m) 5.53 (1H,
t, J ) 6.8), 5.40 (1H, t, J ) 6.9), 4.72 (2H, s), 4.13 (2H, d, J )
1
toluene/EtOAc, 10:1, v/v); H NMR (200 MHz, CDCl3) δ 8.44
(1H, s), 8.25 (1H, d, J ) 8.0), 7.97 (1H, d, J ) 8.0), 7.81 (1H,
s), 7.77 (1H, s), 7.64-7.44 (4H, m), 5.48 (1H, t), 5.40 (1H, t),
4.71 (2H, s), 4.13 (2H, d, J ) 6.8), 2.21-2.07 (4H, m), 1.70
(3H, s), 1.66 (3H, s); 13C NMR (52.3 MHz, CDCl3) δ 198.8,

7744 J . Org. Chem., Vol. 61, No. 22, 1996
Gaon et al.
165.7, 138.9, 138.1, 137.0, 134.2, 133.3, 132.9, 131.1, 131.0,
130.3, 130.2, 129.4, 128.7, 128.6, 124.2, 71.0, 59.4, 38.9, 26.0,
16.3, 14.2; HR-EI MS calcd for C24H26O4 [M]+ 378.1824, found
378.1831. Anal. Calcd for C24H26O4: C, 76.21; H, 6.87.
Found: C, 76.10; H, 6.98.
N-Da n syl-GC(4-BBG)VIA (11a ). Compound 11a was
prepared by reacting 8a with 9 in liquid ammonia as described
by Brown et al.13 The crude product was purified by reversed-
phase HPLC employing a H2O/CH3CN (containing 0.2% TFA)
gradient: FAB MS calcd for C55H70N6NaO11S2 [M + Na]+
1077.4, found 1077.5, calcd C55H69N6Na2O11S2 [M - H + 2Na]+
1099.4, found 1099.4; UV (H2O/CH3CN/TFA, 50/50/0.2) λmax
) 258 nm, ꢀ ) 21 600 M-1‚cm-1, λmax ) 320 nm, ꢀ ) 1780
M-1‚cm-1; fluorescence (H2O/CH3CN/TFA, 50/50/0.2) λex ) 322
nm, λem ) 530 nm, intensity ) 0.21 (relative to N-dansylgly-
cine, 1.0).
N-Da n syl-GC(3-BBG)VIA (11b). Compound 11b was
prepared from 8b and 9 as described for 11a : FAB MS calcd
for C55H70N6NaO11S2 [M + Na]+ 1077.4, found 1077.6, calcd
C55H69N6Na2O11S2 [M - H + 2Na]+ 1099.4, found 1099.6, calcd
C55H69N6O11S2 [M - H]- 1053.4, found 1053.6; UV (H20/CH3-
CN/TFA, 50/50/0.2) λmax ) 254 nm, ꢀ ) 18 100 M-1‚cm-1, λmax
) 320 nm, ꢀ ) 1780 M-1‚cm-1; fluorescence (H2O/CH3CN/TFA,
50/50/0.2) λex ) 322 nm, λem ) 530 nm, intensity ) 0.29
(relative to N-dansylglycine, 1.0).
(E,E)-8-O-(3-Ben zoylben zoyl)-3,7-d im eth yl-2,6-octa d i-
en e 1-Ch lor id e (8b). Compound 8b was prepared from 7b
using the procedure outlined for the synthesis of 8a . The
desired chloride (8b) was obtained as a pale yellow oil (52%):
Rf 0.66 (silica gel, toluene/EtOAc, 10:1, v/v); 1H NMR (200
MHz, CDCl3) δ 8.44 (1H, s), 8.26 (1H, d, J ) 8.0), 7.98 (1H, d,
J ) 7.8), 7.81 (1H, s), 7.78 (1H, s), 7.77-7.38 (4H, m), 5.53-
5.40 (2H, m), 4.71 (2H, s), 4.07 (2H, d, J ) 8.0), 2.21-2.10
(4H, m), 1.72 (6H, s); 13C NMR (52.3 MHz, CDCl3) δ 196.5,
165.7, 142.1, 138.1, 137.2, 134.0, 133.3, 132.9, 131.1, 130.6,
130.3, 130.2, 129.0, 128.7, 128.6, 120.9, 71.0, 41.1, 38.9, 26.0,
16.2, 14.2; HR-EI MS calcd for C24H25ClO3 [M]+ 396.1493,
found 396.1493.
(E,E)-8-O-(3-Ben zoylben zoyl)-3,7-d im eth yl-2,6-octa d i-
en e 1-Dip h osp h a te (3b). The allylic chloride 8b was pyro-
phosphorylated as described for the preparation of 3a to give
3b as a white powder (13 mg, 33%): 1H NMR (300 MHz, D2O,
adjusted to pH 8 with ND4OD) δ 8.18 (1H, s), 8.14 (1H, d, J )
7.8), 7.88 (1H, d, J ) 7.8), 7.66 (1H, m), 7.64 (1H, m), 7.55
(2H, m), 7.42 (2H, m), 5.42 (1H, m), 5.28 (1H, m), 4.78 (2H, s),
4.29 (2H, m), 2.04 (2H, m), 1.93 (2H, m), 1.54 (6H, s); 31P NMR
(121.4 MHz, D2O, adjusted to pH 8 with ND4OD) δ -6.92 (1P,
d, J ) 22), -10.84 (1P, d, J ) 23); UV (H2O), λmax ) 222 nm,
P r ep a r a tion of a n ti-r-Tu bu lin Im m u n oa ffin ity Col-
u m n . Cell line YL1/2 producing anti-R-tubulin was obtained
as a frozen sample, cultured in DMEM/F-12 medium contain-
ing 10% fetal bovine serum to allow the cells to recover from
cryopreservation. The production of anti-R-tubulin by the cells
was measured by ELISA using a horseradish peroxidase
conjugated rabbit anti-rat IgG secondary antibody and purified
R-tubulin as a standard (purified R-tubulin was obtained by
homogenization of a fresh bovine brain, fractionation by
ammonium sulfate precipitation, and chromatography using
DEAE Sephadex). The resulting cells, producing anti-R-
tubulin at approximately 4 µg/mL of media, were weaned from
fetal bovine serum-supplemented medium into protein-free
medium and cultured in large roller bottles. anti-R-Tubulin
was purified by ammonium sulfate fractionation of the culture
medium and dialysis, which yielded approximately 40 mg of
antibody per 400 mL of culture medium. For immunoaffinity
column preparation, purified anti-R-tubulin was dissolved in
binding buffer (10 mM sodium phosphate pH 7.0, 150 mM
NaCl, 10 mM EDTA) and applied to a 1.5 × 10 cm column
containing 5 mL of GammaBind Plus Sepharose resin. The
column was washed with binding buffer, and the amount of
bound antibody was quantitated by comparing the concentra-
tion of the applied solution with the concentration of antibody
in the wash fractions; approximately 24 mg of antibody
remained bound.
P u r ifica t ion of Yea st P r ot ein F a r n esylt r a n sfer a se.
PFTase was purified by a modification of the procedure
described by Mayer et al.16 A 50 mL overnight culture of E.
coli DH5R/pGP114 cells grown in LB media was used to
innoculate 4 L of SB media (32 g of Bactotryptone, 20 g of yeast
extract, 5 g of NaCl, 5 mL of 1 M NaOH) containing ampicillin
(100 µg/mL). The large culture was grown to an OD600 of 0.48,
induced with IPTG (0.2 mM), grown for an additional 12 h,
harvested by centrifugation, flash frozen in N2 (l), and stored
at -80 °C. Approximately 65 g of wet cells were obtained.
For purification, 3.0 g of cells were thawed, suspended in 80
mL of disruption buffer (50 mM Tris‚HCl pH 7.0, 5 mM
2-mercaptoethanol, 1 mM PMSF), and pulse-sonnicated for 2
min. The cell lysate was clarified by centrifugation (23 000g
for 15 min) and dialyzed against disruption buffer (2 × 2 L)
without PMSF and the resulting solution applied to a DE 52
anion exchange column (3 × 30 cm) equilibrated with buffer
A (50 mM Tris‚HCl, pH 7.0, 5 mM MgCl2, 50 µM ZnCl2, 10
mM 2-mercaptoethanol). The column was eluted with a
stepwise gradient consisting of 100 mL aliquots of buffer A
supplemented with 0 mM, 100 mM, 200 mM, and 1 M NaCl.
PFTase eluted from the column in the 200 mM NaCl wash as
determined by the kinetic assay described below. Active
fractions were pooled (25 mL total) and applied to an anti-R-
tubulin immunoaffinity column (described above) that was
equilibrated with buffer B (10 mM sodium phosphate pH 7.0,
150 mM NaCl, 10 mM EDTA, 10 mM 2-mercaptoethanol). The
ꢀ ) 39,100 M-1‚cm-1, λmax ) 258 nm, ꢀ ) 24 100 M-1‚cm-1
;
FAB MS calcd for C24H29O10P2 [M + H]+ 539.0, found 539.2,
calcd for C24H28O10P2Na [M + Na]+ 561.0, found 561.1, calcd
for C24H27O10P2 [M - H]- 537.0, found 537.1.
(E,E)-[r,â(n )32P ]-8-O-(4-Ben zoylben zoyl)-3,7-d im eth yl-
2,6-octa d ien e 1-Dip h osp h a te ([32P ]3a ). Alcohol 7a (2.4 mg,
6.3 µmol) was reacted with anhydrous [32P]H3PO4 (1.2 mg, 13
µmol) in CH3CN (200 µL) containing 20% (v/v) CCl3CN and
triethylamine (2.5 mg, 26 µmol) for 2 h. The volatile compo-
nents from the reaction were then evaporated, and the
resulting residue was purified using a reversed-phase Sep-
pak C18 cartridge and a NH4HCO3/CH3CN step gradient to
yield [32P]3a in 3.3% yield (specific activity 480 Ci/mol); the
concentration of solutions containing [32P]3a were determined
by UV using the wavelengths and extinction coefficients
reported for 3a . The radiochemical purity of [32P]3a was
assessed by thin layer chromatography in 2-propanol/NH4OH/
H2O (6:3:1, v/v/v) followed by phosphorimaging analysis and
was found to be 50%. [32P]3a was further purified by prepara-
tive thin layer chromatography using the above solvent system
to yield material whose radiochemical purity was greater than
90%. Non-radioactive 3a prepared using this procedure and
analyzed by HPLC coeluted with 3a prepared via the chloride.
(E,E)-[r,â(n )32P ]-8-O-(3-Ben zoylben zoyl)-3,7-d im eth yl-
2,6-octa d ien e 1-Dip h osp h a te ([32P ]3b). Compound [32P]3b
was prepared as described for [32P]3a . After reversed-phase
chromatography, [32P]3b was obtained in 1.0% yield (specific
activity 700 Ci/mol) and 56% radiochemical purity. Further
purification by preparative thin layer chromatography yielded
[32P]3b, whose radiochemical purity was greater than 90%.
N-Da n syl-GCVIA (9). N-Dansyl-GCVIA was synthesized
by solid-phase methods using standard protocols for R-N-Boc-
protected amino acids, beginning with N-Boc-Ala Merrifield
resin and employing a p-methoxybenzyl protecting group for
cysteine.15 Dansylation was accomplished by treating the
solid-phase resin with a solution of dansyl chloride in CH2Cl2
in the presence of TEA. The completed peptide was depro-
tected and cleaved from the resin with HF under standard
conditions and purified by reversed-phase HPLC employing a
H2O/CH3CN (containing 0.2% TFA) gradient: FAB MS calcd
for C31H45N6O8S2 [M - H]- 693.3, found 693.4, calcd for
C31H47N6O8S2 [M + H]+ 695.3, found 695.4; fluorescence (H2O/
CH3CN/TFA, 50/50/0.2) λex ) 322 nm, λem ) 530 nm, intensity
) 1.3 (relative to N-dansylglycine, 1.0).
(15) Barany, G.; Merrifield, R. B. In The Peptides; Academic Press:
New York, 1979; Vol. 2; pp 1-284.
(16) Mayer, M. P.; Prestwich, G. D.; Dolence, J . M.; Bond, P. D.;
Wu, H.-y.; Poulter, C. D. Gene 1993, 132, 41-47.

Photoactive Analogs of Farnesyl Pyrophosphate
J . Org. Chem., Vol. 61, No. 22, 1996 7745
column was washed with additional buffer B to remove
contaminating proteins until the A280 returned to base line
followed by elution with buffer B supplemented with 5.0 mM
Asp-Phe to afford purified PFTase (yield: 1.6 mg, specific
activity: 0.86 µmol‚min-1‚mg-1).
P h otolysis Kin etics. Photolysis reactions were conducted
at 4 °C in a UV Rayonet mini-reactor equiped with 8 RPR-
3500° lamps and a circulating platform that allows up to eight
samples to be irradiated simultaneously. All reactions (0.5-1
mL) were performed in silinized quartz test tubes (10 × 45
mm) and contained 52 mM Tris‚HCl, pH 7.0, 5.8 mM DTT, 12
mM MgCl2, 12 µM ZnCl2, 25 mM NH4HCO3, and 3.8 nM
PFTase. Where appropriate, reactions contained 3a (9.1 µM)
or 3b (3.8 µM); these concentrations were chosen to be 10 times
above their calculated Ki values. For substrate protection
experiments, FPP was added to a final concentration of 100
µM. Reactions were photolyzed for up to 12 h, during which
time duplicate samples (50 µL) were removed at various
intervals, placed on ice, and assayed for activity.
P h otolysis Rea ction s w ith [32P ]3a a n d [32P ]3b. All
reactions (100 µL) were performed in silinized quartz test tubes
(10 × 45 mm) and contained 52 mM Tris‚HCl, pH 7.0, 5.8 mM
DTT, 12 mM MgCl2, 12 µM ZnCl2, 25 mM NH4HCO3, and 380
nM PFTase. Each reaction contained [32P]3a (9.1 µM) or [32P]-
3b (3.8 µM); for substrate protection experiments, FPP was
added to a final concentration of 100 µM. Reactions were
photolyzed for 2 h using the apparatus described above.
Loading buffer was then added to each sample, and the
samples were heated to 70 °C for 5-10 min followed by
analysis by SDS-polyacrylamide gel electrophoresis with a 10%
Tris-tricine gel. Gels were silver stained, dried, and subjected
to autoradiography at -80 °C with an intensifying screen for
6-24 h. The relative intensities of photolabeled products were
determined by phosphorimaging analysis of the dried gels.
E n zym e Assa ys. Solutions containing N-dansyl-GCVIA
were prepared by dissolving the solid peptide in buffer (20 mM
Tris‚HCl, pH 7.0 and 10 mM EDTA), and their concentrations
determined by UV absorbance at 340 nm using N-dansylgly-
cine as a standard. Solutions of FPP were prepared by
dissolving the solid in 25 mM NH4HCO3 and their concentra-
tions determined by phosphate analysis as described by Reed
and Rilling employing KH2PO4 as a standard;17 solutions
containing 3a and 3b were prepared and standardized in the
same manner. PFTase was prepared for use by diluting
purified enzyme with buffer (52 mM Tris‚HCl, pH 7.0, 5.8 mM
DTT, 12 mM MgCl2, 12 µM ZnCl2) containing 1 mg/mL bovine
serum albumin. Enzyme assays contained 50 mM Tris‚HCl,
pH 7.0, 10 mM MgCl2, 10 µM ZnCl2, 5.0 mM DTT, 0.040% (w/
v) n-dodecyl-â-D-maltoside, 2.4 µM N-dansyl-GCVIA, PFTase,
FPP, and 3a or 3b, where appropriate, in a final reaction
volume of 500 µL. The assay mixtures were equilibrated to
30 °C, initiated by the addition of PFTase, and monitored
spectrofluorometrically (340 nm excitation and 505 nm emis-
sion) for 300 s. Initial velocities were obtained from linear
regression analysis of the time-dependent fluorescence emis-
sion data using the fluorimeter software.
P r od u ct Stu d ies. Large-scale reactions contained 50 mM
Tris‚HCl, pH 7.0, 10 mM MgCl2, 10 µM ZnCl2, 5.0 mM DTT,
2.4 µM N-dansyl-GCVIA, 45 nM PFTase, and FPP (10 µM),
3a (10 µM), or 3b (10 µM), where appropriate, in a final
reaction volume of 10 mL. The reactions were equilibrated to
30 °C, initiated by the addition of PFTase, and allowed to react
for 1 h. To desalt the samples, each reaction mixture was
applied to a Sep-Pak C18 cartridge, washed with H2O/CH3CN/
TFA (95:5:0.1, v/v/v), eluted with CH3CN/TFA (100:0.1, v/v),
concentrated in vacuo, dissolved in H2O/CH3CN/TFA (50:50:
0.2, v/v/v), and analyzed by HPLC using a C18 reversed-phase
column and a H2O/CH3CN/TFA gradient.
Sta bility Stu d ies. Compound 7b was dissolved in 0.5 mL
of CD3CN/D2O (9:1, v/v) containing 0.2% CD3CO2D. These
conditions were chosen to resemble the acidic conditions used
in the HPLC purification of peptide fragments that would
typically be used to identify photocross-linking sites. The
above solution was incubated at 4 °C for 8 days, rt for 20 days,
and finally 37 °C for 14 days. During this time the ratio of
the integrated areas for the C1 and C8 methylene protons was
1
monitored by H NMR as an indicator of the integrity of the
benzoylbenzoate ester. In the course of this experiment, no
significant cleavage was observed.
En zym e In h ibition Exp er im en ts. To determine if 3a
and 3b were competitive inhibitors of PFTase, a 4 × 6 grid of
duplicate assays were run in which the substrate, FPP, was
maintained at a set of fixed concentrations and the inhibitor
(3a or 3b) concentrations were varied at each FPP concentra-
tion. Concentrations of 1.0, 1.5, 3.0, and 7 µM for FPP and 0,
1.0, 1.7, 2.0, 2.7, and 3.4 µM for 3a and 3b were chosen (based
on IC50 experiments performed first, data not shown); the
PFTase concentration in these experiments was 2.5 nM. The
rates were determined from initial velocity measurements
performed as described above. Ki values were calculated from
Eadie-Hofstee plots obtained from a Macintosh PowerMac
7100 computer running KaleidaGraph (v. 3.0.1) software.
Ack n ow led gm en t. This research was supported by
funds from the American Cancer Society (Grant No. BE-
222 and IN-13-33-47).
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H- and
31P-NMR spectra of 3a and 3b (4 pages). This material is
contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
(17) Reed, B. C.; Rilling, H. C. Biochemistry 1976, 15, 3739-3745.
J O9602736