Synthesis and evaluation of chlorinated substrate analogues for farnesyl diphosphate synthase

Article abstract of DOI:10.1021/jo1024305

Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a cosubstrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of K_M ^3-ClIPP (with DMAPP) and K_M ^3-ClDMAPP (with IPP) were similar to those for IPP and DMAPP; however, values of κ_cat for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate. 2011 American Chemical Society.

Full text of DOI:10.1021/jo1024305

ARTICLE
pubs.acs.org/joc
Synthesis and Evaluation of Chlorinated Substrate Analogues for
Farnesyl Diphosphate Synthase
Nicole A. Heaps†,‡ and C. Dale Poulter*,†
†Department of Chemistry, University of Utah, 315 South 1400 East RM 2020, Salt Lake City, Utah 84112, United States
S Supporting Information
b
ABSTRACT: Substrate analogues for isopentenyl diphosphate
(IPP) and dimethylallyl diphosphate (DMAPP), where the C3
methyl groups were replaced by chlorine, were synthesized and
evaluated as substrates for avian farnesyl diphosphate synthase
(FPPase). The IPP analogue (3-ClIPP) was a cosubstrate when
incubated with dimethylallyl diphosphate (DMAPP) or geranyl
diphosphate (GPP) to give the corresponding chlorinated analo-
gues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate
(3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP
gave 11-ClFPP as the sole product. Values of KM3-ClIPP (with DMAPP) and KM3-ClDMAPP (with IPP) were similar to those for IPP and
DMAPP; however, values of kcat for both analogues were substantially lower. These results are consistent with a dissociative electrophilic
alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to
alkylation of the double bond of the homoallylic substrate.
’ INTRODUCTION
C3 methyl groups of IPP, DMAPP, and GPP.11-15 These
analogues have been used for in vitro enzymatic synthesis of a
variety of biological molecules.15-17 We now report the synthesis
and evaluation of analogues of IPP and DMAPP where the C3
methyl groups in both are replaced by chlorine.
Farnesyl diphosphate (FPP) is a key precursor for synthesis of
isoprenoid metabolites containing three or more isoprene units.1
FPP is synthesized in two steps by addition of dimethylallyl
diphosphate (DMAPP) to isopentenyl diphosphate (IPP) to
give geranyl diphosphate (GPP), followed by addition of GPP to
a second molecule of IPP to provide FPP.2 The reactions are
catalyzed by farnesyl diphosphate synthase (FPPase), a short-
chain elongation enzyme that contains a distinctive R-helical fold
characteristic of members of the isoprenoid synthase super-
family.1,3,4 This group includes E-selective short and long chain
elongation enzymes; the cyclopropanation enzymes squalene
synthase, phytoene synthase, and chrysanthemyl diphosphate
synthase; and mono-, sesqui-, and diterpene cyclases. In FPPase
the hydrocarbon tail of the allylic substrate is located in a
hydrophobic pocket that dictates the ultimate chain length of
the product.1 The diphosphate moiety is bound to active-site
arginine and lysine residues and to three Mg2þ ions stabilized by
two aspartate-rich DDXX(XX)D motifs.5 The hydrocarbon
moiety in IPP lies above the allylic substrate and its diphosphate
moiety is stabilized by a combination of arginine and lysine
residues and tightly bound waters.5
’ RESULTS
Synthesis. The synthesis of the C3 chloro analogue of IPP
(3-ClIPP) is shown in Scheme 1. 3-Buten-1-ol (1-OH) was
converted to the corresponding tosylate (1-OTs) followed by
treatment with sulfuryl chloride and phenyl selenyl chloride.18
The resulting selenoxide was heated with aqueous sodium bicarbo-
nate and treated with hydrogen peroxide to give (2-OTs).18 The
tosylate was treated with tris-tetrabutylammonium hydrogen pyro-
phosphate in acetonitrile19 to give 3-ClIPP in an overall yield of
45%.
Chlorinated DMAPP (3-ClDMAPP) was synthesized in three
steps starting from but-2-yn-1-ol (3-OH) as shown in Scheme 2.
Alcohol 3-OH was converted to 4-OH by treatment with Red-Al
and NCS20 followed by NCS and DMS to give dichloride 4-Cl.19
The allylic chloride was then converted to 3-ClDMAPP using
tris-tetrabutylammonium hydrogen pyrophosphate19 in an over-
all yield of 9% from 3-OH.
Mechanistic studies with fluorinated2,6 and bisubstrate7,8
analogues indicate that the chain elongation reaction is a
dissociative electrophilic alkylation that involves cleavage of the
carbon-oxygen bond in the allylic substrate to form a resonance
stabilized carbocation, alkylation of the double bond in IPP to
generate a tertiary carbocation, and elimination of the pro-R
proton at C2 to generate an E double bond between C2 and
C3 in the product.9,10 FPPase is tolerant to modifications of the
Product Studies. In preliminary experiments, 3-ClIPP and
DMAPP, GPP, or 3-ClDMAPP and 3-ClDMAPP and IPP were
incubated with FPPase in 35 mM HEPES buffer, pH 7.4,
followed by incubation with alkaline phosphatase to hydrolyze
Received: December 23, 2010
Published: February 24, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of 3-ClIPP
The diphosphate products were purified by chromatography on
cellulose and analyzed by 1H, 13C, and 31P NMR spectroscopy. In
1
addition, H-1H COSY, and 1D NOE spectra were used to
determine the stereochemistry of the double bonds in the hydro-
carbon chain. High resolution mass spectra showed an M þ 2
isotope peak due to the presence of chlorine.
Incubations with 3-ClIPP and GPP gave a chlorinated analo-
gue of FPP (3-ClFPP) where the methyl at C3 was replaced by
chlorine (see Scheme 3). Incubations with 3-ClIPP and DMAPP
gave a chlorinated analogue of GPP (3-ClGPP) with a similar
replacement. In this case, chain elongation terminated with the
formation of 3-ClGPP, and longer incubation times and in-
creased substrate and enzyme concentrations did not give
detectable amounts of a doubly chlorinated FPP derivative.
Incubation of 3-ClDMAPP and IPP gave a chlorinated FPP
derivative (11-ClFPP) where the methyl group at C11 was
replaced by chlorine as the sole product.
Scheme 2. Synthesis of 3-ClDMAPP
The stereochemistry of the double bonds in the chlorine-
containing isoprene units of 3-ClGPP, 3-ClFPP, and 11-ClFPP
1
was established from H NOE difference spectra, where NOEs
were recorded as positive peaks and the irradiation frequency
appeared as a negative peak. The difference spectrum for 3-
ClGPP had a strong NOE between the resonances from 2.41 to
2.44 ppm for the methylene protons at C4 and the resonance at
5.84 ppm for the vinyl proton at C2, indicating a Z C2-C3
double bond. A similar NOE was seen between the resonances
from 2.40 to 2.44 ppm and the resonance at 5.86 ppm in the
spectrum for 3-ClFPP. The stereochemistry of the C10-C11
double bond of 11-ClFPP was also determined to be Z due to the
presence of a strong NOE between the methyl group at 2.1 ppm
and the vinyl proton at 5.6 ppm.
Kinetic Studies. Steady-state kinetic constants were mea-
sured for chain elongation with [1-14C]IPP and 3-ClDMAPP
and with 3-ClDMAPP and [1-14C]DMAPP (see Table 2). The
standard acid lability assay was used to determine rates for chain
elongation with [1-14C]IPP.21 Rates for chain elongation with
[1-14C]DMAPP were determined by analyzing assay mixtures by
the diphosphate esters to the corresponding alcohols. The
mixtures were then analyzed by GCMS to determine if the
chlorinated substrates were substrates for chain elongation. As
seen in Table 1, peaks with characteristic masses for chain
elongation products were seen for incubations of 3-ClIPP with
DMAPP and GPP and of 3-ClDMAPP with IPP. In each case,
mass spectra of the putative products had an M þ 2 peak whose
intensity was 30% of M, as expected for the isotopic pattern of
chlorine. Thus, incubation of 3-ClIPP and GPP with FPPase
gave a monochlorinated derivative of FPP and incubation with
DMAPP gave a monochlorinated derivative of GPP. A dichlori-
nated FPP derivative was not detected. Incubation of 3-ClD-
MAPP and IPP with FPPase resulted in the exclusive production
of a monochlorinated FPP derivative.
TLC using autoradiography to quantify radioactivity in substrates
3-ClDMAPP
and products. KM
= 2.1 μM is similar to the value
reported for addition of DMAPP to IPP, while kcat = 0.005 s-1
3-ClIPP
is 100-fold lower.22 The KM
of 6.1 μM is only slightly
higher than the value reported for IPP,23 while kcat = 0.006 s-1 is
substantially lower.
’ DISCUSSION
FPPase can accept a rather wide variety of IPP and DMAPP
analogues as substrates. Numerous studies have shown that the
enzyme is more tolerant of modifications in the allylic substrate
than the homoallyic substrate. The enzyme can utilize DMAPP
Large-scale incubations were then conducted with avian
FPPase in 35 mM sodium phosphate buffer pH 7.4 to facilitate
purification of the diphosphate products for analysis by NMR.
Following the incubations, FPPase was removed by ultrafiltration.
Table 1. GCMS Analysis of Alcohols Obtained by Hydrolysis of Diphosphates Produced by FPPasea
homoallylic substrate
allylic substrate
products
GC tR (min)
m/z
3-ClIPP
3-ClIPP
IPP
GPP
3-ClFOH
3-ClGOH
11-ClFOH
no reaction
39.5
26.7
39.8
242
DMAPP
156 (M - H2O)
224 (M - H2O)
3-ClDMAPP
3-ClDMAPP
3-ClIPP
a FPPase incubations: 35 mM HEPES buffer, pH 7.4, containing 10 mM MgCl2, 5.0 mM β-mercaptoethanol, 200 μM allylic substrate, 200 μM
homoallylic substrate, and 200 μg of avian FPPase in a final volume of 400 μL at 30 °C for 2 h. Phosphatase incubations: 80 μL of 500 mM glycine buffer,
pH 10.5, containing 5 mM ZnCl2 and 40 units of calf mucosa alkaline phosphatase were added, and the mixtures were incubated for an additional 1 h
at 37 °C.
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Scheme 3. Structures of Chlorinated Metabolites Resulting from Incubations of 3-ClIPP and 3-ClDMAPP with IPP, DMAPP, or
GPPa
a Incubations were run in 35 mM sodium phosphate buffer, pH 7.4, containing 10 mM MgCl2, substrate, and 10 mg of avian FPPase in a total volume of
2 mL at 30 °C for 24 h.
Scheme 4. Dissociative Electrophilic Alkylation Mechanism
for Chain Elongation
Table 2. Steady-State Kinetic Constants of Chlorinated
Analogues with Avian FPPasea
varied substrate fixed substrate kcat (s-1
)
KM (μM) kcat/KM (M-1 s-1
)
IPP
DMAPP 1.1b
IPP 1.1
0.6b
1.6c
1.8 ꢀ 106
6.0 ꢀ 105
1.0 ꢀ 103
2.4 ꢀ 103
DMAPP
3-ClIPP
3-ClDMAPP
DMAPP 0.006 ( 0.002 6.1 ( 0.1
IPP 0.005 ( 0.001 2.1 ( 0.2
a Michaelis constants were determined by plotting initial velocity versus
substrate concentration using GRAFIT (Sigma). KM for 3-ClIPP and
3-ClDMAPP are apparent values determined with saturating concen-
trations of the fixed substrate. b Reference 18. c Reference 19.
analogues with the E methyl group replaced by unbranched alkyl
chains of up to C9.12,24 In contrast, it is incapable of utilizing IPP
analogues where the C3 methyl group has been replaced by a
carbon chain longer than n-propyl.14 The ability of FPPase to
accept analogues of both DMAPP and IPP has been utilized for
the stereospecific synthesis of naturally occurring isoprenoid
compounds.16,17
3-ClIPP and 3-ClDMAPP are alternate substrates for chain
elongation with their respective natural cosubstrates. Both
of the analogues are ∼200-fold less active (kcat) than their
natural counterparts and have somewhat elevated KM's—
10-fold for 3-ClIPP and 1.5-fold for 3-ClDMAPP. Incubation
of 3-ClIPP and DMAPP or GPP with FPPase gave the corre-
sponding 3-chloro derivatives of GPP and FPP, while incubation
of 3-ClDMAPP with IPP gave 11-ClFPP. In each case, the
stereochemistry of the newly formed double bonds was the
same as seen for the natural substrates. Turnover was not
detected for incubations with 3-ClIPP and 3-ClDMAPP. We
estimate that chain elongation with both chlorinated analogues is
at least 2000 times slower than for IPP and DMAPP.
3-ClIPP and 3-ClDMAPP can be used for the enzyme-
catalyzed synthesis of 3-ClGPP, 3-ClFPP, 11-ClFPP, and pre-
sumably 7-ClFPP by incubating 3-ClGPP with IPP. A similar
strategy with a combination of FPPase and GGPPase would
permit synthesis of 3-chloro, 7-chloro, 11-chloro, and 15-chloro
analogues of GGPP. These molecules should be valuable tools
for probing the mechanisms of sesquiterpene and diterpene
cyclases and for structural studies of enzymes in the isoprenoid
biosynthetic pathway by X-ray crystallography.
Our kinetic results are consistent with a dissociative elec-
trophilic alkylation mechanism for chain elongation (see
Scheme 4)25 where heterolytic cleavage of the C-O bond in
DMAPP gives an intimate ion pair with the allylic dimethylallyl
cation sandwiched between IPP and PPi. In this scenario, ir-
reversible alkylation to form the tertiary cation would compete with
internal return to DMAPP. KM3-ClDMAPP is only slight larger than
KMDMAPP. The van der Waals radii of chlorine and methyl groups
are similar,26 and apparently the larger bond dipole for the C-Cl
bond27 does not substantially alter binding. However, kcat
3-ClDMAPP
is ∼200-fold smaller than kcatDMAPP, presumably because of a
reduction in k1, the step for carbon-oxygen heterolysis in 3-
ClDMAPP. If one assumes that k1 is rate-limiting for 3-
ClDMAPP, and at least partially rate-limiting for DMAPP, the
changes in kcat are consistent with an electronic effect when the
methyl group (σpþ = -0.311) at C3 in DMAPP is replaced with
chlorine (σpþ = 0.114) in 3-ClDMAPP.28 Related decreasesinkcat
are seen for H, CH2F, CHF2, and CF3 substitutents at C3 of
DMAPP.29 Interestingly, 3-ClIPP shows similar behavior in both
KM and kcat. It is likely that the substantially lower value of kcat
results from an electronic effect for the homoallylic substrate as
well. Although FPPase tolerates a rather broad range of homo-
allylic substrate analogues consistent with the similar values for
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KMIPP and KM3-ClIPP, one cannot exclude the possibility that the
carbon-chlorine bond dipole causes a change in alignment of 3-
ClIPP and DMAPP that places the C3-C4 double bond in 3-
ClIPP in an unfavorable orientation for alkylation and thus
lowers the rate of the reaction. We favor a change in the rate-
limiting step from k1 to k2, alkylation of the double bond in 3-
ClIPP, caused by the chlorine substituent. Attempts to directly
detect internal return (k-1) in FPPase by positional isotope
exchange or internal trapping experiments have not been
successful,30,31 presumably because the conformation of inor-
ganic pyrophosphate in the ion pair is locked in the active site
through coordination with magnesium ions.5 Related experi-
ments were successful in detecting internal return for dimethy-
lallyltryptophan synthase, a prenyl transfer enzyme that does not
bind the magnesium form of DMAPP.32
In conclusion, we describe the synthesis of analogues of IPP
and DMAPP where the methyl at C3 is replaced by chlorine.
These molecules are alternative substrates for FPPase, giving
novel linear chlorinated geranyl and farnesyl analogues. The
kinetic parameters for the analogues are consistent with a
dissociative electrophilic alkylation mechanism for the chain
elongation reaction where the rate-limiting step changes from
carbon-oxygen heterolysis in the allylic substrate for
3-ClDMAPP to addition of the allylic cationic intermediate to
the double bond in 3-ClIPP.
a minimal amount of ion exchange buffer (1:49 (v/v) 2-propanol/25 mM
ammonium bicarbonate) and loaded on to the ammonium form of
Dowex ion-exchange resin. The column was eluted with 2 column
volumes of exchange buffer. The elutant was flash frozen and dried by
lyophilization to yield a white solid. The solid was dissolved in 0.1 M
ammonium bicarbonate; 15 mL of 1:1 (v/v) 2-propanol/acetonitrile
was added; and the mixture was vortexed. The resulting white slurry was
cleared by centrifugation; the supernatant was removed; and the process
was repeated three times. The supernatants were pooled, concentrated,
and dried by dried by lyophilization to yield a white solid. The solid was
chromatographed on cellulose using 70:30 (v/v) 2-propanol/0.1 M
ammonium bicarbonate. Fractions containing the organic diphosphate
were combined, flash frozen, and dried by lyophilization to yield a fluffy
white solid.
3-Chloro-3-butenyl Diphosphate (3-ClIPP). Following the
general procedure, 1.66 g (1.84 mmol) of tris(tetra-n-butylammonium)
hydrogen pyrophosphate and 0.240 g (0.92 mmol) of 2-OTs gave 188
mg (78%) of a fluffy white solid: 1H NMR (D2O) δ 2.71 (t, 2H, J = 6.3
Hz), 4.11 (dd, 2H, J = 6.3, 6.1 Hz), 5.33 (s, 1H), 5.38 (s, 1H) ppm;
13C NMR (D2O) δ 40.0 (d, J = 7.3 Hz), 63.4 (d, J = 5.3 Hz), 115. 2,
139.2; 31P NMR (D2O) δ -6.25 (d, J = 19.5 Hz), -10.24 (d, J = 20.0
Hz); HRMS (FAB) calcd for C4H8ClO7P2 (M - H) 264.9434, found
264.9430.
(Z)-3-Chloro-2-but-en-1-ol (4-OH). To a solution of 0.162 g
(2.3 mmol) of 2-butyn-1-ol in 10 mL of dry THF was added dropwise via
syringe 1.17 mL (3.9 mmol) of Red-Al. The mixture was allowed to stir
at rt for 18 h, cooled to -78 °C before 0.59 g (4.4 mmol) of NCS
dissolved in 5 mL of THF was added dropwise, and allowed to stir at -
78 °C for an additional 1 h. The mixture was stirred at 0 °C for 2 h and
was quenched with saturated Rochelle’s salt. The aqueous layer was
extracted with ether. The organic layers were combined, washed with
brine, dried over MgSO4, and concentrated under vacuum. The residue
was purified by chromatography on silica using 3:2 (v/v) hexane/ether
to yield 96 mg (39%) of a pale yellow oil: 1H NMR (CDCl3) δ 2.08-
2.09 (m, 3H), 3.12 (br s, 1H), 4.19-4.22 (m, 2H), 5.61-5.67 (m, 1H);
13C NMR (CDCl3) δ 26.4, 59.9, 125.1, 133.0; HRMS (EI) calcd for
C4H7OCl (Mþ) 106.0180, found 106.0174.
(Z)-1,3-Dichloro-2-butene (4-Cl). To a solution containing
0.451 of g (3.38 mmol) of N-chlorosuccinimide in 20 mL of dry CH2Cl2
at -30 °C was added 2.1 mL (3.38 mmol) of DMS. The mixture was
briefly allowed to warm to 0 °C and cooled to -40 °C before a solution
of 0.3 g (2.82 mmol) of 4-OH in 2 mL of CH2Cl2 was added. The
reaction mixture was allowed to warm to 0 °C over 1 h and was stirred at
this temperature for an additional 1 h. The mixture was warmed to room
temperature and stirred for 15 min. The mixture was added to 15 mL of
brine and extracted with pentane. The combined organic layers were
washed with brine, dried over MgSO4, filtered, and concentrated to give
185 mg (53%) of a yellow oil. The residue was used in the subsequent
phosphorylation reaction without further purification.
’ EXPERIMENTAL SECTION
3-Buten-1-yl Tosylate (1-OTs). To a solution containing 5.3 g
(27.7 mmol) of p-toluenesulfonyl chloride and 6.8 g (55.5 mmol) of N,
N-dimethylaminopyridine in 25 mL of CH2Cl2 was added 2.0 g (27.7
mmol) of 3-buten-1-ol. The reaction mixture was allowed to stir at room
temperature under N2 for 2 h. Solvent was removed by rotary evapora-
tion, and the resulting white solid was chromatographed on silica using
4:1 (v/v) hexane/ethyl acetate to give 5.8 g (89%) of a colorless oil:
1H NMR (CDCl3) δ 2.37-2.44 (m, 2H), 2.46 (s, 3H), 4.07 (t, 2H, J =
6.6 Hz), 5.05-5.06 (m, 1H), 5.08-5.12 (m, 1H), 5.61-5.75 (m, 1H),
7.33-7.38 (m, 2H), 7.78-7.82 (m, 2H); 13C NMR (CDCl3) δ 21.9,
33.3, 69.6, 118.4, 128.1, 130.0, 132.6, 133.3, 145.0; HRMS (EI) calcd for
C11H15O3S (M þ H) 227.0736, found 227.0735.
3-Chloro-3-butenyl Tosylate (2-OTs). A solution containing
0.5 g (2.2 mmol) of 1-OTs and 0.42 g (2.2 mmol) of phenylselenyl
chloride in 4 mL of dry CHCl3 was allowed to stir under N2 at rt
overnight. Sulfuryl chloride (202 μL, 2.5 mmol) was added dropwise via
syringe, and the reaction mixture was allowed to stir for h at rt. The
mixture was concentrated by rotary evaporation to give a yellow oil.
Benzene and aqueous NaHCO3 were added, and the mixture was heated
at reflux for 4 h. The mixture was allowed to cool, and the organic layer
was shaken with 30% H2O2 until the yellow color disappeared (40 mL
for 30 min). The organic layer was dried over MgSO4, filtered, and
concentrated by rotary evaporation. The residue was purified by
chromatography on silica using 10:1 (v:v) hexane/ethyl acetate to give
625 mg (54%) of a light yellow oil: 1H NMR (CDCl3) δ 2.47 (s, 3H),
2.68 (t, 2H, J = 5.8 Hz), 4.21 (t, 2H, J = 6.0 Hz), 5.23 (s, 1H,), 5.25 (s,
1H), 7.35-7.38 (m, 2H), 7.79-7.82 (m, 2H); 13C NMR (CDCl3) δ
21.8, 38.8, 66.8, 115.8, 128.1, 130.0, 132.8, 136.8, 145.1; HRMS (FAB)
calcd for C11H14SClO3 (M þ H) 261.0353, found 261.0353.
(Z)-3-Chloro-2-butenyl Diphosphate (3-ClDMAPP). Fol-
lowing the general procedure, 2.67 g (2.96 mmol) of tris(tetra-n-
butylammonium) hydrogen pyrophosphate and 0.185 g (1.48 mmol)
of 4-Cl gave 174 mg (45%) of a white solid: 1H NMR (D2O) δ 2.14 (s,
3H), 4.56 (t, 2H, J = 6.9 Hz), 5.83 (t, 1H, J = 5.5 Hz); 13C NMR (D2O) δ
26.1, 63.6(d, J = 5.1 Hz), 122.7(d, J = 7.6 Hz), 134.7; 31P NMR (D2O) δ
-6.75 (d, J = 21.7 Hz), -10.29 (d, J = 21. Hz); HRMS (FAB) calcd for
C4H8ClO7P2 (M - H) 264.9434, found 264.9434.
Protein Expression and Purification. The plasmid for avian
FPPase was transformed into E. coli XA90 cells and plated onto LB
ampicillin plates. Liquid cultures were grown overnight, and 15 mL was
used to inoculate 1.5 L cultures. Cultures were grown at 37 °C to an
OD600 of 0.5 and were induced by addition of IPTG (1 mM). Cultures
were allowed to grow an additional 4-5 h at 37 °C, and cells were
collected by centrifugation at 6000 rpm for 10 min. Cell paste was kept at
General Procedure for Preparation of Diphosphates. To a
stirred mixture of tris(tetra-n-butylammonium) hydrogen pyropho-
sphate (2 equiv) in dry acetonitrile was added via syringe either tosylate
or chloride dissolved in a minimal amount of dry acetonitrile. The
reaction mixture was allowed to stir under N2 for 2 h and then
concentrated by rotary evaporation. The resulting oil was dissolved in
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-80 °C until further use. Frozen cell paste was thawed on ice and
suspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM
imidazole). Cells were lysed by sonication and cellular debris was cleared
by centrifugation at 6000 rpm for 15 min. The supernatant was added to
10 mL of Ni-NTA resin and shaken for 2 h at 4 °C. The resulting slurry
was poured into a fritted glass column and allowed to settle. The column
was washed with two column volumes of lysis buffer, followed by 50 mL
of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH
8.0). Protein was eluted with 100 mL of elution buffer (50 mM
NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) and precipitated
with 33% (w/v) ammonium sulfate. This solution was centrifuged at
7000 rpm for 20 min, and the resulting pellet was dissolved in 5-10 mL
filtration as described above. The filtrates were flash frozen and dried by
lyophilization to give a white powder. The lyophilized solid was and
purified by chromatography on cellulose. The column was eluted with
3 column volumes of 100% 2-propanol, followed by 4:1 (v/v) 2-propa-
nol/0.1 M NH4HCO3. Fractions were analyzed by silica TLC using
p-ansialdehyde. Those containing diphosphate products were pooled,
concentrated at reduced pressure to remove 2-propanol, flash frozen,
and dried by lyophilization to give a white powder. Purified products
were characterized by NMR and mass spectrometry.
(E,E,Z)-11-Chloro-3,7-dimethyldodeca-2,6,10-triene (11-
ClFPP). Following the general procedure for synthesis of chlorinated
metabolites in the section on NMR analysis, 11 mM 3-ClIPP and 12.1
mM GPP were incubated with FPPase to give a white solid: 1H NMR
(D2O) δ 1.63 (s, 3H), 1.72 (s, 3H), 2.10-2.17 (m, 9H), 2.23-2.30
(m, 2H), 4.47 (t, 2H, J = 6.1 Hz), 5.23 (t, 1H, J = 6.4 Hz), 5.47 (t, 1H, J
= 6.6 Hz), 5.53 (t, 1H, J = 6.9 Hz); 13C NMR (D2O) δ 15.7, 16.2, 25.8,
26.2, 27.1, 38.1, 39.4, 63.0 (d, J = 4.7 Hz), 120.5 (d, J = 7.3 Hz), 125.2,
126.3, 130.6, 136.6, 143.2; 31P NMR (D2O) δ -4.92, -9.05; HRMS
(FTMS) calcd for C14H24ClO7P2 (M - H) 401.0691, found 401.0690.
(Z,E)-3-Chloro-7,11-dimethyldodeca-2,6,10-triene (3-
ClFPP). Following the general procedure for synthesis of chlori-
nated metabolites in the section on NMR analysis, 22 mM IPP and
12 mM 3-ClDMAPP were incubated with FPPase to give a white
of dialysis buffer (20 mM Tris HCl, 4 mM DTT at pH 8.0). The
3
solution was transferred to a dialysis cassette (10000 MWCO) and
dialyzed for 3 h in dialysis buffer. The protein was then dialyzed
overnight against this same dialysis buffer and then dialyzed an addi-
tional 4 h in storage buffer (20 mM Tris HCl pH 8.0, 4 mM DTT, 20%
3
glycerol). The protein was stored at -80 °C in 100 μL aliquots until use.
Kinetic Studies. Initial velocities were measured using the acid
lability assay for reactions with [1-14C]IPP and Cl-DMAPP. Incubations
were in 35 mM HEPES buffer, pH 7.4, containing 10 μM [14C]IPP
(30 μCi/μmol), 0.5-50 μM 3-ClDMAPP, 10 mM MgCl2, 10 mM β-ME,
and 1 mg/mL BSA in a total volume of 100 μL. Reactions were initiated by
the addition of enzyme (10 μL) and were incubated at 37 °C for 10 min.
The reaction was quenched by the addition of 200 μL of 4:1 MeOH/HCl,
and incubated for an additional 10 min at 37 °C. One mL of ligroine
(bp 95-110 °C) was added, the mixture was vortexed, and a 0.5 mL sample
of the ligroine layer was counted by liquid scintillation spectrometry. For
incubations with [14C]DMAPP and 3-ClIPP, reactions were run in 35 mM
HEPES buffer, pH 7.4, containing 50 μM [14C]DMAPP (20 μCi/μmol),
0.5-50 μM 3-ClIPP, 10 mM MgCl2, 10 mM β-ME, and 1 mg/mL BSA in
a total volume of 50 μL. Reactions were initiated by the addition of enzyme
(10 μL) and incubated at 37 °C for 10 min. The assay mixtures were
quenched by the addition of 50 μL of MeOH. The resulting mixture was
spotted onto a silica gel TLC plate and developed with chloroform/
pyridine/formic acid/water (30:70:16:10). The plate was allowed to dry
overnight and was visualized by phosphoimaging. Radioactivity was quanti-
fied using Imagequant. Kinetic constants were determined from plots of
initial velocity versus substrate concentration using Grafit 5.
1
solid: H NMR (D2O) δ 1.62 (s, 3H), 1.63 (s, 3H), 1.69 (s, 3H),
2.02-2.04 (m, 2H), 2.09-2.14 (m, 2H), 2.27-2.31 (m, 2H), 2.40-
2.44 (m, 2H), 4.59 (t, 2H, J = 6.5 Hz), 5.17-5.22 (m, 2H), 5.86 (t,
1H, J = 6.4 Hz); 13C NMR (D2O) δ 16.0, 17.6, 25.5, 26.0, 26.5, 39.3,
39.4, 63.4 (d, J = 4.5 Hz), 123.0 (d, J = 7.7 Hz), 123.5, 125.1, 134.2,
138.2, 138.3; 31P NMR (D2O) δ -5.91, -10.04; HRMS (FTMS)
calcd for C14H24ClO7P2 (M - H) 401.0691, found 401.0691.
(Z)-3-Chloro-7-methylocta-2,6-diene (3-ClGPP). Following
the general procedure for synthesis of chlorinated metabolites in the
section on NMR analysis, 16.6 mM DMAPP and 15 mM 3-ClIPP were
incubated with FPPase to give a white solid: 1H NMR (D2O) δ 1.63 (s,
3H), 1.69 (s, 3H), 2.26-2.30 (m, 2H), 2.41-2.44 (m, 2H), 4.60 (t, 2H,
J = 6.4 Hz), 5.17-5.20 (m, 1H), 5.84 (t, 1H, J = 5.8 Hz); 13C NMR
(D2O) δ 17.7, 25.5, 25.9, 39.3, 63.8 (d, J = 3.1 Hz), 122.7 (d, J = 7.3 Hz),
123.2, 135.3, 138.4; 31P NMR (D2O) δ -8.43, -10.51; HRMS
(FTMS) calcd for C9H16ClO7P2 (M - H) 333.0065, found 333.0065.
Product Studies. GC and GC/MS Analysis. Incubations were in
35 mM HEPES buffer, pH 7.4, containing 10 mM MgCl2, 5 mM β-ME, and
200 μM of each substrate in a total volume of 400 μL at 30 °C. After 2 h, 36
μL of 0.5 M glycine buffer, pH 10.6, containing 5 mM ZnCl2 followed by 40
U of alkaline phosphatase, and incubation was continued for 1 h at 37 °C.
Solid NaCl was added, and the mixture was extracted with tert-butyl methyl
ether. Samples were concentrated to 5-10 μL with a gentle stream of N2 and
analyzed by GC and GC-MS on a DB5 capillary column using the following
program: 55 °C for 3 min, followed by a linear gradient (1 °C/min) to 65 °C,
then (2 °C/min) to 120 °C, followed by (10 °C/min) to 230 °C.
’ ASSOCIATED CONTENT
Supporting Information. General methods; H, 13C,
1
S
b
31P,1H NOE-difference, and COSY NMR spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: poulter@chemistry.utah.edu.
NMR Analysis. Avian FPPase was dialyzed against 35 mM Na2HPO4
buffer, pH 7.4, for 24 h to remove HEPES, glycerol, and DTT from the
storage buffer. The resulting enzyme was concentrated by centrifugation
(Centricon, 10000 MWCO). Incubations were in 35 mM Na2HPO4
buffer, pH 7.4, containing 10 mM MgCl2, 10 mg of avian FPPase, and the
indicated concentrations of substrates at 30 °C. The mixture was
incubated for 4 h, after which an additional 2.5 mg of enzyme was
added, followed by another 4 h of incubation. A final 2.5 mg portion of
enzyme was added and the incubation was continued overnight. The
mixture was filtered by centrifugation (Centricon, 10000 MWCO) to
remove enzyme and precipitated salts. The precipitate was washed
several times with 0.1 M NH4HCO3 and filtered as before. The filtrates
were combined, flash frozen and dried by lyophilization to give a white
powder. The solid precipitate stirred overnight with 400 mg of Chelex in
10 mL of 0.1 M NH4HCO3. The remaining precipitate was removed by
Present Addresses
‡Sapphire Energy, 3115 Merryfield Row, San Diego, CA 92121.
’ ACKNOWLEDGMENT
This project was supported by NIH Grant GM 21328. N.A.H.
was supported by a chemistry/biology interface predoctoral
training grant (GM08537).
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