Total synthesis of geranylgeranylglyceryl phosphate enantiomers: Substrates for characterization of 2,3-O-digeranylgeranylglyceryl phosphate synthase

Article abstract of DOI:10.1021/ol0530878

To determine the enantioselectivity of (S)-2,3-di-O-geranylgeranylglyceryl phosphate synthase (DGGGPS) from the thermoacidophilic archaeon Sulfolobus solfataricus, we developed an efficient enantioselective route to the enantiomeric geranylgeranylglyceryl phosphates (R)-GGGP and (S)-GGGP. Previous routes to these substrates involved enzymatic conversions due to the lability of the polyprenyl chains toward common phosphorylation reaction conditions. The synthesis described herein employs a mild trimethyl phosphite/carbon tetrabromide oxidative phosphorylation to circumvent this problem. In contrast to previous results suggesting that only (S)-GGGP can act as the prenyl acceptor substrate, both (R)-GGGP and (S)-GGGP were found to be substrates for DGGGPS.

Full text of DOI:10.1021/ol0530878

ORGANIC
LETTERS
2006
Vol. 8, No. 5
943-946
Total Synthesis of
Geranylgeranylglyceryl Phosphate
Enantiomers: Substrates for
Characterization of
2,3-O-Digeranylgeranylglyceryl
Phosphate Synthase
Honglu Zhang,† Kyohei Shibuya,‡ Hisashi Hemmi,‡ Tokuzo Nishino,‡ and
Glenn D. Prestwich*,†
Department of Medicinal Chemistry, The UniVersity of Utah, 419 Wakara Way,
Suite 205, Salt Lake City, Utah 84108-1257, and Department of Biomolecular
Engineering, Tohoku UniVersity, Aoba-yama 07, Sendai, Miyagi 980-8579, Japan
gprestwich@pharm.utah.edu
Received December 20, 2005
ABSTRACT
To determine the enantioselectivity of (S)-2,3-di-O-geranylgeranylglyceryl phosphate synthase (DGGGPS) from the thermoacidophilic archaeon
Sulfolobus solfataricus, we developed an efficient enantioselective route to the enantiomeric geranylgeranylglyceryl phosphates (R)-GGGP
and (S)-GGGP. Previous routes to these substrates involved enzymatic conversions due to the lability of the polyprenyl chains toward common
phosphorylation reaction conditions. The synthesis described herein employs a mild trimethyl phosphite/carbon tetrabromide oxidative
phosphorylation to circumvent this problem. In contrast to previous results suggesting that only (S)-GGGP can act as the prenyl acceptor
substrate, both (R)-GGGP and (S)-GGGP were found to be substrates for DGGGPS.
All living organisms have been classified into three primary
kingdoms: archaebacteria, eubacteria, and eukaryotes.1
Although the membrane lipids in eubacteria and eukaryotes
are composed of glyceryl esters of fatty acids, archaebacterial
membrane lipids contain isopranyl glyceryl ethers.2,3 The core
structures of archaeal membrane lipids, including diether and
bipolar tetraether lipids, contain fully reduced C20 or C25
prenyl groups.4,5 Biosynthesis of the isoprene moieties in the
core lipids follows the mevalonate pathway used also by
eubacteria and eukaryotes.6-8 The prenyl transfer reactions
responsible for building the isoprenoid diphosphates, e.g.,
geranylgeranyl diphosphate (GGPP), are catalyzed by a
* To whom correspondence should be addressed. Phone: +1-801-585-
9051. Fax: +1-801-585-9053.
(4) Kates, M. Prog. Chem. Fats Other Lipids 1978, 15, 301-342.
(5) Heathcock, C. H.; Finkelstein, B. L.; Aoki, T.; Poulter, C. D. Science
1985, 229, 862-864.
† The University of Utah.
‡ Tohoku University.
(6) De Rosa, M.; De Rosa, S.; Gambacorta, A. Phytochemistry 1977,
16, 1909-1912.
(1) Woese, C. R. Sci. Am. 1981, 244, 98-122.
(2) Langworthy, T. A.; Tornabene, T. G.; Holzer, G. Zentralbl. Bakteriol.,
Mikrobiol. Hyg., Abt. I, Orig. C. 1982, 3, 228-244.
(3) Langworthy, T. A.; Pond, J. L. Syst. Appl. Microbiol. 1986, 7, 253-
257.
(7) De Rosa, M.; Gambacorta, A.; Nicolaus, B. Phytochemistry 1980,
19, 791-793.
(8) Moldoveanu, N.; Kates, M. Biochim. Biophys. Acta 1988, 960, 164-
182.
10.1021/ol0530878 CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/28/2006

Figure 1. Biosynthesis of DGGGP, a key intermediate for archaeal membrane lipids. Key: G-1-P, (S)-glyceryl phosphate; GGGPS, (S)-
GGGP synthase; DGGGPS, DGGGP synthase.
family of prenyltransferases.9-12 As shown in Figure 1, the
biogenesis of the core structure of the archaeal membrane
lipids starts with the prenyl transfer reaction catalyzed by
(S)-3-O-geranylgeranylglyceryl phosphate [(S)-GGGP] syn-
thase, which selectively uses (S)-glyceryl phosphate as the
prenyl acceptor (Figure 1). Then, the product is utilized as
the presumed acceptor substrate for the biosynthesis of (S)-
2,3-di-O-geranylgeranylglyceryl phosphate (DGGGP), an
advanced intermediate of archaeal membrane lipids.13
To date, only an enzyme-assisted synthesis of (S)-GGGP
has been reported.11 An enantiospecific chemical synthesis
of both individual enantiomers of GGGP was required to
validate this biosynthetic hypothesis, in part because of the
acid-sensitive geranylgeranyl group of GGGP. To fully
characterize the substrate selectivity of DGGGP synthase
(DGGGPS), we developed a mild and effective route to the
two GGGP enantiomers. The instability of GGGP indeed
posed significant challenges, and the biological results with
the enantiomers were unexpected.
phorylation of the primary hydroxyl, we first tried a strategy
involving protection of the secondary hydroxyl as a silyl
ether. Thus, both hydroxyl groups of diol 4 were protected
as TBS ethers (TBSCl, imidazole, anhydrous DMF).15,16
Subsequently, the more labile primary TBS ether was
selectively removed at room temperature by using HF‚Py
(HF‚Py/Py/THF ) 1:2:5). Unfortunately, phosphorylation of
the primary alcohol under standard conditions (dimeth-
ylphosphoryl chloride, t-BuOK, CH2Cl2)15,16 failed to give
the desired product because of the lability of the polyene
system.
A second strategy proved more successful. The use of the
trimethyl phosphite/carbon tetrabromide oxidative phospho-
rylation method17 was deemed sufficiently mild to permit
phosphorylation without damage to the geranylgeranyl
moiety. Treatment of diol 4 with 1.1 equiv of CBr4 and 1.2
equiv of P(OMe)3 gave selective phosphorylation of the
primary alcohol to give the protected phosphate 7. Essentially
no bisphosphate product was detected.
The synthesis of (S)-GGGP (8) is summarized in Scheme
1. Treatment of the (2E,6E,10E)-geranylgeraniol 1 with Ph3P/
CBr4 at 25 °C afforded geranylgeranyl bromide 2.14 Next,
(S)-solketal was alkylated with geranylgeranyl bromide by
using KH as base, to give ether 3 in 73% yield.11 The
reported HCl/THF method to remove the acetonide13 resulted
in a complex mixture containing the desired product in low
yield. The desired diol 4 was thus prepared in 75% yield
using p-TsOH in methanol.15,16 To obtain selective phos-
The next challenge in this synthesis was liberation of the
free phosphate monoester from the protected triester. We first
tried TMSBr, a standard deprotecting reagent for removal
of methyl and ethyl groups in the synthesis of acyl-migration-
prone lysophosphatidic acid derivatives.15,16 However, GGGP
did not survive this strong Lewis acid. By using a solution
of TMSBr in 2,4,6-trimethylpyridine (sym-collidine),18-20 we
obtained the desired monophosphate in the acidic form.
Titration with 1 N aq NaOH afforded (S)-GGGP (8) as the
stabilized sodium salt.
(9) Poulter, C. D. In Biochemistry of Cell Walls and Membranes in Fungi;
Kuhn, P. J., Trinci, A. P. J., Jung, M. J., Goosey, M. W., Copping, L. G.,
Eds.; Springer-Verlag: Berlin, Heidelberg, 1990; pp 169-188.
(10) Poulter, C. D. In Biosynthesis of Isopreniod compounds; Rilling,
H. C., Porter, J. W., Spurgeon, S. L., Eds.; Wiley: New York, 1981; Vol.
I, pp 161-224.
To determine the enantioselectivity of DGGGPS, both the
enantiomers (S)-GGGP (8) and (R)-GGGP (12) were re-
(16) Xu, Y.; Qian, L.; Prestwich, G. D. J. Org. Chem. 2003, 68, 5320-
5330.
(11) Zhang, D.; Poulter, C. D. J. Am. Chem. Soc. 1993, 115, 1270-
1277.
(17) Oza, V. B.; Corcoran, R. C. J. Org. Chem. 1995, 60, 3680-3684.
(18) Cermak, D. M.; Wiemer, D. F.; Lewis, K.; Hohl, R. J. Bioorg. Med.
Chem. 2000, 8, 2729-2737.
(12) Ohnuma, S.-i.; Suzuki, M.; Nishino, T. J. Biol. Chem. 1994, 269,
14792-14797.
(13) Hemmi, H.; Shibuya, K.; Takahashi, Y.; Nakayama, T.; Nishino,
T. J. Biol. Chem. 2004, 279, 50197-50203.
(19) Macchia, M.; Jannitti, N.; Gervasi, G.; Danesi, R. J. Med. Chem.
1996, 39, 1352-1356.
(14) Tokumasu, M. A. H.; Hiraga, Y.; Kojima, S.; Ohkta, K. J. Chem.
Soc., Perkin Trans. 1 1999, 489-496.
(20) Magnin, D. R.; Biller, S. A.; Dickson, J. K., Jr.; Logan, J. V.;
Lawrence, R. M.; Chen, Y.; Sulsky, R. B.; Ciosek, C. P., Jr.; Harrity, T.
W.; Jolibois, K. G.; et al. J. Med. Chem. 1995, 38, 2596-2605.
(15) Xu, Y.; Prestwich, G. D. J. Org. Chem. 2002, 67, 7158-7161.
944
Org. Lett., Vol. 8, No. 5, 2006

Scheme 1. Synthesis of (S)-GGGP (8)
quired. Starting with (R)-solketal, (R)-GGGP (12) was
synthesized using the successful route as summarized in
Scheme 2.
DGGGPS is a member of the UbiA prenyltransferase
family that can catalyze the transfer of a prenyl group to its
biological acceptor substrate (S)-GGGP (8). With the enan-
tiomeric substrates (R)-GGGP (12) and (S)-GGGP (8) in
hand, we determined the activity of DGGGPS toward each
of these substrates. From the results of radio HPLC analysis
(Figure 2) and reversed-phase TLC analysis (Figure 3), we
found that the DGGGP and presumably its enantiomer were
formed in the reactions using (S)-GGGP (8) and (R)-GGGP
(12), respectively. In these reactions, the starting reagent
[14C]-GGPP was formed first from [14C]isopentenyl diphos-
phate and (E,E)-farnesyl diphosphate by the activity of
GGPS. Then, [14C]-GGPP was used as the prenyl donor
substrate for DGGGPS. Thus, the results demonstrated that
the C20-prenyl group of GGPP could be transferred to either
of the two GGGP enantiomers by the action of DGGGPS.
(S)-GGGP seems to be marginally preferred in Figure 2,
Scheme 2. Synthesis of (R)-GGGP (12)
Org. Lett., Vol. 8, No. 5, 2006
945

Figure 3. Autoradiogram of TLC from left to right: (1) (S)-GGGP;
(2) (R)-GGGP; (3) without an acceptor substrate. Key: GGGOH,
geranylgeranylglycerol; GGOH, geranylgeraniol; DGGGOH, di-
geranylgeranylglycerol; Ori, origin; S.f., solvent front.
Figure 2. Radio HPLC analysis of 1-butanol extracts from the
enzyme reaction with [14C]-GGPP synthase and DGGGPS, using
0.5 nmol [14C]-isopentenyl diphosphate, 0.5 nmol (E,E)-farnesyl
diphosphate, 0.4 µmol (S)-GGGP (A), or (R)-GGGP (B) as
substrates. Key: GGOH, geranylgeraniol; DGGGOH, digeranyl-
geranylglycerol.
In conclusion, (S)-GGGP and (R)-GGGP were each
synthesized by a five-step procedure starting from the
(2E,6E,10E)-geranylgeraniol and the appropriate enantiomer
of solketal. A regioselective phosphorylation of diol 4 was
achieved using CBr4/P(OMe)3, and the instability problem
of the geranylgeranyl group was circumvented by judicious
selection of mild reaction conditions. The LKC18 reversed-
phase TLC analysis and radio HPLC analysis have shown
that the DGGGPS can catalyze the transfer of a prenyl group
to the secondary hydroxy groups of both (R)-GGGP and (S)-
GGGP.
whereas the (R)-enantiomer appeared to be preferred in
Figure 3. These results are reproducible but qualitative; the
GGGPs produced are quite labile. Nevertheless, to our
surprise, both (R)-GGGP (12) and (S)-GGGP (8) were
accepted at a comparable extent as substrates for DGGGPS.
During the biosynthesis of archaeal membrane lipids, GGGPS
catalyzes the transfer of prenyl groups from GGPP to (S)-
glyceryl phosphate in the formation of (S)-GGGP (8), and
the ether linkage between both (S)-GGGP (8) and another
geranylgeranyl group is formed under the control of DGGGPS.
GGGPSs are known to have strict substrate preferences: (R)-
glyceryl phosphate is a very poor substrate.13,21 Thus, our
results strongly suggest that the chirality of the archaeal
membrane lipid is determined by GGGPS, not by DGGGPS.
However, (R)-GGGP (12) and (S)-GGGP (8) will be
important tools for more detailed analysis of the specific
activity and enantioselectivity of DGGGPS in future studies.
Acknowledgment. We thank Dr. Y. Xu (University of
Utah) and G. Jiang (University of Utah) for many helpful
discussions. We thank the NIH (NS29632 to G.D.P.) and
grants-in-aid from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan for financial support of
this work.
Supporting Information Available: Experimental pro-
cedures and characterization for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(21) Zhang, D.; Poulter, C. D. J. Org. Chem. 1993, 58, 3919-3922.
OL0530878
946
Org. Lett., Vol. 8, No. 5, 2006