A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes

Article abstract of DOI:10.1016/j.bmc.2009.04.034

Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC₅₀ values of 5.8 (meta isomer) and 3.0 μM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K_I of 0.46 μM. Radiolabeled forms of both analogues selectively labeled the β-subunit of ScPFTase. The para isomer was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes.

Full text of DOI:10.1016/j.bmc.2009.04.034

Bioorganic & Medicinal Chemistry 17 (2009) 4797–4805
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A versatile photoactivatable probe designed to label the diphosphate
binding site of farnesyl diphosphate utilizing enzymes
Olivier Henry a, Fernando Lopez-Gallego b, Sean A. Agger b, Claudia Schmidt-Dannert b, Stephanie Sen c,
David Shintani d, Katrina Cornish e, Mark D. Distefano a,
*
a Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, MN 55455, United States
b Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108, United States
c Department of Chemistry, The College of New Jersey, PO Box 7718, Ewing, NJ 08628, United States
d Department of Biochemistry, University of Nevada, Reno, NV 89557, United States
e Yulex Corporation, 37860 W. Smith-Enke Road, Maricopa, AZ 85238, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive
analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide
groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ
FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that
links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moi-
ety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of
the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an
FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones
were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPF-
Received 13 January 2009
Revised 1 April 2009
Accepted 9 April 2009
Available online 22 April 2009
Keywords:
Benzophenone
Farnesyltransferase
Farnesyl diphosphate synthase
Sesquiterpene cyclase
Photoaffinity labeling
Tase) with IC50 values of 5.8 (meta isomer) and 3.0
l
M (para isomer); the more potent analogue, the para
M.
isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a KI of 0.46
l
Radiolabeled forms of both analogues selectively labeled the b-subunit of ScPFTase. The para isomer
was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster far-
nesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a ses-
quiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled
the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demon-
strate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
itself. While such molecules have proved to be useful, they are lim-
ited by the fact that they are only capable of labeling residues lo-
Farnesyl diphosphate (FPP, 1) is a substrate for a large number
of enzymes found in nature. The products of such enzymes include
sesquiterpene natural products,1,2 side-chains of cofactors,3 bio-
polymers such as latex4 and lipidated proteins.5 Given the vast
number of natural products derived from FPP and the medicinally
important properties of many of them, the development of new
probes that can be used to identify FPP-utilizing enzymes is an
important area of inquiry.6 Photoactive analogues of isoprenoid
diphosphates containing either benzophenone,7–16 diazotrifluoro-
propionate,17–23 or azide24,25 groups have been useful for studying
both the enzymes that synthesize FPP as well as those that employ
FPP as a substrate such as protein prenyltransferases. To date, most
photoaffinity labeling analogues of FPP (see Fig. 1, 2 and 3) incor-
porate the light-absorbing functionality into the isoprenoid moiety
cated within the isoprenoid binding site. Moreover, in cases
where the overall size and shape of the active site is highly con-
strained, probes that incorporate bulky modifications in the prenyl
group may not effectively bind. Those limitations led us to design a
new type of FPP analogue (4a and 4b) that would incorporate an
unmodified farnesyl group linked to a diphosphate mimic contain-
ing a photoactive benzophenone moiety. Here we describe the syn-
theses of these two analogues and evaluate their ability to bind and
label several different FPP utilizing enzymes.
2. Results
2.1. Synthesis of farnesyl diphosphate analogue incorporating a
benzophenone photophore into the diphosphate group
The isomeric photoactive analogues 4a and 4b were prepared
using the route summarized in Scheme 1. For the preparation of
* Corresponding author. Tel.: +1 612 624 0544; fax: +1 612 626 7541.
E-mail address: diste001@umn.edu (M.D. Distefano).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.034

4798
O. Henry et al. / Bioorg. Med. Chem. 17 (2009) 4797–4805
O
P
O
P
carbonyldiimidazole in DMF and the purified product (4a) was ob-
tained in 15% yield after ion exchange chromatography followed by
reversed-phase chromatography. Compound 4a was characterized
by 1H NMR, 31P NMR, HR-ESI-MS, and UV spectroscopy. The iso-
meric probe 4b was obtained from an analogous route beginning
with commercially available 4-bromo-methyl-benzophenone
(6b). For the photoaffinity labeling experiments described below,
it was necessary to prepare 4a and 4b in radiolabeled form. In ear-
lier work, we developed a simple method for the preparation of
32P-labeled FPP and related analogues;26,27 a variation of that
method was employed here, shown in Scheme 2, to obtain the de-
sired labeled probes.
O-
O
O
O— O—
1 (FPP)
O
P
O
P
O
O—
O—
O
O
O
O— O—
2
O
O
N2
O
P
O
P
O
F3C
O
O— O—
3
O
P
O
P
First, 32P-labeled farnesyl monophosphate ([32P]9) was synthe-
sized through the reaction of farnesol (10) with [32P]H3PO4 using
CCl3CN as the coupling agent. The radiolabeled product ([32P]9)
was purified using a disposable Sep-Pak reversed-phase column
and then coupled with 8a or 8b in the presence of CDI in DMF to
yield 4a or 4b, respectively. The radiolabeled probes were again
purified using a disposable Sep-Pak reversed-phase column and
their radiochemical purities (>80%) were assessed by TLC followed
by phosphorimaging analysis.
O
O
O
O— O—
4a
O
O
O
P
O
P
O
O— O—
4b
Figure 1. Farnesyl diphosphate (FPP) and photoactive analogues.
2.2. Enzymatic studies with protein farnesyltransferase using
4a and 4b
the meta isomer 4a, commercially available 3-methyl-benzophe-
none 5a was brominated in presence of NBS in CCl4 at reflux to
yield 3-bromo-methyl-benzophenone (6a). The crude material
was then treated in neat trimethylphosphite at reflux to produce
the desired diester 7a in good yield (85% from 5a) followed by
hydrolysis under acidic conditions to afford the unprotected phos-
phonate 8a in 49% yield. Coupling of 8a to farnesyl monophosphate
(9) was achieved through the reaction of the two components with
The utility of the new probes was first evaluated in experiments
with purified Saccharomyces cerevisiae protein farnesyltransferase
(ScPFTase) since that enzyme has been extensively characterized
via structural and enzymological studies.28–32 Importantly, its sub-
strate specificity has been thoroughly investigated including a
large number of isoprenoid diphosphate analogs that have been
examined as inhibitors and alternative substrates.10,11,15,17,33–38
R2
R1
R2
R2
R1
O
P
OCH3
Br
R1
OCH3
NBS
AIBN
CCl4
P(OCH3)3
HCl
Dioxane
5a R1 = PhCO,
7a R1 = PhCO,
6a R1 = PhCO,
R2 = H; crude
6b R1 = H,
Δ
R2 = H
R2 = H; 85%
7b R1 = H,
R2 = PhCO; 90%
R2 = PhCO
R2
R1
R2
R1
O
P
O
O
P
O
O
P
OH
O
O
OH
Farnesyl
phosphate (9), 4a R1 = PhCO,
8a R1 = PhCO,
R2 = H; 49%
8b R1 = H,
2 = H; 15%
4b R1 = H,
R2 = PhCO; 17%
CDI, DMF
R
R2 = PhCO, 49%
Scheme 1. Synthesis of photoaffinity analogs 4a and 4b.
O
P
O
OH
*
O
O
[
32P]H3PO4,
Farnesol (10)
[
32P]9
CCl3CN, Et3N.
CH3CN
R2
R1
[
32P]4a R1 = PhCO,
R2 = H
O
P
O
O
*
P
O
O
NH4
[
32P]4b R1 = H,
R2 = PhCO
O
8a or 8b
CDI, DMF
NH4
Scheme 2. Synthesis of 32P-labeled forms of 4a and 4b. The position of the 32P radiolabel is indicated with a (*).

O. Henry et al. / Bioorg. Med. Chem. 17 (2009) 4797–4805
4799
Compounds 4a and 4b were first analyzed as substrates for
ScPFTase using a continuous fluorescence assay employing the
pentapeptide N-dansyl-GCVIA as the prenyl group acceptor. This
assay monitors the increase in fluorescence of the dansyl group
upon prenylation at the neighboring cysteine residue caused by
the change in hydrophobicity as previously described.39,40 No
observable change in the fluorescence of the pentapeptide was
detected in reactions containing 4a or 4b in place of FPP at concen-
To evaluate their potential as enzyme inhibitors, the rate of
ScPFTase-catalyzed farnesylation of N-dansyl-GCVIA was mea-
sured at a fixed concentration of FPP and varying concentrations
of 4a and 4b. In those reactions, the presence of 4a or 4b attenu-
ated the rate of peptide prenylation, indicating that these com-
pounds are enzyme inhibitors. Plots of enzymatic rate versus
concentration of inhibitor gave IC50 values (tabulated in Table 1)
of 5.8 lM for 4a and 3.0 lM for 4b. To gain additional understand-
trations up to 200
substrates for ScPFTase.
l
M, which suggests that these molecules are not
ing of the mechanism of ScPFTase inhibition by these compounds,
the more potent inhibitor, 4b, was studied in greater detail. The
rate of ScPFTase-catalyzed prenylation of N-dansyl-GCVIA was
measured in the presence of fixed concentrations of the isoprenoid
substrate, FPP, and varying concentrations of 4b. Double reciprocal
plots of those data gave a pattern of lines that intersect on the 1/V
axis, consistent with competitive inhibition with respect to the
substrate FPP (see Fig. 2); this result is consistent with FPP and
4b binding at the same site. Further analysis by the method of
Table 1
IC50 and KI values for inhibition of PFTase with photoactive FPP analogues
Compound
IC50
—
(l
M)
KI (lM)
0.075a
1 (FPP)
2b
3.4
0.72
5.8
3.0
0.61
0.20
N.D.d
0.48
Eadie–Hofstee gave a KI value of 0.48 l
M for ScPFTase.41,42
3c
We next evaluated 32P-labeled forms of 4a and 4b for their abil-
ity to label ScPFTase upon photolysis. In those experiments, each
analogue was photolyzed in the presence of purified ScPFTase
and the resulting reaction mixture was then fractionated by SDS–
PAGE. The total protein present was visualized by staining with
Coomassie Blue and the labeled protein was detected via phos-
phorimaging analysis. Representative photolabeling results using
4a
4b
a
KD from Dolence et al.30
b
c
From Turek et al.11
From Edelstein and Distefano.17
Not determined.
d
[
[
32P]4b are shown in Figure 3. Photolysis of ScPFTase with
32P]4b led to strong labeling of the b-subunit of the enzyme with
16
14
12
10
8
minimal labeling of the a-subunit (Fig. 3, lane 2). The labeling was
almost completely abolished when the photolysis was performed
in the presence of both [32P]4b and excess FPP (competition exper-
iment, Fig. 3, lane 3); no labeling occurred in the absence of irradi-
ation (Fig. 3, lane 1). Similar results were obtained in experiments
with [32P]4a (data not shown).
6
2.3. Labeling studies of farnesyl diphosphate synthases
(FPPSase) using 4b
4
2
As noted above, a number of studies have reported the success-
ful labeling of protein prenyltransferases using photoactive ana-
logues. In contrast, there are relatively few reports of such
probes being used to label FPPSases involved in the biosynthesis
of isoprenoids. Accordingly, we examined the ability of 4b to label
FPPSases from two different sources. First [32P]4b was photolyzed
in the presence of Escherichia coli FPPSase IspA (EcFPPSase)43 under
0
0
0.2 0.4 0.6 0.8
1
1.2
1/[FPP] (1/uM)
Figure 2. Double reciprocal plot showing the competitive inhibition of ScPFTase by
4b. The concentrations of 4b used were as follows: (d) 0 M, (s) 0.2 M, (j)
0.4 M, (h) 0.7 M, (N) 1.0 M, ( ) 1.5 M, (.) 2.0 M.
l
l
l
l
l
D
l
l
Figure 3. Phosphorimaging analysis of photolabeling of ScPFTase with [32P]4b after
SDS–PAGE fractionation. Lane 1: ScPFTase and [32P]4b (29
M), no UV irradiation.
Lane 2: ScPFTase and [32P]4b (29 M), with UV irradiation. Lane 3: PFTase, [32P]4b
(29 M), and FPP (100 M), with UV irradiation.
Figure 4. Phosphorimaging analysis of photolabeling of EcFPPSase with [32P]4b
l
after SDS–PAGE separation. Lane 1: EcFPPSase and
irradiation. Lane 2: EcFPPSase and [32P]4b (29
M), with UV irradiation. Lane 3:
EcFPPSase, [32P]4b (29
M), and FPP (100 M), with UV irradiation.
[ lM), no UV
32P]4b (29
l
l
l
l
l
l

4800
O. Henry et al. / Bioorg. Med. Chem. 17 (2009) 4797–4805
Figure 5. Phosphorimaging analysis of photolabeling of DmFPPSase with [32P]4b
after SDS–PAGE separation. Lane 1: DmFPPSase and [32P]4b (29
M), with UV
M), with UV
l
irradiation. Lane 2: DmFPPSase,
irradiation.
[ lM), and FPP (100 l
32P]4b (29
conditions similar to those used for PFTase described above; re-
sults from those experiments are shown in Figure 4. Photolysis of
EcFPPSase with [32P]4b led to strong labeling of the purified en-
zyme (Fig. 4, lane 2) which was substantially (although not com-
pletely) reduced when the photolysis was performed in the
presence of both [32P]4b and FPP (competition experiment, Fig. 4,
lane 3); no labeling occurred in the absence of irradiation (Fig. 4,
lane 1).
To further examine the utility of probe 4b, labeling experiments
were carried out using DmFPPSase,44 an insect-derived enzyme
from Drosophila melanogaster. Photolysis of purified DmFPPSase
with [32P]4b resulted in labeling of the enzyme (Fig. 5, lane 1);
no labeling was observed without irradiation (data not shown).
When photolysis was performed in the presence of both [32P]4b
and FPP as a competitor (Fig. 5, lane 2), a significant decrease
(46% of the labeling obtained in the absence of the competitor) in
the amount of labeling was observed.
Figure 6. Analysis of the product formed from incubation of 4b with NoSTSase.
Above: Gas chromatographic analysis of reaction. Below: MS the of product peak
(retention time = 11.5 min). Note: It has been previously established that the
product of the cyclization reaction (germacrene A) undergoes thermal rearrange-
ment to b-elemene at the temperature of the GC injection port.45
2.4. Experiments using 4b with a sesquiterpene synthase
Another major class of important FPP-utilizing enzymes is the
sesquiterpene synthases. To evaluate the utility of the diphos-
phate-directed photoprobes developed here to interact with this
class of enzymes, experiments were performed using a sesquiter-
pene synthase from Nostoc sp. strain PCC7120, (NoSTSase);45 that
particular enzyme produces germacrene A from the cyclization of
FPP. Initially, it was assumed that the phosphonophosphate func-
tionality present in 4b would not be processed by NoSTSase (as
was the case with ScPFTase) and that the compound would
hence serve as an inhibitor. Interestingly, addition of 4b to reac-
tions containing purified NoSTSase and FPP did not result in a
decrease in the cyclized product indicating that 4b was not an
inhibitor of the enzyme. Instead, incubation of NoSTSase and
4b alone (without FPP) led to the formation of the same product
(see Fig. 6) obtained using FPP as a substrate, thus demonstrating
that 4b is a bona fide substrate for the enzyme, although the rate
of product formation was approximately 10-fold slower than
with FPP. Based on these promising results, [32P]4b was also
examined for its ability to label the enzyme. Photolysis of NoSTS-
ase with [32P]4b resulted in labeling of the enzyme (see Fig. 7,
lane 1) and significant protection was obtained when FPP was in-
cluded in the photolysis reaction mixture (Fig. 7, lane 2) similar
to that observed with the other enzymes reported here.
Figure 7. Phosphorimaging analysis of photolabeling of NoSTSase with [32P]4b
after SDS–PAGE separation. Lane 1: NoSTSase and
[
32P]4b (29
M), with UV
M), and FPP (100 lM), with UV
l
irradiation. Lane 2: NoSTSase,
irradiation.
[
32P]4b (29
l
2.5. Modeling of 4b in the active sites of FPP-utilizing enzymes
In the design of 4a and 4b, a phosphonate was used to link the
benzophenone unit to the farnesyl phosphate moiety. An impor-
tant result of this is the removal of one oxygen atom relative to far-
nesyl diphosphate. Given that a number of interactions between
the diphosphate group and residues from the protein typically oc-
cur in the active sites of FPP utilizing enzymes, it was decided to
model 4b in the active sites of three of the enzymes employed in
this study. Since no crystal structure ScPFTase exists, the structure
for RnPFTase (Rattus norvegicus) was used instead.46 For that mod-
el the benzophenone phosphonate moiety was appended onto the

O. Henry et al. / Bioorg. Med. Chem. 17 (2009) 4797–4805
4801
farnesyl diphosphate group present in the active site of the binary
(RnPFTaseÁFPP) complex and a conformational search was per-
formed to identify to the lowest energy conformations. The struc-
ture surrounding the diphosphate of FPP from the RnPFTaseÁFPP
complex is shown in Figure 8 (Top Left) with the corresponding re-
gion from the RnPFTaseÁ4b complex (Top Right). Many of the inter-
actions between the distal phosphate (from FPP) and the protein
appear to be retained in the modeled structure of the RnPFTaseÁ4b
aseÁFPP complex is shown in Figure 8 (Middle Left) with the corre-
sponding region from the EcFPPSaseÁ4b complex (Middle Right).
Residues that interact with the distal phosphate (from FPP) and
the phosphonate (from 4b) include K66 R117 and K258; however,
in the case of R117, it should be noted that due to the absence of
one oxygen atom (due to the phosphonate substitution), one
interaction between the terminal guanidino nitrogen and FPP is
missing with 4b along with a longer range interaction with the
internal guanidino nitrogen. Since no crystal structure of the
NoSTSase employed here was available, the structure of a related
enzyme, StSTSase (pentalenene synthase from Streptomyces sp.
UC5319)48 was used; mutants of that enzyme produce germac-
rene.49,50 In this case, modeling was carried out using a docking
complex; that list includes interactions with K164a, H248b, and
Y300b. However, interactions with R291b and K294b are not pres-
ent due to the absence of the one missing oxygen atom. A similar
approach was used to create a model for EcFPP synthase.47 The
structure surrounding the diphosphate of FPP from the EcFPPS-
Figure 8. Modeling of 4b in the active sites of FPP-utilizing enzymes. Top Left: Crystal structure of NrPFTase (1JCR) with bound FPP. Shown, are residues H248b, R291b, K294b,
Y300b, K164 (colored by element; carbon in grey), FPP (colored by element; carbon in green) and Zn (cyan). The yellow dashes illustrate potential hydrogen bonding or
a
electrostatic interactions (within 4 Å) of the distal phosphate. Top Right: Crystal structure of NrPFTase with 4b built on the bound FPP. The color scheme is the same as that for
Top Left. Middle Left: Crystal structure of EcFPPSase (1RQl) with FPP built by modifying the existing bound IPP. Shown, are residues K66, R116, R117, K258 (colored by
element; carbon in grey), FPP (colored by element; carbon in green), and Mg (purple). The yellow dashes illustrate potential hydrogen bonding or electrostatic interactions
(within 4 Å) of the distal phosphate. Middle Right: Crystal structure of EcFPPSase with 4b built on the bound IPP. The color scheme is the same as that for Middle Left. Bottom
Left: Crystal structure of StSTSase (Pentalenene synthase, 1PS1) with FPP docked in the active site of the enzyme. Shown, are residues R173, N219 (colored by element; carbon
in grey), FPP (colored by element; carbon in green), and Mg (purple). The yellow dashes illustrate potential hydrogen bonding or electrostatic interactions (within 4 Å) of the
distal phosphate. Bottom Right: Crystal structure of StSTSase with 4b docked in the active site of the enzyme. The color scheme is the same as that for Bottom Left.

4802
O. Henry et al. / Bioorg. Med. Chem. 17 (2009) 4797–4805
algorithm. The structure surrounding the diphosphate of FPP from
the StSTSaseÁFPP complex is shown in Figure 8 (Bottom Left) with
the corresponding region from the StSTSaseÁ4b complex (Bottom
Right). In the case of StSTSase, most of the interactions between
the diphosphate group and the enzyme are mediated by two Mg2+
cations; all of these interactions are preserved in the StSTSaseÁ4b
model. However, several interactions between the distal phosphate
and R173 are not present in the StSTSaseÁ4b complex.
their reduced affinity, 4a and 4b still bind with significant avidity.
The KI using 4b with ScPFTase (0.48
M) is only sixfold higher than
the KD (0.075 M) for FPP binding to ScPFTase. Interestingly, the
decrease affinity for ScPFTase caused by the phosphonate substitu-
tion is comparable to that resulting from modest modification of
the isoprenoid itself (see Table 1). These results suggest that phos-
phonate substitutions may be useful for the design of other probes
designed to target FPP-utilizing enzymes.
l
l
3. Discussion
4. Experimental
4.1. General
To create a probe that would be capable of labeling residues in
proximity to the diphosphate binding site of enzymes that employ
FPP as a substrate, two photoactive molecules, 4a and 4b, were
prepared in five (4a) or four steps (4b) from farnesyl monophos-
phate (9) and suitably functionalized phosphonate precursors.
These compounds were competitive inhibitors of ScPFTase with re-
All reagents were obtained from Aldrich. Analytical TLC was
performed on precoated (0.25 mm) silica gel 60F-254 plates pur-
chased from E. Merck. Flash chromatography silica gel (60–120
mesh) was obtained from E. M. Science. HPLC analysis was carried
out using a Beckman Model 127/166 instrument equipped with a
diode array UV detector. Analytical separations employed a Rainin
Dynamax-60 ŠC18 column (4.6 mm  25 cm with a 2 cm guard
column). GC/MS analysis was carried out on a Varian 3800 gas
chromatograph linked to an ion-trap mass spectrometer employ-
spect to FPP with IC50 values less than 6
fests a KI of 0.48 M. In general, those values are comparable to
lM; analogue 4b mani-
l
those obtained earlier with photoactive FPP analogues such as 2
and 3 (see Table 1) that incorporate the photophore into the
isoprenoid moiety itself. Thus it appears that this new class of iso-
prenoid probes retain significant affinity for their cognate binding
sites despite modification of one of the diphosphate oxygen atoms.
Since the last step in the synthesis is the coupling of farnesyl
monophosphate to a benzophenone phosphonate fragment, the
preparation of radiolabeled forms of the probes can be easily
accomplished from commercially available terpenoid precursors;
accordingly, we prepared 32P-labeled forms of 4a and 4b and eval-
uated their ability to label ScPFTase. Both of these probes undergo
crosslinking reactions with ScPFTase upon photolysis at 350 nm
resulting in predominant labeling of the b-subunit of this heterodi-
meric enzyme; that labeling is abolished when photolysis is per-
formed in the presence of the natural ligand, FPP. These results
are consistent with the isoprenoid binding site being composed
of residues primarily located on the b-subunit as evidenced from
numerous X-ray crystal structures containing bound isoprenoid
analogues.10,11,37,51,52 Overall, these results demonstrate that 4a
and 4b bind to an FPP-utilizing enzyme in a predictable fashion
similar to results obtained with other photoprobes. Moreover,
ing
(30 m  0.25 mm inner diameter, 1.5
(g) as a carrier. Solid phase micro extraction (SPME) fibers
(100 m polydimethylsiloxane fiber) were obtained from Supelco.
Phosphorimaging analysis was performed with Molecular
a
HP-1ms (Agilent Tech. Inc.) capillary column
l
m film thickness) with He
l
a
Dynamics 445 SI Phosphorimager. Dowex 50W-X8 resin was ob-
tained from Bio-Rad and Sep-Pak columns were purchased from
Waters. [32P]H3PO4 (specific activity 8500-9120 Ci/mmol) was pur-
chased from DuPont NEN. N-dansyl-GCVIA was synthesized by Dr.
Dan Mullen in the Department of Chemistry, University of Minne-
sota. The enzymes used in this study were purified as previously
described for yeast PFTase,15 EcFPPSase,43 DmFPPSase,44 and
NpSTSase.45
4.2. Chemical synthesis
4.2.1. 3-Bromomethyl-benzophenone (6a)
To
a
solution of 3-methyl-benzophenone (5a, 100 mg,
the absence of crosslinking to the
a-subunit indicates that these
0.51 mmol) in CCl4 (7.0 mL) was added N-bromosuccinimide
(NBS, 102 mg, 0.57 mmol) and azobisisobutyronitrile (AIBN,
0.52 mg, 0.02 mmol). The mixture was allowed to react at reflux
for 24 h. The solvent was evaporated under vacuum and the crude
material was used in the next reaction without purification. 1H
NMR (300 MHz, CDCl3) d = 4.52 (s, 2H), 7.42–7.52 (m, 3H), 7.57–
7.63 (m, 2H), 7.69–7.73 (ddd, 1H, J = 1.5, 1.5, 7.5 Hz) 7.78–7.84
(m, 3H). 13C NMR (75 MHz, CDCl3 d = 32.7, 128.5, 128.9, 130.1,
130.5, 132.7, 133.0, 137.3, 138.2, 138.3, 196.0. HRMS (ESI) (m/z):
[M+Na]+ calcd for C14H11BrONa 296.9891 and 298.9870, found
296.9876 and 298.9861.
probes manifest selectivity in their photoaffinity labeling reactions.
Having established this, we then examined their interactions with
several other types of enzymes that use FPP as a substrate or gen-
erate it as a product. FPPSases from E. coli and D. melanogaster are
both labeled by 4b upon photolysis. Since FPPSases from insect
sources are involved in the biosynthesis of certain pheromones
and juvenile hormones, these probes should be useful in functional
studies of such proteins. Compound 4b also labels a sesquiterpene
synthase from Nostoc sp. strain PCC7120 upon photolysis and inter-
estingly, this probe is an alternative substrate for that enzyme. Gi-
ven the large number of sesquiterpene synthases present in nature,
these new probes should be highly useful for identifying sesquiter-
pene synthases from new sources. They may also be helpful in ana-
lyzing FPP binding to previously uncharacterized enzymes and for
mapping their active sites. We are currently employing these mol-
ecules to assist in the identification of FPP-utilizing enzymes from
a variety of sources. Finally, we note that molecular modeling of
the complexes between various FPP-utilizing enzymes and 4b sug-
gests that these enzymes are able to interact with the phosphonate
present in 4b in agreement with the studies reported here. How-
ever, the substitution of carbon in place of oxygen (compare the
distal phosphate in FPP with the phosphonate in 4b) does elimi-
nate some specific interactions between the probe and the various
enzymes that results in decreased affinity. Significantly, despite
4.2.2. (3-Benzoyl-benzyl)-phosphonic acid dimethyl ester (7a)
A solution of crude 3-bromo-methyl-benzophenone (crude 6a,
estimated to be 0.51 mmol, prepared as described above) was dis-
solved in P(OCH3)3 (5.0 mL) and refluxed for 12 h. Excess trimethyl
P(OCH3)3 was evaporated under vacuum and the resulting crude
material was purified via silica gel chromatography (hexanes/
EtOAc, 1:1, v/v) to yield 131 mg (85% from 5a). 1H NMR
(300 MHz, CDCl3) d = 3.16 (d, 2H, J = 22 Hz), 3.62 (d, 6H,
J = 11 Hz), 7.32–7.40 (m, 4H), 7.46–7.51 (m, 1H), 7.66–7.71 (m,
4H). 13C NMR (75 MHz, CDCl3) d = 32.6 (d, J = 138 Hz), 52.9 (d,
J = 6.3 Hz), 128.3, 128.6 (d, J = 3.1 Hz), 128.7 (d. J = 3.7 Hz), 130.0,
131.2 (d, J = 6.8 Hz), 131.8 (d, J = 9.5 Hz), 132.5, 133.7 (d,

O. Henry et al. / Bioorg. Med. Chem. 17 (2009) 4797–4805
4803
J = 5.8 Hz), 137.4, 137.9 (d, J = 2.6 Hz), 196.2. 31P NMR (121 MHz,
CDCl3) d = 28.9.
J = 22.2 Hz), 7.46–7.53 (m, 4H), 7.60–7.65 (m, 1H), 7.70–7.74 (m,
4H). 13C NMR (75 MHz, CDCl3) d = 34.9 (d, J = 134 Hz), 128.3,
129.7, 129.8, 129.9, 130.2, 130.2, 132.6, 135.7, 137.4, 138.2 197.2.
31P NMR (121 MHz, CDCl3) d = 23.6. HRMS (ESI) (m/z): [MÀH]À
calcd for C14H12O4P 275.0479, found 275.0466.
4.2.3. (3-Benzoyl-benzyl)-phosphonic acid (8a)
A solution of dimethyl ester 7a (200 mg, 0.66 mmol) was re-
fluxed in an aqueous solution of 4 N HCl (7 mL) and dioxane
(7 mL) over 12 h. The reaction mixture was extracted with EtOAc
(20 mL, 4 times) and the resulting product (89 mg, 49%) was
deemed to be of sufficient purity to use in the next step without
additional purification. 1H NMR (300 MHz, CDCl3) d = 3.02 (d, 2H,
J = 21 Hz), 7.27–7.36 (m, 3H), 7.43–7.47 (m, 3H), 7.57 (s, 1H),
7.64 (d, 2H, J = 7.2 Hz). 13C NMR (75 MHz, CDCl3) d = 34.1 (d,
J = 134 Hz), 128.2, 130.0, 131.2 (d, J = 6.3 Hz), 132.7, 133.4 (d,
J = 9.0 Hz), 134.0 (d, J = 5.9 Hz), 137.1, 137.4, 197.4. 31P NMR
(121 MHz, CDCl3) d = 24.9. HRMS (ESI) (m/z): [MÀH]À calcd for
C14H12O4P 275.0479, found 275.0461.
4.2.7. 4-Benzoylbenzylphosphonic ((2E,6E)-3,7,11-trimethyl
dodeca-2,6,10-trienyl phosphoric) anhydride (4b)
The procedure for the synthesis of 4b was identical to that for
4a. Starting from 90 mg (0.33 mmol) of the phosphonic acid 8b,
33 mg (17% yield) of the desired product 4b was obtained. The
product eluted at 36 min (HPLC with same conditions than 4a).
1H NMR (300 MHz, CDCl3) d = 1.34 (s, 3H), 1.35 (s, 3H), 1.39 (s,
3H), 1.43 (s, 3H), 1.71–1.82 (m, 8H), 3.0 (d, 2H, J = 22.2 Hz), 4.11
(dd, 2H, J = 6.3 Hz), 4.83–4.84 (m, 2H), 5.11 (t, 1H, J = 6.6 Hz),
7.23–7.28 (m, 4H), 7.35–7.38 (m, 1H), 7.47–7.53 (m, 4H). 31P
NMR (121 MHz, CDCl3)
d
= À9.6 (d, J = 25.3 Hz), 12.1 (d,
4.2.4. 3-Benzoylbenzylphosphonic ((2E,6E)-3,7,11-trimethyl
dodeca-2,6,10-trienyl phosphoric) anhydride (4a)
J = 25.9 Hz). HRMS (ESI) (m/z): [MÀH]À calcd for C29H37O7P2
559.2020, found 559.2076. UV
e
= 17.8 mMÀ1 cmÀ1 at 272 nm.
Phosphonic acid 8a (90 mg, 0.33 mmol) and CDI (79 mg,
0.39 mmol) were dissolved in DMF (1.5 mL) and allowed to react
at room temperature under argon for 2 h, at which time farnesyl
phosphate (9, cyclohexylamine salt) was added (130 mg,
0.326 mmol). The mixture was allowed to react for 7 days with
stirring at room temperature. At that point, the reaction was
quenched with water (5 mL) and extracted with EtOAc (10 mL, 4
times). The combined organic layers were dried over Na2SO4, fil-
tered, and concentrated to afford yellow oil. It was then dissolved
in a solvent mixture composed of 50% CH3CN and 50% aqueous
25 mM NH4HCO3/isopropanol (50:1, v/v). This solution was ap-
plied to an ionic exchange column (Dowex 50X8-400,
2.6 Â 4.5 cm), previously washed with aqueous NH4OH and equil-
ibrated to pH 6.0 with aqueous 25 mM NH4HCO3/isopropanol
(50:1, v/v). The column was eluted with additional aqueous
25 mM NH4HCO3/isopropanol (50:1, v/v) and the fractions contain-
ing the desired compound were concentrated by lyopholization. Fi-
nal purification was accomplished via reversed-phase HPLC using
25 mM NH4HCO3 (solvent A) and CH3CN (solvent B) and the fol-
lowing elution profile performed at 5.0 mL/min: 5.0 min 0% solvent
B; linear gradient from 0% to 60% solvent B over 40 min. Under
these conditions the desired product eluted at 37 min. The solvent
mixture was removed by lyopholization to afford 29 mg (15%) of a
white solid. 1H NMR (500 MHz, CDCl3) d = 1.51 (s, 3H), 1.52 (s, 3H),
1.55 (s, 3H), 1.63 (s, 3H), 1.92–1.98 (m, 8H), 3.1 (d, 2H, J = 21.6 Hz),
4.22 (dd, 2H, J = 5.7 Hz), 5.01–5.03 (m, 2H), 5.22 (t, 1H, J = 6.5 Hz),
7.27–7.34 (m, 2H), 7.39–7.44 (m, 2H), 7.52–7.54 (m, 2H), 7.70–7.75
(m, 3H). 31P NMR (202 MHz, CDCl3) d = 11.5 (d, J = 23.1 Hz), À10.3
(d, J = 22.6 Hz). HRMS (ESI) (m/z): [MÀH]À calcd for C29H37O7P2
4.2.8. [32P]-4-Benzoylbenzylphosphonic ((2E,6E)-3,7,11-
trimethyldodeca-2,6,10-trienyl phosphoric) anhydride ([32P]4b)
The synthesis of [32P]4b accomplished in two steps. First, [32P]-
labeled farnesyl monophosphate was prepared via phosphoryla-
tion of farnesol using a procedure similar to that we have previ-
ously employed for the preparation of isoprenyl diphosphates.
Carrier free
H3PO4 (1.6 mg, 20
drous H3PO4 was added a solution of triethylamine (4.1 mg,
40 mol) in CH3CN (50 L). Farnesol (4.4 mg, 20 mol) in 200 lL
[
32P]-H3PO4 (5 mCi) was diluted with unlabeled
mol) and lyophilized. To the resulting anhy-
l
l
l
l
of CH3CN containing 20% of CCl3CN (v/v) was then added and the
reaction was allowed to stir under N2 for 2 h after which time
the volatile components were removed under a stream of N2. The
crude material was purified with a Sep-Pac reversed phase C18 col-
umn using a stepwise gradient of CH3CN and 25 mM NH4HCO3 and
the fractions containing
[
32P]9 (determined by TLC: i-PrOH/
NH4OH/H2O, 6:3:1 v/v/v, Rf = 0.62, visualized by phosphoroimag-
ing analysis) were pooled and lyophilized under vacuum. The ben-
zophenone phosphonic acid derivative (8b, 5.5 mg, 20
dissolved in anhydrous DMF (400 L), under Ar (g) at room tem-
perature. CDI (3.9 mg, 24 mol) was added and the resulting acti-
lmol) was
l
l
vation reaction was left stirring 2 h before it was added to the
lyophilized [32P]9. The reaction was allowed to react for 2 days at
which time the DMF was removed under a stream of N2 (g). The
crude material was purified with a Sep-Pak reversed phase C18 col-
umn using a stepwise gradient of CH3CN and 25 mM NH4HCO3. The
product eluted at 30% of CH3CN (specific activity 197 Ci/mol). The
radiochemical purity was assessed by TLC (i-PrOH/NH4OH/H2O,
6:3:1 v/v/v, Rf = 0.7, 81% of product) followed by phosphoroimag-
ing analysis.
559.2020, found 559.2042. UV
e
= 15.7 mMÀ1 cmÀ1 at 260 nm.
4.2.5. (4-Benzoyl-benzyl)-phosphonic acid dimethyl ester (7b)
The procedure employed here was identical to that used for the
preparation of 7a. Starting from 300 mg (1.1 mmol) of the bromide
6b, 298 mg (90% yield) of the desired diester product 7b was ob-
tained. 1H NMR (300 MHz, CDCl3) d = 3.16 (d, 2H, J = 22 Hz), 3.62
(d, 6H, J = 11 Hz), 7.32–7.40 (m, 4H) 7.46–7.51 (m, 1H) 7.66–7.71
(m, 4H). 13C NMR (75 MHz, CDCl3) d = 32.9 (d, J = 137 Hz), 52.9
(d, J = 6.9 Hz), 128.3, 129.7 (d, J = 6.8 Hz), 129.9, 130.4 (d,
J = 3.1 Hz), 132.4, 136.2 (d, J = 3.7 Hz), 136.4 (d, J = 9.5 Hz), 137.5,
196.1. 31P NMR (121 MHz, CDCl3) d = 28.2.
4.3. Biological experiments
4.3.1. Farnesyltransferase assays
IC50 values were determined for 4a and 4b with ScPFTase by
running duplicate assays with FPP and N-dansyl-GCVIA maintained
at 2.4
lM and 4a or 4b at concentrations of 0, 1, 2, 3, 5, 10 and
20 M. An enzyme concentration of 5.0 nM ScPFTase was em-
l
ployed. IC50 values were calculated from a direct fit of the rate ver-
sus inhibitor concentration data using KaleidaGraph software.
Based on those results, a more systematic study was performed
with 4b using a 4 Â 6 grid of duplicate assays with FPP maintained
4.2.6. (4-Benzoyl-benzyl)-phosphonic acid (8b)
The procedure employed here was identical to that used for the
preparation of 8a. Starting from 200 mg (0.66 mmol) of the diester
7b, 90 mg (49% yield) of the desired phosphonic acid product 8b
was obtained. 1H NMR (300 MHz, CDCl3) d = 3.24 (d, 2H,
at fixed concentrations (1, 2, 5, and 7.0
tions of 4b (0, 0.2, 0.4, 0.7, 1, 1.5 and 2
l
M) and varying concentra-
lM for 4b). In those exper-
iments, the concentration of ScPFTase was 5 nM, and the
concentration of N-Ds-GCVIA was fixed at 2.4 M. The rates were
l

4804
O. Henry et al. / Bioorg. Med. Chem. 17 (2009) 4797–4805
determined from the initial velocity measurements and KI value
Acknowledgment
calculated from an Eadie–Hofstee plot.41,42
This research was supported by the National Institutes of
Health Grant GM58442 (M.D.D.), the National Science Foundation
Grant DBI 0321690 (D.S.) and funds from Yulex Corp.
4.3.2. Photolabeling reactions
All photolysis reactions were conducted at 4 °C in a UV Rayonet
mini-reactor equipped with six RPR-3500 Å lamps and a circulat-
ing platform that allows up to eight samples to be irradiated simul-
Supplementary data
taneously. All reactions (100
quartz test tubes (10 Â 75 mm) and contained 52 mM TrisÁHCl,
pH 7.0, 5.8 mM DTT, 12 mM MgCl2, 12 M ZnCl2, 25 mM NH4HCO3
and the targeted enzyme at 50 g/mL. Where appropriate, reac-
tions contained [32P]4a (29 M) or [32P]4b (29
M); these concen-
lL) were performed in silinized
1H NMR spectra for 6a, 7a, 7b, 8a, 8b, 4a, and 4b, 31P NMR spec-
tra for 6a, 7a, 7b, 8a, 8b, 4a, and 4b, HR-ESI-MS spectra for 4a and
4b and HPLC chromatograms for 4a and 4b are included. Stereo
images of the models shown in Figure 8 are included along with
several tables that summarize important distances within the ac-
tive site of each enzyme model. Pymol files for each of the enzyme
models are also provided. Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.bmc.2009.04.034.
l
l
l
l
trations were chosen to be 10 times above their experimentally
measured KI values (for ScPFTase). For substrate protection exper-
iments, FPP was added to a final concentration of 100
tions were photolyzed for 6 h using the apparatus described
above. Loading buffer [50 L; 4.0% SDS, 12% glycerol (w/v),
lM. Reac-
l
50 mM TrisÁHCl, 2.0% 2-mercaptoethanol (v/v), 0.01% bromophenol
blue] was then added to each sample, and the samples were heated
to 70 °C for 20 min followed by analysis by SDS–polyacrylamide
gel electrophoresis using a 12% Tris–Tricine gel. Gels were first
stained with Comassie Blue or Sypro Orange to visualize the pro-
teins followed by phosphorimaging analysis to assess the extent
of crosslinking that was subsequently quantified via volume inte-
gration of the labeled protein bands (after subtraction of any back-
ground signal).
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