Synthesis of geranyl S-thiolodiphosphate. A new alternative substrate/inhibitor for prenyltransferases.

Article abstract of DOI:10.1021/ol006055n

The tris(tetra-n-butylammonium) salt of thiopyrophosphate 5 was prepared from trimethyl phosphate in four steps. Treatment of geranyl bromide with 5 gave an 80% yield of geranyl S-thiolodiphosphate (6). Thiolodiphosphate 6 is substantially less reactive than geranyl diphosphate (7) in the prenyl transfer reaction catalyzed by farnesyl diphosphate synthase and is a good inhibitor of the enzyme.

Full text of DOI:10.1021/ol006055n

ORGANIC
LETTERS
2
000
Vol. 2, No. 15
287-2289
Synthesis of Geranyl
S-Thiolodiphosphate. A New Alternative
Substrate/Inhibitor for
2
Prenyltransferases
Richard M. Phan and C. Dale Poulter*
Department of Chemistry, UniVersity of Utah, Salt Lake City, Utah 84112
poulter@chemistry.chem.utah.edu
Received May 12, 2000
ABSTRACT
The tris(tetra-n-butylammonium) salt of thiopyrophosphate 5 was prepared from trimethyl phosphate in four steps. Treatment of geranyl bromide
with 5 gave an 80% yield of geranyl S-thiolodiphosphate (6). Thiolodiphosphate 6 is substantially less reactive than geranyl diphosphate (7)
in the prenyl transfer reaction catalyzed by farnesyl diphosphate synthase and is a good inhibitor of the enzyme.
Prenyltransferases catalyze the electrophilic alkylation of
electron-rich acceptor substrates by the hydrocarbon moiety
in allylic isoprenoid diphosphates.1 Some of the more
common acceptors are carbon-carbon double bonds (syn-
form farnesyl diphosphate (FPP). FPP is required for the
biosynthesis of numerous essential metabolites, including
ubiquinones, sterols, and dolichols, and FPPase activity is
apparently present in all organisms.
1c
thesis of isoprenoid chains), aromatic rings (synthesis of
2
respiratory quinones), amino groups (modification of
3
4
tRNAs), and sulfhydryl moieties (modification of proteins).
The products of prenyl transfer reactions are ultimately
converted into over 30 000 naturally occurring isoprenoid
compounds. Farnesyl diphosphate synthase (FPPase) is the
5
best characterized of the prenyltransferases. The enzyme
catalyzes two sequential chain elongation reactions. Isopen-
tenyl diphosphate (IPP) is first condensed with dimethylallyl
diphosphate (DMAPP) to form geranyl diphosphate (GPP,
The chain elongation reaction is a stepwise electrophilic
1c
alkylation. The carbon-oxygen bond in the allylic diphos-
phate is cleaved and the resulting allylic carbocation alkylates
the double bond in IPP to form a tertiary carbocation. In the
final step a proton, originally at C2 of IPP, is removed to
7), followed by condensation of GPP with a second IPP to
(
1) (a) Poulter, C. D.; Rilling, H. C. Acc. Chem. Res. 1978, 11, 307-
3
13. (b) Poulter, C. D.; Mash, E. A.; Argyle, J. C.; Muscio, O. J.; Rilling,
H. C. J. Am. Chem. Soc. 1979, 101, 6761-6763. (c) Poulter, C. D.; Rilling,
H. C. In Biosynthesis of Isoprenoid Compounds; Porter, J. W., Spurgeon,
S. L., Eds.; John Wiley and Sons: New York, 1981; pp 162-224.
6
give FPP. Several different analogues of the allylic sub-
strates, in which the hydrocarbon moiety is attached to a
(
2) Ashby, M. N.; Edwards, P. A. J. Biol. Chem. 1990, 265, 13157-
1
3164.
(
3) Caillet, J.; Droogmans, L. J. Bacteriol. 1988, 170, 4147-4152.
(6) (a) Rilling, H. C.; Block, K. J. Biol. Chem. 1959, 29, 311-314. (b)
Conforth, J. W. Angew. Chem., Int. Ed. Engl. 1968, 7, 903-911. (c) Lynen,
F.; Eggerer, H.; Henning, U.; Kessel, I. Angew. Chem. 1958, 70, 738-
742. (d) Poulter, C. D.; Argyle, J. C.; Mash, E. A. J. Am. Chem. Soc. 1977,
99, 957-959.
(4) Mayer, M. P.; Prestwich, G. D.; Dolence, J. M.; Bond, P. D.; Wu,
H.; Poulter, C. D. Gene 1993, 132, 41-47.
5) Tarshis, L. C.; Yan, M.; Poulter, C. D.; Sacchettini, J. C. Biochemistry
994, 33, 10871-10877.
(
1
1
0.1021/ol006055n CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/06/2000

1
3
diphosphate mimic, have been synthesized. These include
bromo derivative with inorganic pyrophosphate. We based
our synthesis of 6 on the displacement procedure, assuming
that the nucleophilicity of the sulfur atom in the thiopyro-
phosphate nucleophile would be sufficiently dominate to give
7
8
phosphonophosphates, phosphonophosphinates, and bis-
9
phosphonates, where methylene groups replace the oxygen
between phosphorus and carbon and the bridging oxygen
between the two phosphorus atoms. Both classes of com-
pounds are inhibitors, and the bisphosphonates are alternate
substrates as well. The lower binding affinities these
molecules typically have relative to the normal substrates
1
4
the thiolo derivative as the sole regioisomer.
The synthesis of thiopyrophosphate (5) is outlined in
Scheme 1. Trimethyl phosphate was heated at 90-100 °C
have been attributed to differences in the pK
a
s of the
phosphate and phosphonate groups.10 Difluoromethylene
Scheme 1
10
a
analogues have more closely matched pK s, but the fluorine
atoms introduce steric interactions not found in the normal
substrates.
Thiophosphate derivatives are widely used as inhibitors
11
and alternate substrates for nucleotides. For these applica-
tions, the analogues typically replace substrates for phos-
phoryl transfer reactions where bonds are being made and
broken at the phosphorus atom. Model solvolysis studies with
allylic isoprenoid thiolo- and thionophosphates, where the
bond to the allylic moiety is cleaved, suggest substantially
different patterns of reactivity.12 For simple dimethylallyl
derivatives, the reactivity of the thiono isomer is similar to
that of the corresponding phosphate, while the thiolo isomer
with 1 equiv of tetrabutylammonium hydroxide (TBAH) for
6
is approximately 10 -fold less reactive. These results translate
2
4 h to give dimethyl phosphate in 95% yield. We used
to enzyme-catalyzed reactions, where the P(1) thiono ana-
logue of GPP is a good alternate substrate for FPPase with
steady-state kinetic parameters similar to those of the GPP
TBAH for the hydrolysis because the resulting salt is highly
1
3
soluble in organic solvents and can be used directly in the
next steps without having to exchange the counterion.
Treatment of 2 with dimethyl thiophosphochloridate gave
tetramethyl thiolodiphosphate 3 in a 30% yield. The tetra-
methyl ester was unstable to the reaction conditions. The
best yields were obtained when the reaction was conducted
at -35 °C for 30 min, and the mixture was immediately
1
2
itself. On the basis of the model studies, we reasoned that
the thiolo isomer would be substantially less reactive in the
prenyl transfer reaction but that its binding affinity would
be similar to that of the natural substrate. We now describe
a synthesis of tris(tetrabutylammonium)thiopyrophosphate (5)
with sulfur in a nonbridging position, the subsequent
preparation of geranyl S-thiolodiphosphate (6), and prelimi-
nary studies of 6 as a substrate for FPPase.
transferred to a silica column to separate 3 from unreacted
31
2
. The proton-decoupled P NMR spectrum of 3 was an
AB quartet with doublets at 55.6 and -13.8 ppm (J ) 20
Hz) for P(S) and P(O), respectively.
The methyl groups in tetraester 3 were removed at -35
C in 97% yield using a 5-fold excess of trimethyliodosilane
°
(TMSI). Initial attempts using trimethylbromosilane (TMSBr)
or TMSI generated in situ by treating TMSBr with NaI gave
poorer yields. The mechanism for dealkylation, displacement
of iodide from TMSI by the phosphoryl oxygen or thiophos-
phoryl sulfur followed by a second displacement at the
Allylic isoprenoid diphosphates are most easily prepared
by a direct displacement of the corresponding chloro or
15
(
7) (a) Corey, E. J.; Volante, R. R. J. Am. Chem. Soc. 1976, 98, 1291-
methyl groups by iodide, predicts formation of thiolo-TMS
1
293. (b) Cane, D. E.; Yang, G.; Xue, Q.; Shim, J. H. Biochemistry 1995,
diphosphate 4. The TMS derivative was too unstable to be
3
4, 2471-2479.
31
chromatographed, but a proton-decoupled P NMR spectrum
of the reaction mixture had a four-line pattern consistent with
an AB quartet with peaks at -32.64 and 29.94 ppm (J )
16.5 Hz) for P(O) and P(S) resonances, respectively. Diphos-
phate 4 was desilylated by slow addition of 40% (v/v)
(
8) McClard, R. W.; Fujita, T. S.; Stremler, K. E.; Poulter, C. D. J. Am.
Chem. Soc. 1987, 109, 5544-5545.
9) Stremler, K. E.; Poulter, C. D. J. Am. Chem. Soc. 1987, 109, 5542-
544.
10) (a) Blackburn, G. M.; England, D. A.; Kolkmann, F. J. Chem. Soc.,
(
5
(
Chem. Commun. 1981, 930-932. (b) Blackburn, G. M.; Kent, D. E.;
Kolkmann, F. J. Chem. Soc., Chem. Commun. 1981, 1188-1190. (c)
Blackburn, G. M.; Eckstein, F.; Kent, D. E.; Perree, T. D. Nucleosides
Nucleotides 1985, 4, 165-167.
(13) (a) Davisson, V. J.; Woodside, A. B.; Poulter, C. D. Method
Enzymol. 1984, 110, 130-144. (b) Davisson, V. J.; Woodside, A. B.; Neal,
T. R.; Stremler, K. E.; Poulter, C. D. J. Org. Chem. 1986, 51, 4768-4778.
(14) (a) Chojnowski, J.; Cypryk, M.; Fortuniak, W.; Michalski, J. Synth.
Commun. 1977, 683-686. (b) Almasi, L. In Sulfur in Organic and Inorganic
Chemistry; Senning, A., Ed.; Marcel Dekker: New York, 1971.
(15) (a) Blackburn, G. M.; Ingleson, K. J. Chem. Soc., Chem. Commun.
1978, 870-871. (b) Zygmunt, J.; Kafarski, P.; Mastalerz, P. Synthesis 1978,
609.
(
11) (a) Goody, R. S.; Eckstein, F. J. Am. Chem. Soc. 1971, 93, 6252.
(
b) Burgers, B. M. J.; Ecksteinm F. Proc. Natl. Acad. Sci. U.S.A. 1978, 75,
4
3
798-4800. (c) Richard, J. P.; Frey, P. A. J. Am. Chem. Soc. 1982, 104,
476-3481. (d) Eckstein, F. Annu. ReV. Biochem. 1985, 54, 367-402. (e)
Gish, G.; Eckstein, F. TIBS 1989, 14, 97-100. (g) Eckstein, F.; Ludwig, J.
J. Org. Chem. 1991, 56, 1777-1783.
(12) Poulter, C. D.; Mautz, D. S. J. Am,. Chem. Soc. 1991, 113, 4895-
4
903.
2288
Org. Lett., Vol. 2, No. 15, 2000

tetrabutylammonium hydroxide to give the isomer of thio-
pyrophosphate with sulfur in a nonbridging position. Thio-
pyrophosphate 5 is sensitive to strongly basic conditions. The
pH of the solution was monitored as the TMS groups were
removed, and the addition of tetrabutylammonium hydroxide
was stopped at pH 7-7.5, where 5 is a trianion. The tris-
M
conditions. Although IC50 and K are not strictly comparable
kinetic constants, the somewhat larger values for IC50 for 6
may reflect the larger C-S and P-S bonds in the thio
analogue as well as differences in the C-S-P and C-O-P
bond angles. Preliminary studies also indicate that 6 is a
substrate for FPPase, although the thiolo analogue is
substantially less reactive than GPP. Recombinant FPPase
(tetra-n-butylammonium) salt was extracted into benzene, and
solvent was removed at reduced pressure to give a viscous
pale yellow oil. A proton-decoupled 31P NMR showed an
AB quartet with resonances at 36.88 and -7.68 ppm (J )
14
was incubated with [ C]IPP (20 µM, 10 µCi/µmol) and 6
or GPP (50 µM), and the rates of the reactions were
17
determined by the standard acid lability assay. Under
30 Hz) for P(S) and P(O), respectively. The resulting salt
14
conditions in which the condensation of [ C]IPP and GPP
was used directly for the thiolophosphorylation of geranyl
bromide without additional purification.
gave 2471 dpm in a 10 min incubation, no detectable
14
formation of product was seen for [ C]IPP (20 µM, 10 µCi/
µmol) and thio analogue 6 (50 µM). When the concentration
on FPPase was increased 100-fold and the time of the
incubation was lengthened 10-fold, 130 dpm were seen above
background. When extrapolated back to the normal assay
Geranyl S-thiolodiphosphate was prepared by the protocol
1
3
of Davisson et al. with minor modifications (see Scheme
2). Geranyl bromide was added dropwise to a well-stirred
5
conditions, the reaction of IPP with 6 is > 10 times slower
Scheme 2
than that for GPP.
1
4
FPPase (34 µg) was incubated with [ C]IPP (20 µM, 55
µCi/µmol) and 6 (50 µM) in 200 µL of the assay buffer at
1
8
3
7 °C overnight (20 h). A parallel incubation in which 6
was replaced by 7 was performed for 10 min. A 10 µL
portion of each sample was co-spotted with cold FPP on a
silica gel 60 F254 TLC plate, which was developed with 25:
1
5:4:2 (v/v/v) CHCl
products were detected by phosphorimaging, a single new
spot (R ) 0.37), corresponding to FPP, was seen. In the
3
/methanol/water/acetic acid. When the
f
1
4
incubation with 7, all of the [ C]IPP was consumed, while
only 4% of the IPP radioactivity appeared in FPP for the
incubation with 6. Thus, thiolodiphosphate 6 is a slow
reacting alternative substrate for GPP that binds with similar
affinity to avian FPPase.
solution of 2.3 equiv of 5 in CH CN at -30 °C over a period
3
of approximately 30 min. The salt was then passed through
+
a short ion exchange column (NH
4
form), and the material
was chromatographed on cellulose to afford geranyl S-
thiolodiphosphate (6) in 80% yield. The proton-decoupled
In summary, we describe a new useful synthesis of tris-
(tetra-n-butylammonium)thiopyrophosphate (5), the subse-
quent synthesis of geranyl S-thiolodiphosphate (6), and
preliminary results demonstrating that the analogue for GPP
is a slowly reacting alternate substrate. This procedure should
provide a practical route to thiolodiphosphate esters that can
be synthesized by the displacement protocol, including a wide
variety of isoprenoid and nucleotide derivatives useful in
31
P NMR spectrum of 6 had a characteristic AB pattern with
doublets (J ) 29 Hz) at 7.97 and -7.43 ppm for P(S) and
1
31
P(O), respectively. In an H-coupled P spectrum, each peak
of the doublet at 7.97 ppm was further split into triplets (J
1
)
8.6 Hz) by the C(1) protons in the geranyl moiety. An H
NMR spectrum of 6 confirmed the thiolo structure. The C(1)
methylene protons in GPP appear as a triplet (coupling to
the proton at C(2) and the P(1) phosphorus) at 4.46 ppm. In
11
biological studies.
6
the C(1) protons also appear as a triplet at 3.5 ppm, as
expected for the C-S-P linkage.
Acknowledgment. This work was supported by NIH
Grant GM 21328. R.M.P. is an NIH Predoctoral Trainee,
GM 08573.
Although sulfur is substantially more nucleophilic than
oxygen, we used a 2.3-fold excess of 5 in the displacement
reaction with geranyl bromide to guard against competing
displacement by one of the four anionic oxygens in the tris-
salt. Under these conditions geranyl S-thiolodiphosphate (6)
was the only product.
Supporting Information Available: Complete experi-
mental procedures for the synthesis of compounds 2-6 and
conditions for acid-labile activity assay. This material is
available free of charge via the Internet at http://pubs.acs.org.
We conducted preliminary biochemical experiments with
6
as a substrate for the chain elongation reaction catalyzed
16
by recombinant avian FPPase. As anticipated, the thiolo-
diphosphate is a good inhibitor of the enzyme with IC50
2
OL006055N
)
GPP
8 µM. In comparison, K
M
) 4.5 µM under similar
(
(
17) The acid-lability assay is described in the Supporting Information.
18) The reaction mixture was prepared in the same manner as the assay
(16) Reed, B. C.; Rilling, H. C. Biochemistry 1975, 14, 50-54.
mixture for the acid-lability assay.
Org. Lett., Vol. 2, No. 15, 2000
2289