Farnesyl diphosphate analogues with Aryl moieties are efficient alternate substrates for protein farnesyltransferase

Article abstract of DOI:10.1021/bi3011362

Farnesylation is an important post-translational modification essential for the proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTas

Full text of DOI:10.1021/bi3011362

Article
pubs.acs.org/biochemistry
Farnesyl Diphosphate Analogues with Aryl Moieties Are Efficient
Alternate Substrates for Protein Farnesyltransferase
Thangaiah Subramanian,† June E. Pais,§,∥ Suxia Liu,†,⊥ Jerry M. Troutman,†,@ Yuta Suzuki,§
Karunai Leela Subramanian,† Carol A. Fierke,§ Douglas A. Andres,† and H. Peter Spielmann*,†,‡
†Department of Molecular and Cellular Biochemistry and ‡Department of Chemistry, Markey Cancer Center, and Kentucky Center
for Structural Biology, University of Kentucky, Lexington, Kentucky 40536-0084, United States
§Departments of Chemistry and Biological Chemistry, University of Michigan, 930 North University, Ann Arbor, Michigan
48109-1055, United States
S
* Supporting Information
ABSTRACT: Farnesylation is an important post-translational modification essential for the proper localization and function of
many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein
farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual
isoprene moieties to examine some of the structural and electronic properties of the transfer of an analogue to the peptide
catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple-turnover
activity depends on the analogue structure. Analogues in which the first isoprene is replaced with a benzyl group and an analogue
in which each isoprene is replaced with an aryl group are good substrates. In sharp contrast with the steady-state reaction, the
single-turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased
chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single-turnover rate
constant for alkylation does depend on the Ca1a2X peptide sequence. These results suggest that the isoprenoid transition-state
conformation is preferred over the inactive E·FPP·Ca1a2X ternary complex conformation. Furthermore, these data suggest that
the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the
active site binding pocket and therefore might be a useful site for the design of novel inhibitors.
reaction mechanism may provide useful insights for developing
PFIs.17
umerous membrane-associated proteins require post-
Ntranslational farnesylation catalyzed by protein farnesyl-
transferase (FTase) for proper function. FTase catalyzes the
transfer of a 15-carbon farnesyl group from farnesyl
diphosphate 1 (FPP) to a conserved cysteine in the C-terminal
Ca1a2X motif of a range of proteins, including the oncoprotein
H-Ras (C refers to the cysteine, a to any aliphatic amino acid,
and X to any amino acid).1−8 The covalently attached
isoprenoid increases the protein’s hydrophobicity, promotes
membrane localization, and enhances protein−protein inter-
actions.9−11 The clinical development of farnesyltransferase
inhibitors (FTIs) as anticancer therapeutics has been hampered
by alternative prenylation of some FTase substrates by the
related prenyltransferase geranylgeranyl transferase type I
(GGTase I) when FTase activity is limited.12 This has spurred
the development of alternative FTase-transferable lipids
incapable of supporting normal prenyl group functions [prenyl
function inhibitors (PFIs)].13−16 Defining the isoprenoid
chemical features that affect each step of the transferase
The FTase kinetic mechanism is complex (Figure 1), and the
enzyme rarely exists in the free, unbound form during the
catalytic cycle. The FTase kinetic mechanism is thought to be
functionally ordered, and efficient catalysis occurs when FPP
first binds to the enzyme forming the enzyme·FPP complex
(E·FPP) followed by Ca1a2X substrate association in which the
active site zinc ion directly coordinates the cysteine thiolate to
form a ternary complex (E·FPP·Ca1a2X) that is inactive based
on the crystal structure.18−21
Models based on structural, mutagenesis, and computational
studies in which a conformational rearrangement of the first
two FPP isoprene units translocates the reactive isoprenoid C1
5.4 Å across the active site within reacting distance (2.4 Å) of
the thiolate to form an active substrate conformation
Received: August 22, 2012
Published: September 18, 2012
© 2012 American Chemical Society
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reactivity to investigate isoprenoid molecular features that
contribute to substrate binding, recognition, and catalysis by
FTase (Figure 3 and Table 1). The analogues vary with respect
to the size and electronic properties of one, two, or all three
isoprene groups. We used analogues that stabilize the
carbocationic character of C1 to further examine the
dissociative character of the transition state.
We have characterized FTase kinetic properties with these
analogues and describe unexpected results with regard to their
effect on catalysis. We measured single-turnover (STO) rate
constants to examine the role of intrinsic chemical reactivity
and steric bulk on the FPP conformational rearrangement and
the chemical step [kobs (Figure 1)]. Surprisingly, the STO rate
constant is the same as that of FPP for all analogues tested,
indicating that FTase-catalyzed peptide alkylation is very
tolerant of the increased steric bulk in all three isoprene
positions and that the rate of peptide alkylation is not limited
by the chemical reactivity of the first isoprene in FPP. Within
the series tested here, the hydrophobicity of the analogues does
not limit the observed rate of thioether formation. However,
the rate of peptide alkylation is dependent on the sequence of
the C-terminal residue in the Ca1a2X motif. The steady-state
data show that while some analogues are fairly good FTase
substrates, others have kcat/KMisoprenoid values that are decreased
up to 475-fold. These data indicate that a step subsequent to
farnesylation, such as product dissociation, is sensitive to the
structure of the farnesyl moiety.
Figure 1. Basic kinetic pathway for FTase. Path A depicts FPP-
stimulated product release and path B Ca1a2X peptide-stimulated
product release. The dashed box encloses rate constants included in
the observed single-turnover rate constant (kobs).
(E·FPP·Ca1a2X*) have been proposed.22−24 A variety of
experiments suggest that the transfer of a farnesyl group to
Ca1a2X thiol proceeds by a nucleophilic (SN2, associative)
mechanism with electrophilic (SN1, dissociative) character,
proceeding through a highly polar “exploded” transition state
with considerable (C−1)−O bond cleavage and modest (C1)−
S bond formation, partial positive charge on C−1, and partial
negative charges on the zinc-coordinated sulfur and on the
Mg2+-coordinated diphosphate leaving group (Figure 2).25−27
EXPERIMENTAL PROCEDURES
■
Materials and Methods. The peptides TKCVIM, GCVLS,
and dansylated GCVLS were synthesized and purified by high-
pressure liquid chromatography to more than 90% purity by
Sigma-Genosys (The Woodlands, TX), and the molecular
masses of peptides were confirmed by electrospray mass
spectrometry. 7-Diethylamino-3-({[(2-maleimidyl)ethyl]-
amino}carbonyl)coumarin (MDCC) was purchased from
Molecular Probes (Eugene, OR). Farnesyl diphosphate
(FPP), purine nucleoside phosphorylase (PNPase), 7-methyl-
guanosine (MEG), and inorganic pyrophosphatase from baker's
yeast (PPiase) were purchased from Sigma-Aldrich (St. Louis,
MO). All other chemicals used were reagent grade.
Figure 2. FTase-catalyzed peptide alkylation by FPP. The transition
state is stabilized by positive charge delocalization on the allylic and
benzylic systems of FPP and the analogues.
All synthetic organic reactions except for resin preparation
were performed in PTFE tubes using a Quest 210 apparatus
manufactured by Argonaut Technologies. All RP-HPLC was
performed on an Agilent 1100 HPLC system equipped with a
microplate autosampler, a diode array, and a fluorescence
detector. N-Dansyl-GCVLS was purchased from Peptidogenics
(San Jose, CA). Spectrofluorometric analyses were performed
in 96-well flat bottom, nonbinding surface, black polystyrene
plates (Corning, excitation wavelength of 340 nm, emission
wavelength of 505 nm with a 10 nm cutoff) with a SpectraMax
GEMINI XPS fluorescence well-plate reader. Absorbance
readings were determined using a Cary UV−vis spectropho-
tometer. All assays were performed at minimum in triplicate
where the average values are reported with a one standard of
deviation error. Reaction temperature refers to the external
bath. All solvents and reagents were purchased from VWR (EM
Science-Omnisolv high purity) and Aldrich respectively, and
used as received. Merrifield-Cl resin was purchased from
Argonaut Technologies. Synthetic products were purified by
silica gel flash chromatography (EtOAc/hexane) unless
otherwise noted. RP-HPLC purification of lipid diphosphates
was conducted using a Varian Dynamax, 10 μm, 300 Å, C-18
Recent computational studies suggest that the transition-state
structure may depend on the structure of the peptide
substrate.28 The final step in the reaction cycle is product
release, which is stimulated by binding of either a Ca1a2X
peptide (path B) or FPP (path A) to form either the E·peptide
or E·FPP complex.29 The product release pathway depends on
the sequence of the peptide.22,23,30
Because FPP is capable of binding to the free enzyme, the
E·Ca1a2X complex, and the E·product complex, a better
understanding of the binding interactions between the active
site and FPP is needed and is of particular interest for the
design of PFIs. FPP analogues have been employed to study the
FTase mechanism and the interactions among the isoprenoid,
enzymes, and the Ca1a2X peptide as well as the biological
function of the post-translational modification. The reactivity of
FPP analogues depends both on the isoprenoid structure and
on the peptide substrate sequence.15,31−33
We report the synthesis and reactivity of FPP analogues with
a range of steric demands and increased intrinsic chemical
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Figure 3. FPP and analogue structures.
Table 1. Kinetic Constants for the FTase-Catalyzed Reaction of FPP and Analogues with Ca1a2X Peptides
a
b
steady-state kinetic parameters for alkylation of dns-GCVLS
single-turnover rate constant kobs (s−1
)
isoprenoid
kcat (s−1
)
KM (μM)
kcat/KM (μM−1 s−1
192
)
GCVLS
TKCVIM
TKCVIF
0.26
1 (FPP)
0.29 0.01
0.0021 0.0001
0.43 0.01
1.5 0.2
0.49 0.08
6.0 0.7
4
3.8
4.1
4.0
3.1
3.1
3.7
4.0
2.8
3.5
4.2
4.0
3.8
4.0
3.9
4.0
6.8
5.8
5.4
4.3
4.7
5.0
6.0
4.1
5.0
6.4
6.3
6.2
6.4
6.0
6.2
c
2
4.3 0.5
72 16
ND
c
3
ND
d
c
c
4
0.03 0.002
ND
ND
0.21
0.22
d
c
c
5
0.20 0.01
ND
ND
c
6
0.12 0.01
5
2
30 10
ND
7
0.017 0.003
0.55 0.09
30
5
0.28
0.24
d
c
c
8
0.05 0.01
ND
ND
c
9
0.22 0.04
5
2
40 20
ND
c
c
c
10
11
12
13
14
15
>0.05
>15
1.4 0.2
0.4 0.01
ND
c
0.0013 0.0001
3.1 0.8
ND
ce
,
c
c
ND
ND
ND
0.29
c
0.0031 0.0001
0.053 0.002
0.29 0.01
0.2 0.1
0.5 0.1
1.5 0.2
15
0
ND
c
110 20
ND
c
300 100
ND
a
Steady-state experiments were performed using dansylated peptides and measuring the time-dependent change in fluorescence.44 Final solutions
contained 25 nM FTase, 5 μM dns-GCVLS, varying concentrations (1−20 μM) of FPP/analogue, 50 mM Heppso (pH 7.8), 5 mM MgCl2, and 2
b
mM TCEP. Assays were conducted at 25 °C. Single-turnover rate constant, measured using an MDCC-PBP/PPiase58 assay on a stopped-flow
fluorimeter. Measurements were repeated two or three times with a <10% error. Final solutions contained 400 nM FTase, 100 nM FPP/analogue, 25
μM peptide, 5 μM MDCC-PBP, 34 units/mL PPiase, 50 mM Heppso (pH 7.8), 5 mM MgCl2, and 2 mM TCEP. Assays were conducted at 25 °C.
c
d
a
Not determined. Steady-state experiments were performed as described in footnote except that kcat was determined by varying the dns-GCVLS
e
concentration (1−10 μM) at a saturating analogue concentration (20 μM). The reaction was too slow to measure by the steady-state assay.
(10 mm × 250 mm) column and eluted with a gradient mobile
phase and a flow rate of 4 mL/min (90% A from 0 to 3 min, 0%
A from 3 to 18 min, 0% A from 18 to 20 min, and 90% A from
20 to 23 min) and monitored at 254 and 210 nm. 1H NMR and
1
13C NMR spectra of alcohols were obtained in CDCl3 and H
and 31P NMR spectra of diphosphates in D2O with a Varian
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Inova spectrometer operating at 400 MHz (1H) and 161.8
MHz (31P). Chemical shifts are reported in parts per million
from the CDCl3 internal peak at 7.27 ppm for 1H (TSP, 0 ppm
for 1H; H3PO4 as an external reference, 0 ppm for 31P). ESI-MS
was performed at the University of Kentucky Mass Spectra
Facility. Positive and negative ion electrospray ionization (ESI)
mass spectra were recorded on a Thermo Finnigan LCQ
instrument with sample introduction by direct infusion.
General Procedure for Mitsunobu Coupling (18a, 18b,
22a−c, 25a−g, 29a, and 29b). DEAD (40% in toluene, 1.2
equiv) was added dropwise to a stirred solution of alcohol (1
equiv), phenol (1.2 equiv), and Ph3P (1.2 equiv) in THF at 0
°C and the mixture stirred for 1 h. After the reaction mixture
had been allowed to warm to room temperature and stirred
overnight, it was diluted with saturated NaHCO3, concentrated,
and extracted with CH2Cl2 (twice). The organic extracts were
dried (MgSO4), filtered, and concentrated. Silica gel column
chromatography of the oily residue gave the desired
compounds.
1H), 4.30 (m, 1H), 4.41 (s, 2H), 4.61 (t, J = 3.6 Hz, 2H), 4.92
(s, 2H), 5.73 (t, J = 6.0 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H),
6.95−7.10 (m, 5H), 7.15−7.20 (2H).
(E)-2-({3-Methyl-4-[3-(phenoxymethyl)phenoxy]but-2-en-
1-yl}oxy)tetrahydro-2H-pyran 25b. One hundred milligrams
1
of 23a gave 117 mg of 25b (87% yield): H NMR (CDCl3) δ
1.49−1.59 (m, 5H), 1.72 (s, 3H), 1.77−1.85 (m, 1H), 3.46−
3.52 (m, 1H), 3.83−3.88 (m, 1H), 4.06−4.12 (m, 1H), 4.26−
4.31 (m, 1H), 4.40 (s, 2H), 4.60 (t, J = 4.0 Hz, 1H), 5.00 (s,
2H), 5.72−5.75 (m, 1H), 6.79−6.85 (m, 1H), 6.90−6.98 (m,
4H), 7.20−7.28 (m, 4H).
(E)-2-[(4-{3-[(4-Fluorophenoxy)methyl]phenoxy}-3-meth-
ylbut-2-en-1-yl)oxy]tetrahydro-2H-pyran 25c. One hundred
1
milligrams of 23a gave 117 mg of 25c (82% yield): H NMR
(CDCl3) δ 1.48−1.69 (m, 5H), 1.77−1.81 (m, 1H), 1.78 (s,
3H), 3.47−3.50 (m, 1H), 3.83−3.88 (m, 1H), 4.08−4.11 (m,
1H), 4.28 (dd, J = 6.4, 12.4 Hz, 1H), 4.41 (s, 2H), 4.60 (t, J =
3.6 Hz, 2H), 4.93 (s, 2H), 5.73 (t, J = 6.0 Hz, 1H), 6.90 (d, J =
8.8 Hz, 2H), 7.14−7.18 (m, 3H), 7.30 (d, J = 8.8 Hz, 2H).
(E)-2-[(4-{3-[(3-Fluorophenoxy)methyl]phenoxy}-3-meth-
ylbut-2-en-1-yl)oxy]tetrahydro-2H-pyran 25d. One hundred
(E)-2-{[3-Methyl-4-(3-phenoxyphenoxy)but-2-en-1-yl]oxy}-
tetrahydro-2H-pyran 18a. One gram of 16 gave 1.6 g of 18a
1
1
(84% yield): H NMR (CDCl3) δ 1.43−1.59 (m, 5H), 1.66−
milligrams of 23a gave 117 mg of 25d (81% yield): H NMR
1.72 (m, 1H), 1.76 (s, 3H), 1.77−1.85 (m, 1H), 3.47−3.50 (m,
1H), 3.83−3.88 (m, 1H), 4.06−4.12 (m, 1H), 4.26−4.31 (m,
1H), 4.40 (s, 2H), 4.61 (t, J = 3.6 Hz, 1H), 4.75 (s, 2H), 5.74
(t, J = 6.4 Hz, 1H), 6.79−6.81 (m, 2H), 6.88−6.98 (m, 3H),
7.20−7.28 (m, 4H).
(CDCl3) δ 1.48−1.69 (m, 5H), 1.77−1.81 (m, 1H), 1.76 (s,
3H), 3.46−3.51 (m, 1H), 3.83−3.88 (m, 1H), 4.06−4.11 (m,
1H), 4.26−4.31 (m, 1H), 4.40 (s, 2H), 4.60 (t, J = 4.0 Hz, 2H),
4.97 (s, 2H), 5.72−5.75 (m, 1H), 6.72−6.75 (m, 1H), 6.83−
6.97 (m, 6H), 7.24−7.27 (m, 1H).
(E)-2-{[3-Methyl-4-(4-phenoxyphenoxy)but-2-en-1-yl]oxy}-
tetrahydro-2H-pyran 18b. One gram of 16 gave 1.72 g of 18b
(E)-2-({4-[2-Ethoxy-5-(phenoxymethyl)phenoxy]-3-methyl-
but-2-en-1-yl}oxy)tetrahydro-2H-pyran 25e. One hundred
1
(91% yield): H NMR (CDCl3) δ 1.48−1.60 (m, 4H), 1.67−
1
milligrams of 23c gave 77 mg of 25e (77% yield): H NMR
1.74 (m, 1H), 1.76 (s, 3H), 1.79−1.84 (m, 1H), 3.46−3.51 (m,
1H), 3.83−3.88 (m, 1H), 4.07−4.12 (m, 1H), 4.20−4.32 (m,
1H), 4.38 (s, 2H), 4.60 (t, J = 3.2 Hz, 1H), 5.72−5.75 (m, 1H),
6.84−6.95 (m, 6H), 6.99−7.03 (m, 1H).
(CDCl3) δ 1.41 (t, J = 7.2 Hz, 3H), 1.48−1.69 (m, 5H), 1.76
(s, 3H), 1.77−1.81 (m, 1H), 3.46−3.51 (m, 1H), 3.82−3.88
(m, 1H), 4.06−4.11 (m, 1H), 4.26 (dd, J = 6.4, 12.4 Hz, 1H),
4.46 (s, 2H), 4.58 (t, J = 2.8 Hz, 1H), 4.93 (s, 2H), 5.70−5.73
(m, 1H), 6.84−6.96 (m, 6H), 7.23−7.28 (m, 2H).
(E)-2-[(4-{2-Ethoxy-5-[(2-fluorophenoxy)methyl]phenoxy}-
3-methylbut-2-en-1-yl)oxy]tetrahydro-2H-pyran 25f. One
hundred milligrams of 23c gave 120 mg of 25f (80% yield):
1H NMR (CDCl3) δ 1.42 (t, J = 7.2 Hz, 3H), 1.48−1.69 (m,
5H), 1.76 (s, 3H), 1.77−1.81 (m, 1H), 3.46 (dd, J = 4.4, 10.4
Hz, 1H), 3.82−3.87 (m, 1H), 4.05−4.12 (m, 3H), 4.25 (dd, J =
6, 12.4 Hz, 1H), 4.46 (s, 2H), 4.58 (t, J = 2.4 Hz, 1H), 5.02 (s,
2H), 5.71 (t, J = 7.6 Hz, 1H), 6.95−7.10 (m, 4H), 6.82−6.90
(m, 3H).
(E)-3-({2-Methyl-4-[(tetrahydro-2H-pyran-2-yl)oxy]but-2-
en-1-yl}oxy)benzaldehyde 22a. Five hundred milligrams of 16
1
gave 630 mg of 22a (81% yield): H NMR (CDCl3) δ 1.46−
1.60 (m, 4H), 1.60−1.72 (m, 1H), 1.76 (s, 3H), 1.77−1.81 (m,
1H), 3.45−3.51 (m, 1H), 3.82−3.87 (m, 1H), 4.06−4.11 (m,
1H), 4.26−4.31 (m, 1H), 4.45 (s, 2H), 4.60 (t, J = 6.8 Hz, 1H),
5.73−5.77 (m, 1H), 7.15−7.18 (m, 1H), 7.36−7.37 (m, 1H),
7.40−7.44 (m, 2H), 9.79 (s, 1H).
(E)-4-({2-Methyl-4-[(tetrahydro-2H-pyran-2-yl)oxy]but-2-
en-1-yl}oxy)benzaldehyde 22b. Five hundred milligrams of 16
1
gave 619 mg of 22b (69% yield): H NMR (CDCl3) δ 1.42−
(E)-2-[(3-Methyl-4-{3-[(4-phenoxyphenoxy)methyl]-
phenoxy}but-2-en-1-yl)oxy]tetrahydro-2H-pyran 25g. One
hundred milligrams of 23c gave 123 mg of 25g (78% yield):
1H NMR (CDCl3) δ 1.45−1.60 (m, 6H), 1.65−1.75 (m, 2H),
1.78 (s, 3H), 1.79−1.82 (m, 1H), 3.44−3.50 (m, 1H), 3.82−
3.90 (m, 1H), 4.06−4.12 (m, 1H), 4.26−4.34 (m, 1H), 4.41 (s,
2H), 4.58−4.62 (m, 1H), 4.98 (s, 2H), 5.72−5.78 (m, 1H),
6.78−6.80 (m, 1H), 6.83−7.06 (m, 8H), 7.20−7.30 (m, 4H).
(E)-3-[(3,7-Dimethylocta-2,6-dien-1-yl)oxy]benzaldehyde
1.55 (m, 7H), 1.57−1.70 (m, 1H), 1.75 (s, 3H), 1.77−1.84 (m,
1H), 3.44−3.49 (m, 1H), 3.80−3.86 (m, 1H), 4.08−4.18 (m,
3H), 4.24−4.29 (m, 1H), 4.54 (s, 2H), 4.57 (t, J = 7.6 Hz, 1H),
5.71−5.75 (m, 1H), 6.91−7.00 (m, 1H), 7.34−7.38 (m, 2H),
9.79 (s, 1H).
(E)-4-Ethoxy-3-({2-methyl-4-[(tetrahydro-2H-pyran-2-yl)-
oxy]but-2-en-1-yl}oxy)benzaldehyde 22c. Five hundred milli-
1
grams of 16 gave 690 mg of 22c (85% yield): H NMR
(CDCl3) δ 1.46−1.60 (m, 4H), 1.61−1.72 (m, 1H), 1.76 (s,
3H), 1.77−1.84 (m, 1H), 3.45−3.51 (m, 1H), 3.81−3.87 (m,
1H), 4.05−4.10 (m, 1H), 4.27−4.31 (m, 1H), 4.47 (s, 2H),
4.59 (t, J = 3.6 Hz, 1H), 5.72−5.76 (m, 1H), 6.98 (d, J = 8.8
Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 9.84 (s, 1H).
(E)-2-[(4-{4-[(3-Fluorophenoxy)methyl]phenoxy}-3-meth-
ylbut-2-en-1-yl)oxy]tetrahydro-2H-pyran 25a. One hundred
milligrams of 23b gave 104 mg of 25a (82.5% yield): 1H NMR
(CDCl3) δ 1.40−1.70 (m, 5H), 1.77−1.81 (m, 1H), 1.78 (s,
3H), 3.47−3.50 (m, 1H), 3.83−8.88 (m, 1H), 4.08−4.12 (m,
1
29a. One gram of geraniol gave 1.6 g of 29a (95% yield): H
NMR (CDCl3) δ 1.57 (s, 3H), 1.64 (s, 3H), 1.72 (s, 3H),
2.03−2.11 (m, 4H), 4.56 (s, 2H), 4.58 (s, 2H), 5.04−5.08 (m,
1H), 5.44−5.48 (m, 1H), 7.15−7.18 (m, 1H), 7.37−7.42 (m,
3H), 9.94 (s, 1H).
(E)-4-[(3,7-Dimethylocta-2,6-dien-1-yl)oxy]benzaldehyde
1
29b. One gram of geraniol gave 1.6 g of 29b (78% yield): H
NMR (CDCl3) δ 1.57 (s, 3H), 1.64 (s, 3H), 1.72 (s, 3H),
2.02−2.14 (m, 4H), 4.60 (d, J = 6.8 Hz, 2H), 5.03−5.06 (m,
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1H), 5.43−5.46 (m, 1H), 6.97 (d, J = 8.8 Hz, 2H), 7.80 (d, J =
8.8 Hz, 2H), 9.85 (s, 1H).
3.46−3.51 (m, 1H), 3.82−3.87 (m, 1H), 4.06−4.11 (m, 1H),
4.26−4.31 (m, 1H), 4.41 (s, 2H), 4.59 (t, J = 3.6 Hz, 1H), 4.64
(d, J = 4.4 Hz, 2H), 5.72−5.75 (m, 1H), 6.80−6.92 (m, 1H),
6.90−6.92 (m, 2H), 7.21−7.25 (m, 1H).
(E)-[4-({2-Methyl-4-[(tetrahydro-2H-pyran-2-yl)oxy]but-2-
en-1-yl}oxy)phenyl]methanol 23b: 1H NMR (CDCl3) δ
1.40−1.44 (m, 3H), 1.48−1.56 (m, 5H), 1.65−1.71 (m, 1H),
1.76 (s, 3H), 3.45−3.48 (m, 1H), 3.82−3.87 (m, 1H), 4.02−
4.13 (m, 2H), 4.24−4.28 (m, 1H), 4.45 (s, 2H), 4.56−4.58 (m,
3H), 5.71 (t, J = 7.2 Hz, 1H), 6.79−6.90 (m, 4H).
(E)-[4-Ethoxy-3-({2-methyl-4-[(tetrahydro-2H-pyran-2-yl)-
oxy]but-2-en-1-yl}oxy)phenyl]methanol 23c: 1H NMR
(CDCl3) δ 1.48−1.56 (m, 5H), 1.68−1.73 (m, 2H), 1.76 (s,
3H), 1.77−1.85 (m, 1H), 3.46−3.51 (m, 1H), 3.83−3.88 (m,
1H), 4.06−4.12 (m, 1H), 4.27−4.32 (m, 1H), 4.40 (s, 2H),
4.59−4.61 (m, 3H), 5.72−5.75 (m, 1H), 6.87 (d, J = 8.8 Hz,
2H), 7.25 (d, J = 8.8 Hz, 2H).
General Procedure for THP Ether Removal. THP ether
and 5% (w/v) PPTS were stirred in dry MeOH overnight. The
reaction mixture was evaporated, taken up in ethyl acetate,
washed with saturated NaHCO3 and brine, dried (MgSO4),
filtered, and evaporated. Chromatographic purification of the
residue gave alcohol in quantitative yield.
1
(E)-3-Methyl-4-(3-phenoxyphenoxy)but-2-en-1-ol 19a: H
NMR (CDCl3) δ 1.75 (s, 3H), 4.25 (d, J = 6.8 Hz, 2H), 4.40 (s,
2H), 5.72−5.77 (m, 1H), 6.55−6.60 (m, 2H), 6.64 (ddd, J =
0.8, 2.4, 8.4 Hz, 1H), 7.00−7.03 (m, 2H), 7.08−7.12 (m, 1H),
7.18−7.22 (m, 1H), 7.30−7.35 (m, 2H).
1
(E)-3-Methyl-4-(4-phenoxyphenoxy)but-2-en-1-ol 19b: H
NMR (CDCl3) δ 1.75 (s, 3H), 4.22 (d, J = 6.8 Hz, 2H), 4.36 (s,
2H), 5.72−5.77 (m, 1H), 6.55−6.60 (m, 2H), 6.64 (ddd, J =
0.8, 2.4, 8.4 Hz, 1H), 6.85−6.90 (m, 4H), 7.00−7.03 (m, 1H),
7.08−7.12 (m, 1H), 7.30−7.35 (m, 2H).
(E)-{3-[(3,7-Dimethylocta-2,6-dien-1-yl)oxy]phenyl}-
methanol 30a: 1H NMR (CDCl3) δ 1.58 (s, 3H), 1.65 (s, 3H),
1.71 (s, 3H), 2.05−2.09 (m, 4H), 4.52 (d, J = 6.4 Hz, 2H), 4.64
(s, 2H), 5.07 (t, J = 6.8 Hz, 1H), 5.46 (t, J = 8.0 Hz, 1H), 6.81−
6.83 (m, 1H), 6.89−6.92 (m, 2H), 7.22−7.26 (m, 1H).
(E)-{4-[(3,7-Dimethylocta-2,6-dien-1-yl)oxy]phenyl}-
(E)-4-{4-[(3-Fluorophenoxy)methyl]phenoxy}-3-methyl-
but-2-en-1-ol 26a: 1H NMR (CDCl3) δ 1.78 (s, 3H), 4.20 (d,
J = 5.6 Hz, 2H), 4.42 (s, 2H), 5.05 (s, 2H), 5.78−5.80 (m, 1H),
6.81−6.90 (m, 1H), 6.90−7.10 (m, 5H), 7.28−7.35 (m, 3H).
(E)-3-Methyl-4-[3-(phenoxymethyl)phenoxy]but-2-en-1-ol
1
1
26b: H NMR (CDCl3) δ 1.78 (s, 3H), 4.20 (d, J = 5.6 Hz,
methanol 30b: H NMR (CDCl3) δ 1.58 (s, 3H), 1.65 (s,
2H), 4.42 (s, 2H), 5.05 (s, 2H), 5.78−5.80 (m, 1H), 6.81−6.90
3H), 1.71 (s, 3H), 2.04−2.11 (m, 4H), 4.51 (d, J = 7.2 Hz,
2H), 4.59 (d, J = 6.0 Hz, 2H), 5.05−5.08 (m, 1H), 5.44−5.48
(m, 1H), 6.88 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H).
General Procedure for Diphosphate Synthesis.
Ph3PCl2 (2 equiv) in dry CH3CN was added dropwise to a
cooled (0 °C) solution of the alcohol (1 equiv) in CH3CN (5
mL) and the mixture stirred for 2 h. The reaction mixture was
concentrated and filtered through a pad of silica gel. The
chlorides were sufficiently pure to proceed to the next step. [(n-
Bu)4N]3HP2O7 (5 equiv) in CH3CN was then added to the
solution of chloride in CH3CN at 0 °C, and the solution was
allowed to warm to room temperature and stirred overnight.
The reaction mixture was concentrated and washed with Et2O.
The organic extracts were discarded, and the residue was
suspended in 4 mL of ion exchange buffer [25 mM NH4HCO3
in 2% (v/v) i-PrOH/water]. The resultant white solution was
loaded onto a pre-equilibrated 6 cm × 50 cm column of Dowex
(m, 1H), 6.90−7.10 (m, 5H), 7.28−7.35 (m, 3H).
(E)-4-{3-[(4-Fluorophenoxy)methyl]phenoxy}-3-methyl-
1
but-2-en-1-ol 26c: H NMR (CDCl3) δ 1.54 (s, 3H), 1.76
(brs, 1H), 4.22 (d, J = 5.6 Hz, 2H), 4.40 (s, 2H), 4.97 (s, 2H),
5.74−5.79 (m, 1H), 6.83−7.00 (m, 7H), 7.23−7.27 (m, 1H).
(E)-4-{3-[(3-Fluorophenoxy)methyl]phenoxy}-3-methyl-
1
but-2-en-1-ol 26d: H NMR (CDCl3) δ 1.42 (t, J = 7.2 Hz,
3H), 1.77 (s, 3H), 4.07 (q, J = 6.8, 14 Hz, 2H), 4.21 (t, J = 5.6
Hz, 2H), 4.45 (s, 2H), 4.94 (s, 2H), 5.74−5.78 (m, 1H), 6.83−
6.96 (m, 6H), 7.23−7.28 (m, 2H).
(E)-4-[2-Ethoxy-5-(phenoxymethyl)phenoxy]-3-methylbut-
1
2-en-1-ol 26e: H NMR (CDCl3) δ 1.41 (t, J = 7.2 Hz, 3H),
1.76 (s, 3H), 4.07(q, J = 6.8, 14 Hz, 2H), 4.20 (t, J = 6.0 Hz,
2H), 4.45 (s, 2H), 5.02 (s, 2H), 5.75−5.78 (m, 1H), 6.82−6.91
(m, 3H), 6.95−7.08 (m, 4H).
(E)-4-{2-Ethoxy-5-[(2-fluorophenoxy)methyl]phenoxy}-3-
methylbut-2-en-1-ol 26f: 1H NMR (CDCl3) δ 1.40 (t, J = 7.2
Hz, 3H), 1.80 (s, 3H), 4.0 (q, J = 6.8, 14 Hz, 2H), 4.10 (t, J =
6.0 Hz, 2H), 4.40 (s, 2H), 5.00 (s, 2H), 5.78 (t, J = 14 Hz, 1H),
6.80−7.05 (m, 7H).
(E)-3-Methyl-4-{3-[(4-phenoxyphenoxy)methyl]phenoxy}-
but-2-en-1-ol 26g: 1H NMR (CDCl3) δ 1.76 (s, 3H), 4.22 (t, J
= 5.6 Hz, 2H), 4.42 (s, 2H), 5.00 (s, 2H), 5.75−5.80 (m, 1H),
6.84−6.87 (m, 1H), 6.91−7.04 (m, 9H), 7.23−7.29 (m, 3H).
{3-[(3-Phenoxyphenoxy)methyl]phenyl}methanol 36: 1H
NMR (CDCl3) δ 4.70 (s, 2H), 5.02 (s, 2H), 6.90−6.96 (m,
5H), 7.00−7.04 (m, 1H), 7.23−7.25 (m, 2H), 7.36−7.42 (m,
4H).
General Procedure for Aldehyde Reduction. NaBH4 (2
equiv) was added to aldehyde (1 equiv) in EtOH at 0 °C and
the mixture stirred for 3−6 h. The mixture was diluted with
water and extracted with CH2Cl2 (twice). The organic extracts
were dried (MgSO4), filtered, and evaporated. The silica gel
column chromatographic purification of the oily residue gave
the alcohol in quantitative yield.
+
AG 50W-X8 (100−200 mesh) cation exchange resin (NH4
form). The flask was washed with buffer (2 × 2 mL) and loaded
onto the column before elution with 150 mL of ion exchange
buffer. The eluent was lyophilized to yield a white solid. This
solid was dissolved in a 25 mM solution of NH4HCO3 buffer (4
mL), purified by RP-HPLC (retention time of ∼7 min), and
lyophilized to give the diphosphate as a white powder.
(2E,6E)-3,7-Dimethyl-8-(2,3,5,6-tetrafluorophenoxy)octa-
2,6-dien-1-diphosphate 3 (26% in two steps from the
Mitsunobu reaction product):14 1H NMR (D2O) δ 1.50 (s,
3H), 1.60 (s, 3H), 1.83−1.87 (m, 2H), 1.98−2.04 (m, 2H),
4.27 (t, J = 6.4 Hz, 2H), 4.49 (s, 2H), 5.23 (t, J = 7.6 Hz, 1H),
5.36 (t, J = 6.8 Hz, 1H), 6.87−6.95 (m, 1H); 31P NMR (D2O)
δ −5.90 (d, J = 22 Hz, 1P), −9.49 (d, J = 22 Hz, 1P);
LRMS(EI) (M+ − H+) 477, (M+) 478
(E)-3-Methyl-4-(3-phenoxyphenoxy)but-2-en-1-diphos-
phate 4. One hundred milligrams of 19a gave 42.7 mg of 4
(23% yield): 1H NMR (D2O) δ 1.63 (s, 3H), 4.40 (s, 2H), 4.64
(s, 2H), 5.63−5.69 (m, 1H), 6.85−6.89 (m, 1H), 6.97−7.01
(m, 1H), 7.21−7.25 (m, 2H); 31P NMR (D2O) δ −9.7 (d, J =
22 Hz, 1P), 8.76 (d, J = 22 Hz, 1P); LRMS(EI) (M+ − H+)
429, (M+) 430.
(E)-[3-({4-[(Tetrahydro-2H-pyran-2-yl)oxy]but-2-en-1-yl}-
1
oxy)phenyl]methanol 23a: H NMR (CDCl3) δ 1.48−1.57
(m, 5H), 1.62−1.72 (m, 2H), 1.76 (s, 3H), 1.79−1.84 (m, 1H),
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Biochemistry
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(E)-3-Methyl-4-(4-phenoxyphenoxy)but-2-en-1-diphos-
phate 5. One hundred milligrams of 19b gave 53 mg of 5 (28%
yield): 1H NMR (D2O) δ 1.58 (s, 3H), 4.35 (bs, 2H), 4.62 (s,
2H), 5.58 (t, J = 5.6 Hz, 1H), 6.50−6.54 (m, 2H), 6.65 (ddd, J
= 0.8, 2.4, 8.4 Hz, 1H), 6.91−6.94 (m, 2H), 7.03−7.07 (m,
1H), 7.14−7.18 (m, 1H), 7.25−7.30 (m, 2H); 31P NMR (D2O)
δ −9.7 (d, J = 20.8 Hz, 1P), −8.51 (d, J = 20.8 Hz, 1P);
LRMS(EI) (M+ − H+) 429, (M+) 430.
3H), 1.47 (s, 3H), 1.93−1.99 (m, 4H), 4.47 (d, J = 6.8 Hz,
2H), 5.31 (t, J = 6.0 Hz, 1H), 4.97 (t, J = 6.4 Hz, 1H), 6.80 (d, J
= 8.8 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H); 31P NMR (D2O) δ
−10.02 (d, J = 22.0 Hz, 1P), −8.00 (d, J = 22.0 Hz, 1P);
LRMS(EI) (M+ − H+) 419, (M+) 420.
(E)-{4-[(3,7-Dimethylocta-2,6-dien-1-yl)oxy]phenyl}-
methanyldiphosphate 14. One hundred milligrams of 30b
1
gave 32.0 mg of 14 (18% yield): H NMR (D2O) δ 1.42 (s,
(E)-4-{4-[(3-Fluorophenoxy)methyl]phenoxy}-3-methyl-
but-2-en-1-diphosphate 6. Fifty milligrams of 26a gave 20 mg
3H), 1.47 (s, 3H), 1.56 (s, 3H), 1.94−2.02 (m, 4H), 4.49 (d, J
= 8.0 Hz, 2H), 4.80 (d, J = 6.0 Hz, 2H), 4.98 (t, J = 7.2 Hz,
1H), 5.33 (t, J = 6.0 Hz, 1H), 6.76−6.78 (m, 1H), 6.94−6.96
(m, 2H), 7.16−7.20 (m, 1H); 31P NMR (D2O) δ −9.78 (d, J =
22.0 Hz, 1P), −5.93 (d, J = 22.0 Hz, 1P); LRMS(EI) (M+ −
H+) 419, (M+) 420.
1
of 6 (22% yield): H NMR (D2O) δ 1.59 (s, 3H), 4.35 (t, J =
7.2 Hz, 2H), 4.40 (s, 2H), 5.00 (s, 2H), 5.61 (t, J = 6.8 Hz,
1H), 6.83−7.00 (m, 6H), 7.17−7.21 (m, 3H); 31P NMR (D2O)
δ −9.35 (d, J = 22.0 Hz, 1P), −9.82 (d, J = 22.0 Hz, 1P);
LRMS(EI) (M+ − H+) 429, (M+) 430.
(E)-3-Methyl-4-[3-(phenoxymethyl)phenoxy]but-2-en-1-di-
phosphate 7. Fifty milligrams of 26b gave 24 mg of 7 (28%
yield): 1H NMR (D2O) δ 1.58 (s, 3H), 4.33 (t, J = 5.6 Hz, 2H),
4.35 (s, 2H), 4.94 (s, 2H), 5.61 (t, J = 6.4 Hz, 1H), 6.80−6.92
(m, 2H), 7.15−7.19 (m, 2H); 31P NMR (D2O) δ −5.8 (d, J =
20.0 Hz, 1P), −9.39 (d, J = 20.0 Hz, 1P); LRMS(EI) (M+ −
H+) 443, (M+) 444.
{ 3 - [ ( 3 - P h e n o x y p h e n o x y ) m e t h y l ] p h e n y l } -
methanyldiphosphate 15. Fifty milligrams of 36 gave 23 mg
1
of 15 (27% yield): H NMR (D2O) δ 1.73 (s, 3H), 4.97 (s,
2H), 5.82 (d, J = 6.4 Hz, 2H), 4.97 (s, 2H), 6.51−6.53 (m,
2H), 6.68 (dd, J = 2.0, 9.6 Hz, 2H), 6.87−6.89 (m, 2H), 7.05 (t,
J = 7.6 Hz, 1H), 7.15−7.34 (m, 7H); 31P NMR (D2O) δ −7.10
(d, J = 22 Hz, 1P), 9.87 (d, J = 22 Hz, 1P); LRMS(EI) (M+ −
H+) 465, (M+) 466.
(E)-4-{3-[(4-Fluorophenoxy)methyl]phenoxy}-3-methyl-
2-({3-[(3-Phenoxyphenoxy)methyl]benzyl}oxy)tetrahydro-
2H-pyran 35. To the cooled 0 °C solution of m-hydroxyphenol
33 (500 mg, 2.68 mmol) in dry DMF was added 60% sodium
hydride in mineral oil (129 mg, 3.2 mmol). The resultant
solution was stirred for 1 h at 0 °C and for 2 h at room
temperature. 2-{[3-(Chloromethyl)benzyl]oxy}tetrahydro-2H-
pyran (645 mg, 2.68 mmol) was added dropwise and the
mixture stirred overnight. The reaction was quenched with
water, the mixture extracted with methylene chloride, and the
organic phase washed with water (thrice). The combined
organic extracts were washed with brine, dried over MgSO4,
concentrated, and purified by silica gel column chromatography
(hexanes, ethyl acetate) to yield oily residue 35 (980 mg, 93%):
1H NMR (CDCl3) δ 1.47−1.81 (m, 2H), 1.80−1.88 (m, 1H),
3.49−3.55 (m, 1H), 3.86−3.92 (m, 1H), 4.49 (d, J = 12.0 Hz,
2H), 4.68 (t, J = 3.6 Hz, 1H), 4.78 (d, J = 12.4 Hz, 2H), 5.00 (s,
2H), 5.57−6.62 (m, 2H), 6.70 (dd, J = 2.4, 8.4 Hz, 1H), 6.98−
7.01 (m, 2H), 7.06−7.10 (m, 1H), 7.17−7.28 (m, 1H), 7.29−
7.39 (m, 6H).
Preparation of MDCC-PBP. The purification and labeling
of the His6-tagged A197C phosphate binding protein (PBP)
with the coumarin fluorophore MDCC were performed as
described previously.27,34 The final MDCC-labeled PBP was
dialyzed against 50 mM Hepes (pH 7.8), 2 mM TCEP, 0.5
unit/mL purine nucleoside phosphorylase, and 15 mM 7-
methylguanosine (“phosphate mop”) to remove any residual
phosphate by forming ribose 1-phosphate. The low-molecular
weight species of the “Pi mop” are removed by exchanging the
buffer with 50 mM Hepes (pH 7.8) and 2 mM TCEP using
Amicon Ultra centrifugal filter devices (10000 molecular weight
cutoff). The purity of the labeled protein was confirmed by
sodium dodecyl sulfate−polyacrylamide gel electrophoresis
(SDS−PAGE). The protein concentration and yield are
determined by the absorbance at 280 nm using a molecular
weight of 35276 and a calculated extinction coefficient of 64204
M−1 cm−1, and protein stocks were stored at −80 °C.
but-2-en-1-diphosphate 8. Fifty milligrams of 26c gave 21 mg
1
of 8 (25% yield): H NMR (D2O) δ 1.60 (s, 3H), 4.37 (t, J =
6.8 Hz, 1H), 4.40 (s, 2H), 5.0 (s, 2H), 5.63 (t, J = 7.2 Hz, 1H),
6.84−7.14 (m, 7H), 7.18−7.22 (m, 2H); 31P NMR (D2O) δ
−7.48 (d, J = 20.0 Hz, 1P), −9.55 (d, J = 20.0 Hz, 1P);
LRMS(EI) (M+ − H+) 461, (M+) 462.
(E)-4-{3-[(3-Fluorophenoxy)methyl]phenoxy}-3-methyl-
but-2-en-1-diphosphate 9. Fifty milligrams of 26d gave 22 mg
1
of 9 (28% yield): H NMR (D2O) δ 1.58 (s, 3H), 4.33−4.35
(m, 4H), 4.94 (s, 2H), 5.61 (t, J = 6.0 Hz, 1H), 6.80−6.92 (m,
5H), 7.15−7.19 (m, 3H); 31P NMR (D2O) δ −5.74 (d, J = 19.6
Hz, 1P), −9.40 (d, J = 19.6 Hz, 1P); LRMS(EI) (M+ − H+)
461, (M+) 462.
(E)-4-[2-Ethoxy-5-(phenoxymethyl)phenoxy]-3-methylbut-
2-en-1-diphosphate 10. Fifty milligrams of 26e gave 31 mg of
1
10 (36% yield): H NMR (D2O) δ 1.15 (t, J = 7.2 Hz, 3H),
1.56 (s, 3H), 3.92 (q, J = 6.8, 14 Hz, 2H), 4.33 (t, J = 6.4 Hz,
2H), 4.37 (s, 2H), 4.86 (s, 2H), 5.56 (t, J = 6.0 Hz, 1H), 6.82−
6.93 (m, 6H), 7.13−7.17 (m, 2H); 31P NMR (D2O) δ −7.03
(d, J = 19.5 Hz, 1P), −9.39 (d, J = 19.5 Hz, 1P); LRMS(EI)
(M+ − H+) 487, (M+) 488.
(E)-4-{2-Ethoxy-5-[(2-fluorophenoxy)methyl]phenoxy}-3-
methylbut-2-en-1-diphosphate 11. Fifty milligrams of 26f
1
gave 18 mg of 11 (22% yield): H NMR (D2O) δ 1.16 (t, J =
7.2 Hz, 3H), 1.57 (s, 3H), 3.91 (q, J = 6.8, 14.0 Hz, 3H), 4.33
(t, J = 6.4 Hz, 2H), 4.37 (s, 2H), 4.86 (s, 2H), 5.56 (t, J = 6.4
Hz, 1H), 6.82−6.85 (m, 5H), 6.93 (s, 1H), 7.13−7.17 (m, 2H);
31P NMR (D2O) δ −7.03 (brs, 1P), −9.59 (brs, 1P);
LRMS(EI) (M+ − H+) 505, (M+) 506.
(E)-3-Methyl-4-{3-[(4-phenoxyphenoxy)methyl]phenoxy}-
but-2-en-1-diphosphate 12. Fifty milligrams of 26g gave 21
mg of 12 (26% yield): 1H NMR (CDCl3) δ 1.20 (t, J = 7.6 Hz,
3H), 1.60 (s, 3H), 3.93 (q, J = 7.2, 14 Hz, 2H), 4.37 (t, J = 6.4
Hz, 2H), 4.40 (s, 2H), 4.94 (s, 2H), 5.59 (t, J = 6.0 Hz, 1H),
6.80−6.84 (m, 3H), 6.92−7.10 (m, 4H); 31P NMR (D2O) δ
−9.59 (brs, 1P), −7.81 (brs, 1P); LRMS(EI) (M+ − H+) 535,
(M+) 536.
Preparation of WT FTase. Recombinant rat protein FTase
expression and purification were conducted in Escherichia coli
BL21(DE3) FPT/pET23a cells as described previously.23,35
The purified FTase was determined by SDS−PAGE to be
>90% pure. The protein was dialyzed at 4 °C against 50 mM
(E)-{3-[(3,7-Dimethylocta-2,6-dien-1-yl)oxy]phenyl}-
methanyldiphosphate 13. One hundred milligrams of 30a
1
gave 34.5 mg of 13 (19% yield): H NMR (D2O) δ 1.42 (s,
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Biochemistry
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RP
Fmax
Hepes (pH 7.8) and 2 mM TCEP and stored at −80 °C. The
concentration of FTase was determined by active site titration
as previously described.23
V =
(3)
where P is the concentration of the limiting substrate and Fmax
is the amplitude in fluorescence measured from the end point
of each experiment.
Single-Turnover Kinetics. The single-turnover rate
constant was determined by measuring the release of
diphosphate (PPi) using a fluorescently labeled phosphate
binding protein (MDCC-PBP) coupled with PPi cleavage by
inorganic pyrophosphatase (PPiase), as described by Pais et
al.27 FTase was preincubated with FPP or an analogue for >15
min at room temperature and then rapidly mixed with a peptide
solution containing MDCC-PBP and PPiase. The following
final concentrations were used: 800 nM FTase, 200 nM FPP/
analogue, 25 μM peptide, 5 μM MDCC-PBP, 34 units/mL
PPiase, 50 mM Heppso (pH 7.8), 5 mM MgCl2, and 2 mM
TCEP. Experiments were conducted at 25 °C using a KinTek
model SF-2001 stopped-flow apparatus (KinTek Corp., Austin,
TX) to detect an increase in fluorescence upon binding of
inorganic phosphate to MDCC-PBP (λex = 430 nm; λem = 450
nm; Corion LL-450-F cutoff filter). The stopped-flow syringes
and mixing chamber were preincubated prior to experiments in
buffer containing a Pi mop [50 mM Heppso (pH 7.8), 5 mM
MgCl2, 2 mM TCEP, 0.5 unit/mL PNPase, and 15 μM MEG].
At least five kinetic traces were averaged, and the single-
turnover rate constant (kobs) was determined by fitting eq 1 to
the data
The values of the steady-state kinetic parameters kcat,
isoprenoid
KMisoprenoid, and kcat/KM
were calculated from a fit of
the Michaelis−Menten equation to the initial V versus [S] data.
RESULTS
■
Design and Synthesis of Aryl-Substituted Isoprenoid
Diphosphate. FPP analogues 2−15 (Figure 3) were designed
to probe aspects of isoprenoid C1 reactivity, the isoprene
conformational rearrangement, and the steric constraints of the
FTase mechanism. We had previously established that an
aniline or phenoxy group is an isostere for the terminal
isoprene of FPP and that a range of substituent groups are
tolerated by FTase to give transferable analogues under steady-
state conditions. Analogues 2−15 were designed to test the
extent that FPP could be altered and still allow transfer
catalyzed by FTase. The chemical reactivity of C1 was increased
by replacing the first isoprene with a benzyl moiety in
analogues 13−15 (Table 1 and Schemes 1 and 2). Solvolysis
a
Scheme 1
obst
Fl = ΔFl × e−k + Flmax
(1)
where Fl is the observed fluorescence at <450 nm at time t, ΔFl
is the amplitude, and Flmax is the fluorescence end point. The
single-turnover rate constant (kobs) was derived from a
reversible two-step kinetic mechanism proceeding from the
E·FPP·CaaX complex shown in the dashed box in Figure 1
using eq 1.
kconfkchem
kconf + k−conf + kchem
kobs
=
(2)
Steady-State Kinetics. The steady-state kinetic parameters
isoprenoid
kcat, KMisoprenoid, and kcat/KM
were determined from the
dependence of the initial velocity on the concentration of FPP
or an analogue at a saturating dansylated peptide (dns-GCVLS)
concentration, or kcat was determined from the dependence of
the initial velocity on the concentration of dns-GCVLS at a
saturating isoprenoid concentration. The initial velocity was
measured from the time-dependent increase in fluorescence
intensity (λex = 340 nm; λem = 520 nm) upon farnesylation of
dansylated GCVLS, as described previously. Reactions were
initiated by the addition of FTase (final concentration of 25
nM) to solutions containing 5 μM dns-GCVLS, varying
concentrations (1−20 μM) of FPP/analogue, 50 mM Heppso
(pH 7.8), 5 mM MgCl2, and 2 mM TCEP at 25 °C. For
measurements at a fixed isoprenoid concentration, reactions
were initiated by the addition of FTase (final concentration of
25 nM) to solutions containing varying concentrations (1−10
μM) of dns-GCVLS, 20 μM isoprenoid, 50 mM Heppso (pH
7.8), 5 mM MgCl2, and 2 mM TCEP at 25 °C. The
fluorescence intensity over time is measured for the first 10% of
the reaction, using a Polarstar Galaxy fluorescence plate reader
(BMG Laboratory Technologies, Durham, NC). The initial
velocity of the reaction in fluorescence units per second (R) is
converted to the velocity of the product formed in micromolar
per second (V) using eq 3
a
(a) DEAD, Ph3P, THF; (b) PPTS, MeOH; (c) Ph3PCl2, CH3CN;
(d) (n-Bu4)3HP2O7, CH3CN.
studies suggest benzyl isoprenoids 13−15 are expected to be 5
and 20 times more reactive than the corresponding allylic
isoprenoids in SN1- and SN2-type reactions, respectively.36,37
The terminal isoprene was replaced by an aromatic group in
analogues 2−12 and 15 but retained in analogues 13 and 14.
The second isoprene in analogues 4−12 and 15 was replaced
by a series of substituted phenyl groups. In analogues 4−12, the
first isoprene unit was retained and the steric demands and the
number of rotatable bonds relative to FPP were varied.
Analogue 12 is longer than the other FPP analogues and was
designed to mimic the 20-carbon geranylgeranyl diphosphate
(GGPP). GGPP is an alternative FTase substrate for some
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a
a
Scheme 2
Scheme 3
a
(a) DEAD, Ph3P, THF; (b) NaBH4, EtOH; (c) Ph3PCl2, CH3CN;
(d) (n-Bu4)3HP2O7, CH3CN.
hydroxybenzaldehyde followed by reduction of aldehydes 29a
and 29b with NaBH4 gave the corresponding alcohols 30a and
30b, respectively. The desired diphosphates 13 and 14 were
obtained by conversion of these alcohols as described above.
Synthesis of analogue 15 where all of the isoprene units are
replaced by aromatic moieties is shown in Scheme 4. Previously
described chloride 3242 was coupled with the sodium salt of m-
phenoxyphenol followed by removal of the THP protecting
group, chlorination, and diphosphorylation as described above.
a
Scheme 4
a
(a) DEAD, Ph3P, THF; (b) NaBH4, EtOH; (c) DEAD, Ph3P, THF;
(d) PPTS, MeOH; (e) Ph3PCl2, CH3CN; (f) (n-Bu4)3HP2O7,
CH3CN.
peptide sequences but is a nanomolar inhibitor of the enzyme
with others.38−40
Analogues 2 and 3 were prepared as previously reporte-
d.14,41Analogues 4 and 5 were prepared by Mitsunobu coupling
of either m- or p-phenoxyphenol with alcohol 16 to give THP-
protected isoprenols 18a and 18b (Scheme 1). Removal of the
THP ether with PPTS in methanol afforded alcohols 19a and
19b that were converted to the corresponding chlorides 20a
and 20b, respectively, using Ph3PCl2 in acetonitrile.14 The
allylic chlorides were diphosphorylated with (n-Bu4)3HP2O7 to
give diphosphates 4 and 5 in moderate yield.
The preparation of FPP analogues 6−12 is shown in Scheme
2 and involved Mitsunobu coupling of alcohol 16 with the
appropriate hydroxybenzaldehyde to give aldehydes 22a−c that
were reduced with NaBH4 to the corresponding benzyl alcohols
23a−c, respectively. A second Mitsunobu reaction with the
requisite substituted phenols then afforded THP ethers 25a−g.
These protected isoprenoid analogues were converted to the
desired diphosphates 6−12, respectively, as described above.
Synthesis of FPP analogues 13 and 14 where the first
isoprene moiety was replaced with a benzyl group is shown in
Scheme 3. Mitsunobu coupling of geraniol with either m- or p-
a
(a) NaH, THF; (b) PPTS, MeOH; (c) Ph3PCl2, CH3CN; (d) (n-
Bu4)3HP2O7, CH3CN.
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FTase-Catalyzed Alkylation of GCVLS Depends on the
Isoprenoid Donor Structure. A fluorescent assay was used to
determine the effect of the FPP analogue structure on multiple-
The Single-Turnover Rate Constant for GCVLS
Alkylation Is the Same for Structurally Divergent
Isoprenoid Diphosphates. We measured the STO rate
constant (kobs) for FTase-catalyzed alkylation of GCVLS by
FPP and analogues 2−15 using a fluorescence-based assay that
detects the release of pyrophosphate following alkyl transfer
under conditions of excess FTase and peptide and limiting
isoprenoid diphosphate analogue.45 The observed rate constant
for alkylated peptide product formation (kobs), indicated in
Figure 1, measures formation of the E·product complex from
the ternary E·FPP·peptide complex and includes both the
chemical farnesylation step (kchem) and the proposed FPP
rotational rearrangement (kconf) to form the active substrate
conformation (eq 3).26,27,31 Surprisingly, the observed rate
constant under STO conditions (kobs) for all of the analogues is
very similar to that measured for FPP (Table 1). The
discrepancy between the effects of these analogues on the
single-turnover and steady-state kinetics suggests that different
rate-limiting steps are being measured by the two methods; in
particular, product dissociation may contribute significantly to
the measured values for the steady-state kinetics.
turnover kinetics catalyzed by FTase. The steady-state kinetic
isoprenoid
parameters kcat for 1−15 and KM
of FPP and analogues
2, 3, 6, 7, 9−11, and 13−15 with dansylated GCVLS peptide
(dns-GCVLS) were measured (Table 1). KM
isoprenoid
was
measured by varying the FPP or analogue concentration in
the presence of a saturating peptide concentration, and kcat was
measured by varying the isoprenoid concentration in the
presence of a saturating peptide concentration or by varying the
peptide concentration in the presence of a saturating analogue
concentration.43 The dns-GCVLS peptide corresponds to the
well-characterized Ca1a2X sequence of H-Ras.21,39,44
FTase catalyzed transfer of the analogues to dns-GCVLS
with an efficiency equal to or lower than that of FPP, with the
exception of the GGPP mimetic 12 for which no turnover was
detected. For the FPP analogues in which the third isoprene is
replaced with substituted aniline groups, the steady-state kinetic
parameters depend in a complex manner on the size and ring
position of the substituents, and the sequence of the Ca1a2X
peptide. For example, analogue 2 is a poor donor for
modification of the dansyl-GCVLS peptide but is a good
substrate with the dansyl-GCVIM peptide. The steady-state
rate constant kcat/KMisoprenoid is termed the “specificity constant”
for the reaction of a peptide with different isoprenoids and can
be used to determine how efficiently the FPP analogue is
The Rate Constant for Alkylation Depends on the
Ca1a2X Peptide Sequence. To delineate the dependence of
the STO rate constants on peptide structure, we also measured
the reactivity of analogues 2−15 for TKCVIM and TKCVIF
(Table 1). TKCVIM is the K-Ras Ca1a2X peptide, and the STO
rate constant for farnesylation is nearly 2 times greater (FPP;
k
obs = 6.5 s−1) than the value for GCVLS (FPP; kobs = 3.4 s−1).
transferred to the peptide.15,30,31 Relative to that of FPP, the
These rate constants are in good agreement with those
previously reported.31,46 TKCVIF is the Ca1a2X peptide from
TC21 and is normally a substrate for both FTase and GGTase
I. The STO rate constant for farnesylation of TKCVIF
catalyzed by FTase is 25-fold lower (FPP; kobs = 0.27 s−1)
than the value for TKCVIM. Once again, the STO rate
constants for the structurally dissimilar analogues 2−15 were
identical to that of FPP for each of the three peptides examined
(Table 1). Notably, GGPP mimetic 12 reacts as efficiently as
FPP with all three peptides even though modification by this
analogue under steady-state turnover conditions is undetect-
able. These results indicate that the conformational rearrange-
ment and the farnesylation step are insensitive to the structural
differences between FPP and analogues 2−15 (Table 1).
Rather, the STO rate constant for peptide alkylation depends
on the structure of the peptide, particularly the C-terminal
residue in the Ca1a2X motif. The uniformly high reactivity of
FPP and analogues 2−15 suggests a facile conformational
change in the analogue hydrocarbon chains to achieve the
transition state, consistent with recent computational studies.28
Furthermore, the conformational rearrangement of the
isoprenoid diphosphate in the active site to achieve the reactive
conformation is independent of the product release step.
isoprenoid
value of kcat/KM
for the analogues decreases up to 475-
fold. Analysis of the steady-state parameters reveals that rate
constant kcat decreases for the analogues 1.3−220-fold relative
to that of FPP, while the value of KMisoprenoid varies from 0.2 to
9.2 μM, which is within a 7.5-fold difference of that of FPP
(KMisoprenoid = 1.5 μM). Therefore, the decreases in kcat roughly
isoprenoid
follow the same trends observed for kcat/KM
.
The efficiency of the multiple-turnover reaction depends on
analogue structure. Notably, a meta or para linkage geometry of
the second isoprene substitution does not appear to alter the
isoprenoid
value of kcat/KM
for modification of peptides catalyzed
by FTase. For example, compounds 9 (meta) and 6 (para) have
very similar efficiencies. Furthermore, the reactivity of
analogues with dns-GCVLS does not depend on having the
same number of rotatable bonds as FPP. Analogue 15 has seven
rotatable bonds compared with eight in FPP. Clearly, the
rotatable bonds in these analogues can adopt conformations
sufficient to achieve the reactive ternary complex and the
transition state. In contrast, the FTase-catalyzed efficiency of
alkylation of peptides with analogues 10 and 11 is severely
compromised, suggesting that increased steric bulk on the lipid
reduces either the peptide affinity or the rate constant for
product formation.
DISCUSSION
Analogues in Which the α-Isoprene Is Replaced with
a Benzyl Group Can Be Transferred by FTase. Benzyl
■
Increased Isoprenoid Chemical Reactivity Does Not
Alter the Rate Constant for Peptide Alkylation. The
electronic structure of the first isoprene unit is thought to be
critical for the FTase-catalyzed transfer to the peptide. 3-Vinyl-
FPP is an efficient alternative substrate for FTase and is likely
to have an increased level of delocalization of any cationic
character in the transition state.47,48 The structure of the first
isoprenoid is also an important factor in efficient multiple
turnovers as both the 2Z isomer of FPP and 3-allyl-FPP are
potent inhibitors of FTase, although they are electronically
analogues 13−15 are surprisingly good substrates, with kcat/
isoprenoid
KM
values that are within a factor of 3 of that of FPP
(Table 1). Remarkably, analogue 15 is almost as good a
substrate as FPP for FTase despite having no isoprene units in
the structure. Aryl analogue 13 shows that most of the reactivity
of FPP is retained by substituting a benzyl group for the first
isoprene. This is the first time FTase has been shown to
catalyze the transfer of an isoprenoid with a non-allylic
diphosphate to a Ca1a2X peptide.
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almost identical to FPP. It is unknown whether these two
molecules alkylate peptide substrates under STO condi-
tions.47,48 The reactivity of C1 in benzyl analogues 13−15 is
increased relative to that of FPP. The solvolytic reactivity of p-
geranyloxybenzyl diphosphate 13 is higher than that of meta-
substituted 14 and 15 because of enhanced stabilization of the
delocalized positive charge that develops on the aromatic ring
in the transition state (Figure 2B).37 The identical STO
reactivity for analogues 13−15 indicates that enhanced
stabilization of the carbocationic aspects of the transition
state does not contribute to an increased observed rate constant
for peptide alkylation catalyzed by FTase. These results
contrast with the substantial decrease in reactivity caused by
analogues indicates that substantial conformational freedom to
achieve the transition state is available to the lipid in the
confines of the active site and that any barriers to achieving the
transition state are small. These observations indicate that the
decreased steady-state turnover rate for the analogues most
likely reflects alterations in the product dissociation step.
The Isoprenoid Structure Mainly Affects Product
Release. The decoupling of isoprenoid structure and intrinsic
chemical reactivity from the STO rate constant kobs, while
retaining a profound isoprenoid structural dependence on the
rate of product release, kcat, indicates that the alkylation reaction
depends mainly on the structure of the peptide,46 while product
release depends on the structure of both the peptide and the
isoprenoid.30,31 Equation 2 demonstrates the contribution of
conformational rearrangement kconf and chemical step kchem to
STO rate constant kobs. Inspection of eq 2 suggests that either
kconf and k−conf are unchanged by the range of structural
modifications explored here or kchem is sufficiently small to be
the rate-determining step for all of the substrates. This latter
conclusion is consistent with computational studies suggesting
that the barrier to achieving the transition state going from the
E·FPP·Ca1a2X complex to the E·FPP·Ca1a2X* complex is on
the order of 1 kcal/mol for CVIM to 2.5 kcal/mol for CVLM
compared to ∼20 kcal/mol for the chemical step.24,28,50
Mg2+ binding accelerates FTase-catalyzed alkylation of
GCVLS and TKCVIF by FPP by 700- and 100-fold,
respectively. The Mg2+ cofactor may stabilize both the active
substrate conformation and the developing charge on the
pyrophosphate leaving group.51−53 The FPP pyrophosphate
leaving group in the inactive (E·FPP·Ca1a2X) complex is bound
in the FTase PPi binding pocket comprised of positively
charged residues.19,22,54−57 Conformational rearrangement of
the FPP isoprenoid chain moves the pyrophosphate leaving
group out of the PPi binding pocket and creates a Mg2+ binding
site that stabilizes the active complex (E·FPP·Ca1a2X*), which
is then followed by the attack of the zinc-bound thiolate
nucleophile on C1. For each CaaX peptide, the observed rate
constant, kobs, for analogues 2−15 were identical to that of the
natural substrate FPP. The insensitivity of kobs to the increased
chemical reactivity of analogues 13−15 indicates that the rate
of chemical step kchem for each of the three peptides was not
limited by the intrinsic reactivity of the allylic C1 electrophile.
Furthermore, the insensitivity of kobs to the structure of the
analogues for all three peptides suggests that there is abundant
room in the active site for the proposed conformational
rearrangement of the isoprenoid to take place. Consistent with
this model, the uniform observed values of kobs for FPP and
analogues 2−15 could be accommodated by correlated changes
in chemical reactivity that compensate for an isoprenoid
structure-dependent decrease in the ratio of the equilibrium
constant for the conformational change (kconf/k−conf). However,
there is no increase in kobs relative to that of FPP for the more
reactive benzyl analogues 13−15, suggesting that kchem depends
on the CaaX peptide sequence and does not vary with
isoprenoid structure.
electron withdrawing fluorine substitution at the C3 methyl of
26,27
FPP on both steady-state turnover and kchem
.
Isoprenoid hydrophobicity is uncorrelated with efficiency in
multiturnover reactions,14 although hydrophobicity of the
peptide substrates affects reactivity.31,46 The lack of any
isoprenoid structural effect for analogues 2−15 on the STO
rates suggests that interactions between the isoprenoid and the
amino acid residues lining the walls of the active site are not
particularly sensitive to the polarity of the chain.
Reaction Steps Involved in FTase Isoprenoid Diphos-
phate Selectivity. For FTase, the steady-state parameter kcat/
isoprenoid
KM
normally includes the rate constants for reaction
steps for formation of the E·FPP·Ca1a2X complex, including
peptide binding to E·FPP, and the first irreversible step of
diphosphate dissociation (Figure 1).30,31,45 However, because
FPP also enhances product dissociation, the measured value of
kcat/KMisoprenoid likely includes these steps, as well.29 In contrast,
peptide alkylation catalyzed by FTase under single-turnover
conditions (with a saturating enzyme concentration) monitors
only steps that occur after peptide binding through diphosphate
release [kobs (dashed box, Figure 1)].45 Therefore, FTase-
catalyzed single-turnover kinetics in the presence of a limiting
amount of isoprenoid diphosphate isolates the alkyl transfer
step (kchem) and presumed isoprenoid conformational change
CaaX
(kconf) from the dissociation of the alkylated peptide [koff
(Figure 1)] that is frequently the rate-limiting step for kcat
under multiple-turnover conditions.18,26,49 The lack of any
isoprenoid structural effect, yet a strong Ca1a2X peptide
sequence effect, on kobs suggests that the chemical step and/
or the proposed conformational rearrangement of the FPP
depends on how the Ca1a2X peptide binds to FTase. The clear
differences in kobs for the three peptides suggest that subtle
differences in the Ca1a2X−FTase binding interactions alter the
rate of the proposed isoprenoid conformational rearrangement
or alter the chemical reactivity of the thiolate nucleophile.
Increased Isoprenoid Bulk Does Not Alter the Rate
Constant for GCVLS Alkylation. Previous measurements
demonstrated that the secondary kinetic isotope effect (KIE)
for the reaction of FPP with peptides under STO conditions
catalyzed by FTase varies with the structure of the peptide; the
3
α secondary H KIE observed for TKCVIF is large (1.13
0.01) but decreases to near unity for GCVLS.27 The original
interpretation of this result was that the conformational change
became the rate-limiting step for farnesylation of GCVLS under
STO conditions. However, recent computational studies from
the Merz group suggest that the ΔG⧧ for the conformational
step is much smaller than that of the alkylation step, even for
GCVLS, and that the change in the secondary KIE reflects
alterations in the transition-state structure due to changes in the
peptide binding mode. The similar STO kobs for all of the
Implications for the Development of Alternative
FTase Substrates That Block Prenylated Protein
Function (PFIs). This study tested the sensitivity of the
chemical step to changes in the electronic structure of the lipid
electrophile and the sensitivity of the conformational rearrange-
ment required to achieve the reactive conformation to changes
in the lipid structure. There are four primary conclusions. (1)
Increases in the chemical reactivity of the lipid electrophile do
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not increase kobs, and (2) increased steric bulk in the isoprene
chain does not decrease kobs. (3) kobs depends on the Ca1a2X X-
residue, and (4) in contrast, the steady-state rate constants for
turnover are highly dependent on lipid structure. The ability of
FTase to transfer analogues with no isoprene units opens the
door to a much wider range of alternative structures as
potential PFIs. These results suggest that as long as the
chemical reactivity of the analogue C1 is at least as reactive as
the allylic group of FPP, efficient S-alkylation is possible and
largely unaffected by the chemical structure and bulk of the
isoprenoid chain. There must be limits to the size of
transferrable lipid moieties, as the FTase pocket has finite
dimensions. However, the limits were not reached by the
analogues investigated in this study. In contrast to the
structure-independent rate of cysteine alkylation, product
release was highly dependent on the structure of the analogue.
Previously, our lab and others have noted a Ca1a2X sequence-
dependent interplay in the turnover of FPP analogues.30,32
Despite investigation of almost 200 analogues with a wide range
of chemical structures, no clear structure−activity relationship
has emerged. Broadly, large analogues like 10−12 are
inefficiently turned over. Furthermore, these data suggest that
the farnesyl binding site in the exit groove may be significantly
more selective for the farnesyl diphosphate substrate than the
active site binding pocket and therefore might be a useful site
for the design of novel inhibitors. Alternatively, the
conformation of the isoprenoid in the E·product complex
may block efficient binding of the new analogue diphosphate in
the exit groove or is inhibited from rearranging to put the
alkylated isoprenoid into the exit groove.
diethylazodicarboxylate; DMF, dimethylformamide; THF,
tetrahydrofuran; PPTS, pyridinium-p-toluene sulfonate; H-
Ras, Harvey-Ras; K-Ras, Kirsten-Ras; DTT, dithiothreitol;
RP-HPLC, reverse phase high-performance liquid chromatog-
raphy; PBP, phosphate binding protein; kcat, turnover number;
KMisoprenoid, Michaelis−Menten constant for FPP or an
analogue; MDCC, diethylamino-3-[N-(2-maleimidoethyl)-
carbamoyl]coumarin; PNPase, polynucleotide phosphorylase;
TCEP, tris(2-carboxyethyl)phosphine hydrochloride.
REFERENCES
■
(1) Fu, H. W., and Casey, P. J. (1999) Enzymology and biology of
CaaX protein prenylation. Recent Prog. Horm. Res. 54, 315−342.
(2) Casey, P. J., and Seabra, M. C. (1996) Protein prenyltransferases.
J. Biol. Chem. 271, 5289−5292.
(3) Adjei, A. A. (2003) An overview of farnesyltransferase inhibitors
and their role in lung cancer therapy. Lung Cancer 41 (Suppl. 1), S55−
S62.
(4) Rowinsky, E. K., Windle, J. J., and Von Hoff, D. D. (1999) Ras
protein farnesyltransferase: A strategic target for anticancer therapeutic
development. J. Clin. Oncol. 17, 3631−3652.
(5) Zhang, F. L., and Casey, P. J. (1996) Influence of metal ions on
substrate binding and catalytic activity of mammalian protein
geranylgeranyltransferase type-I. Biochem. J. 320 (Part 3), 925−932.
(6) Sousa, S. F., Fernandes, P. A., and Ramos, M. J. (2005)
Unraveling the mechanism of the farnesyltransferase enzyme. J. Biol.
Inorg. Chem. 10, 3−10.
(7) Vergnes, L., Peterfy, M., Bergo, M. O., Young, S. G., and Reue, K.
(2004) Lamin B1 is required for mouse development and nuclear
integrity. Proc. Natl. Acad. Sci. U.S.A. 101, 10428−10433.
(8) Konstantinopoulos, P. A., Karamouzis, M. V., and Papavassiliou,
A. G. (2007) Post-translational modifications and regulation of the
RAS superfamily of GTPases as anticancer targets. Nat. Rev. Drug
Discovery 6, 541−555.
(9) Reents, R., Wagner, M., Schlummer, S., Kuhlmann, J., and
Waldmann, H. (2005) Synthesis and application of fluorescent ras
proteins for live-cell imaging. ChemBioChem 6, 86−94.
(10) Rando, R. R. (1996) Chemical biology of isoprenylation/
methylation. Biochem. Soc. Trans. 24, 682−687.
(11) Figueroa, C., Taylor, J., and Vojtek, A. B. (2001) Prenylated Rab
acceptor protein is a receptor for prenylated small GTPases. J. Biol.
Chem. 276, 28219−28225.
(12) Whyte, D. B., Kirschmeier, P., Hockenberry, T. N., Nunez-Oliva,
I., James, L., Catino, J. J., Bishop, W. R., and Pai, J. K. (1997) K- and
N-Ras are geranylgeranylated in cells treated with farnesyl protein
transferase inhibitors. J. Biol. Chem. 272, 14459−14464.
(13) Roberts, M. J., Troutman, J. M., Chehade, K. A., Cha, H. C.,
Kao, J. P., Huang, X., Zhan, C. G., Peterson, Y. K., Subramanian, T.,
Kamalakkannan, S., Andres, D. A., and Spielmann, H. P. (2006)
Hydrophilic anilinogeranyl diphosphate prenyl analogues are Ras
function inhibitors. Biochemistry 45, 15862−15872.
(14) Subramanian, T., Liu, S., Troutman, J. M., Andres, D. A., and
Spielmann, H. P. (2008) Protein farnesyltransferase-catalyzed
isoprenoid transfer to peptide depends on lipid size and shape, not
hydrophobicity. ChemBioChem 9, 2872−2882.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental procedure for the synthesis of all new
1
compounds; H NMR spectra of 19a, 19b, 26a−g, 30a, 30b,
1
and 35; and H, 13P, and LR mass spectra of 3−15. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: hps@uky.edu.
■
Present Addresses
∥Department of Biochemistry, University of Wisconsin, 433
Babcock Dr., Madison, WI 53706.
⊥Immunology Institute, Shandong University School of
Medicine, Jinan, Shandong, People’s Republic of China.
@Chemistry Department, University of North Carolina, 9201
University City Blvd., Charlotte, NC 28223-0001.
Funding
Supported by National Institutes of Health Grants R01
GM66152 to H.P.S. and R01 GM40602 to C.A.F.
Notes
(15) Troutman, J. M., Subramanian, T., Andres, D. A., and
Spielmann, H. P. (2007) Selective modification of CaaX peptides
with ortho-substituted anilinogeranyl lipids by protein farnesyl
transferase: Competitive substrates and potent inhibitors from a
library of farnesyl diphosphate analogues. Biochemistry 46, 11310−
11321.
(16) Placzek, A. T., Hougland, J. L., and Gibbs, R. A. (2012)
Synthesis of frame-shifted farnesyl diphosphate analogs. Org. Lett. 14,
4038−4041.
(17) Nguyen, U. T., Goody, R. S., and Alexandrov, K. (2010)
Understanding and exploiting protein prenyltransferases. ChemBio-
Chem 11, 1194−1201.
The authors declare no competing financial interest.
ABBREVIATIONS
■
FTase, farnesyltransferase; AGPP, 8-anilinogeranyl diphos-
phate; FPP, farnesyl diphosphate; TLC, thin layer chromatog-
raphy; GGTase I, geranylgeranyl transferase; FTI, farnesyl-
transferase; Ca1a2X, tetrapeptide sequence cysteine aliphatic
amino acid-aliphatic amino acid-X (serine, glutamine, or
methionine); GGPP, geranylgeranyl diphosphate; DEAD,
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(18) Furfine, E. S., Leban, J. J., Landavazo, A., Moomaw, J. F., and
Casey, P. J. (1995) Protein farnesyltransferase: Kinetics of farnesyl
pyrophosphate binding and product release. Biochemistry 34, 6857−
6862.
(19) Long, S. B., Casey, P. J., and Beese, L. S. (1998) Cocrystal
structure of protein farnesyltransferase complexed with a farnesyl
diphosphate substrate. Biochemistry 37, 9612−9618.
(20) Huang, C. C., Casey, P. J., and Fierke, C. A. (1997) Evidence for
a catalytic role of zinc in protein farnesyltransferase. Spectroscopy of
Co2+-farnesyltransferase indicates metal coordination of the substrate
thiolate. J. Biol. Chem. 272, 20−23.
(38) Shao, Y., Eummer, J. T., and Gibbs, R. A. (1999) Stereospecific
synthesis and biological evaluation of farnesyl diphosphate isomers.
Org. Lett. 1, 627−630.
(39) Chehade, K. A., Kiegiel, K., Isaacs, R. J., Pickett, J. S., Bowers, K.
E., Fierke, C. A., Andres, D. A., and Spielmann, H. P. (2002)
Photoaffinity analogues of farnesyl pyrophosphate transferable by
protein farnesyl transferase. J. Am. Chem. Soc. 124, 8206−8219.
(40) Henriksen, B. S., Zahn, T. J., Evanseck, J. D., Firestine, S. M.,
and Gibbs, R. A. (2005) Computational and conformational evaluation
of FTase alternative substrates: Insight into a novel enzyme binding
pocket. J. Chem. Inf. Model. 45, 1047−1052.
(21) Pompliano, D. L., Rands, E., Schaber, M. D., Mosser, S. D.,
Anthony, N. J., and Gibbs, J. B. (1992) Steady-state kinetic mechanism
of Ras farnesyl:protein transferase. Biochemistry 31, 3800−3807.
(22) Long, S. B., Casey, P. J., and Beese, L. S. (2002) Reaction path
of protein farnesyltransferase at atomic resolution. Nature 419, 645−
650.
(23) Bowers, K. E., and Fierke, C. A. (2004) Positively charged side
chains in protein farnesyltransferase enhance catalysis by stabilizing the
formation of the diphosphate leaving group. Biochemistry 43, 5256−
5265.
(24) Cui, G., and Merz, K. M., Jr. (2007) Computational studies of
the farnesyltransferase ternary complex part II: The conformational
activation of farnesyldiphosphate. Biochemistry 46, 12375−12381.
(25) Dolence, J. M., and Poulter, C. D. (1995) A mechanism for
posttranslational modifications of proteins by yeast protein farnesyl-
transferase. Proc. Natl. Acad. Sci. U.S.A. 92, 5008−5011.
(26) Huang, C., Hightower, K. E., and Fierke, C. A. (2000)
Mechanistic studies of rat protein farnesyltransferase indicate an
associative transition state. Biochemistry 39, 2593−2602.
(27) Pais, J. E., Bowers, K. E., and Fierke, C. A. (2006) Measurement
of the α-secondary kinetic isotope effect for the reaction catalyzed by
mammalian protein farnesyltransferase. J. Am. Chem. Soc. 128, 15086−
15087.
(28) Yang, Y., Wang, B., Ucisik, M. N., Cui, G., Fierke, C. A., and
Merz, K. M., Jr. (2012) Insights into the mechanistic dichotomy of the
protein farnesyltransferase peptide substrates CVIM and CVLS. J. Am.
Chem. Soc. 134, 820−823.
(29) Tschantz, W. R., Furfine, E. S., and Casey, P. J. (1997) Substrate
binding is required for release of product from mammalian protein
farnesyltransferase. J. Biol. Chem. 272, 9989−9993.
(30) Troutman, J. M., Andres, D. A., and Spielmann, H. P. (2007)
Protein farnesyl transferase target selectivity is dependent upon
peptide stimulated product release. Biochemistry 46, 11299−11309.
(31) Hougland, J. L., Lamphear, C. L., Scott, S. A., Gibbs, R. A., and
Fierke, C. A. (2009) Context-dependent substrate recognition by
protein farnesyltransferase. Biochemistry 48, 1691−1701.
(32) Reigard, S. A., Zahn, T. J., Haworth, K. B., Hicks, K. A., Fierke,
C. A., and Gibbs, R. A. (2005) Interplay of isoprenoid and peptide
substrate specificity in protein farnesyltransferase. Biochemistry 44,
11214−11223.
(33) Krzysiak, A. J., Aditya, A. V., Hougland, J. L., Fierke, C. A., and
Gibbs, R. A. (2010) Synthesis and screening of a CaaL peptide library
versus FTase reveals a surprising number of substrates. Bioorg. Med.
Chem. Lett. 20, 767−770.
(41) Chehade, K. A., Andres, D. A., Morimoto, H., and Spielmann,
H. P. (2000) Design and synthesis of a transferable farnesyl
pyrophosphate analogue to Ras by protein farnesyltransferase. J. Org.
Chem. 65, 3027−3033.
(42) Naghipour, A., Sabounchei, S. J., Morales-Morales, D.,
Hernandez-Ortega, S., and Jensen, C. M. (2004) Synthesis of a new
class of unsymmetrical PCP′ pincer ligands and their palladium(II)
complexes: X-ray structure determination of PdCl{C6H3-2-CH2PPh2-
6-CH2PBu2t}. J. Organomet. Chem. 689, 2494−2502.
(43) Micali, E., Chehade, K. A., Isaacs, R. J., Andres, D. A., and
Spielmann, H. P. (2001) Protein farnesyltransferase isoprenoid
substrate discrimination is dependent on isoprene double bonds and
branched methyl groups. Biochemistry 40, 12254−12265.
(44) Cassidy, P. B., Dolence, J. M., and Poulter, C. D. (1995)
Continuous fluorescence assay for protein prenyltransferases. Methods
Enzymol. 250, 30−43.
(45) Pais, J. E., Bowers, K. E., Stoddard, A. K., and Fierke, C. A.
(2005) A continuous fluorescent assay for protein prenyltransferases
measuring diphosphate release. Anal. Biochem. 345, 302−311.
(46) Hartman, H. L., Hicks, K. A., and Fierke, C. A. (2005) Peptide
specificity of protein prenyltransferases is determined mainly by
reactivity rather than binding affinity. Biochemistry 44, 15314−15324.
(47) Zahn, T. J., Whitney, J., Weinbaum, C., and Gibbs, R. A. (2001)
Synthesis and evaluation of GGPP geometric isomers: Divergent
substrate specificities of FTase and GGTase I. Bioorg. Med. Chem. Lett.
11, 1605−1608.
(48) Rawat, D. S., Krzysiak, A. J., and Gibbs, R. A. (2008) Synthesis
and biochemical evaluation of 3,7-disubstituted farnesyl diphosphate
analogues. J. Org. Chem. 73, 1881−1887.
(49) Mathis, J. R., and Poulter, C. D. (1997) Yeast protein
farnesyltransferase: A pre-steady-state kinetic analysis. Biochemistry 36,
6367−6376.
(50) Cui, G., Wang, B., and Merz, K. M., Jr. (2005) Computational
studies of the farnesyltransferase ternary complex part I: Substrate
binding. Biochemistry 44, 16513−16523.
(51) Pickett, J. S., Bowers, K. E., and Fierke, C. A. (2003)
Mutagenesis studies of protein farnesyltransferase implicate aspartate
β352 as a magnesium ligand. J. Biol. Chem. 278, 51243−51250.
(52) Pickett, J. S., Bowers, K. E., Hartman, H. L., Fu, H. W., Embry,
A. C., Casey, P. J., and Fierke, C. A. (2003) Kinetic studies of protein
farnesyltransferase mutants establish active substrate conformation.
Biochemistry 42, 9741−9748.
(53) Saderholm, M. J., Hightower, K. E., and Fierke, C. A. (2000)
Role of metals in the reaction catalyzed by protein farnesyltransferase.
Biochemistry 39, 12398−12405.
(54) Long, S. B., Hancock, P. J., Kral, A. M., Hellinga, H. W., and
Beese, L. S. (2001) The crystal structure of human protein
farnesyltransferase reveals the basis for inhibition by CaaX
tetrapeptides and their mimetics. Proc. Natl. Acad. Sci. U.S.A. 98,
12948−12953.
(34) Hirshberg, M., Henrick, K., Haire, L. L., Vasisht, N., Brune, M.,
Corrie, J. E., and Webb, M. R. (1998) Crystal structure of phosphate
binding protein labeled with a coumarin fluorophore, a probe for
inorganic phosphate. Biochemistry 37, 10381−10385.
(35) Zimmerman, K. K., Scholten, J. D., Huang, C. C., Fierke, C. A.,
and Hupe, D. J. (1998) High-level expression of rat farnesyl:protein
transferase in Escherichia coli as a translationally coupled heterodimer.
Protein Expression Purif. 14, 395−402.
(36) Mu, Y. Q., Omer, C. A., and Gibbs, R. A. (1996) On the
stereochemical course of human protein-farnesyl transferase. J. Am.
Chem. Soc. 118, 1817−1823.
(37) March, J. (1968) Advanced Organic Chemistry, 2nd ed., McGraw-
Hill, New York.
(55) Park, H. W., Boduluri, S. R., Moomaw, J. F., Casey, P. J., and
Beese, L. S. (1997) Crystal structure of protein farnesyltransferase at
2.25 angstrom resolution. Science 275, 1800−1804.
(56) Reid, T. S., Terry, K. L., Casey, P. J., and Beese, L. S. (2004)
Crystallographic analysis of CaaX prenyltransferases complexed with
substrates defines rules of protein substrate selectivity. J. Mol. Biol. 343,
417−433.
8318
dx.doi.org/10.1021/bi3011362 | Biochemistry 2012, 51, 8307−8319

Biochemistry
Article
(57) Strickland, C. L., Windsor, W. T., Syto, R., Wang, L., Bond, R.,
Wu, Z., Schwartz, J., Le, H. V., Beese, L. S., and Weber, P. C. (1998)
Crystal structure of farnesyl protein transferase complexed with a
CaaX peptide and farnesyl diphosphate analogue. Biochemistry 37,
16601−16611.
(58) Hougland, J. L., and Fierke, C. A. (2009) Getting a handle on
protein prenylation. Nat. Chem. Biol. 5, 197−198.
8319
dx.doi.org/10.1021/bi3011362 | Biochemistry 2012, 51, 8307−8319