Synthesis and Enzymatic Studies of Isoprenoid Thiolo Bisubstrate Analogues

Article abstract of DOI:10.1021/acs.joc.6b00664

Chain elongation prenyltransferases catalyze the addition of the hydrocarbon moiety of allylic isoprenoid diphosphates to the carbon-carbon double bond in isopentenyl diphosphate (IPP) in the primary building reactions in the isoprenoid biosynthetic pathway. Bis-O-diphosphate analogues 3-OPP/OPP, 4-OPP/OPP, and 5-OPP/OPP and bis-thiolodiphosphate bisubstrate analogues 3-SPP/SPP, 4-SPP/SPP, and 5-SPP/SPP were synthesized. The analogues 4-OPP/OPP, 5-OPP/OPP, 4-SPP/SPP, and 5-SPP/SPP were excellent competitive inhibitors of avian farnesyl diphosphate synthase with K_I = 1.0 ± 0.12 μM, K_I = 0.5 ± 0.2 μM, K_I = 0.7 ± 0.3 μM, and K_I = 2.9 ± 0.27 μM, respectively, whereas, analogues 3-OPP/OPP and 3-SPP/SPP displayed mixed type inhibition with K_I = 1.4 μM and K_I = 5.5 μM, respectively.

Full text of DOI:10.1021/acs.joc.6b00664

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Article
Synthesis and Enzymatic Studies of Isoprenoid Thiolo Bisubstrate Analogs
Gurusankar Ramamoorthy, Richard Man Phan, and C Dale Poulter
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b00664 • Publication Date (Web): 03 May 2016
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Synthesis and Enzymatic Studies of Isoprenoid Thiolo Bisubstrate
Analogs
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Gurusankar Ramamoorthy, Richard M. Phan,† C. Dale Poulter*
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States
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Mailing address:
315 South 1400 East RM 2020
Department of Chemistry
University of Utah
Salt Lake City, UT 84112
Phone: (801) 581ꢀ6685
FAX: (801) 581ꢀ4391
Eꢀmail: poulter@chem.utah.edu
† Present Address: U.S. Army Dugway Proving Ground, Building 4153, Dugway,
UT 84022, United States
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Abstract
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Chain elongation prenyltransferases catalyze the addition of the hydrocarbon moiety of
allylic isoprenoid diphosphates to the carbonꢀcarbon double bond in isopentenyl
diphosphate (IPP) in the primary building reactions in the isoprenoid biosynthetic
pathway. BisꢀOꢀdiphosphate analogs 3-OPP/OPP, 4-OPP/OPP and 5-OPP/OPP and
bisꢀthiodiphosphate bisubstrate analogs 3-SPP/SPP, 4-SPP/SPP and 5-SPP/SPP were
synthesized. The analogs 4-OPP/OPP, 5-OPP/OPP, 4-SPP/SPP, and 5-SPP/SPP were
excellent competitive inhibitors of avian farnesyl diphosphate synthase with KI = 1.0 ±
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0.12 ꢀM, KI = 0.5 ± 0.2 ꢀM, KI = 0.7 ± 0.3 ꢀM, and KI = 2.9 ± 0.27 ꢀM, respectively;
whereas, analogs 3-OPP/OPP and 3-SPP/SPP displayed mixed type inhibition with KI =
1.4 ꢀM and KI = 5.5 ꢀM, respectively.
XPP
Inhibition of FPP synthesis
16
PPX
R
X = O,O/S,S
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OPP
4
PPO
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0.4
-1
ꢀ
1/GPP (
M
)
Gallus gallus FPPase
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Introduction
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Prenyltransferases catalyze electrophilic alkylation of electronꢀrich double bonds
or electronegative atoms by isoprenoid allylic diphosphate esters to create new carbonꢀ
carbon or carbonꢀheteroatom bonds.1 The enzymes alkylate a broad spectrum of
nucleophilic substrates, including diꢀ and trisubstituted double bonds (chain elongation
and cyclopropanation prenyltransferases),2 aromatic rings (aromatic prenyltransferases),
and heteroatoms in amino acids (tyrosine, serine etc.),3 peptides (ComQꢀrelated bacterial
aggregation pheromones), proteins (protein prenyltransferases), and nucleic acids
(nitrogen atoms of tRNA and AMP).4,5 To date, over 67000 isoprenoid metabolites,
originating from prenyltransfer reactions, have been identified according the dictionary of
natural products,6 which are important for a variety of cellular functions.
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Chainꢀelongation polyprenyltransferases catalyze the stepwise electrophilic
alkylation of the carbonꢀcarbon double bond in isopentenyl diphosphate (IPP) by
isoprenoid allylic diphosphates to give longer chain polyprenyl diphosphate compounds.7
The most studied chain elongation prenyltransferase is farnesyl diphosphate synthase
(FPPase), a critical enzyme found in all kingdoms of life. FPPase catalyzes the sequential
addition of two molecules IPP, first to dimethylallyl diphosphate (DMAPP) to give
geranyl diphosphate (GPP) and then to GPP to give farnesyl diphosphate (FPP).8 These
reactions are dissociative electrophilic alkylations with the stepwise addition of
dimethylallyl and geranyl allylic cations to the IPP double bond.9 FPPase synthesizes the
substrates for branch point enzymes for the biosynthesis of sterols,10 hopanoids,11
sesquiterpenes,12 heme A,13 and longer chain polyisoprenoid diphosphates.8 The enzyme
has become a target for antiꢀinfective, antitumor, malignant bone disease, antiꢀparasitic,
and cholesterolꢀlowering drug discovery.14,15
Several groups have reported novel inhibitors of FPPase, including fluoroꢀ and
alkylꢀmodified analogs of the allylic diphosphates,16 ammonium analogs (carbocation
mimics),17 phosphonophosphonates,18 phosphinoꢀphosphinates,19 bisꢀphosphonates and
nitrogenꢀcontaining bisꢀphosphonates.20 While bisꢀphosphonate inhibitors are used to
treat diseases related to FPPase, including different bone disorders such as metabolic
bone disease, osteoporosis, hypercalcimia, Paget’s disease, and tumor bone metastases,
they often suffer from poor binding abilities and undesirable side effects.21 Isoprenoid
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allylic thiolodiphosphate analogs of DMAPP and GPP, which are substantially less
reactive than their diphosphate counterparts, are potent inhibitors of FPPase.22 We found
that bisubstrate analogs, where IPP was tethered to DMAPP or GPP, were alternative
substrates for the enzyme with kinetic constants similar to those of the natural
substrates.23 These observations led us to design the new class of thiolo bisubstrate
inhibitors of prenyltransfer reactions, which may also be useful for Xꢀray studies of the
enzymeꢀanalog complexes. We now describe the synthesis of thiolo bisubstrate analogs
of IPP linked to Eꢀ2ꢀbutenyl diphosphate, DMAPP, and GPP and inhibition studies with
FPPase.
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Results and Discussion
Our interest in the substrate selectivity of FPPase, led us to synthesize two types
of bisubstrate analogs constructed from IPP and allylic diphosphate fragments.
Compounds 1-OPP/OPP and 2-OPP/OPP (Figure 1) are double bond isomers where C4
of IPP is linked to the Eꢀ or Zꢀmethyl group in DMAPP by a oneꢀcarbon bridge.24 These
analogs are substantially less reactive than IPP and DMAPP and give a mixture of
products that suggests more than one conformers of the substrates are bound in the
enzymeꢀsubstrate complex. In contrast, another class of bisubstrate analogs represented
by compounds 3-OPP/OPP and 4-OPP/OPP tether the C3ꢀmethyl group in IPP directly
to the Zꢀmethyl group at C3 in DMAPP and GPP, respectively These bisꢀdiphosphates
are excellent substrates for FPPase that give novel 7ꢀmembered cyclic products with
kinetic constants similar to those for IPP and DMAPP. The bisubstrate analogs are
apparently bound in a single conformation in the FPPase•3-OPP/OPP and FPPase•4-
OPP/OPP complexes.23
We used two approaches for designing inhibitors for FPPase based on the
structures of 3-OPP/OPP and 4-OPP/OPP but are less reactive. The first is based on the
observation that 2ꢀbutenyl analogs, where a methyl group at Cꢀ3 in DMAPP is replaced
with hydrogen, are 102–103 are less reactive than DMAPP.25 The second approach is
based on our work with thiolodiphosphate analogs of DMAPP and GPP, which are
substantially less reactive than the corresponding diphosphates and are excellent
competitive inhibitors of FPPase.22 Accordingly, we synthesized the diphosphate and
thiolodiphosphate bisubstrate analogs lacking a methyl group in the DMAPP fragment
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(5-OPP/OPP and 5-SPP/SPP) and where the oxygens between C1 and phosphorus in the
allylic DMAPP and GPP fragments are replaced by sulfur (3-SPP/SPP and 4-SPP/SPP).
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Figure 1. Structures of bisꢀdiphosphate and bisꢀthiodiphosphate analogs
Synthesis of tris(tetra-n-butylammonium) thiodiphosphate. The synthesis of
thiolo bisubstrate analogs was based on our previous synthesis of isopentenyl
thiolodiphosphate, dimethylallyl thiolodiphosphate, geranyl thiolodiphosphate, farnesyl
thiolodiphosphate, and geranylgeranyl thiolodiphosphate. This strategy relied on the
installation of suitable leaving groups in the allylic and homoallylic precursors and
regioselective displacement by the nucleophilic sulfur in tris(tetraꢀnꢀbutylammonium)
thiopyrophosphate (9).22
Thiopyrophosphate 9 was prepared by modifying the procedure described earlier
(Scheme 1).22 In the original synthesis, treatment of salt 7 with dimethyl
chlorothiophosphate gave a 30% yield of anhydride 8. We found that addition of a
catalytic amount of N,N’ꢀdimethylaminopyridine (DMAP) increased the yield of
anhydride 8 to 60%. This modification was used to synthesize multigram quantities of 9.
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Scheme 1. Synthesis of tris(tetraꢀnꢀbutylammonium) thiodiphosphate
Synthesis of diphosphate and thiolodiphosphate analogs. Synthesis of the
isopentenyl/Zꢀ2ꢀbutenyl bisubstrate carbon skeleton (Scheme 2) began with the selective
mono THPꢀprotection of commercially available cisꢀbutꢀ2ꢀenꢀ1,4ꢀdiol (10-OH/OH). A
CoreyꢀKim allylic bromination of 10-OH/OTHP at –30 ºC gave bromide 10-Br/OTHP
in good yield.26 The bromide was used for the Cꢀselective alkylation of the dianion of 3ꢀ
methylꢀ3ꢀbuteneꢀ1ꢀol to give a 60% yield of 5-OTHP/OH.27 Removal of the THP group
with PPTS gave a 77% yield of 5-OH/OH.
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5
OH
OTHP
DHP, PPTS
61%
HO
HO
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10-OH/OH
10-OH/OTHP
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NBS, DMS
70%
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OTHP
HO
HO
Br
OH
TMEDA, n-BuLi
60%
5-OH/OH
10-Br/OTHP
PPTS
77%
THPO
OH
5-OTHP/OH
Scheme 2. Synthesis of diol 5-OH/OH
Syntheses of the carbon skeletons for the IPP/DMAPP and IPP/GPP bisubstrate
analogs are shown in Scheme 3. α,βꢀUnsaturated esters 11-OTHP and 12-OTHP were
synthesized as previously described23 and treated with DIBAL to the give corresponding
mono protected diols 3-OH/OTHP and 4-OH/OTHP. The THP protecting group was
removed with PPTS to give diols 3-OH/OH and 4-OH/OH, respectively.
EtO
O
HO
DIBAL
OTHP
OTHP
R
R
11-OTHP, R = CH3
12-OTHP, R = C6H11
3-OH/OTHP, R = CH3 (88%)
4-OH/OTHP, R = C6H11 (90%)
PPTS
HO
OH
R
3-OH/OH, R = CH3 (88%)
4-OH/OH, R = C6H11 (90%)
Scheme 3. Synthesis of diols 3-OH/OH and 4-OH/OH
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Based on our previous experience with isoprenoid diphosphates28 and
thiodiphosphates,22,23 we selected allylic chloride/bromide and homoallylic tosylate
leaving groups for the phosphorylation reactions required to make bisꢀthiolodiphosphates.
To this end, 5-OTHP/OH was converted to 5-Cl/OTs by treatment with pꢀTsCl/DMAP,
removal of the THP protecting group with PPTS, and treatment of the resulting allylic
alcohol with NCS/DMS to give 5-Cl/OTs. In preliminary experiments, 5-Cl/OTs, when
treated with inorganic thiopyrophosphate 9, unexpectedly gave cyclic thiolodiphosphate
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5-cSPP, as determined from its NMR and mass spectra (Scheme 4). The Hꢀdecoupled
31PꢀNMR spectrum of the crude cyclic thiolodiphosphate showed an AX quartet with
resonances at 8.0 ppm and –7.0 ppm for P(S) and P(O), respectively. In the 1H spectrum
of the crude mixture, the proton at C8 appeared as a quartet at 3.4 ppm as the result of an
overlapping doublet of triplets and the two protons at C7, while the protons at C1
appeared as a triplet at 4.3 ppm as a result of overlapping double of doublet patterns from
couplings to 31P(O) and the proton at C2. In addition, the negative ion electrospray (ES)
mass spectrum of 5-cSPP gave a peak at m/z 313.3 [M–H+]– (C9H15P2O6S). The
structure of 5-cSPP indicates that the tosylate in 5-Cl(Br)/OTs was displaced first by the
more nucleophilic sulfur, followed by intramolecular displacement of halogen by the
oxygen nucleophile. In contrast, treatment of 5-Cl/OTs with tris(tetraꢀnꢀ
butylammonium)pyrophosphate (OPPi) gave 5-OPP/OPP.23,24
Scheme 4. Phosphorylation/thiolophosphorylation reactions of chloride/tosylate and
dibromide derivatives of 5-OH/OH.
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After some experimentation, we discovered that dibromide 5-Br/Br, obtained by
treatment of 5-OH/OH with Nꢀbromosuccinimide and PPh3 gave linear bisꢀ
thiolodiphosphate 5-SPP/SPP in 39% yield. Formation of the bisꢀthiolodiphosphate
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depended on the slow addition of 5-Br/Br to an excess of 9 in acetonitrile. A Hꢀ
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decoupled 31PꢀNMR spectrum (Figure 2b) of 5-SPP/SPP has two overlapping AX
quartets for the P(S) and P(O) phosphorus atoms in the thiolodiphosphate moieties. The
coupling constants for between phosphorus atoms in both of the quartets are identical
(JP–P = 29.2 Hz). The 31P signals for the thiolophosphate moieties are clearly resolved and
resonate at 8.36 and 7.89 ppm, while the signals for the phosphate moieties give
1
overlapping signals at −6.76 and −6.83 ppm (Figure 2b). The H NMR spectrum for 5-
SPP/SPP (Figure 2a) has an overlapping doublet of triplets (JP–H = 9.0 Hz and JH–H = 6.0
Hz) at 2.93 ppm for the C8 protons and a doublet of doublets (JP–H = 9.0 Hz and JH–H
=
6.0 Hz) at 3.49 ppm for the C1 protons. A highꢀresolution mass spectrum of 5-SPP/SPP
had a molecular ion peak at m/z = 530.9244, corresponding a molecular formula
C9H20O12NaP4S2 (M+Na)+.
a)
b)
3.6
3.4
3.2
3.0
2.
0
5
0
-5
-1
c)
d)
0
5
0
-5
-10
3.6
3.4
3.2
3.0
2.8
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Figure 2. H and 31P NMR spectra of 4-SPP/SPP and 5-SPP/SPP. 5-SPP/SPP: (a) H
NMR spectrum of the C1 and C8 methylene protons; (b) 31P NMR spectrum showing
1
resonances for P(S) and P(O). 4-SPP/SPP: (c) H NMR spectrum of the C1 and C8
methylene protons. (d) 31P NMR spectrum showing resonances for P(S) and P(O).
However dibromide 5-Br/Br was not suitable for synthesis of the corresponding
bisꢀdiphosphate under similar conditions but instead gave cyclic diphosphate 5-cOPP!
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The structure of 5-cOPP was deduced from its NMR and mass spectra. The Hꢀ
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decoupled 31P NMR spectrum of crude 5-cOPP had an AX quartet at 7.0 ppm and –10.0
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8
9
1
31
ppm. The Hꢀcoupled P spectrum was used to assign the resonance at 7.0 ppm to the
phosphate attached to C1 and the downfield resonance to the phosphate attached to C8. In
1
the HꢀNMR spectrum, the C8 protons gave an apparent quartet at 4.0 ppm, while the
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protons at C1 at 4.3 ppm gave an apparent doublet of triplets. The negative ion ES mass
spectrum of 5-cOPP had a peak at m/z 297.3 [M–H+]– (C9H15P2O7), which confirmed the
presence of 5-cOPP. At this point we do not have a satisfactory explanation for why the
combined effects of differences in nucleophilicity for sulfur and oxygen and in the
leaving group reactivities for tosylate and the halides lead to formation of the various
combination of linear and cyclic products shown in Scheme 4. Since the binding pockets
for the diphosphate moieties in IPP and the allylic substrate are separated by over 6 Å in
the active site of FPPase, the cyclic diphosphates cannot mimic the binding interactions
seen in the FPPaseꢀsubstrate complex. As a result, we purified the materials sufficiently
to obtain 31P and high resolution mass spectra but did not optimize yields or obtain highly
purified samples.
Bisꢀthiolodiphosphates 3-SPP/SPP and 4-SPP/SPP were synthesized from diols
3-OH/OH and 4-OH/OH as described for 5-SPP/SPP. Cyclic products were not seen
when the dibromides were slowly added to an excess of 9 in acetonitrile. Conversely, a
substantial amount of cyclic product was observed when 9 was added to an acetonitrile
solution of the dibromides. While we typically purify diphosphate and bisdiphosphate
esters by chromatography on cellulose,23,24,28 the procedure was not suitable for the
purification of bisꢀthiolodiphosphates. Several attempts using 0.1 M NH4HCO3 in
combination with either 2ꢀpropnolꢀacetonitrile or 1ꢀbutanolꢀTHF were unsuccessful.
Ultimately, pure samples of 3-SPP/SPP, 4-SPP/SPP, and 5-SPP/SPP were obtained by
chromatography on DEAE SephacelTM (a weak anion exchanger). The 1H and 31P NMR
spectra of 3-SPP/SPP (SI Figures S16 and 18) and 4-SPP/SPP (Figures 2c and 2c) were
similar to those of 5-SPP/SPP.
Kinetic Studies. Inhibition studies with avian FPPase were conducted for
bisubstrate analogs 3-OPP/OPP, 3-SPP/SPP, 4-OPP/OPP, 4-SPP/SPP, 5-OPP/OPP
and 5-SPP/SPP. Initial velocities were measured by the standard acidꢀlability assay.29
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Although the C9 and C15 analogs were competitive inhibitors against GPP for avian
FPPase (Figures S35 and S36), with inhibition constants in the low micromolar/high
nanomolar range (Table 1), the C10 bisubstrate analogs showed a mixed type inhibition
behavior (Figures S33 and S34).
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No.
Inhibitors
Inhibition constant
KI (µM)
1.
2.
3.
4.
5.
6.
7.
GSPP
24.8 ± 1.94
1.4
3-OPP/OPP
3-SPP/SPP
4-OPP/OPP
4-SPP/SPP
5-OPP/OPP
5-SPP/SPP
5.5
1.0 ± 0.12
0.7 ± 0.3
0.5 ± 0.2
2.9 ± 0.27
Table 1. Inhibition constants (KI) of thiolo substrates and bisubstrates
These values are slightly lower than the related KM values for chain elongation of
GPP and GSPP to FPP measured under same conditions.22 The small difference between
binding of the normal substrates and the bisubstrate analogs of IPP and DMAPP (or GPP)
are most likely the result of slight difference in the topology of the two classes of
molecules in the active site. The normal substrates are bound in the active site of FPPase
through strong electrostatic interactions between three tightly bound Mg2+ ions with
highly conserved aspartate residues and strong hydrogen bonds to lysine and arginine
residues, while the methyl (or homogeranyl) moieties are inserted into a hydrophobic
pocket.8a Previously, we reported that diphosphate residues of 3-OPP/OPP and 4-
OPP/OPP are bound in the same locations as the normal substrates; however, their
conformations and the conformations of the carbon atoms are distorted to accommodate
the linkage between the isopentenyl and allylic units. These distortions would be
expected to have a negative impact on binding affinity. Replacement of the oxygen
between carbon and phosphorus with sulfur appears to have little impact on the binding
while reducing the reactivity of the allylic electrophile by >106ꢀfold.22
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Conclusion
Unreactive bisꢀthiolodiphosphate bisubstrate analogs were constructed where the
isopentenyl moiety in IPP was fused to allylic Zꢀ2ꢀbutenyl, dimethylallyl, and geranyl
moieties. The general strategy was based on a oneꢀpot displacement of leaving groups
attached to terminal methylene carbons in the fused isopentenyl (homoallylic) and allylic
units. A homoallylic tosylate/allylic chloride combination gave a linear bisꢀdiphosphate
when treated with inorganic tris(tetraꢀnꢀbutylammonium)pyrophosphate and a cyclic
thiolodiphosphate treated with tris(tetraꢀnꢀbutylammonium)thiopyrophosphate. In
contrast, when both leaving groups were bromide, treatment with inorganic tris(tetraꢀnꢀ
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butylammonium)pyrophosphate gave
a
cyclic diphosphate and tris(tetraꢀnꢀ
butylammonium)thiopyrophosphate gave a linear bisꢀthiolodiphosphate. The bisubstrate
analogs were strong competitive inhibitors against GPP for avian FPPase. The analogs
are excellent candidates for coꢀcrystallization of chain elongation prenyltransferases with
occupancy in the IPP and allylic regions of the active site. The ability to synthesize linear
bisdiphosphate esters by the proper choice of leaving groups provides a practical
approach to this class of biologically important molecules.
Experimental Section
General Methods and Materials: All the air and moisture sensitive reactions were
performed using anhydrous solvents and under nitrogen atmosphere in ovenꢀdried
glassware (100 °C). The solvents Et2O, THF, CH3CN, CH2Cl2 were dried by passing
through a column of activated alumina. The organic compounds were purified by silica
gel flash column chromatography (230ꢀ400 mesh, 60 Å), and the glassꢀbacked TLC
plates (Silica Gel 60 Å F254) were used for the thinꢀlayer chromatographic analysis. The
spots on the TLC plates were visualized by using phosphomolybdic acid stain. Dowex
AG 50WꢀX8 cationꢀexchange resin was purchased as hydrogen form and exchanged to
the active ammonium form by washing with 3 M ammonium hydroxide solution prior to
use. All the bisthiodiphosphates were purified by Cellulose flash column chromatography
using DEAE SephacelTM (weak anion exchanger). The thinꢀlayer chromatographic
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analysis of bisthiodiphosphates was performed on the glassꢀbacked cellulose TLC plates
and the spots were visualized using 5ꢀsulfosalisylic acidꢀferric chloride spray. All the 1Hꢀ,
31
13Cꢀ and PꢀNMR spectra were recorded at room temperature and chemical shifts were
reported in δ ppm (parts per million) values. 1HꢀNMR spectra were recorded at 300 MHz,
and were referenced using the residual CHCl3 singlet at 7.26 ppm or HDO singlet at 4.80
ppm from the deuterated NMR solvents. 13CꢀNMR spectra were recorded at 75 MHz and
125 MHz, and were referenced using the residual CHCl3 triplet at 77.23 ppm from the
deuterated NMR solvents. 13CꢀNMR spectra recorded in D2O were unreferenced. 31Pꢀ
NMR spectra were recorded at 121.5 MHz, and the 31PꢀNMR chemical shifts were
referenced to 85% H3PO4 as an external reference. HRMSꢀESI data were recorded on
LCꢀTOF mass spectrometer.
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Radioactivity was measured in Cytoscint scintillation cocktail. Concentrations of
diphosphate compounds were analyzed by the phosphate analysis method30 before using
in the enzyme assays. [14C]ꢀIPP was purchased from Amersham (now GE Healthcare)
and PerkinꢀElmer.
Tetramethyl thiodiphosphate (9). In a flameꢀdried flask, a solution of 12.8 g of tetraꢀnꢀ
butylammonium dimethyl phosphate (7) (34.5 mmol) and 0.4 g of N,Nꢀdimethylꢀ4ꢀ
aminopyridine (3.48 mmol) in 15 mL of acetonitrile was cooled to −35 °C before
dropwise addition of 4.5 mL of dimethyl chlorothiophosphate (5.9 g, 36.7 mmol). The
mixture was allowed to stir at −35 °C for 30 min and warmed to 0 °C over 30 min, and
immediately loaded onto a silica gel column and run with 2 column volumes of CH2Cl2
before the product was eluted with 50% EtOACꢀCH2Cl2. Solvents were removed under
reduced pressure to give 5.5 g (60%) of a clear oil.
(Z)-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-ol (10-OH/OTHP). To a solution of
cisꢀbutꢀ2ꢀeneꢀ1,4ꢀdiol 10-OH/OH (1.2 g, 13.64 mmol) and 3,4ꢀdihydroꢀ2Hꢀpyran (1.09
g, 12.95 mmol) in CH2Cl2 (30 mL) was added 35 mg of PPTS (0.136 mmol). The mixture
was allowed to stir for 16 h at rt; diluted with 20 mL of CH2Cl2 washed with saturated
aqueous NaHCO3, water, and brine, and dried over anhydrous Na2SO4. CH2Cl2 was
removed at reduced pressure, and the resulting residue was chromatographed by flash
column chromatography on silica gel, eluting with 30% Et2O/hexanes to give 1.45 g
(61%) of a colorless oil; TLC (Rf 0.5, 50% EtOAc/hexanes); 1HꢀNMR (300 MHz, CDCl3)
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δ ppm 5.84–5.80 (m, 1H), 5.73–5.65 (m, 1H), 4.67 (t, 1H, J = 3.0 Hz), 4.29–4.09 (m,
4H), 3.81–3.48 (m, 1H), 3.56–3.48 (m, 1H), 2.39 (bs, 1H), 1.85–1.49 (m, 6H); 13CꢀNMR
(75 MHz, CDCl3) δ ppm 132.6, 128.3, 97.7, 62.6, 62.3, 58.5, 30.6, 25.5, 19.4; HRMS
(ESI+) calculated for C9H16O3Na (M+Na)+ m/z = 195.0997, found: 195.0999.
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(Z)-2-((4-bromobut-2-en-1-yl)oxy)tetrahydro-2H-pyran (10-Br/THP). A solution of
1.24 g (6.97 mmol) of Nꢀbromosuccinimide in 20 mL of CH2Cl2 was chilled to 0 °C, after
which 0.43 g (6.97 mmol) of dimethyl sulfide was added slowly. The resulting white
slurry was allowed to stir at 0 °C for 10 min before the temperature was lowered to –30
°C. A solution of 1.0 g (5.81 mmol) of 10-OH/OTHP in 10 mL of CH2Cl2 was added
slowly over a period of 10 min. The mixture was diluted with 10 mL of CH2Cl2, washed
water and brine, dried over Na2SO4, and concentrated under reduced pressure. The
residue was purified by silica gel flash chromatography, eluting with 10% Et2O/hexanes
1
to afford 0.96 g (70%) of a colorless oil; TLC (Rf 0.75, 20% EtOAc/hexane); HꢀNMR
(300 MHz, CDCl3) δ ppm 5.94–5.84 (m, 1H), 5.76–5.68 (m, 1H), 4.64 (t, 1H, J = 3.0
Hz), 4.36–4.29 (m, 1H), 4.19–4.13 (m, 1H), 4.03 (d, 2H, J = 8.1 Hz), 3.90–3.83 (m, 1H),
13
3.57–3.50 (m, 1H), 1.85–1.50 (m, 6H); CꢀNMR (75 MHz, CDCl3) δ ppm 131.1, 128.5,
98.2, 62.5, 62.1, 30.7, 26.8, 25.6, 19.6; HRMS (ESI+) calculated for C9H15O2NaBr
(M+Na)+ m/z = 257.0153, found: 257.0156.
(Z)-3-methylene-8-((tetrahydro-2H-pyran-2-yl)oxy)oct-6-en-1-ol (5-OH/OTHP). A
solution of 3.9 mL (9.79 mmol) of nꢀbutyl lithium (2.5 M in hexane) in 10 mL Et2O was
cooled to 0 °C while 1.8 mL (11.7 mmol) of tetramethylethylenediamine (TMEDA) was
added slowly over a 5ꢀmin period. The mixture was allowed to stir 0 °C for 1 h before
0.34 g (3.92 mmol) of 3ꢀmethylꢀ3ꢀbutenꢀ1ꢀol in 2 mL Et2O was added slowly at 0 °C
over a period of 10 min. The resulting solution was allowed to stir at 0 °C for 1 h,
allowed to warm to room temperature, and vigorously stirred for 6 h. The yellow mixture
was then cooled to –78 °C, and a solution of 0.92 g (3.91 mmol) of 10-Br/OTHP in Et2O
was slowly added over a period of 10 min. The reaction mixture was allowed to stir at
–78 °C for 2 h, then allowed to warm to room temperature with stirring for an additional
16 h. The dark brown mixture was quenched by a slow addition ammonium chloride at 0
°C followed by the addition of water and the layers were separated. The aqueous layer
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was extracted with diethyl ether and the combined organic layers were washed with water
and brine, dried over Na2SO4 and concentrated at reduced pressure. The residue was
purified by silica gel flash chromatography eluted using 30% EtOAc/hexanes to afford
1
0.67 g (60%) of a light brown oil; TLC (Rf 0.45, 50% EtOAc/hexanes); HꢀNMR (300
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MHz, CDCl3) δ ppm 5.62–5.52 (m, 2H), 4.86 (s, 1H), 4.83 (s, 1H), 4.62 (t, 1H, J = 3.0
Hz), 4.25 (dd, 1H, J = 6.0, 12.0 Hz), 4.09–4.03 (m, 1H), 3.90–3.83 (m, 1H), 3.70 (dd, 2H,
J = 6.3, 12.0 Hz), 3.54–3.47 (m, 1H), 2.28 (t, 2H, J = 6.6 Hz), 2.25–2.21 (m, 2H), 2.09
13
(dd, 2H, J = 6.9, 8.1 Hz), 1.88–1.48 (m, 6H); CꢀNMR (75 MHz, CDCl3) δ ppm 145.5,
132.9, 126.5, 112.1, 98.1, 62.8, 62.4, 60.5, 39.3, 35.7, 30.8, 25.9, 25.6, 19.7; HRMS
(ESI+) calculated for C14H24O3Na (M+Na)+ m/z = 263.1623, found: 263.1624.
(Z)-6-methyleneoct-2-ene-1,8-diol (5-OH/OH). To a solution of 240 mg (0.96 mmol)
of 5-OH/OTHP in 5 mL of EtOH, was added 25 mg (0.096 mmol) of PPTS. The mixture
was allowed to stir at for 16 h. Solvent was removed on rotary evaporator; the residue
was dissolved in EtOAc and washed with sat. Na2CO3, water, and brine; dried over
anhydrous Na2SO4; and concentrated under reduced pressure. The residue was purified
by silica gel flash column chromatography, eluted with 70% EtOAc/hexane to afford 115
mg (77%) of a light yellow oil; TLC (Rf 0.20, 50% EtOAc/hexanes); 1HꢀNMR (300 MHz,
CDCl3) δ ppm 5.65–5.57 (m, 1H), 5.54–5.45 (m, 1H), 4.85 (s, 1H), 4.84 (s, 1H), 4.16 (d,
2H, J = 6.6 Hz), 3.71 (dd, 2H, J = 6.3, 12.0 Hz), 2.28 (t, 2H, J = 6.6 Hz), 2.25–2.20 (m,
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2H), 2.10 (t, 2H, J = 7.2 Hz), 2.01 (bs, 2H); CꢀNMR (75 MHz, CDCl3) δ ppm 145.4,
132.1, 129.1, 112.1, 60.5, 58.5, 39.0, 35.8, 25.8; HRMS (ESI+) calculated for C9H16O2Na
(M+Na)+ m/z = 179.1048, found: 179.1052.
6-Methylene-oct-2(Z)-enyl-1,8-S,S-bis-thiolodiphosphate (5-SPP/SPP). A solution of
134 mg (0.86 mmol) of 5-OH/OH and 382 mg (2.15 mmol) Nꢀbromosuccinimide in 5
mL dichloromethane was cooled at 0 °C before 560 mg (2.15 mmol) of PPh3 was added
slowly over a 5 min period. After 30 min, the ice bath was removed, and the solution
was allowed to stir at room temperature for 16 h. The mixture was concentrated on
rotary evaporator, then diluted with 20 mL of hexane. The resulting white precipitate
was removed by filtration. The solvent was removed in vacuo to give a brown oil. The
product was immediately used in the next step without further purification.
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The oil was dissolved in 1 mL acetonitrile and added slowly over a 5 min period
to a cold (0 °C) stirred solution of 2.37 g (2.58 mmol) of trisꢀ(tetraꢀnꢀ
butylammonium)hydrogen thiopyrophosphate 9 in 3 mL of acetonitrile. The solution was
allowed to stir at 0 °C for 30 min and at room temperature for 3 h, after which the solvent
was removed by rotatory evaporator. The residue was dissolved in 2 mL of exchange
buffer (25 mM ammonium bicarbonate containing 2% (v/v) of isopropanol) and was
passed through a 2x30 cm column containing DOWEX AG 50WꢀX8 cationꢀexchange
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+
resin (NH4 form). The product was eluted with two column volumes of exchange
buffer, and the eluent was lyophilized to afford a white solid. The powder was dissolved
in a minimal amount of 10 mM NH4HCO3 and chromatographed on a DEAE SephacelTM
(weak anion exchanger) cellulose flash column chromatography using a gradient 10–500
mM ammonium bicarbonate. Thiolodiphosphateꢀcontaining fractions were visualized by
5ꢀsulfosalisylic acidꢀferric chloride spray, pooled, and lyophilized to give 240 mg (45%)
of a white powder; TLC (cellulose, Rf 0.65, 5.0:2.5:2.5 (v/v/v) 50 mM NH4HCO3/THF/1ꢀ
propanol); 1HꢀNMR (300 MHz, D2O) 5.66–5.47 (m, 2H), 4.86 (s, 1H), 4.73 (s, 1H), 3.49
(dd, 2H, J = 6.0 Hz, 9.0 Hz), 2.93 (dt, 2H, J = 6.0 Hz, 9.0 Hz), 2.38 (t, 2H, J = 9.0 Hz),
2.25–2.21 (m, 2H), 2.12–2.07 (m, 2H); 13CꢀNMR (75 MHz, D2O) 148.4, 132.9, 126.4
(JC–P = 7.5 Hz), 110.6, 36.5 (JC–P = 7.5 Hz), 34.8, 28.3 (JC–P = 3.0 Hz), 26.9 (JC–P = 3.0
Hz), 24.7; 31PꢀNMR (121.5 MHz, D2O) 8.36 (d, 1P, JP–P = 29.2 Hz), 7.89 (d, 1P, JP–P
=
29.2 Hz), −6.76 (d, 1P, JP–P = 29.2 Hz), −6.83 (d, 1P, JP–P = 29.2 Hz); HRMS (ESI+)
calculated for C9H20O12NaP4S2 (M+Na)+ m/z = 530.9244, found: 530.9244.
(Z)-3-methyl-6-methylene-8-((tetrahydro-2H-pyran-2-yl)oxy)oct-2-en-1-ol
(3-
OH/OTHP). To a solution of 250 mg (0.85 mmol) of α,βꢀunsaturated ester 11-OTHP in
5 mL of toluene at −78 °C, was slowly added 1.4 mL (2.11 mmol) of 1.5 M DIBAL in
toluene. The resulting mixture allowed to stir for 2 h under N2 before addition of 10 mL
of saturated sodium potassium tartrate, followed by water and EtOAc. The mixture was
allowed to warm to room temperature and then vigorously stirred until the layers
separated (45–60 min). The aqueous layer was extracted with EtOAc and the combined
organic layers were washed with water and brine and dried over anhydrous Na2SO4.
Solvent was removed under reduced pressure and the residue was purified by silica gel
flash column chromatography, eluting with 50% EtOAc/hexane to give 185 mg (86%) of
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a light yellow oil; TLC (Rf 0.4, 30% EtOAc/hexanes); HꢀNMR (300 MHz, CDCl3) δ
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ppm 5.43 (t, 1H, J = 6.6 Hz), 4.80 (s, 1H), 4.79 (s, 1H), 4.59 (dd, 1H, J = 2.7, 4.2 Hz),
4.09 (d, 2H, J = 6.0 Hz), 3.90–3.82 (m, 2H), 3.55–3.47 (m, 2H), 2.33 (t, 2H, J = 6.9 Hz),
2.24–2.18 (m, 2H), 2.14–2.09 (m, 2H), 1.74 (d, 3H, J = 1.2 Hz), 1.83–1.65 (m, 2H),
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1.60–1.46 (m, 4H); CꢀNMR (75 MHz, CDCl3) δ ppm 146.6, 139.7, 124.8, 111.0, 99.1,
64.5, 62.6, 59.1, 36.1, 35.3, 30.9, 30.7, 25.6, 23.6, 19.8; HRMS (ESI+) calculated for
C15H26O3Na (M+Na)+ m/z = 277.1780, found: 277.1782.
(Z)-3-methyl-6-methyleneoct-2-ene-1,8-diol (3-OH/OH). To a solution of 160 mg
(0.63 mmol) of 3-OH/OTHP in 3 mL of EtOH, was added 16 mg (0.063 mmol) of PPTS.
The resulting mixture was allowed to stir at rt under nitrogen for 16 h and then
concentrated by rotary evaporation. The residue was dissolved in EtOAc; washed with
saturated Na2CO3, water, brine; and dried over anhydrous Na2SO4. Solvent was removed
at reduced pressure and the residue was purified by silica gel flash chromatography,
eluted using 70% EtOAc/hexanes to afford 91 mg (88%) of a light yellow oil; TLC (Rf
1
0.3, 50% EtOAc/hexanes); HꢀNMR (300 MHz, CDCl3) δ ppm 5.43 (t, 1H, J = 6.0 Hz),
4.86 (s, 1H), 4.84 (s, 1H), 4.08 (d, 2H, J = 6.0 Hz), 3.72 (dd, 2H, J = 6.0, 9.0 Hz), 2.29 (t,
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2H, J = 6.0 Hz), 2.22–2.19 (m, 2H), 2.13–2.08 (m, 2H), 2.02 (bs, 1H), 1.74 (s, 3H); Cꢀ
NMR (75 MHz, CDCl3) δ ppm 145.9, 139.6, 124.9, 111.9, 60.5, 59.0, 39.1, 34.6, 30.6,
23.5; HRMS (ESI+) calculated for C10H18O2Na (M+Na)+ m/z = 193.1204, found:
193.1205.
(Z)-3-methyl-6-methyleneoct-2-ene-1,8-bis-thiolodiphosphate (3-SPP/SPP). The
procedure described for 5-SPP/SPP was used to synthesize 3-SPP/SPP from 40 mg (0.24
mmol) of 3-OH/OH to give 55 mg (39%) of a white powder; TLC (cellulose, Rf 0.54,
5:2.5:2.5 (v/v/v) 50 mM NH4HCO3/THF/1ꢀpropanol); 1HꢀNMR (300 MHz, D2O) 5.45 (t,
1H, J = 6.0 Hz), 4.89 (s, 1H), 4.87 (s, 1H), 3.49 (dd, 2H, J = 9.0 Hz, J = 9.0 Hz), 2.97 (dt,
2H, J = 6.0 Hz, J = 6.0 Hz), 2.44 (t, 2H, J = 6.0 Hz), 2.28−2.11 (m, 4H), 1.72 (s, 3H);
13CꢀNMR (125 MHz, D2O) 148.7, 140.1, 121.2 (JC–P = 7.5 Hz), 110.1, 36.3 (JC–P = 7.5
31
Hz), 33.4, 29.4, 28.1 (JC–P = 3.8 Hz), 27.4 (JC–P = 3.8 Hz), 22.4; PꢀNMR (121.5 MHz,
D2O) 8.35 (d, 1P, JP–P = 29.2 Hz), 8.0 (d, 1P, JP–P = 29.2 Hz), −6.72 (d, 1P, JP–P = 29.2
Hz), −6.77 (d, 1P, JP–P = 29.2 Hz); HRMS (ESI+) calculated for C10H21O12P4S2 (M−H)−
m/z = 520.9425, found: 520.9428.
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Z)-7-methyl-3-(3-methylene-5-((tetrahydro-2H-pyran-2-yl)oxy)pentyl)octa-2,6-dien-
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1-ol (4-OH/OTHP). Alcohol 4-OH/OTHP was prepared from 420 mg (1.15 mmol) of
12-OTHP and 1.9 mL (2.89 mmol) of 1.5 M DIBAL in toluene using the procedure
described for 3-OH/OTHP to give 335 g (90%) of a colorless oil; TLC (Rf 0.45, 50%
EtOAc/hexanes); 1HꢀNMR (300 MHz, CDCl3) δ ppm 5.43 (t, 1H, J = 7.2 Hz), 5.10–5.07
(m, 1H), 4.80 (s, 1H), 4.79 (s, 1H), 4.59 (dd, 1H, J = 2.1, 4.2 Hz), 4.13 (d, 2H, J = 7.2
Hz), 3.90–3.82 (m, 2H), 3.55–3.47 (m, 2H), 2.33 (t, 2H, J = 6.0 Hz), 2.23–2.18 (m, 2H),
2.12–2.04 (m, 6H), 1.82–1.70 (m, 2H), 1.68 (s, 3H), 1.60 (s, 3H), 1.58–1.48 (m, 4H); 13Cꢀ
NMR (75 MHz, CDCl3) δ ppm 146.7, 143.3, 132.0, 124.2, 124.1, 111.9, 99.1, 66.5, 62.6,
59.2, 36.9, 36.2, 35.8, 30.9, 29.3, 26.8, 25.9, 25.6, 19.8, 17.9; HRMS (ESI+) calculated
for C20H34O3Na (M+Na)+ m/z = 345.2406, found: 345.2414.
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(Z)-6-methylene-3-(4-methylpent-3-en-1-yl)oct-2-ene-1,8-diol (4-OH/OH).
The
procedure described for 3-OH/OH was used to convert 335 mg (1.04 mmol) of 4-
1
OH/OTHP into 223 mg (90%) a colorless oil; TLC (Rf 0.4, 50% EtOAc/hexanes); Hꢀ
NMR (300 MHz, CDCl3) δ ppm 5.44 (t, 1H, J = 7.2 Hz), 5.14–5.06 (m, 1H), 4.87 (s, 1H),
4.85 (s, 1H), 4.12 (d, 2H, J = 7.2 Hz), 3.73 (t, 2H, J = 6.0 Hz), 2.30 (t, 2H, J = 6.0 Hz),
2.22–2.18 (m, 2H), 2.12–2.03 (m, 6H), 1.76 (bs, 2H), 1.68 (s, 3H), 1.60 (s, 3H); 13Cꢀ
NMR (75 MHz, CDCl3) δ ppm 146.0, 143.2, 132.1, 124.3, 124.0, 112.0, 60.5, 59.1, 39.1,
36.8, 35.1, 29.2, 26.8, 25.9, 17.9; HRMS (ESI+) calculated for C15H26O2Na (M+Na)+ m/z
= 261.1831, found: 261.1831.
(Z)-6-methylene-3-(4-methylpent-3-en-1-yl)oct-2-ene-1,8-bis-thiolodiphosphate (4-
SPP/SPP). Using the procedure described for 3-SPP/SPP, 83 mg (0.35 mM) of 4-
OH/OH was converted into 110 mg (41%) of a white powder; TLC (cellulose, Rf 0.34,
5:2.5:2.5 (v/v/v) 50 mM NH4HCO3/THF/1ꢀpropanol); 1HꢀNMR (300 MHz, D2O) 5.42 (t,
1H, J = 6.0 Hz), 5.22−5.14 (m, 1H), 4.90 (s, 1H), 4.87 (s, 1H), 3.52 (dd, 2H, J = 9.0 Hz, J
= 12.0 Hz), 2.98 (dt, 2H, J = 9.0 Hz, J = 12.0 Hz), 2.44 (t, 2H, J = 6.0 Hz), 2.28−2.23 (m,
2H), 2.15−2.08 (m, 6H), 1.66 (s, 3H), 1.60 (s, 3H); 13CꢀNMR (75 MHz, D2O) 148.8,
143.9, 133.8, 124.3, 121.0 (JC–P = 7.5 Hz), 110.4, 36.4 (JC–P = 7.5 Hz), 36.0, 34.1, 28.5
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(JC–P = 3.8 Hz), 27.9 (JC–P = 3.0 Hz), 27.8, 25.9, 24.9, 17.1; PꢀNMR (121.5 MHz, D2O)
8.85 (d, 1P, JP–P = 27.9 Hz), 8.40 (d, 1P, JP–P = 27.9 Hz), −8.83 (d, 1P, JP–P = 29.2 Hz),
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−8.91 (d, 1P, JP–P = 29.2 Hz); HRMS (ESI+) calculated for C15H30O12NaP4S2 (M+Na)+
m/z = 613.0027, found: 613.0027.
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(Z)-3-methylene-8-((tetrahydro-2H-pyran-2-yl)oxy)oct-6-en-1-yl-4-methylbenzene-
sulfonate (5-OTHP/OTs). Using a previously described procedure,23 26 mg (0.11 mmol)
of 5-OTHP/OH was converted into 34 mg (79%) of a colorless oil; TLC (Rf 0.3, 20%
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EtOAcꢀhexanes); HꢀNMR (300 MHz, CDCl3) δ ppm 7.77 (d, 2H, J = 9.0 Hz), 7.33 (d,
2H, J = 9.0 Hz), 5.59–5.44 (m, 2H), 4.80 (s, 1H), 4.72 (s, 1H), 4.61 (t, 2H, J = 6.0 Hz),
4.23 (dd, 2H, J = 6.0 and J = 12.0 Hz), 4.10 (t, 2H, J = 9.0 Hz), 4.07–4.00 (m, 1H), 3.92–
3.80 (m, 1H), 3.54–3.44 (m, 1H), 2.44 (s, 3H), 2.34 (t, 2H, J = 9.0 Hz), 2.16 (dd, 2H, J =
6.0 and J = 12.0 Hz), 1.98 (dd, 2H, J = 6.0 and J = 9.0 Hz), 1.82–1.70 (m, 2H), 1.60–1.45
(m, 4H) ; 13CꢀNMR (75 MHz, CDCl3) δ ppm 144.9, 143.4, 133.3, 132.5, 130.0 (2C),
128.1 (2C), 126.7, 112.5, 98.1, 68.8, 62.8, 62.4, 35.8, 35.3, 30.8, 25.7, 25.6, 21.8, 19.7;
HRMS (ESI+) calculated for C21H30O5SNa (M+Na)+ m/z = 417.1712, found: 417.1711.
(Z)-8-hydroxy-3-methyleneoct-6-en-1-yl 4-methylbenzenesulfonate (5-OH/OTs).
Using a previously described procedure,23 40 mg (0.068 mmol) of 5-OTHP/OTs was
converted into 20 mg (63%) of a colorless oil. TLC (Rf 0.4, 50% v/v EtOAc/hexanes);
1HꢀNMR (300 MHz, CDCl3) δ ppm 7.79 (d, 2H, J = 9.0 Hz), 7.35 (d, 2H, J = 9.0 Hz),
5.65–5.57 (m, 1H), 5.50–5.41 (m, 1H), 4.81 (s, 1H), 4.74 (s, 1H), 4.16 (d, 2H, J = 6.0
Hz), 4.12 (t, 2H, J = 6.0 Hz), 2.45 (s, 3H), 2.36 (t, 2H, J = 6.0 Hz), 2.17 (dd, 2H, J = 9.0
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and J = 15.0 Hz), 2.01 (dd, 2H, J = 9.0 and J = 15.0 Hz); CꢀNMR (75 MHz, CDCl3) δ
ppm 145.0, 143.5, 133.3, 131.9, 130.0 (2C), 129.3, 128.1(2C), 112.7, 68.9, 58.7, 36.0,
35.3 25.7, 21.9; HRMS (ESI+) calculated for C16H22O4SNa (M+Na)+ m/z = 333.1137,
found: 333.1138.
6-Methylene-oct-2(Z)-enyl-1,8-bis-diphosphate (5-OPP/OPP). Using a previously
described procedure,23 100 mg (0.32 mmol) of 5-OH/OTs was converted into 103 mg
(55%) of a hygroscopic white solid; TLC (cellulose, Rf 0.5, 2.5:2.5:5 (v/v/v) THF/1ꢀ
1
propanol/0.1 M NH4HCO3); HꢀNMR (300 MHz, D2O) δ ppm 5.74−5.59 (m, 2H), 4.92
(s, 1H), 4.91 (s, 1H), 4.54 (t, 2H, J = 6.0 Hz), 4.06 (q, 2H, J = 6.0 Hz), 2.41 (t, 2H, J =
13
6.0 Hz), 2.37–2.26 (m, 2H), 2.22–2.13 (m, 2H); CꢀNMR (75 MHz, D2O) δ ppm 146.7,
134.1, 125.8 (d, JCꢀP = 7.5 Hz), 111.0, 64.3 (d, JCꢀP = 7.5 Hz), 61.8 (d, JCꢀP = 5.5 Hz),
36.3 (d, JCꢀP = 7.5 Hz), 35.2, 25.1; 31PꢀNMR (121 MHz, D2O) δ ppm −5.95 (d, 1P, JPꢀP
=
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20.7 Hz), −5.91 (d, 1P, JPꢀP = 21.9 Hz), −9.88 (d, 1P, JPꢀP = 21.9 Hz), −10.1 (d, 1P, JPꢀP
=
21.9 Hz); HRMS (FTMS/ESI+) calculated for C9H20O14P4Na (M+Na)+ m/z = 498.9701,
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8
found: 498.9695.
Kinetic studies. The acid lability assay was used to determine kinetic constants for avian
FPPase.29 Each assay contained 70 ng (C9 and C15 bisubstrates) or 20 ng (C10
bisubstrates) of purified enzyme, 10 ꢀM [14C]IPP (10 ꢀCi/ꢀmol), and varied
concentrations of GPP and bisꢀdiphosphates (3-OPP/OPP, 4-OPP/OPP, or 5-OPP/OPP)
or thiolodiphosphates (3-SPP/SPP, 4-SPP/SPP, or 5-SPP/SPP), in 40 mM BHDA
buffer, pH 7.3, containing 20 mM BME, 2 mM MgCl2, and 2 mg/mL BSA in a total
volume of 200 ꢀL. The assay mixture was incubated at 37 °C for 10 min and then
quenched with 200 ꢀL of methanol/HCl (4:1, v/v), followed by incubated for an
additional 10 min before extraction with 1 mL of ligroin. The radioactivity in a 0.5 mL
sample of the organic layer was measured by liquid scintillation spectrometry.
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ASSOCIATED CONTENT
Supporting Information: 1Hꢀ, 13Cꢀ, and 31PꢀNMR spectra of all the new compounds and
inhibition data of bisubstrate analogs. This material is available for free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Address: 325 South 1400 East RM 2020, Salt Lake City, UT 84112
Telephone: (801) 581ꢀ6685; Eꢀmail: poulter@chem.utah.edu
Present Address
† Present Address: U.S. Army Dugway Proving Ground, Building 4153, Dugway,
UT 84022, United States
Notes
The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
This research was supported by the NIH grants GM25521 and GM093342
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