Synthesis, Properties, and applications of diazotrifluropropanoyl- containing photoactive analogs of farnesyl diphosphate containing modified linkages for enhanced stability

Article abstract of DOI:10.1111/j.1747-0285.2009.00914.x

Photoactive analogs of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogs that contain diazotrifluoropropanoyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analog (3) was synthesized in ³²P-labeled form from geraniol in seven steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [ ³²P]3 demonstrate that this compound labels the β-subunits of both farnesyltransferase and geranylgeranyltransferase (types 1 and 2). However, the amide-linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester-linked analog 4 was prepared in six steps from geraniol. Computational analysis and X-ray crystallographic studies suggest that 4 binds to protein farnesyl transferase (PFTase) in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase, and a ³²P-labeled form selectively photocrosslinks the β-subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene-producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [³²P]4 manifests significant selectivity and should hence be useful for identifying novel FPP-utilizing enzymes in crude protein preparations.

Full text of DOI:10.1111/j.1747-0285.2009.00914.x

ª 2009 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2009.00914.x
Chem Biol Drug Des 2010; 75: 51–67
Research Article
Synthesis, Properties, and Applications of
Diazotrifluropropanoyl-Containing Photoactive
Analogs of Farnesyl Diphosphate Containing
Modified Linkages for Enhanced Stability
substrate for PFTase, and a 32P-labeled form selec-
tively photocrosslinks the b-subunit of ScPFTase as
well as E. coli farnesyldiphosphate synthase and a
germacrene-producing sesquiterpene synthase
from Nostoc sp. strain PCC7120 (a cyanobacterial
source). Finally, nearly exclusive labeling of ScPF-
Tase in crude E. coli extract was observed, suggest-
ing that [32P]4 manifests significant selectivity and
should hence be useful for identifying novel FPP-
utilizing enzymes in crude protein preparations.
Marisa L. Hovlid1, Rebecca L. Edelstein1,
Olivier Henry1, Joshua Ochocki1, Amanda
DeGraw1, Stepan Lenevich1, Trista Talbot1,
Victor G. Young1, Alan W. Hruza2,
Fernando Lopez-Gallego3, Nicholas P.
Labello4, Corey L. Strickland2, Claudia
Schmidt-Dannert3 and Mark D.
Distefano1,*
1Department of Chemistry, University of Minnesota, Minneapolis,
MN 55455, USA
Key words: diazotrifluoropropanoyl, farnesyl diphosphate, germac-
rene synthase, photoaffinity labeling, prenyltransferase, protein prenyla-
tion, sesquiterpene synthase
2Department of Structural Chemistry, Schering-Plough Research
Institute, Kenilworth, NJ 0703, USA
3Department of Biochemistry, Molecular Biology and Biophysics,
University of Minnesota, St. Paul, MN 55108, USA
4Minnesota Supercomputing Institute, University of Minnesota,
Minneapolis, MN 55455, USA
Abbreviations: BSA, bovine serum albumin; CPK, bovine Corey-
Pauling-Koltun; DATFP, diazotrifluoropropanoyl; DEAD, diethyl azodicarb-
oxylate; DEPT, Distortionless Enhancement by Polarization Transfer; DFT,
Density functional theory; DTT, dithiothreitol; EcFPPSase, E. coli farnesyl
diphosphate synthase; EDTA, ethylenediaminetetraacetic acid; ESI-MS,
electrospray ionization mass spectrometry; ESI-TOF, High resolution time
of flight; EtOAc, Ethyl acetate; FAB-MS, fast atom bombardment mass
spectrometry; FPP, farnesyl diphosphate; FT-IR, Fourier transform infrared
spectrum; GGPP, geranylgeranyl diphosphate; HPLC, high performance
liquid chromatography; HR-ESI-MS, High resolution electrospray ioniza-
tion mass spectrometry; HR-FAB-MS, High resolution fast atom bom-
bardment mass spectrometry; HsPGGTase I, H. sapiens protein
geranylgeranyltransferase type 1; NMR, nuclear magnetic resonance;
NoSTSase, Nostoc sp. strain PCC7120 sesquiterpene synthase; PFTase,
protein farnesyl transferase; PPTS, pyridinium p-toluenesulfonate;
QM-MM, quantum mechanics molecular mechanics; RnPGGTase II,
R. norvegicus protein geranylgeranyltransferase type 2; RnPFTase, R.
norvegicus protein farnesyltransferase; ScPFTase, S. cerevisiae protein
farnesyltransferase; SDS–PAGE, sodium dodecylsulfate polyacrylamide
gel electrophoresis; THF, Tetrahydrofuran; THP, Tetrahydropyran.
*Corresponding Author: Mark D. Distefano, diste001@umn.edu
Photoactive analogs of farnesyl diphosphate (FPP)
are useful probes in studies of enzymes that
employ this molecule as a substrate. Here, we
describe the preparation and properties of two new
FPP analogs that contain diazotrifluoropropanoyl
photophores linked to geranyl diphosphate via
amide or ester linkages. The amide-linked analog
(3) was synthesized in 32P-labeled form from
geraniol in seven steps. Experiments with Saccha-
romyces cerevisiae protein farnesyltransferase
(ScPFTase) showed that 3 is an alternative sub-
strate for the enzyme. Photolysis experiments with
[
32P]3 demonstrate that this compound labels the
b-subunits of both farnesyltransferase and geranyl-
geranyltransferase (types 1 and 2). However, the
amide-linked probe 3 undergoes a rearrangement
to a photochemically unreactive isomeric triazo-
lone upon long term storage making it inconve-
nient to use. To address this stability issue, the
ester-linked analog 4 was prepared in six steps
from geraniol. Computational analysis and X-ray
crystallographic studies suggest that 4 binds to
protein farnesyl transferase (PFTase) in a similar
fashion as FPP. Compound 4 is also an alternative
Received 13 August 2009, revised 23 October 2009 and accepted for
publication 24 October 2009
Farnesyl diphosphate (FPP, 1, see Figure 1) is an important metabo-
lite in the biosynthesis of a variety of molecules including sesquit-
erpenes (1,2) and the side chains of a number of cofactors;(3)
FPP also serves as the source of prenyl groups that are appended
to proteins (4). Photoactive analogs of FPP have been useful in
Editor’s invited manuscript to celebrate the 4th Anniversary of Chemical Biology & Drug Design.
51

Hovlid et al.
synthesis of 4, an analog based on a 6,7 dihydrogeranyl framework
that replaces the allylic ester present in 2 with an alkyl ester; that
design is based on earlier work with azide-containing alternative
substrates for ScPFTase (26,27). Computational analysis and X-ray
crystallographic studies suggest that 4 binds to protein farnesyl
transferase (PFTase) in a similar fashion as FPP. Compound 4 is also
an alternative substrate for ScPFTase and labels a number of FPP-
utilizing enzymes including protein farnesyltransferase, farnesyl
diphosphate synthase and a sesquiterpene cyclase. Of particular
significance, photolysis of crude E. coli extracts expressing
ScPFTase, in the presence of 4, results in labeling of the enzyme
suggesting that this probe should be useful for identifying other
FPP-utilizing enzymes present in complex protein mixtures.
O
P
O
P
O
O
O–
O–
O–
O–
O– O–
Farnesyl Diphosphate (1)
N2
O
P
O
P
O
C-8
F3C
O
O
O– O–
O
O
O
Allylic Ester (2)
Amide (3)
N2
O
P
O
P
H
N
O
F3C
O
O
O– O–
Materials and Methods
N2
O
P
O
P
F3C
O
O
General
C-6
Alkyl Ester (4)
O– O–
All trans-geraniol, dimethylallyl alcohol, t-butyl hydroperoxide,
H2SeO3, salicylic acid, hydrazine monohydrate, toluene sulfonic acid,
and trichloroacetonitrile were obtained from Aldrich (St. Lous, MO).
All synthetic reactions were performed at room temperature in open
air, with magnetic stirring unless noted otherwise. TLC analysis was
performed on precoated (250 lm) silica gel 60 F-254 plates from
Merck (Whitehouse Station, NJ), and the plates were visualized by
UV light at 254 nm or KMnO4 staining. Flash chromatography silica
gel (60–120 mesh) was obtained from Mallickrodt Baker Inc., Paris,
KY, USA. Dowex 50W-X8 resin and silver staining kits were obtained
from Bio-Rad (Hercules, CA), Sep-Pak columns were purchased from
Waters (Millford, MA), and Amplify was from Amersham Life
Science Amersham (Piscataway, NJ). [32P]H3PO4 (specific activity
8500–9120 Ci ⁄ mmol) was purchased from DuPont NEN Dupont NEN
(Boston, MA). CH2Cl2 and CH3CN were dried with an M. Braun
solvent purification system. Deuterated solvents were used as
obtained from Cambridge Isotope Laboratories Inc. Cambridge Iso-
tope (Andover, MA). 1H-NMR spectra were obtained at 300 MHz
and 13C-NMR at 75 MHz. All nuclear magnetic resonance (NMR)
spectra were acquired on Varian (Palo Alto, CA) instruments at
25 ꢀC. Chemical shifts are reported in ppm with J values reported in
Hz. IR readings were taken using NaCl polished plates. HPLC was
performed on a Beckman 127 ⁄ 166 instrument equipped with a
Linear Instruments LC305 fluorescence detector. Phosphor imaging
analysis was performed with a Molecular Dynamics 445 SI Phosphor
imager. N-dansyl-GCVIA was synthesized by Dr. Dan Mullen in the
Department of Chemistry, University of Minnesota. ScPFTase (21),
H. sapiens protein geranylgeranyltransferase type 1 (HsPGGTase I),
E. coli farnesyl diphosphate synthase (EcFPPSase) (28), and Nostoc
sp. strain PCC7120 sesquiterpene synthase (NoSTSase) (29) were
purified as previously described. R. norvegicus protein geranyl-
geranyltransferase type 2 (RnPGGTase II) was a generous gift from
Dr. Miguel Seabra (Imperial College, London).
Figure 1: Farnesyl diphosphate and related diazotrifluoropropa-
noyl-containing photoprobes.
identifying proteins and enzymes that use this molecule as a sub-
strate. Hence, a variety of photocrosslinking groups have been used
to prepare FPP analogs for use in such experiments including diazo-
trifluropropionates (DATFP) (5–12), benzophenones (13–22), and aryl
azides (23,24). While DATFP-based probes do suffer from the disad-
vantage that they require activation at lower wavelengths than ben-
zophenone-containing and aryl azide-containing reagents, they also
have the advantage that they are intrinsically smaller than most
other types of photoactive moieties; in particular, a DATFP group is
an excellent mimic of a single isoprene unit. For that reason, Baba
and Allen synthesized compound 2 and initially employed it in stud-
ies with FPP synthase (6–8). In subsequent years, this compound
has been used extensively by a number of researchers in experi-
ments with protein prenyltransferases (5,9,11,25).
One feature of compound 2 that makes it suboptimal for photoaf-
finity labeling experiments is the ester linkage that connects the
photoactive DATFP moiety with the isoprenoid skeleton. Because of
the allylic nature of this ester, cleavage between the two parts of
the molecule can occur by nucleophilic addition to either the ester
carbonyl or the allylic position (C-8). This causes problems in the
synthesis but more critically, it can result in loss of the radioactive
label (usually present at C-1) in subsequent crosslinking experi-
ments. To circumvent this problem, we decided to explore the syn-
thesis and use of DATFP-containing analogs that incorporate more
stable linkages between the crosslinking group and isoprenoid. In
this paper, we first describe the preparation and use of an amide-
linked DATFP analog (3); the design of that compound is based on
earlier work with amide-linked benzophenone-containing probes
(19). While this new compound functions as an alternative substrate
of Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase)
and selectively labels the b-subunit of all three known protein pren-
yltransferases, its diazo amide moiety undergoes a rearrangement
to a photochemically inert triazoline over time in aqueous solution.
This property limits the utility of this probe. Next, we describe the
(E,E)-8-N-(2-Diazo-3,3,3-trifluoropropionamido)-
3,7-dimethyl-2,6-octadienyl-1-diphosphate (3).
Alcohol 9 (3.1 mg, 10 lmol) was reacted with anhydrous H3PO4
(2.0 mg, 20 lmol, prepared by lyophilization over P2O5) in CH3CN
(255 lL) containing 25% (v ⁄ v) CCl3CN and triethylamine (40 lmol)
52
Chem Biol Drug Des 2010; 75: 51–67

DATFP-Containing Photoactive FPP Analogs
for 2 h at rt under N2. The volatile components were then evapo-
rated, and the resulting residue was partially purified by reversed-
(E)-[a,b(n)32P]-8-O-(2-Diazo-3,3,3-
trifluoropropanoyloxy)-3,7-dimethyl-2-octeny-1-
diphosphate ([32P]4)
phase chromatography on
a Sep-Pak C18 cartridge with an
NH4HCO3 ⁄ CH3CN step gradient from 0–100% CH3CN; pure product
eluted at 25% CH3CN. The fractions containing the desired pyro-
phosphate product were concentrated and redissolved in 25 mM
NH4HCO3, and the final concentration was determined by UV absor-
bance (236 nm, extinction coefficient = 14 000 per cm ⁄ M) (30).
Diphosphate 3 was obtained in 8% yield, and the purity was deter-
mined to be 93% diphosphate, 7% monophosphate by reversed-
Radiolabeled [32P]4 was synthesized using a procedure similar to
that described for unlabeled 4. Briefly, a solution of 1% H3PO4
(v ⁄ v) (110 lL, 20 lmol) was prepared in a 10 mL round bottom
flask and lyophilized overnight as previously described. Compound
17 (3.1 mg, 10 lmol) was dissolved in 50 lL CH3CN and added to
the reaction flask containing the anhydrous H3PO4. To this solution,
a mixture of CH3CN ⁄ CCl3CN 20% (v ⁄ v) (200 lL) was added, fol-
lowed by Et3N (5.6 lL). The reaction was stirred in the dark under
an Ar atmosphere for 2 h followed by evaporation under a stream
of N2 (g) to yield a yellow residue. The resulting material was then
dissolved in 25 mM NH4HCO3 (5 mL) and applied to a reversed-
phase Sep-Pak cartridge equilibrated in 25 mM NH4HCO3. The col-
umn was eluted with a step gradient of CH3CN (10% steps,
4 · 1.0 mL fractions per step), and the composition of the fractions
was determined by TLC (i-PrOH ⁄ NH4OH ⁄ H2O, 6:3:1 v ⁄ v ⁄ v) followed
by phosphor imaging analysis. After evaporation of the product-con-
taining fractions (30% CH3CN) and redissolving in 25 mM NH4HCO3,
[32P]4 (specific activity: 275 Ci ⁄ mol) was obtained. The radio-
chemical purity was determined to be 24%, as assessed by TLC
(i-PrOH ⁄ NH4OH ⁄ H2O, 6:3:1, v ⁄ v ⁄ v), followed by phosphor imaging
analysis; Impurities that were present include the corresponding
monophosphate (36%), the triphosphate (11%), and inorganic phos-
phate (9%). The remaining 20% is likely a dimer formed by conden-
sation of two monophosphates. The concentration of [32P]4 was
determined by UV spectroscopy using the extinction coefficient
(e236 = 14 000 per cm ⁄ M) previously reported for the diazoester
fragment (30).
1
phase HPLC analysis. H-NMR (300 MHz, D2O, pH 8.0): d = 1.53 (s,
3H), 1.56 (s, 3H), 2.00–2.05 (m, 4H), 3.65 (s, 2H), 4.13 (t, 2H,
J = 6.0), 5.26–5.30 (m, 2H); High resolution fast atom bombardment
mass spectrometry (HR-FAB-MS) calcd for C13H10N3O8P2F3Na
[M+Na]+ 488.0576, found 488.0609
(E,E)-[a,b(n)32P]-8-N-(2-Diazo-3,3,3-
trifluoropropionamido)-3,7-dimethyl-2,6-
octadienyl-1-diphosphate ([32P]3)
Alcohol
9 (2.0 mg, 6.3 lmol) was reacted with anhydrous
[32P]H3PO4 (1.2 mg, 13 lmol) prepared as previously described in
CH3CN (200 lL) containing 20% (v ⁄ v) CCl3CN and triethylamine
(2.5 mg, 26 lmol) for 2 h (31). The volatile components were then
evaporated, and the resulting residue was purified using
a
reversed-phase Sep-Pak C18 cartridge with an NH4HCO3 ⁄ CH3CN
step gradient. The product ([32P]3) eluted from the Sep Pak car-
tridge in fractions containing 20% and 25% CH3CN. After evapora-
tion and redissolving in 25 mM NH4HCO3, [32P]3 (specific activity:
1.25 Ci ⁄ mmol) was obtained, giving a 7% yield. The radiochemical
purity was determined to be 40%, as assessed by thin layer chro-
matography in isopropanol ⁄ NH4OH ⁄ H2O (6:3:1, v ⁄ v ⁄ v), followed by
phosphor imaging analysis.
(E,E)-8-N-(2-Diazo-3,3,3-trifluoropropionamido)-
3,7-dimethyl-1-tetrahydropyranyl-2,6-octadiene
(8)
(E)-8-O-(2-Diazo-3,3,3-trifluoropropanoyloxy)-
3,7-dimethyl-2-octenyl-1-diphosphate (4)
The amine 6 was prepared by reacting phthalimide 5 (300 mg,
0.78 mmol) with NH2NH2•H2O (62 mg, 1.2 mmol) in EtOH (10 mL)
at rt for 16 h. The reaction was filtered and used directly in the
next step of the synthesis without further purification. Amide 8
was prepared by reacting crude 6 (110 mg, 0.43 mmol) with 7
(100 mg, 0.58 mmol) in dry pyridine (0.5 mL) at 0 ꢀC. The reac-
tion was stirred in the dark for 30 min under N2, followed by
an additional 2 h at rt. It was then concentrated in vacuo, redis-
solved in Ethyl acetate (EtOAc), and washed sequentially with
0.10 N HCl, 1.0 M NaHCO3, and aqueous saturated NaCl. The
organic layer was dried over MgSO4, filtered, concentrated under
reduced pressure, and the crude product was purified by flash
chromatography on silica (toluene ⁄ EtOAc, 4:1, v ⁄ v), giving 8 in
18% yield (30 mg, 77 lmol). Rf = 0.44 (silica gel, toluene ⁄ EtOAc,
4:1, v ⁄ v); 1H-NMR (500 MHz, CDCl3) d 1.52–1.81 (m, 6H), 1.62
(s, 3H), 1.67 (s, 3H), 2.05–2.09 (m, 2H), 2.13–2.18 (m, 2H), 3.49–
3.53 (m, 1H), 3.85–3.91 (m, 1H), 3.88 (d, 2 H, J = 8.5), 3.98–4.04
(m, 1H), 4.21–4.27 (m, 1H), 4.61–4.63 (m, 1H), 5.28–5.38 (m, 2H);
13C-NMR (75.4 MHz, CDCl3, Distortionless Enhancement by Polari-
zation Transfer (DEPT)) d 14.38, 16.39 (primary C); 19.62, 25.49,
25.99, 30.71, 39.08, 47.45, 62.31, 63.64 (secondary C); 97.92,
121.03, 126.95 (tertiary C); 122.22, 125.79, 131.20, 139.56,
158.94 (quaternary C); 19F-NMR (282.2 MHz, CDCl3) d )55.77.
Anhydrous phosphoric acid was prepared by lyophilization of a solu-
tion of 1% H3PO4 (v ⁄ v) (3.8 mL, 0.64 mmol). To that residue was
added CH3CN (1.6 mL), and Et3N (180 lL, 1.3 mmol) followed by com-
pound 17 (100 mg, 0.32 mmol), dissolved in 3.2 mL of a solution of
CCl3CN in CH3CN (20%, v ⁄ v). The reaction was allowed to proceed in
the dark under an N2 atmosphere with magnetic stirring for 2 h. The
solvent was then removed by rotory evaporation, and the residue was
redissolved in 10 mL of 25 mM NH4HCO3. The product was purified by
reversed-phase HPLC with buffer A (25 mM NH4OH) and buffer B
(CH3CN) over seven injections, using a linear gradient from 0–35%
buffer B over 35 min. Samples (2.0 mL) were injected into a 5 mL
loop, onto
a Phenomex Luna C18 semi-preparative column
(250 · 10 mm). Monitoring at a wavelength of 236 nm, the desired
product eluted at 22–26% solvent B. The product-containing fractions
1
were lyophilized to yield 23 mg (16%) of a white powder. H-NMR
(300 MHz, D2O) d 0.77 (d, J = 6.6 Hz, 3H), 1.05–1.40 (m, 6H), 1.55 (s,
3H), 1.63–1.80 (m, 1H), 1.90 (t, J = 7.5 Hz, 2H), 3.99 (t, J = 6.9 Hz,
2H), 4.14 (t, J = 6.3 Hz, 2H), 5.31 (t, J = 6.0 Hz, 1H); 31P-NMR
(121 MHz, CDCl3) )5.93 (d, J = 22.0 Hz, 1P), )0.91 (d, J = 22.0 Hz,
1P); HR-MS High resolution time of flight (ESI-TOF) calcd for
C13H20F3N2O9P2 [M-H]) 467.0596, found 467.0594.
Chem Biol Drug Des 2010; 75: 51–67
53

Hovlid et al.
(E,E)-8-N-(2-Diazo-3,3,3-trifluoropropionamido)-
3,7-dimethyl-2,6-octadien-1-ol (9)
trifluoropropanoyl chloride (DATFP-Cl, 7, 85 mg, 0.49 mmol) was
then added, and the mixture was cooled to 0 ꢀC and allowed to
react with stirring, in the dark, for 30 min followed by an additional
2 h at rt. The solvent (pyridine) was removed in vacuo, and the
resulting residue dissolved in Et2O and washed sequentially with
0.10 M HCl, 1.0 M NaHCO3, and aqueous saturated NaCl. The
organic layer was dried over MgSO4, filtered, concentrated under
reduced pressure, and the resulting crude product was purified by
flash chromatography (hexanes: EtOAc, 6:1) affording compound 16
Alcohol 9 was prepared by reacting protected DATFP ester (8,
50 mg, 0.13 mmol) with TsOH (1.3 mg, 6.9 lmol) in MeOH (13 mL)
in the dark at rt for 20 h. The reaction mixture was then concen-
trated under reduced pressure and purified by flash chromatography
on silica (toluene ⁄ EtOAc 4:1, v ⁄ v) giving the pure product in 67%
yield (26 mg, 85 lmol). Rf = 0.15 (silica gel, toluene ⁄ EtOAc, 4:1,
1
v ⁄ v); H-NMR (300 MHz, CDCl3) d 1.62 (s, 3H), 1.66 (s, 3H), 2.04–
1
2.09 (m, 2H), 2.13–2.18 (m, 2H), 3.87 (d, 2H, J = 5.7), 4.14 (d, 2H,
J = 6.9), 5.25–5.30 (m, 1H), 5.36–5.42 (m, 1H), 5.45 (broad s, 1H);
13C-NMR (75.4 MHz, CDCl3, DEPT): d 14.38, 16.12 (primary C);
25.74, 38.94, 47.41, 59.31 (secondary C); 124.01, 126.50 (tertiary C);
122.22, 125.79, 131.19, 138.78, 159.03 (quaternary C); 19F-NMR
(282.2 MHz, CDCl3) d )55.77; HR-FAB-MS calcd for C13H18N3O2F3Na
[M+Na]+ 328.1249, found 328.1259.
in 88% yield. H-NMR (300 MHz, CDCl3) d 0.95 (d, 2H, J = 6.6 Hz),
1.12–1.64 (m, 12H), 1.73 (s, 3H), 1.80 (m, 2H), 2.03 (t, 2H,
J = 7.5 Hz), 3.52 (m, 1H), 3.91 (m, 1H), 4.05 (m, 2H), 4.16 (dd, 1H,
J1 = 5.7 Hz, J2 = 10 Hz), 4.24 (dd, 1H, J1 = 6.6 Hz, J2 = 12 Hz),
4.64 (t, 1H, J = 4.2 Hz), 5.37 (t, 1H, J = 6.9 Hz); 13C-NMR (75 MHz,
CDCl3) d 16.33, 16.73, 19.69, 24.77, 25.55, 30.77, 32.58, 32.76,
39.69, 62.38, 63.71, 70.80, 97.97, 120.88, 121.73, 124.54, 140.04,
161.10; 19F-NMR (282 MHz, CDCl3) d )58.02 (s, 3F); Fourier trans-
form infrared spectrum (FT-IR) (neat) 1320 (vs), 1352 (vs), 1394 (s),
1732 (vs), 2136 (vs), 2344 (w), 2361 (w), 2869 (s), 2939 (vs) per cm;
HR-MS (ESI-TOF) calcd for C18H27F3N2NaO4 [M+Na]+ 483.2441,
found 483.2444.
2,2¢-((2E,6E)-2,6-dimethylocta-2,6-diene-
1,8-diyl)diisoindoline-1,3-dione (10)
Compound 5 (150 mg, 0.40 mmol) and pyridinium p-toluenesulfonate
(PPTS) (98 mg, 0.4 mmol) were dissolved in EtOH (4.8 mL) and stirred
at 55 ꢀC for 4 h. The solvent was removed in vacuo, redissolved in
Et2O (5 mL) and washed with half-saturated brine to remove the cata-
lyst. The ether solution was dried over Na2SO4 and evaporated to
yield 83 mg of the desired deprotected alcohol in (70% yield).
Rf = 0.30 (silica gel, hexane ⁄ EtOAC, 5:2, v ⁄ v); 1H NMR (300 MHz,
CDCl3): d 1.60 (s, 3H), 1.63 (s, 3H), 2.04–2.08 (m, 2H), 2.11–2.19 (m,
2H), 4.10 (d, 2H, J = 6.3), 4.17 (s, 2H), 5.24–5.35 (m, 2H), 7.68–7.75
(m, 2H), 7.82–7.87 (m, 2H); High resolution electrospray ionization
mass spectrometry (HR-ESI-MS) calcd for C18H21NaNO3 [M+Na]+
322.1419, found 322.1397. The previously mentioned deprotected
alcohol (80 mg, 0.27 mmol) was combined with phthalimide (45 mg,
0.27 mmol) and PPh3 (68 mg, 0.27 mmol) in 2.2 mL Tetrahydrofuran
(THF). To the stirred solution was added diethyl azodicarboxylate
(DEAD) (6 lL, 0.27 mmol) in 0.6 mL THF dropwise over 30 min. The
reaction mixture was stirred for 18 h at rt followed by a second addi-
tion of PPh3 (42 mg, 0.15 mmol) and DEAD (3.0 lL, 0.15 mmol). The
reaction was stirred overnight at rt and quenched with hexane
(5.0 mL) to precipitate the reaction byproducts. The filtrate was evap-
orated, and the residue was purified by flash chromatography (silica
gel, hexane ⁄ EtOAC, 5:2, v ⁄ v) to yield compound 10 (63 mg, 54%) as
a white solid. Crystallization of the product was achieved by adding
5.5 mL CH3OH and heating in an oil bath with swirling until the solid
was completely dissolved. The flask was removed from the heat and
left undisturbed, to cool to rt. Crystals formed over a period of 4 h.
Rf = 0.50, silica gel, hexane ⁄ EtOAC, 5:2, v ⁄ v; 1H NMR (300 MHz,
CDCl3): d 1.59 (s, 6H), 1.98–2.03 (m, 2H), 2.07–2.12 (2H), 4.16 (s, 2H),
4.26 (d, 2H, J = 7.2), 5.24 (t, 1H, J = 6.3), 5.32 (t, 1H, J = 6.3), 7.69–
7.33 (m, 4H), 7.83–7.87 (m, 4H); HR-ESI-MS calcd for C26H24NaN2O4
[M+Na]+ 451.1634, found 451.1648.
(E)-8-O-(2-Diazo-3,3,3-trifluoropropanoyloxy)-
3,7-dimethyl-2-octen-1-ol (17)
Alcohol 16 (135 mg, 0.34 mmol) was dissolved in 10 mL of abso-
lute EtOH. After adding PPTS (12 mg, 50 lmol), the flask was
sealed with a rubber septum and stirred in the dark for 6 h at
60 ꢀC. The product was purified by flash chromatography (hexanes:
EtOAc, 5:1, v ⁄ v) and obtained in 90% yield. 1H-NMR (300 MHz,
CDCl3) d 0.95 (d, 3H, J = 6.6 Hz), 1.10–1.56 (m, 6H), 1.67 (s, 3H),
1.81–1.84 (m, 1H), 2.01 (t, 2H, J = 7.8 Hz), 4.05 (dd, 1H, J1 =
6.6 Hz, J2 = 11 Hz), 4.15 (m, 3H), 5.41 (t, 1H, J = 6.9 Hz); 13C-NMR
(75 MHz, CDCl3) d 15.83, 16.37, 24.45, 32.26, 32.36, 39.29, 59.04,
70.52, 120.72, 123.43, 124.29, 139.12, 160.97; 19F-NMR (282 MHz)
d )58.01 (s, 3F); FT-IR (neat) 1319 (vs), 1351 (vs), 1396 (vs), 1701
(vs), 2135 (vs), 2857 (s), 2925 (vs), 2962 (vs), 3371 (b) per cm;
HR-MS (ESI-TOF) calcd for C13H19F3N2NaO3 [M+Na]+ 331.1240,
found 331.1240.
1-(2-Diazo-3,3,3-trifluoropropionyloxy)-3-
methyl-2-butene (18)
Dimethylallyl alcohol (100 mg, 1.16 mmol) was dissolved in freshly
distilled pyridine (0.5 mL) and stirred until homogenous. A solution
of DATFP-Cl (7, 239 mg, 1.39 mmol) in pyridine (1.0 mL) was slowly
added, and the reaction allowed to proceed at 0 ꢀC in the dark for
2.5 h. The pyridine solvent was removed in vacuo, and the product
was redissolved in Et2O. The organic layer was then washed
sequentially with 0.1 M HCl, 1 M NaHCO3, and brine, dried over
Na2SO4 and concentrated. Purification was accomplished by flash
chromatography (hexanes: EtOAc, 3:1) producing 258 mg (64%) of a
1
pale yellow oil. H-NMR (300 MHz, CDCl3) d 1.73 (s, 3H), 1.77 (s,
(E)-8-O-(2-Diazo-3,3,3-trifluoropropanoyloxy)-
3,7-dimethyl-1-O-tetrahydropyranyl-2-octene
(16)
Pyridine (3 mL) was added to alcohol 15 (100 mg, 0.39 mmol), and
the reaction flask was sealed with a rubber septum. 2-diazo-3,3,3-
3H), 3.74 (d, 2H, J = 7.2 Hz), 5.35 (tq, 1H, J1 = 1.5 Hz, 8.7 Hz);
13C-NMR (75 MHz, CDCl3) d 18.04, 25.77, 62.82, 117.76, 121.00,
124.57, 136.14, 160.92; 19F-NMR (282 MHz, CDCl3) d )58.26 (s, 3F);
FT-IR (neat) 1313 (vs), 1355 (vs), 1384 (vs), 1448 (m), 1723 (vs), 2137
(vs), 2341 (w), 2360 (w), 2309 (m), 2977 (m) per cm.
54
Chem Biol Drug Des 2010; 75: 51–67

DATFP-Containing Photoactive FPP Analogs
1-(2-Diazo-3,3,3-trifluoropropionyloxy)-3-
methyl-3-butene (19)
ther purified by reversed-phase HPLC using a Varian Microsorb C18
analytical column (250 · 4.6 mm). Elution was performed using a
linear gradient from 100% solvent A to 100% solvent B (solvents
described earlier) over 40 min with a flow rate of 1 mL ⁄ min. The
product was collected, lyophilized, and analyzed by MS. In cases
where the reaction kinetics were monitored by HPLC, aliquots of
500 lL were removed at varying time intervals from large scale
reactions, flash frozen in N2 (l), and stored at )20 ꢀC prior to
analysis. The samples were then thawed and analyzed by reversed-
phase HPLC as described earlier.
Compound 19 was prepared as described for 18 starting from
1
isopentenyl alcohol in 66% yield. H-NMR (300 MHz, CDCl3) d 1.76
(s, 3H), 2.39 (t, 2H, J = 6.9 Hz), 4.37 (t, 2H, J = 6.9 Hz), 4.74 (s,
1H), 4.83 (s, 1H); 13C-NMR (75 MHz, CDCl3) d 22.33, 36.74, 64.11,
112.87, 120.92, 124.50, 141.01, 160.85; 19F-NMR (282 MHz, CDCl3)
d )58.20 (s, 3F); FT-IR (neat) 1329 (vs), 1344 (vs), 1392 (vs), 1456
(m), 1721 (vs), 2136 (vs), 2256 (m), 2359 (w), 2973 (m), 3081 (m),
3434 (b) per cm.
Stability studies of 18 and 19
Photolabeling of prenyltransferases with
Compound 18 (11 mg, 0.05 mmol) was added to an 8 -inch thin-
walled NMR tube and dissolved by the addition of 0.7 mL of mildly
acidic solution (CD3CN ⁄ D2O 10% ⁄ trifluoroacetic acid (TFA)-d 0.1%,
v ⁄ v ⁄ v); a separate NMR tube containing 18 (11 mg, 0.05 mmol)
dissolved in a mildly basic solution (CD3CN ⁄ D2O 10% ⁄ ND4OD 0.1%,
v ⁄ v ⁄ v) was also prepared. The samples were then heated directly
in the NMR tubes in a 100 ꢀC water bath for 4 h followed by H-
NMR analysis. Experiments with 19 in acidic or basic solutions
were prepared and analyzed in a similar manner.
[
32P]DATFP-Amide ([32P]3)
For experiments with ScPFTase, purified enzyme (4 lg) was incu-
bated in 50 mM HEPES (pH 7.5), 1.0 mM DTT, 5.0 mM MgCl2,
10 lM ZnCl2, 15 lM [32P]3 (dissolved in 25 mM NH4HCO3), and
30 lM FPP (where appropriate, dissolved in 25 mM NH4HCO3) or
the equivalent volume of 25 mM NH4HCO3 in a final volume of
75 lL. Samples were irradiated at 254 nm at 4 ꢀC for 3 min in
quartz test tubes (10 · 45 mm) using a UV Rayonet mini-reactor
equipped with 4 RPR-2540 ꢀ lamps and a circulating platform.
The samples were heated to 70 ꢀC for 5 min in loading buffer
(4% SDS, 12% glycerol (w ⁄ v), 50 mM Tris, 2% mercaptoethanol
(v ⁄ v), 0.01% Serva blue G, pH 6.8) and analyzed by SDS–PAGE
with a 10% Tris-tricine gel. The gel was fixed by slow shaking
in isopropanol ⁄ H2O ⁄ acetic acid (25:65:10, v ⁄ v ⁄ v) for 20 min. It
1
Enzymatic substrate and inhibition studies
monitored by fluorescence spectroscopy
The photoactive isoprenoids 3 and 4 were studied as possible sub-
strates for ScPFTase by monitoring the increase in fluorescence of
a peptide substrate as it becomes prenylated as previously
described (32,33). For experiments with the yeast enzyme, the assay
conditions used were 50 mM Tris HCl (pH 7.0), 10 mM MgCl2,
10 lM ZnCl2, 5.0 mM dithiothreitol (DTT), 0.040% (w ⁄ v) n-dodecyl-
b-D-maltoside, 2.0 lM N-dansyl-GCVIA, isoprenoid diphosphate (FPP,
3 or 4, 10 lM), and ScPFTase (24–92 nM) in a final volume of
500 lL. For experiments with the mammalian enzyme, similar condi-
tions were employed except that N-dansyl-GCVLS (2.4 lM) was
used as a substrate. Enzymatic reactions were initiated by the addi-
tion of PFTase and monitored for 300–3000 seconds (340 nm excita-
tion, 505 emission) to determine the rate. For IC50 determinations,
fluorescence assays were performed in triplicate as described ear-
lier except that reactions contained 2.0 lM FPP along with 3 or 4
at varying concentrations. The IC50 values were determined from a
direct plot of rate versus inhibitor concentration.
was then exposed to
a phosphor imaging screen for 3 h,
followed by silver staining. Experiments with HsPGGTase were
performed as described earlier except that geranylgeranyl diphos-
phate (30 lM final concentration) was used instead of FPP to
demonstrate substrate protection. Experiments with RnPGGTase II
were also performed as described earlier except that ZnCl2 was
omitted from the reaction buffer. Geranylgeranyl diphosphate
(30 lM final concentration) was used, instead of FPP, to show
substrate protection. In all cases, enzyme concentrations were
determined via the method of Bradford using bovine serum
albumin (BSA) as a standard (34).
Photolabeling of enzymes with [32P]DATFP-
Dihydroester ([32P]4)
Samples of [32P]4 (5 lM) were photolyzed in 52 mM Tris HCl, pH 7,
5.8 mM DTT, 12 mM MgCl2, 12 mM ZnCl2, and 25 lM FPP dissolved
in either 25 mM NH4HCO3 or the equivalent volume of 25 mM
NH4HCO3 (where appropriate) in a final volume of 100 lL. In exper-
iments employing purified enzymes (ScPFTase, EcFPPSase and NoS-
TSase), the final concentrations of the proteins were 0.05 mg ⁄ mL;
for experiments with crude E. coli lysate containing ScPFTase, a
final protein concentration of 0.81 mg ⁄ mL was employed. Enzyme
concentrations were determined via the method of Bradford using
BSA as a standard (34). Samples were irradiated for 5 min using
the apparatus described earlier. After photolysis, 50 lL of loading
buffer (see the previous text) was added to each sample before
heating to 60 ꢀC for 20 min. Analysis was accomplished via electro-
phoresis using a 12% Tris-glycine SDS–PAGE gel and subsequent
staining with Sypro Orange followed by phosphor imaging to visual-
ize the radiolabeled bands.
Enzymatic substrate studies monitored by
HPLC
To confirm the enzymatic modification of N-dansyl-GCVIA by 3 and
4, larger scale reactions (10–15 mL) were performed using the con-
ditions reported for the fluorescence assays described earlier except
that the detergent was omitted (detergent is usually included to
solubilize the product, not for enzyme activity). In each case, the
reaction was initiated with enzyme and incubated at 30 ꢀC for 6–
24 h. The reaction mixture was then applied to a Sep Pak cartridge
equilibrated in solvent C (5% CH3CN, 95% H2O, 0.1% TFA, v ⁄ v ⁄ v);
product was eluted with solvent B (100% CH3CN, 0.1% TFA, v ⁄ v).
The fluorescent fractions were concentrated, redissolved in 50%
solvent A (100% H2O, 0.1% TFA, v ⁄ v) and 50% solvent B, and fur-
Chem Biol Drug Des 2010; 75: 51–67
55

Hovlid et al.
Docking of 4 in the active sites of
R. norvegicus protein farnesyltransferase
(RnPFTase)
generate the molecular electrostatic potential (MEP), the electrostatic
potential was plotted on an isodensity surface (Molecular orbital
(MO) = 0.02 and density = 0.02) using Gaussian view. The minimum
and maximum potentials were set to )0.05 and 0.2, respectively.
Calculations of total polar surface area and LogP were performed
using ChemBioDraw Ultra (v. 11.0.1) CambridgeSoft (Cambridge, MA).
For modeling compound 4 in the active site of RnPFTase (pdb file
1JCR) (35), docking was performed using Glide (Schrodinger, version
5.5). A standard precision docking parameter was set, and 10 000
ligand poses per docking were run. Five resulting conformations
with the lowest docking score for each enantiomer (R-4 and S-4)
were chosen. Each one of those was then subjected to quantum
mechanics molecular mechanics (QM-MM) using QSITE (Schrodinger,
(New York, NY) version 5.5). The three conformations with the over-
all lowest energy were chosen for display for each enantiomer.
Results and Discussion
Synthesis of DATFP-amide 3
The amide-containing analog 3 was prepared using the route
shown in Scheme 1. Amine 6 was prepared as previously described
X-Ray crystallography studies of RnPFTase
Crystals of the RnPFTase:4 complexes were prepared by soaking 4
into preformed crystals using methods previously described (36).
X-ray diffraction data for the PFTase:4 complex was collected on a
Rigaku FRE rotating anode generator equipped with VariMax HR
optics and a Raxis-IV++ image plate detector. The detector set at
150 mm, data were collected in 200 contiguous 0.30ꢀ oscillation
images each exposed for 5 min. The data extend to 2.05 ꢀ resolution
and have a Rmerge of 6.1% with a 3.8-fold multiplicity. The structure
was refined using autoBUSTER Global Phasing Limited (Cambridge,
UK) to an Rfactor of 18.1% and an Rfree of 21.5%. The coordinates for
the structure have been deposited in the Protein Data Bank (3KSL).
Electrostatic potential calculations of 1 and 4
Structures of 1 and 4 were generated in Gaussian View, and the
geometries were optimized at the b3lyp ⁄ 6-31G(d) level using Gauss-
ian 03 (M.J. Frisch, G.W. Trucks, H.B. Schlegel et al., Gaussian 03,
Revision E.01, Gaussian, Inc., Wallingford, CT, 2004). The extended
conformation of each molecule was produced by constraining the dis-
tance between remote atoms. A map of electrostatic potential and a
map of the electron density were generated for each molecule. To
Figure 2: Structure of compound 10 determined by X-ray crys-
tallography to establish the E stereochemistry of the C-6 alkene.
C-10
C-7
O
O
O
C-
3
N
N
OH
O
O
N
1) PPTs
EtOH
2) Ph3P, DEAD
Phthalimide
3 steps
C-2
C-8 C-6
5
O
O
10
Geraniol
O
N2H4•H2O
EtOH
N2
O
O
P
H
N
P
H2N
F3C
O
O
O–
N2
O
O
O– O–
6
3
O
Cl
Pyridine
F3C
O
7
H3PO4 (s)
CCl3CN, TEA
CH3CN
N2
N2
H
H
N
N
F3C
O
O
F3C
OH
PPTs
EtOH
8
9
O
O
N2
O
P
O
H
N
P
F3C
O
O
O–
H3PO4 (s)
CCl3CN, TEA
CH3CN
O– O–
3
O
Scheme 1: Synthesis of amide-linked photoaffinity analog 3.
56
Chem Biol Drug Des 2010; 75: 51–67

DATFP-Containing Photoactive FPP Analogs
H
N2
N
N N
H
N-Dansyl-GCVIA
N
N
R
F3C
R
F3C
O
O
O
O
O
H
N
H
N
S
OH
N
N
N
H
O
Figure 3: Rearrangement of diazotrifluoropropanoyl-amides to
H
H
O
O
O
SH
triazolones.
(19) and acylated with DATFP-Cl (30) (7) in pyridine to produce the
protected intermediate 8. That compound was subsequently depro-
tected and phosphorylated using the method of Cramer (37,38) to
yield a mixture mono and diphosphates. Pure diphosphate 3 was
obtained by reversed-phase HPLC and characterized by 1H-NMR,
MS, and UV ⁄ vis spectrophotometry. While the regioselectivity of
geraniol hydroxylation with SeO2 has been established in other
cases (39), we elected to confirm this in the Tetrahydropyran (THP)-
protected version by converting intermediate 5 to the corresponding
bis-phthalimide derivative 10. That transformation was accom-
plished by THP removal followed by subsequent Mitsunobu reaction
(40,41) to yield 10 whose structure was then confirmed via x-ray
crystallography. The E stereochemistry of the 6,7 alkene is apparent
in the structure of this derivative shown in Figure 2. For photolabel-
ing experiments, radiolabeled 3 was prepared via phosphorylation
of alcohol 9 with [32P]-H3PO4 as previously described (42). Solutions
of 3 in aqueous NH4HCO3 are stable for several weeks when
stored at 4 ꢀC. However, storage for longer periods of time results
ScPFTase
1, 2, 3 or 4
N
O
O
O
H
N
H
N
S
OH
N
N
H
N
H
O
H
O
O
O
S
R
= R, 11
= R, 12
= R, 13
= R, 14
N2
O
F3C
F3C
F3C
O
N2
N2
H
N
in
a change in the UV spectrum from kmax = 238 nm to
O
O
kmax = 250 nm with no change in the mass spectrum of the com-
pound; these observations are indicative of isomerization of the
diazo amide to the corresponding triazolone (see Figure 3) (43,44).
Thus, for maximal crosslinking efficiency compound 3 must be used
within one month of its synthesis.
O
Scheme 2: Structures of Saccharomyces cerevisiae protein
farnesyltransferase modified N-dansyl-GCVIA peptide products. 11:
peptide product from reaction with farnesyl diphosphate (1); 12:
peptide product from reaction with allylic ester 2; 13: peptide prod-
uct from reaction with amide 3; 14: peptide product from reaction
with alkyl ester 4.
Enzymatic studies with PFTase using DATFP-
amide 3
Initially, a continuous fluorescence assay was used to determine
whether or not the new photoaffinity analog 3 is a substrate for
A
B
700
0.04
600
0.03
0.02
0.01
0
(a) FPP
500
400
300
200
100
0
(b) Amide 3
0
500 1000 1500 2000 2500 3000
Time (seconds)
0
20
40
[3] (µ M)
60
80
Figure 4: Evaluation of amide 3 as an alternative substrate and inhibitor of Saccharomyces cerevisiae protein farnesyltransferase (ScPF-
Tase). (A) Reaction between N-dansyl-GCVIA and farnesyl diphosphate (FPP) or amide 3 catalyzed by ScPFTase monitored by fluorescence
spectroscopy. (a) Kinetic data using FPP as a substrate. (b) Kinetic data using amide 3 as a substrate. Fluorescence was monitored at 30 ꢀC
by excitation at 340 nm and emission at 505 nm. (B). Inhibition of ScPFTase-catalyzed farnesylation of N-dansyl-GCVIA by amide 3. Reactions
contained 2.0 lM FPP, 2.0 lM N-dansyl-GCVIA, and 3 at varying concentrations and were monitored using a continuous spectrofluorometric
assay. Each point is the average of 2–3 determinations with the error bars indicating the standard error for each measurement.
Chem Biol Drug Des 2010; 75: 51–67
57

Hovlid et al.
ScPFTase. Incubation of the substrate peptide N-dansyl-GCVIA with
FPP resulted in a rapid increase in dansyl group fluorescence (see
Figure 4A, trace a) because of the production of the farnesylated
product 11 (see Scheme 2). Replacing the natural substrate, FPP,
with 10 lM amide 3 in the assay resulted in a similar time-depen-
dent increase in fluorescence, suggesting that the analog is a
substrate for the enzyme (see Figure 4A, trace b); interestingly, the
fluorescence plateaued at a lower value than that obtained with FPP
suggesting that the prenylated product 13 is less fluorescent than
11. However, importantly, the fluorescence increased at a much
slower rate with 10 lM 3 than with 10 lM FPP and much longer
reaction times (3000 seconds) were required for significant conver-
sion to product compared with 200 seconds for complete conversion
to the farnesylated peptide product. Also, a significantly higher
concentration of enzyme (92 nM) was used, compared with 2.5 nM
normally added for assays with FPP as the substrate. Based on those
numbers, we estimate that 3 functions some 250-fold times slower
than FPP as a substrate for ScPFTase. To verify that 3 is a substrate
for the enzyme, the product from a large-scale enzyme reaction
(15 mL) was purified by reversed-phase HPLC and analyzed by MS.
The fast atom bombardment mass spectrometry (FAB-MS) spectrum
showed a [M+H]+ peak of 982.5 and a [M+Na]+ peak at 1004.6,
corresponding to the expected peptide product (13).
Photolysis experiments with DATFP-amide 3
Amide 3 was first evaluated for its ability to label ScPFTase. UV
irradiation of ScPFTase in the presence of 15 lM [32P]3 at 254 nm
for 1 min resulted in preferential labeling of the 43 kDa b subunit
(Figure 5, Lane 3¢); inclusion of 30 lM FPP in the reaction mixture
gave substantial protection from labeling (Lane 4¢, 3% of the cross-
linking observed in Lane 3¢). This selective labeling of the b-subunit
is consistent with previous results with DATFP esters and suggests
that the isoprenoid moiety interacts predominantly with the b-sub-
unit when it binds to the enzymatically active a,b-heterodimer. Sim-
ilar results were obtained with HsPGGTase I where UV irradiation
of purified enzyme in the presence of 15 lM [32P]DATFP-GPP amide
at 254 nm for 1 min resulted in preferential labeling of the 43 kDa
b subunit (Figure 6, Lane 2¢); inclusion of 30 lM geranylgeranyl
diphosphate (GGPP) in the reaction mixture resulted in substantial
protection from labeling (Lane 3¢, 5% of the crosslinking observed
in Lane 2¢). It is interesting to note that a small amount of labeling
of the a-subunit was also observed with this enzyme. This may
reflect an alternative binding mode for the probe; crystallographic
studies suggest that a second isoprenoid binding site exists in PGG-
Tase that is involved in interactions with the prenylated product
(45). Finally, we explored the photolysis of Rattus norvegicus protein
geranylgeranyltransferase type II (RnPGGTase II) in the presence of
3 because there have been no reported studies of photoaffinity
labeling of the type II enzyme with DATFP-based probes. UV irradia-
tion of RnPGGTase II in the presence of 10 lM [32P]3 resulted in
preferential labeling of the 38 kDa b subunit (Figure 7, Lane 2¢). As
was observed in the cases noted earlier including 30 lM GGPP in
the reaction mixtures gave substantial protection from labeling
(Lane 3¢, 9% of the crosslinking observed in Lane 2¢). These results
give direct evidence for the involvement of the b subunit of
Because DATFP-amide 3 appeared to be a significantly slower sub-
strate than FPP for ScPFTase, the analog was evaluated as an
inhibitor of the reaction between FPP and the peptide substrate
N-dansyl-GCVIA. An IC50 value of 50 lM (see Figure 4B) was
obtained for the inhibition of ScPFTase by 3 that is significantly
higher than the value of 0.72 lM measured for the corresponding
ester-linked compound 2 (5). This higher value suggests lower affin-
ity for the amide-containing analog and may reflect the more rigid
nature of the amide-linked analog versus ester-linked analog.
Figure 5: Analysis of photolabeling of Saccharomyces cerevisiae
protein farnesyltransferase with diazotrifluoropropanoyl-amide
([32P]3) by SDS–PAGE. Lanes 1 and 1¢: molecular weight standards.
Lanes 2 and 2¢: sample containing protein farnesyl transferase
(PFTase) and [32P]3, no UV irradiation. Lanes 3 and 3¢: PFTase irradi-
ated in the presence of [32P]3. Lanes 4 and 4¢: PFTase irradiated in
the presence of [32P]3 and farnesyl diphosphate (substrate). Lanes
1, 2, 3, and 4 show the silver-stained proteins. Lanes 1¢, 2¢, 3¢, and
4¢ show the radiolabeled proteins.
Figure 6: Analysis of photolabeling of H. sapiens protein gera-
nylgeranyltransferase type 1 (HsPGGTase I) with diazotrifluoropropa-
noyl-amide ([32P]3) by SDS–PAGE. Lanes 1 and 1¢ contain samples
of PGGTase I and [32P]3 that were not irradiated. Lanes 2 and 2¢
contain samples of HsPGGTase I irradiated at 254 nm in the pres-
ence of [32P]3. Lanes 3 and 3¢ contain samples of PGGTase I irradi-
ated in the presence of [[32P]3 and farnesyl diphosphate (substrate).
Lanes 1, 2, and 3 show the silver-stained proteins. Lanes 1¢, 2¢,
and 3¢ show the radiolabeled proteins.
58
Chem Biol Drug Des 2010; 75: 51–67

DATFP-Containing Photoactive FPP Analogs
geraniol followed by allylic oxidation and selective reduction of the
a,b-unsaturated aldehyde afforded 15 as previously described (26).
That alcohol was acylated with DATFP-Cl (7), deprotected and phos-
phorylated using the method of Cramer (38). For photolabeling
experiments, radiolabeled 4 was prepared via phosphorylation of
alcohol 17 with [32P]H3PO4 using a procedure previously described
for related analogs (31). It should be noted that 4 contains a stere-
ogeneic center (C-7) and is hence chiral. No attempt was made to
resolve the enantiomers, and thus all experiments were performed
with racemic material.
Stability studies of alkyl and allylic DATFP
esters
To investigate the stability of the dihydro isoprene unit in 4 com-
pared with its allylic counterpart present in 2, five carbon DATFP-
containing model compounds were synthesized. Compound 18
(Scheme 4) contains a DATFP moiety linked to an allylic alcohol to
model compound 2 while 19 lacks the allylic alcohol and is hence
designed to mimic 4. Model compounds 18 and 19 were synthe-
sized by reaction of DATFP-Cl (7) with dimethylallyl alcohol or iso-
pentenyl alcohol, respectively.
Figure 7: Analysis of photolabeling of R. norvegicus protein ger-
anylgeranyltransferase type 2 (RnPGGTase II) with diazotrifluoroprop-
anoyl-amide ([32P]3) by SDS–PAGE. Lanes 1 and 1¢: PGGTase II and
[32P]3, no UV irradiation. Lanes 2 and 2¢: RnGGPTase II and [32P]3,
1 min UV irradiation. Lanes 3 and 3¢: RnPGGTase II, [32P]3, and ger-
anylgeranyl diphosphate, 1 min UV irradiation. Lanes 1, 2, and 3
show the silver-stained proteins. Lanes 1¢, 2¢, and 3¢ show the
radiolabeled proteins.
As a test of stability, compounds 18 and 19 were treated with a
solvent mixture consisting of CD3CN ⁄ D2O (9 ⁄ 1, v ⁄ v) containing 0.1%
TFA-d. That mixture was used here because it is representative of
what is typically employed for peptide extraction from gels and sub-
sequent chromatographic fractionation. Deuterated solvents were
used to allow direct analysis of the reaction mixtures to be per-
RnGGPTase II in isoprenoid substrate binding that is consistent with
crystallographic data on this system (46,47). Also, because these
experiments were carried out in the absence of the escort protein,
REP-1, the results indicate that this protein is not necessary for iso-
prenoid substrate binding.
1
formed by H-NMR. Portions of the spectral data from 18 and 19
prior to reaction are shown Figure 8 (panels A and C, respectively).
Treatment of allylic ester 18 in the solvent mixture described earlier
at 100 ꢀC for 4 h resulted in substantial hydrolysis to produce primar-
ily tertiary alcohol 21 together with traces of several other products
including allylic alcohol 20 as evidenced from the H-NMR.
Synthesis of dihydroester 4
While the amide linkage present in 3 did contribute to its enhanced
stability, the rearrangement of the diazo amide to the triazolone
shown in Figure 3 limited the utility of this compound. Thus, we
undertook the synthesis of dihydroester-linked analog 4 that was
prepared using the route shown in Scheme 3. THP protection of
NMR spectrum (Figure 8B) of the reaction mixture; in addition to
unreacted starting material, the observation of three doublet of
N2
Pyridine
HO
O
O
O
OH
F3C
O
O
N2
3 steps
15
16
O
Cl
Geraniol
F3C
N2
O
7
N2
O
P
O
P
O
O
F3C
OH
F3C
O
O
O–
PPTs
EtOH
H3PO4 (s)
CCl3CN, TEA
CH3CN
O– O–
4
17
O
O
OH Ha
Hc
O
Ha Ha
Ha Ha
OH
19
+
CF3
CF3
Hb
O
CD3CN/D20/
0.1% CF3CO2D
or 0.1% ND4OD
Hb
N2
N2
Hb
20
17
O
Ha Ha
O
Ha Ha
OH
21
CD3CN/D20/
0.1% CF3CO2D
or 0.1% ND4OD
18
Scheme 3: Synthesis of dihydroester-linked photoaffinity analog 4.
Chem Biol Drug Des 2010; 75: 51–67
59

Hovlid et al.
OH Ha
O
Ha Ha
Ha Ha
OH
20
+
CF3
Hb
O
CD3CN/D2O/
0.1% CF3CO2D
or 0.1% ND4OD
Hc
Hb
N2
Hb
21
18
Hd
Hd Hd
O
Ha Ha
Ha Ha
OH
22
Hc
CF3
O
CD3CN/D2O/
0.1% CF3CO2D
or 0.1% ND4OD
Hb Hb
Hc
N2
19
Scheme 4: Diazotrifluoropropanoyl-containing allylic ester and alkyl ester model compounds and their putitive hydrolysis products.
doublets at 4.92, 5.13, and 5.96 ppm confirms the presence of 21.
In contrast, incubation of the homoallylic ester 19 under identical
conditions resulted in no changes in the 1H NMR spectrum (Fig-
ure 8D) of the corresponding reaction mixture (compare Figure 8C
with Figure 8D); no evidence for the formation of 22 was observed.
These results indicate that 19 is substantially more stable than 18
under these mildly acidic conditions and suggest that crosslinked
products derived from reactions with probe 4 (homoallylic ester)
should be significantly more resistant to hydrolytic degradation than
products obtained with allylic ester 2.
Enzymatic studies with PFTase using
dihydroester 4
To evaluate dihydroester 4 as a substrate, we initially utilized the
continuous fluorescence assay employed to study amide 3. Those
A
0 h
B
4 h
160
140
120
100
80
160
140
120
100
80
O
Ha Ha
CF3
18
Ha
18
Ha
O
Hb
N2
18
Ha Ha
18 Hb,
20 Hb
18 Hb
OH
20
21
21
21
Hc
60
Hb
60
20
Ha
Hb
Ha
40
40
OH Ha
20
20
Hb
0
0
Hc
21
6.00 5.50 5.00 4.50 4.00
Shift (ppm)
6.00 5.50 5.00 4.50 4.00
Shift (ppm)
0 h
4 h
C
D
160
160
140
120
100
80
140
120
100
80
Hd
Hd Hd
S
S
O
Ha Ha
Hc
CF3
19
Ha
19
Ha
19
Hd
O
19
Hd
S
S
Hb Hb
Hc
N2
S
S
19
Ha Ha
OH
22
19
Hc
19
Hb
19
Hc
19
Hb
60
60
40
40
20
20
0
0
6.00 5.00 4.00 3.00 2.00 1.00 0.00
Shift (ppm)
6.00 5.00 4.00 3.00 2.00 1.00 0.00
Shift (ppm)
Figure 8: 1H-NMR spectra of diazotrifluoropropanoyl-containing model esters obtained under mild acidolytic conditions. Spectra on the left
are the compounds before heating, and spectra on the right are the compounds after heating at 100 ꢀC for 4 h. (A) Compound 18 treated
under acidic conditions (CD3CN ⁄ D20 10% ⁄ trifluoroacetic acid (TFA)-d 0.1%) before heating. (B) Compound 18 treated under acidic conditions
(CD3CN ⁄ D20 10% ⁄ TFA-d 0.1%) after heating to 100 ꢀC for 4 h. The appearance of three new doublets of doublets after heating indicates the
presence of 21. (C) Compound 19 treated under acidic conditions (CD3CN ⁄ D20 10% ⁄ TFA-d 0.1%) before heating. (D) Compound 19 treated
under acidic conditions (CD3CN ⁄ D20 10% ⁄ TFA-d 0.1%) after heating to 100 ꢀC for 4 h. Compound 19 shows no visible change in the
1H-NMR spectrum after 4 h. In panels (C) and (D), peaks labeled 'S' are from D2O, p-xylene (internal standard for integration), and CD3CN.
60
Chem Biol Drug Des 2010; 75: 51–67

DATFP-Containing Photoactive FPP Analogs
A
B
70
60
50
40
30
20
10
0
35
30
25
20
15
10
5
N-dansyl-GCVIA
(2)
N-dansyl-GCVIA
(2)
14
(1)
11
(1)
0
20
25
30
35
40
45
20
25
30
35
40
45
Time (min)
Time (min)
Figure 9: Reversed-phase HPLC analysis with fluorescence detection of reactions between N-dansyl-GCVIA and farnesyl diphosphate (FPP)
(1) or dihydroester 4 catalyzed by Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase). (A) N-dansyl-GCVIA (2.4 lM) incubated
with FPP (1, 10 lM) and ScPFTase (24 nM). Chromatogram 1 (bottom): Reaction before the addition of the enzyme with the peptide eluting at
tR = 25.7 min. Chromatogram 2 (top): Reaction after 5 min with the prenylated peptide (11) appearing at tR = 41.5 min. (B) N-dansyl-GCVIA
(2.4 lM) incubated with dihydroester (4, 10 lM) and ScPFTase (24 nM). Chromatogram 1 (bottom): Reaction before the addition of the enzyme
with the peptide substrate at tR = 25.7 min. Chromatogram 2 (top): Reaction after 23 h with the prenylated peptide (14) appearing at
tR = 36.0 min and some starting material still present.
experiments gave similar results as were observed with 3, suggest-
ing that compound 4 was a slow alternative substrate for ScPF-
Tase. However, unambiguous interpretation of the data was made
difficult because of slow, time-dependent perturbations of the fluo-
rescence of the putative prenylated peptide product (14, see
Scheme 2). It has been previously noted that the fluorescence assay
is sensitive to small changes in the reaction conditions (32,33) and
that some isoprenoid analogs that are alternative substrates modu-
late the fluorescence of the starting peptide and product (48,49).
Hence, we elected to monitor the enzymatic reactions by a method
that is less prone to such artifacts. Accordingly, the incorporation of
4 into a peptide substrate was monitored by reversed-phase HPLC
using fluorescence detection. When the natural substrate FPP
(10 lM) was incubated with N-dansyl-GCVIA (2.4 lM) and ScPFTase
(24 nM), near complete conversion (93%) to the prenylated peptide
product (11) was observed within 5 min (Figure 9A). The reaction
of 4 (10 lM) with N-dansyl-GCVIA (2.4 lM) and ScPFTase (24 nM)
to yield 14, however, was still not complete after 23 h (Figure 9B).
Based on peak integrations, prenylation of the dansylated peptide
with the unnatural substrate 4 was 77% complete after 23 h, mak-
ing it approximately 300 times slower than the same reaction with
FPP. The lower rate observed with 4 is comparable to that observed
with 2 and 3 (see the previous text). However, it should be noted
that if extensive conversion is desired, the lower efficiency of incor-
poration manifested by these compounds can generally be compen-
sated for by the employment of higher enzyme concentrations and
longer reaction times. Moreover, methods have been developed for
the purification of both prenylated peptides (conventional reversed-
phase HPLC) and proteins (cyclodextrin affinity chromatography) (50).
Thus, this lower efficiency of incorporation does not represent a
serious limitation for applications with these analogs even if the
goal is to produce proteins or peptides that incorporate these pho-
toactive isoprenoids.
inhibitor of the reaction between FPP and the peptide substrate
N-dansyl-GCVIA. An IC50 value of 30 lM (see Figure 10) was
obtained for the inhibition of ScPFTase by 4. That value is 1.7-fold
lower than that for 3 but still significantly higher than that
observed for 2 (0.7 lM) (5); these data show how sensitive the
enzyme is to even small perturbations in the isoprenoid structure.
This result is perhaps not surprising because both the first and the
second isoprenoid units from FPP undergo conformational changes
as the enzyme proceeds from the ternary complex to the product
complex (51).
0.04
0.03
0.02
0.01
0
0
100
200
300
400
[4] (µM)
Figure 10: Inhibition of Saccharomyces cerevisiae protein farne-
syltransferase-catalyzed farnesylation of N-dansyl-GCVIA by dihydro-
ester 4. Reactions contained 2.0 lM farnesyl diphosphate, 2.0 lM
N-dansyl-GCVIA, and 4 at varying concentrations and were moni-
tored using a continuous spectrofluorometric assay. Each point is
the average of three determinations with the error bars indicating
the standard error for each measurement.
Because dihydroester 4 appeared to be a significantly slower sub-
strate than FPP for ScPFTase, the analog was evaluated as an
Chem Biol Drug Des 2010; 75: 51–67
61

Hovlid et al.
A
B
C
Figure 11: Analysis of photolabeling of purified farnesyl diphosphate (FPP)-utilizing enzymes with dihydroester [32P]4. For all samples,
photolysis reactions were fractionated by SDS–PAGE followed by phosphor imaging analysis to allow visualization of the radiolabeled pro-
teins. In each case, Lane 1 contains enzyme irradiated in the presence of [32P]4 while Lane 2 contains enzyme irradiated in the presence of
[32P]4 and FPP (substrate). Where shown, Lane 3 contains [32P]4 incubated in the presence of protein without irradiation. Panel A: Analysis of
photolabeling of purified protein farnesyl transferase. Panel B: Analysis of photolabeling of purified E. coli farnesyl diphosphate synthase.
Panel C: Analysis of photolabeling of purified Nostoc sp. strain PCC7120 sesquiterpene synthase.
A
B
Photolysis experiments with dihydroester 4
To compare the properties of dihydroester 4 and amide 3, com-
pound 4 was evaluated for its ability to label ScPFTase. UV irradia-
tion of ScPFTase in the presence of 15 lM [32P]4 at 254 nm for
1 min resulted in preferential labeling of the 43 kDa b subunit (see
Figure 11A, Lane 1). Inclusion of 30 lM FPP in the reaction mixture
gave substantial protection from labeling, reducing the amount of
crosslinking to 20% of that obtained in the absence of FPP (Lane
2); no labeling was observed in unphotolyzed reaction mixtures
(Lane 3). This selective labeling of the b-subunit is similar to what
is observed in experiments with 2 and 3. We next wanted to
explore the ability of 4 to label other FPP-utilizing enzymes to gain
insight into the scope of this probe. Thus, E. coli farnesyldiphos-
phate synthase (EcFPPSase) was irradiated in the presence of
[32P]4, and the reaction analyzed in a manner analogous to that
employed above with ScPFTase. The results of this experiment are
shown in Figure 11B; Lane 1 shows significant labeling of the pro-
tein while Lane 2 shows a decrease in labeling (29% of the label-
ing observed in Lane 1) when the photolysis was performed in the
presence of both [32P]4 and FPP as a competitor. Similar results
were obtained in labeling experiments with a germacrene-produc-
ing, sesquiterpene synthase from a cyanobacterial source, Nostoc
sp. strain PCC7120 (NoSTSase). Lane 1 (Figure 11C) illustrates the
labeling of the enzyme by [32P]4 while Lane 2 shows the results
when the photolysis was performed in the presence of both [32P]4
and FPP; a significant (although not as great as for the other
enzymes studied) decrease in labeling (58% of the labeling
observed in Lane 1) was again observed when the reaction was
carried out in the presence of the competitor (FPP). Finally, to evalu-
ate the specificity of [32P]4, photolabeling was performed on a com-
plex mixture of proteins containing ScPFTase present at low
concentration. We routinely purify ScPFTase by expressing the
genes in E. coli; based on activity assays, we estimate that ScPF-
Tase is present in crude soluble extract as 1% of the total protein.
Figure 12: Selective photolabeling of ScyPFTase in crude E. coli
extract using dihydroester 4. Analysis of photolabeling of Saccharo-
myces cerevisiae protein farnesyltransferase (ScPFTase) with [32P]4
by SDS–PAGE. Lanes 1 and 1¢: Molecular weight standards. Lanes
2 and 2¢: Crude ScPFTase irradiated in the presence of [32P]4. Lanes
3 and 3¢: Crude ScPFTase irradiated in the presence of [32P]4 and
farnesyl diphosphate (substrate). Lanes 4 and 4¢: Sample containing
crude yPFTase and [32P]4, no UV irradiation. Panel A: Sypro Orange-
stained proteins. Panel B: Radiolabeled proteins visualized via phos-
phor imaging analysis.
Hence, we considered this protein mixture to be a useful bench-
mark for examining the specificity of these probes. Accordingly,
crude E. coli extract containing ScPFTase was photolyzed in the
presence of 5.0 lM [32P]4; results of those experiments are shown
in Figure 12. Lane 2 shows the large number of different proteins
present in E. coli crude extract while lane 2¢ reveals only one major
photocrosslinked product corresponding to the ScPFTase b-subunit
and a few additional faint bands. As was noted earlier with photo-
lysis reactions employing purified ScPFTase, inclusion of FPP in the
62
Chem Biol Drug Des 2010; 75: 51–67

DATFP-Containing Photoactive FPP Analogs
photolysis mixture results in almost complete elimination of phot-
olabeling (Lane 3¢). Thus, it appears that [32P]4 can selectively label
ScPFTase selectively even in the presence of a large number of
other proteins.
the positioning of the diphosphate groups were observed in the
docked R-poses compared to the docked S-poses. Collectively,
these docking experiments predict that dihydroester 4 binds to
RnPFTase in the FPP binding site in a fashion similar to FPP
although the significant variations observed in the docked struc-
tures make a specific conformation difficult to assign.
Docking analysis of dihydroester 4 with
RnPFTase
As noted earlier, excellent selectivity of labeling was obtained
with dihydroester 4. However, it is clear that 4 binds to ScPF-
Tase with significantly less affinity than does FPP; a rough esti-
mation of this difference can be obtained from a comparison of
the IC50 of 4 reported here for ScPFTase (IC50 = 30 lM) versus
the KD of FPP for PFTase (KD = 75 nM) (52). To gain insight into
why 4 might bind to ScPFTase with attenuated affinity, docking
experiments were performed in which the two enantiomers of
dihydroester 4 were docked into the structure of RnPFTase. For
each enantiomer, an ensemble of poses was obtained; inspection
of those structures indicated that the diazoester moiety was
twisted significantly from a planar conformation. Consequently,
the top five poses for each enantiomer were further optimized
by QM-MM calculations. Interestingly, the rank order (based on
energy) of the structures obtained following QM-MM energy min-
imization was different than the order obtained directly from the
docking (based on docking scores). In all subsequent discussion,
the term 'docked' poses refers to docked poses following QM-
MM energy minimization. In general, there was greater variation
in the docked R-poses (Figure 13A) compared to the docked S-
poses (Figure 13B) with the greatest variation occurring in the
position of the DATFP group. The best structures for each of the
two enantiomers preserved the 180 ꢀ dihedral angle between
the DATFP-carbonyl and azide groups. Significant deviations in
Crystallographic analysis of dihydroester 4
bound to RnPFTase
Because the docking experiments described earlier suggested that
compound 4 would bind in the active site of PFTase in a similar
manner as FPP, we sought to confirm this by obtaining a structure
of the analog bound to RnPFTase; hence, 4 was soaked into crys-
tals of RnPFTase and the structure determined by X-ray crystallogra-
phy. Comparison of the protein in the structure of the RnPFTase:4
complex and the RnPFTase:FPP:SCH66336 ternary complex shows a
0.2 ꢀ r.m.s. deviation for 667 of the 716 C-alpha protein atoms,
indicating that no significant domain movements have occurred
(36,53). The most significant side chain movement close to 4 is
Tyr166a, which rotates 75ꢀ degrees about chi1 resulting in a 5.5ꢀ
movement of the tyrosine hydroxyl (Figure 14C). The electron den-
sity resulting from the presence of 4 is shown in Figure 14A;
because it was not possible to unambiguously determine whether
one or both enantiomers were bound, models for both were fit into
the density. A superposition of these models with the previously
determined structure for bound FPP is presented in Figure 14B
along with a view of the enzyme active site showing both the ana-
log, FPP and the protein surface (Right). In general, the analog (4)
mimics FPP well. The diphosphate group and first isoprenoid units
of 4 closely mirror the corresponding positions in FPP although C-1
in the analog is displaced 0.2 ꢀ for the S-enantiomer and 0.9 ꢀ for
A
B
D
C
Figure 13: Results of docking experiments with dihydroester 4 and RnPFTase. Top Left (A): Top 3 poses for the R enantiomer of 4 (line
representations) docked into RnPFTase. The position of farnesyl diphosphate (FPP) (stick representation) bound to RnPFTase determined via
crystallography (pdb code: 1JCR) is shown for comparison. Top Right (B): Top 3 poses for the S enantiomer of 4 docked into RnPFTase. Bottom
Left (C): Comparison of highest-scoring docked pose for the R enantiomer of 4 (line representation) with the structure of the protein-bound
R enantiomer determined via crystallography (stick representation, green carbons) and FPP (stick representation, yellow carbons). Bottom Right
(D): Comparison of highest-scoring docked pose for the S enantiomer of 4 (line representation) with the structure of the protein-bound
S enantiomer determined via crystallography (stick representation) and FPP (stick representation, yellow carbons). Colors: C (line representa-
tions: grey, stick representations: yellow [FPP], green [crystallographic 4), F (line representations: green, stick representations: white), N (blue),
O (red), P (line representations: purple, stick representations: orange). As noted in the text, the term 'docked' pose used here refers to the
docked poses that have been subjected to energy minimization.
Chem Biol Drug Des 2010; 75: 51–67
63

Hovlid et al.
A
B
C
Y166a
Figure 14: Structure of dihydroester 4 bound to RnPFTase determined by X-ray crystallography. (A) Electron density for dihydroester 4
(mesh) contoured at 1.0 r. The structures of the two enantiomers of 4 (shown in stick representations) are fit within the electron density.
(B) Structures of the two enantiomers of 4 superimposed with the structure of farnesyl diphosphate (FPP) (green) bound to RnPFTase.
(C) Active site of RnPFTase showing structures of the two enantiomers of 4 superimposed with the structure of FPP (green). The insert shows
the difference in conformation for Y166a and H210a between the structures of 4 and FPP. Colors: C (yellow), N (blue), O (red), a-subunit
(grey), b-subunit (light blue), Zn (orange).
the R-enantiomer. Additionally, the DATFP group of 4 is positioned
similarly to the third isoprenoid unit of FPP although it is also dis-
placed approximately 1.0 ꢀ to one side. The most significant differ-
ence lies in the position of the second isoprenoid unit within the
analog. Introduction of the sp3 hybridized centers at C-6 and C-7
along with the ester linkage present in the analog results in a sig-
nificant rotation of the second isoprenoid unit; however, one note
of caution that should be acknowledged concerning the structure of
the middle isoprenoid element is that the electron density in that
region is not well defined (see Figure 14A) making definitive struc-
tural assignment difficult. It is interesting to compare the conforma-
tion of 4 obtained from crystallography with the best poses
obtained from docking. A comparison for the R-enantiomer of 4 is
shown in Figure 13C and for the S-enantiomer in Figure 13D. The
best docked pose of S-4 is closer to the conformation of crystallo-
graphically determined FPP than to crystallographically determined
S-4; in contrast, the best docked pose of R-4 differs significantly
from the crystallographically determined conformations of both FPP
and R-4. These results suggest that while the docking experiments
do correctly predict the overall orientation of the bound analog,
they are less accurate in predicting the precise conformation of the
bound compound. This may reflect that fact that no specific hydro-
gen bonds or polar interactions occur between the protein and the
ligand (except for the diphosphate moiety). Hence, the energetics
are dominated by non-directional Van der Waals interactions.
Comparison of electrostatic potential
calculations of FPP and dihydroester 4
While the docking calculations described earlier in conjunction with
the crystallographic experiments demonstrate that compound 4 can
be accommodated into the active site of PFTase, they do not
A
B
C
D
Figure 15: CPK models and molecular electrostatic potential (MEP) maps calculated for farnesyl diphosphate (FPP) and dihydroester 4.
Above: CPK model (A) and MEP (B) for FPP. Below: CPK model (C) and MEP (D) for 4. For MEP maps, potentials were plotted on a surface of
constant density (density = 0.02). Electronegative areas are shown in red, and electropositive areas are shown in blue.
64
Chem Biol Drug Des 2010; 75: 51–67

DATFP-Containing Photoactive FPP Analogs
explain why the affinity of 4 for the enzyme is so much weaker
than that of FPP. To gain insight into that question, Density func-
tional theory (DFT) calculations were employed to calculate the
molecular electrostatic potential maps for FPP and 4 presented in
Figure 15 (Panels B and D); standard Corey-Pauling-Koltun (CPK)
models are also provided in Figure 15 (Panels A and C) and serve
to illustrate that the overall size of the DATFP photophore (compare
Figure 15A and 15C) is similar to that of the third isoprene unit in
FPP. Clearly, based on geometric considerations alone, 4 and FPP
are quite similar. However, inspection of the molecular electrostatic
potential maps shows how much these molecules differ (compare
Figures 15B and 15D). Note the regions of significant negative
potential (red and yellow) in the surface of 4 because of the car-
bonyl oxygen and fluorine atoms. Similarly, significant positive
potential is observed because of the presence of the diazo group
Acknowledgments
This research was supported by the National Institutes of Health
Grants GM58442 (M.D.D.) and GM080299 (C.S.-D.) and by a Fellow-
ship from the Beckman Scholars Program (M. L. H.). This work was
carried out in part using computing resources at the University of
Minnesota Supercomputing Institute, instrumentation from the Uni-
versity of Minnesota Center for Mass Spectrometry and Proteomics,
and facilities of the Masonic Cancer Center.
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In an effort to create photoactive analogs of FPP with enhanced
stability, two new compounds have been prepared. Probe 3 con-
tains an amide-linked DATFP moiety is an alternative substrate for
ScPFTase and covalently modifies a number of prenyltransferases
upon photolysis. However, 3 rearranges to a photochemically inac-
tive triazolone limiting its potential utility. Probe 4 employs an alkyl
ester in lieu of the allylic ester found in 2; that substitution renders
the linkage between the DATFP group and the isoprenoid signifi-
cantly more stable to acidic and basic conditions. Compound 4 is
an alternative substrate for ScPFTase. Computer assisted docking
and crystallographic analysis indicate that 4 binds to RnPFTase in a
manner similar (although not identical) to the natural substrate FPP;
those suggest that 4 is a reasonable surrogate for a farnesyl group.
Photolysis of 4 in the presence of a several different types of FPP-
utilizing enzymes results in crosslinking; of particular note, photoly-
sis of 4 in crude E. coli extract expressing ScPFTase results in
selective labeling of that enzyme even though it is present as
approximately 1% of the total protein. We are currently employing
4 in experiments designed to identify proteins involved in latex and
sesquiterpene biosynthesis. Finally, the ability to introduce 4 into
peptides, established here, suggests that it should be possible to
prepare peptides and proteins that incorporate photoactive isopre-
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these experiments in living cells (57). Efforts to accomplish this are
currently under way.
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