Synthesis and enzymatic phosphorylation of a photoactivatable dolichol analogue

Article abstract of DOI:10.1021/ja9721677

The synthesis of the photochemical probes 6 and 7 is described. These photoprobes are analogues of dolichol and dolichol phosphate, obligatory intermediates in the N-linked glycosylation pathway in the endoplasmic reticulum. The synthesis of 6 and 7 follo

Full text of DOI:10.1021/ja9721677

10992
J. Am. Chem. Soc. 1997, 119, 10992-10999
Synthesis and Enzymatic Phosphorylation of a Photoactivatable
Dolichol Analogue
D. Grassi, V. Lippuner, M. Aebi,† J. Brunner,‡ and A. Vasella*,§
Contribution from the Institutes of Microbiology, Biochemistry, and Organic Chemistry, Swiss
Federal Institute of Technology, CH-8092 Zu¨rich, Switzerland
ReceiVed June 30, 1997X
Abstract: The synthesis of the photochemical probes 6 and 7 is described. These photoprobes are analogues of
dolichol and dolichol phosphate, obligatory intermediates in the N-linked glycosylation pathway in the endoplasmic
reticulum. The synthesis of 6 and 7 follows a new strategy. It involves the sequential alkylation of a monoterpenoid
hydroxysulfonyl dianion with allyl chlorides. The photoreactive group, a 3-(trifluoromethyl)-3-aryldiazirine, was
connected to the hydroxylated methyl group of the â-prenyl unit of the fully assembled polyprenyl chain. The
photoactivatable dolichol analogue 6 is a substrate for dolichol kinase from yeast membranes, an essential enzyme
involved in the N-linked glycosylation pathway.
Dolichols1 (1), a family of polyisoprenoid alcohols, are found
Dol-P-P (3) is the membrane anchor and carrier of the
oligosaccharide GlcNAc2Man9Glc3, which is assembled in a
stepwise manner,16 UDP-GlcNAc,19 GDP-Man, Dol-P-Man, and
Dol-P-Glc serving as glycosyl donors.5,16 The Dol-P-P-bound
oligosaccharide GlcNAc2Man9Glc3 is finally transferred to the
amido nitrogen of an Asn residue of the triplet Asn-Xaa-Ser-
(Thr) of nascent acceptor proteins.20
in all eukaryotic cells and have also been identified in Archae-
bacteria.2 Dolichols (1) occur as mixtures differing only in the
number (n ) 9-20)3 of (E)-isoprene units. The number of
isoprene units shows a Gaussian distribution around a mean
value, which appears to increase with the complexity of the
organism.4
Dolichol (Dol, 1), dolichol phosphate (Dol-P, 2), and dolichol
pyrophosphate (Dol-P-P, 3) are involved in the biosynthesis of
glycoproteins in eukaryotes and prokaryotes.
Dol-P (2) is also involved in the process of GPI-anchoring
in eukaryotes and in O-linked glycosylations in fungal cells.21-27
In Archaebacteria, Dol-P (2) is used in the biosynthesis of
cell surface glycoproteins.28
To investigate the distribution of Dol (1) and Dol-P (2) in
the ER and their possible topological relation to specific proteins
recognizing these lipids, we decided to pursue photoaffinity
labeling29,30 using the radiolabeled Dol and Dol-P analogues 4
and 5, respectively (Figure 1). As a first step toward this goal
we report a new strategy for the preparation of functionalized,
long-chain dolichols, leading to the synthesis and characteriza-
tion of the Dol analogue 6 and the corresponding phosphate 7.
We also demonstrate that the photoactivatable dolichol deriva-
tive 6 is a substrate for dolichol kinase from yeast.
In eukaryotes, N-linked glycosylation of secretory and
membrane proteins occurs at the endoplasmic reticulum (ER)
and follows a highly conserved pathway.5 In this pathway
dolichol kinase phosphorylates the free alcohol 16-13 to produce
Dol-P (2), an obligatory intermediate,14-16 which in turn is a
substrate for several enzymes involved in oligosaccharide
assembly. The concentration of Dol-P (2) in the ER is believed
to be rate limiting in these glycosylations.17,18
† Institute of Microbiology.
‡ Laboratory of Biochemistry.
§ Laboratory of Organic Chemistry.
X Abstract published in AdVance ACS Abstracts, November 1, 1997.
(1) Pennock, J. F.; Hemming, F. W.; Morton, R. A. Nature 1960, 186,
470-472.
Results and Discussion
(2) Lechner, J.; Wieland, F.; Sumper, M. J. Biol. Chem. 1985, 260, 8984-
8989.
(3) Hemming, F. W. Biochem. Cell Biol. 1992, 70, 377-381.
(4) Quesada-Allue, L. A.; Parodi, A. J. Biochem. J. 1983, 212, 123-
128.
Design of the Photolabeling Reagents 4-7. The proper
design of a coenzyme-mimicking photolabeling reagent should
solve the problem of introducing a photoactivatable group in a
(5) Kornfeld, R.; Kornfeld, S. Annu. ReV. Biochem. 1985, 54, 631-664.
(6) Allen, C. M.; Muth, J. D.; Gildersleeve, N. Biochim. Biophys. Acta
1982, 712, 33-41.
(17) Carson, D. D.; Lennarz, W. J. Proc. Natl. Acad. Sci. U.S.A. 1979,
76, 5709-5713.
(7) Ardail, D.; Lerme´, F.; Louisot, P. Biochim. Biophys. Acta 1990, 1024,
131-138.
(18) Mills, J. T.; Adamany, A. M. J. Biol. Chem. 1978, 253, 5270-
5273.
(8) Gandhi, C. R.; Keenan, R. W. J. Biol. Chem. 1983, 258, 7639-
7643.
(19) Kean, E. L. J. Biol. Chem. 1991, 266, 942-946.
(20) Marshall, R. D. Biochem. Soc. Symp. 1974, 40, 17-26.
(21) Tanner, W.; Lehle, L. Biochim. Biophys. Acta 1987, 906, 81-99.
(22) Bretthauer, R. K.; Wu, S. Arch. Biochem. Biophys. 1975, 167, 151-
160.
(9) Heller, L.; Orlean, P.; Adair, W. L., Jr. Proc. Natl. Acad. Sci. U.S.A.
1992, 89, 7013-7016.
(10) Itami, T.; Mueck, R. L.; Keenan, R. W. Biochim. Biophys. Acta
1988, 960, 374-381.
(23) Soliday, C. L.; Kolattukudy, P. E. Arch. Biochem. Biophys. 1979,
197, 367-378.
(11) Scher, M. G.; Sumbilla, C. M.; Waechter, C. J. J. Biol. Chem. 1985,
260, 13742-13746.
(24) Gold, M. H.; Hahn, H. J. Biochemistry 1976, 15, 1808-1814.
(25) Babczinski, P.; Tanner, W. Biochem. Biophys. Res. Commun. 1973,
54, 1119-1124.
(12) Sumbilla, C.; Waechter, C. J. Methods Enzymol. 1985, 111, 471-
482.
(13) Szkopinska, A.; Swiezewska, E.; Chojnacki, T. Int. J. Biochem.
1992, 24, 1151-1157.
(14) Verbert, A. In Glycoproteins; Montreuil, J., Schachter, H., Vliegen-
hart, J. F. G., Eds.; Elsevier: Amsterdam, 1995; pp 145-152.
(15) Hemming, F. W. In Glycoproteins; Neuberger, A., Van Deenen, L.
L. M., Eds.; Elsevier: Amsterdam, 1995; Vol. 29a, pp 127-199.
(16) Abeijon, C.; Hirschberg, C. B. TIBS 1992, 17, 32-36.
(26) Kruszewska, J.; Messner, R.; Kubicek, C. P.; Palamarczyk, G. J.
Gen. Microbiol. 1989, 135, 301-307.
(27) Sharma, C. B.; Babczinski, P.; Lehle, L.; Tanner, W. Eur. J.
Biochem. 1974, 46, 35-41.
(28) Lechner, J.; Wieland, F. Annu. ReV. Biochem. 1989, 58, 173-194.
(29) Fleming, S. A. Tetrahedron 1995, 51, 12479-12520.
(30) Brunner, J. Annu. ReV. Biochem. 1993, 62, 483-514.
S0002-7863(97)02167-7 CCC: $14.00 © 1997 American Chemical Society

A PhotoactiVatable Dolichol Analogue
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10993
Figure 2.
transformed into 11 and 12, and chain extended toward the
unsaturated (“left”) end, using building blocks derived from 12
and the farnesyl sulfone 13 to give a pentadecaprenyl derivative.
This was debenzylated and desulfonylated under conditions of
the Birch reaction and chain extended toward the saturated
(“right”) end, using a building block derived from the sesquit-
erpenoid analogue of 8 and the allyl chloride 14.
This strategy has been applied to the synthesis of other long-
chain prenol derivatives, allowing one to study the behavior of
prenols of defined chain length and geometry in biological
systems.39-41 Despite its advantages, the strategy suffers from
a significant drawback. The reductive cleavage of the C-S
bonds is accompanied by double bond migration and leads to
isomeric byproducts (e15%) from which the desired isomer is
separated only with considerable difficulty.37,42
We envisaged a shorter synthesis free of this drawback. For
this, we conceived a linear synthesis, starting at the saturated
hydroxyl terminus while still using the alkylation of sulfonyl
anions as one of the key steps. However, we decided to use
phenyl sulfones instead of p-tolyl sulfones, to avoid the
deprotonation of the tolylsulfonyl group with the strong bases
required in the coupling steps.43,44 The synthesis from the
reduced end obviates the use of benzyl protecting groups. Both
the removal of all sulfonyl groups in one step at the end of the
chain assembly and the use of the hydroxysulfone 21 (Scheme
1) rather than the corresponding THP derivative should further
reduce the number of steps. In addition, desulfonylation with
LiBEt3H in the presence of a Pd catalyst45 should proceed
without formation of isomeric byproductes.46
The synthesis started with the preparation of the acrylate 20
and the hydroxy sulfone 21 (Scheme 1). The acrylate 20 was
prepared from (S)-citronellol (15). Ozonolysis of the triisopro-
pylsilyl ether 16 afforded aldehyde 17 in 65-83% yield.
Baylis-Hillman reaction of 17 with methyl acrylate (18) yielded
62-79% of acrylate 19 but proceeded very slowly.47 Acety-
lation led to the desired building block 20 as an approximately
Figure 1.
position that leads to specific labeling of the protein without
interfering with the recognition. The inability of mutant Chinese
hamster ovary cells to reduce the R-isoprene unit of polyprenols
to form Dol results in a severe alteration of N-linked glycosy-
lation,31 suggesting an essential role of the saturated unit in the
recognition of Dol. Therefore, this recognition site should be
present in the photoactivatable analogue, and the photolabile
functionality should be close to this segment to increase the
labeling specificity. To trace the photoadducts, the photoprobe
must bear a reporter group, preferentially a radioisotope with a
high specific radioactivity, such as 125I.
Dolichol analogues 4/5 are expected to satisfy these require-
ments. The chain length of 11 isoprene units was chosen, since
authentic Dol of this chain length (Dol-11) is a good substrate
for dolichol kinase in mammalian cells13 and Dol-P-11 is readily
accepted by mammalian Dol-P-Man synthase13 and by UDP-
GlcNAc:Dol-P N-acetylglucosamine-1-P transferase from yeast.32
A 3-(trifluoromethyl)-3-aryldiazirine was chosen as photo-
labile substituent. In the absence of UV light, this diazirine is
stable under a wide range of physical and chemical conditions.
It is stable to temperatures up to 80 °C, strong bases, strong
acids, oxidizing conditions, and mild reducing agents.33 Upon
irradiation with light of a wavelength of around 350 nm, the
diazirine is rapidly photolyzed to generate N2 and a carbene
capable of reacting with the full range of functional groups
occurring in biomolecules, including nonactivated C-H bonds.33
Synthesis of the Photoactivatable, Non-radiolabeled Doli-
chol Analogue 6 and the Corresponding Phosphate 7. The
syntheses of Dol (1) have been reviewed.34-36 The longest
dolichol, Dol-20, the main component of human dolichols, has
been prepared by Sato et al. in a convergent way37,38 (Figure
2). Their synthesis is based on the preparation of 10 from the
sulfone 8 and the allyl chloride 9. The product 10 was
(39) Jaenicke, L.; Siegmund, H.-U. Biol. Chem. Hoppe-Seyler 1986, 367,
787-795.
(40) Jaenicke, L.; Van Leyen, K.; Siegmund, H.-U. Biol. Chem. Hoppe-
Seyler 1991, 372, 1021-1026.
(31) Beck, P. J.; Gething, M.-J.; Sambrook, J.; Lehrman, M. A. Somat.
Cell Mol. Genet. 1990, 16, 539-548.
(41) Siegmund, H.-U. Dissertation Thesis, Ko¨ln, Germany, 1986.
(42) Sato, K.; Miyamoto, O.; Inoue, S.; Furusawa, F.; Matsuhashi, Y.
Chem. Lett. 1984, 1105-1108.
(32) Palamarczyk, G.; Lehle, L.; Mankowski, T.; Chojnacki, T.; Tanner,
W. Eur. J. Biochem. 1980, 105, 517-523.
(33) Weber, T.; Brunner, J. J. Am. Chem. Soc. 1995, 117, 3084-3095.
(34) Veselovsky, V. V.; Grigorieva, N. Y.; Moiseenkov, A. M. Chem.
Phys. Lipids 1989, 51, 147-157.
(43) Muraoka, M.; Itoi, T.; Yamamoto, T. J. Chem. Res. S 1982, 256-
257.
(44) Madaj, E. J., Jr.; Snyder, D. M.; Truce, W. E. J. Am. Chem. Soc.
1986, 108, 3466-3469.
(45) Mohri, M.; Kinoshita, H.; Inomata, K.; Kotake, H. Chem. Lett. 1985,
451-454.
(46) Bouzbouz, S.; Kirschleger, B. Synthesis 1994, 714-718.
(47) Baylis, A. B.; Hillman, M. E. D. Verfahren zur Herstellung von
Acrylverbindungen (Acrylic Compounds). In German Offenlegungs-schrift
2155113; 10 May 1992, US Pat. Appl. 87591; Celanese Corporation:
Germany, 1972; pp 1-16.
(35) Moiseenkov, A. M. In Organic Synthesis: Modern Trends; Chizhov,
O., Ed.; Blackwell Scientific Publishers: Oxford, 1986; pp 151-160.
(36) Grigor’eva, N. Y.; Pinsker, O. A. Russ. Chem. ReV. 1994, 62, 169-
183.
(37) Sato, K.; Miyamoto, O.; Inoue, S.; Matsuhashi, Y.; Koyama, S.;
Kaneko, T. J. Chem. Soc., Chem. Commun. 1986, 1761-1762.
(38) Inoue, S.; Kaneko, T.; Takahashi, Y.; Miyamoto, O.; Sato, K. J.
Chem. Soc., Chem. Commun. 1987, 1036-1037.

10994 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Grassi et al.
Scheme 1a
a (a) TIPSCl, imidazole, CH2Cl2; (b) 1. O3, CH2Cl2, -78° 2. Me2S (65-83% from 15); (c) DABCO (62-79%); (d) Ac2O, pyridine, DMAP,
CH2Cl2 (90-95%); (e) BuLi, THF, DMTP, -78° (69%); (f) MsCl, LiCl, collidine, DMF, 0°; (g) BuLi, THF, DMTP, -78° (52% from 22); (h)
MsCl, LiCl, collidine, DMF, 0° (92%); (i) BuLi, THF, DMTP, -78° (76%); (k) MsCl, LiCl, collidine, 0° (89%); (l) BuLi, THF, DMTP, -78°
(66%); (m) DIBAH, CH2Cl2, -78° (78%); (n) LiEt3BH, [PdCl2(dppp)], THF, 0° (43-51%); (o) 32, benzene, NaOHaq, TBAI; (p) THF, TBAF
(56% from 31); (q) POCl3, NEt3, hexane (68%).
1:1 mixture of two diastereoisomers. The hydroxysulfone 21
was prepared according to Sato et al.41,48
alkylate the anion derived from the farnesyl sulfone 28, yielding
66% of the tetrasulfone 29.
Alkylation with 20 of the dianion derived from 21 afforded
69% of the ester 22. There was no advantage in protecting the
hydroxyl group of 21, since coupling of either the alcohol or
its THP derivative with 20 proceeded with similar yields. The
(E)-configuration of the acrylic double bond was evidenced by
the typical chemical shift (6.83 ppm) of the acrylic H.49 The
alcohol 22 was transformed into the allyl chloride 23. Alky-
lation of the dianion derived from 21 with 23 led to the triterpene
24 in an overall yield of 52% from 22. Deoxyhalogenation of
24 gave allyl chloride 25, which was treated with the same
dianion derived from 21 to afford the tetraterpene 26 (70% from
24). The chloride 27, obtained in 89% yield, was used to
Desulfonylation of 29 with LiBEt3H in the presence of [PdCl2-
(dppp)] (dppp ) 1,3-bis(diphenylphosphino)propane) was ac-
companied by partial (50%, NMR) 1,4-reduction of the acrylic
ester function. Therefore, 29 was first reduced with diisobu-
tylaluminium hydride (DIBAH) to give the alcohol 30 (78%)
and then desulfonylated with LiBEt3H in the presence of 30
mol % [PdCl2(dppp)].45,46 Although the desulfonylation was
incomplete, we isolated 43-51% of 31, uncontaminated by
isomeric byproducts.
Benzyl iodide 32 is available by treating the known alcohol
3333 with methyltriphenoxyphosphonium iodide.50 Alkylation
of allylic alcohol 31 with 32 under phase transfer conditions
led to 34, which was desilylated with tetrabutylammonium
(48) Sato, K.; Miyamoto, O.; Inoue, S.; Furusawa, F.; Matsuhashi, Y.
Chem. Lett. 1983, 725-728.
(49) Brouwer, H.; Stothers, J. B. Can. J. Chem. 1972, 50, 601-611.
(50) Shih, L. B.; Bayley, H. Anal. Biochem. 1985, 144, 132-141.

A PhotoactiVatable Dolichol Analogue
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10995
Figure 3. Enzymatic phosphorylation of dolichol analogue 6. Membranes derived from dolichol kinase overexpressing yeast cells and [γ-32P]-CTP
were incubated under assay conditions; lipids were extracted and subjected to reversed phase HPLC. 32P-radioactivity and/or absorption at 250 nm
were monitored. (A) Analysis of extract from an assay containing only yeast membrane lipids; Dol-14-32P, Dol-15-32P, and Dol-16-32P give rise to
distinct peaks. (B) Analysis of extract from an assay containing yeast membranes (dolichol kinase and endogenous dolichols) plus Dol analogue 6.
After incubation, extracted lipids were mixed with synthetic, nonradioactive reference compound 7. Enzymatically phosphorylated 6 (1, detected
by radioactivity) coelutes with synthetic reference compound 7 (detected by UV absorbance, dashed line). The second UV signal (2) derives from
6 added to the kinase assay. The radio signals with higher retention time derive from Dol-14-32P, Dol-15-32P, Dol-16-32P, and Dol-17-32P.
fluoride (TBAF) in THF, yielding the desired photoactivatable
dolichol derivative 6 (56% from 31). It was phosphorylated51
with phosphorous oxychloride, followed by aqueous workup.
The product 7 was purified by ion exchange and size exclusion
chromatography to give the phosphate 7 in 68% yield.
Experimental Section
General Methods. 1H, 13C, and 31P NMR spectra were recorded at
200-500 MHz on Varian instruments, chemical shifts are reported in
ppm, and coupling constants (J) are given in hertz. IR spectra were
recorded on a Perkin Elmer 1600 Series FT-IR spectrometer, FAB mass
spectra were recorded on a VG-SABSEQ, and ESI mass spectra on a
Finnigan MAT TSQ 7000 instrument. Thin-layer chromatography
(TLC) was performed on precoated silica gel 60 F254 plates (Merck,
Darmstadt, Germany), column chromatography was performed with
silica gel 60 (230-400 mesh) from Fluka (Buchs, Switzerland), gel
filtration was performed with Sephadex LH-20 from Pharmacia Biotech
(Uppsala, Sweden), and ion exchange chromatography was performed
with DEAE Sepharose CL-6B from Pharmacia Biotech. Dolichol-11
was purchased from Sigma (St. Louis, MO). (E,E)-Farnesol (>98%)
and (S)-citronellol (ee >98%) were gifts from R. K. Mu¨ller, F.
Hoffmann-La Roche AG (Basel, Switzerland). [γ-32P]-CTP (25 Ci/
mmol, 10 mCi/mL) was purchased from ICN (Irvine, CA). For
quantification of 32P, â-counting system LS 180S from Beckman was
used, with IRGA-SAFE PLUS (Packard) as scintillation fluid. Plasmid
pSec5920 was a gift from R. Schekman, University of California,
Berkeley.
Enzymatic Phosphorylation of Dolichol Analogue 6. To
test if photoprobe 6 is a substrate for dolichol kinase, we
incubated 6 with membranes9 derived from dolichol kinase
overproducing yeast cells, in the presence of [γ-32P]-CTP as
phosphoryl donor. Since the membrane extract contains en-
dogenous yeast dolichols, the phosphorylation of 6 competes
with the phosphorylation of the endogenous substrate.
In the absence of 6, HPLC analysis (Figure 3A, radio
detection) of the lipid extract from the kinase assay showed a
characteristic pattern of 32P-labeled yeast dolichol phosphates
(Dol-14, Dol-15, Dol-16).52 When 6 was added, the chromato-
gram of the lipid extract showed an additional radio signal
possessing the same retention time as the chemically synthesised
phosphate 7 (Figure 3B, 1). The second UV-detected signal
(2) is derived from excess photoprobe 6, not phosphorylated in
the assay. These results provide strong evidence that the
dolichol analogue 6 is phosphorylated by dolichol kinase in the
membrane preparation.
Dolichol Kinase Assay. Microsomal membranes were prepared
according to Knauer and Lehle53 from 4 L cultures with the following
modifications. Unless otherwise specified, all steps were performed
at 4 °C. Cells from Saccharomyces cereVisiae strain DBY746
transformed with the plasmid pSec5920, which contains the S.
cereVisiae dolichol kinase gene, were grown at 30 °C in synthetic
minimal medium supplemented with adenine, histidine, uracil, and
tryptophan according to Sherman54 to OD546nm ) 1.0. The cells were
pelleted, washed once with water, resuspended in 300 mL buffer
containing 50 mM Tris HCl (pH 7.69), 10 mM 2-mercaptoethanol and
protease inhibitors (1 mM PMSF, 2 µg/mL each of pepstatin A,
chymostatin, antipain, aprotonin), and broken in a bead beater (Biospec
Products, U.S.A.) at 0 °C using six 1 min pulses with 1 min intervals.
Cell wall and unlysed cells were removed by centrifugation in a Sorvall
GS3 rotor at 3000 rpm, and the supernatant was recentrifuged at 22000
rpm in a Beckman Ti45 rotor for 30 min. The membrane pellet was
washed once in the same buffer. The membranes were resuspended
in 3 mL buffer containing 50 mM Tris HCl (pH 7.5), 10 mM
2-mercaptoethanol, and 30% glycerol and were homogenized using a
Potter homogenizer yielding a concentration of 14 µg/µL protein as
determined by the BioRad protein assay using bovine serum albumin
To evaluate the substrate properties of 6, the apparent affinity
of this lipid for dolichol kinase was compared to that of Dol-
11, as described in the Experimental Section. Dol-11 was
chosen as reference, since it contains the same number of
isoprene units as 6 and is a good substrate for dolichol kinase.13
Although the photoprobe 6 is a substrate for the kinase, it proved
approximately 170 times less stimulatory to the the kinase
activity than Dol-11, indicating that the additional photoacti-
vatable functionality interferes with the phosphorylation of 6.
We therefore assume that the photolabile group is located close
to the active site of the kinase in the enzyme-substrate complex
and that the radiolabeled analogue of 6 (4) is likely to be useful
for studying the active site of this enzyme. In addition, these
results open the possibility to specifically label other Dol or
Dol-P recognizing enzymes with the photoprobes 4 and 5.
(51) Danilov, L. L.; Chojnacki, T. FEBS Lett. 1981, 131, 310-312.
(52) Szkopinska, A.; Nowak, L.; Swiezewska, E.; Palamarczyk, G. Arch.
Biochem. Biophys. 1988, 266, 124-131.
(53) Knauer, R.; Lehle, L. FEBS Lett. 1994, 344, 83-86.
(54) Sherman, F. Methods Enzymol. 1991, 194, 3-21.

10996 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Grassi et al.
(BSA) as a standard. Aliquots of extract were frozen in liquid nitrogen
and stored at -80 °C.
(d), 29.02 (t), 19.43 (q), 18.05 (6q), 12.00 (3d). EI-MS: m/z 285 (0.5,
M+), 243 (100), 213 (13), 201 (63), 187 (24), 171 (24), 157 (15), 145
(19), 131 (67), 119 (15), 115 (13), 103 (40), 101(13), 95 (23), 87 (12),
81 (13), 75 (33), 69 (14), 61 (19), 59 (12). Anal. Calcd for C16H34O2-
Si (286.53): C, 67.07; H, 11.96. Found: C, 66.90; H, 11.90.
Dolichol kinase activity was assayed using a modified procedure of
Heller et al.9 A known amount of 6 (8.4-170 µg) or Dol-11 (0.038-
0.77 µg) dissolved in CHCl3 or CCl4 was placed in a pointed test tube,
and the solvent was evaporated under reduced pressure. Nikkol (10
µL, 4%, BL-8SY, Nikko Chemicals Co LTD, Tokyo) was added and
mixed with the dolichol derivative using a Hamilton syringe followed
by incubation at 37 °C for 30 min and sonification for 5 min. Buffer
was added, and the enzymatic reaction was started by the addition of
300 µg of membrane protein. The final volume was 100 µL, containing
0.05 M TrisHCl (pH 7.5), 0.01 M UTP, 0.03 M CaCl2, 15 µCi [γ-32P]-
CTP (25 Ci/mmol, 0.6 nmol), 0.9 nmol unlabeled CTP (total 15 µm
CTP), and 0.4% nikkol. The mixture was incubated at 24 °C for 20
min, and the reaction was stopped by the addition of 750 µL of KOH
in MeOH (1 M) followed by a 25 min incubation at 37 °C to hydrolyze
alkali-labile lipids (especially phosphatidic acid9). Water (1 mL) was
added, and the lipids were extracted with CHCl3/MeOH (2:1, 3 × 2.3
mL). The combined organic phases were washed with 1 mL of the
upper phase of a mixture containing CHCl3/MeOH/H2O 2.6:1.3:1 and
0.02 M KCl. An aliquot of the organic phase (1 mL) was transferred
into a scintillation vial and evaporated, and the activity (cpm) was
determined. The remainder (4 mL) was evaporated under a stream of
N2, dissolved in 2-propanol/MeOH/H2O 40:60:5 (20 mM H3PO4, 250
µL) filtered through a Teflon filter (Chromafil, Typ O-20/3, Macherey-
Nagel, Germany) and subjected to HPLC analysis.55
High-Pressure Liquid Chromatography (HPLC). HPLC55 was
performed on a Merck system with a radiochromatography detector
(series A-500) from Packard, using a Hypersil ODS 3 column (100 ×
4.6 mm) from Supelco (Buchs, Switzerland). Solvents were degassed
by sonification for 10 min. Dol-P derivatives were eluted with
2-propanol/MeOH/H2O 40:60:5 containing 20 mM H3PO4 for 25 min.
After each run, the column was regenerated with acetonitrile for 10
min. The flow rate was 1 mL/min, and the injection volume was 40-
80 µL. The apparent affinity of 6 for dolichol kinase was compared
to that of Dol-11. Reversed phase HPLC allowed the separation of
the different Dol-P species, and their relative amount was determined
by integration of the peak area. The ratio of Dol-P formed by
phosphorylation of the exogenous/endogenous substrate was determined
by dividing the peak area of the endogenous Dol-P (phosphate of 6 or
Dol-11-P) by the sum of the peak areas of the yeast dolichol phosphates.
In order to obtain a 1:5 ratio of exogenous/endogenous Dol-P, the
concentration of 6 in the kinase assay had to be about 170 times higher
than the one of Dol-11 (505 µM, 3 µM respectively).
Methyl (1′RS,4′S)-2-[1-Hydroxy-4-methyl-6-(triisopropylsilyloxy)-
hexyl]acrylate (19). A mixture of 17 (6.708 g, 23.4 mmol), methyl
acrylate (18, 4.059 mL, 47.1 mmol), and 1,4-diazabicyclooctane (533
mg, 4.75 mmol) was stirred at 25 °C for 37 d. Evaporation of excess
methyl acrylate and flash column chromatography (hexane/EtOAc 10:
1) of the residue gave 19 (5.431 g, 62% yield) as a colorless oil.
TLC: Rf (hexane/EtOAc 10:1) 0.11. IR (CHCl3): 3608w, 3007w,
2945s, 2867s, 1713s, 1653w, 1628w, 1463m, 1441m, 1382w, 1337w,
1158m, 1098s, 1069m, 1014w, 996w, 960w, 883m cm-1 1H NMR
.
(300 MHz, CDCl3): δ 6.23 (d, J ) 1, 1H), 5.80 (m, 1H), 4.38-4.34
(m, 1H), 3.79 (s, OMe), 3.74-3.67 (m, 2H), 2.58-2.52 (m, 1H), 1.71-
1.43 (m, 5H), 1.43-1.23 (m, 2H), 1.14-1.02 (m, 21H), 0.90 (d, J )
6.5, Me). 13C NMR (75 MHz, CDCl3, 1:1.1 mixture): δ 142.5/142.00
(2s), 125.1/125.0 (2t), 72.3/72.1 (2d), 61.6 (t), 51.9 (q), 40.1/39.9 (2t),
33.7 (2t), 33.2/33.1 (2t), 29.4/29.3 (2d), 19.7/19.6 (2q), 18.1 (6q), 12.0
(3d). EI-MS: m/z 341 (5), 329 (6), 181 (10), 149 (84), 131 (43), 125
(15), 121 (100), 119 (42), 115 (34), 107 (12), 103 (23), 95 (19), 93
(40), 87 (17), 83 (19), 81 (13), 79 (17), 77 (12), 75 (38), 73 (12), 69
(15), 61 (25), 59 (27, 55 (17). Anal. Calcd for C20H40O4Si (372.63):
C, 64.47; H, 10.82. Found: C, 64.44; H, 10.83.
Methyl (1′RS,4′S)-2-[1-Acetoxy-4-methyl-6-(triisopropylsilyloxy)-
hexyl]acrylate (20). Pyridine (3.35 mL, 41.5 mmol), acetic anhydride
(2.4 mL, 25.4 mmol), and 4-(dimethylamino)pyridine (33.5 mg, 0.27
mmol) were added at 24 °C to a solution of 19 (3.056 g, 8.2 mmol) in
CH2Cl2 (86 mL). The mixture was stirred for 2 h and then poured
into a saturated aq solution of NH4Cl (20 mL). The organic phase
was washed with CuSO4 (20% in H2O, 2 × 15 mL) and brine (3 × 20
mL). Drying over Na2SO4 and evaporation, followed by flash column
chromatography (hexane/EtOAc 10:1), gave 20 (3.121 g, 92% yield)
as a colorless oil. TLC: Rf (hexane/Et2O 10:1) 0.20. IR (CHCl3):
2945s, 2867s, 1729s, 1632w, 1463m, 1440m, 1371m, 1248s, 1159m,
1100s, 1068m, 1028m, 996m, 959m, 908w, 883m, 657w cm-1 1H
.
NMR (200 MHz, CDCl3): δ 6.28 (br s, 1H), 5.76-5.75 (m, 1H), 5.62-
5.55 (m, 1H), 3.78 (s, OMe), 3.75-3.64 (m, 2H), 2.09 (s, Ac), 1.88-
1.01 (m, 28H), 0.88 (d, J ) 6.2, Me). 13C NMR (75 MHz, CDCl3,
1:1.2 mixture): δ 170.3 (s), 166.1 (s), 140.5/140.4 (2s), 125.5/125.3
(2t), 72.3/72.2 (2d), 61.6/61.6 (2t), 52.1 (q), 40.1/39.9 (2t), 32.6/32.5
(2t), 31.9/31.8 (2t), 29.4/29.3 (2d), 21.1 (q), 19.7/19.5 (2q), 18.1 (6q),
12.0 (3d). FAB-MS (NOBA): m/z 415 (10, M+), 371 (31), 355 (13),
313 (15), 312 (34), 311 (100), 181 (10), 174 (10), 173 (50). Anal.
Calcd for C22H42O5Si (414.67): C, 63.73; H, 10.21. Found: C, 63.85;
H, 10.45.
(3S)-3,7-Dimethyloct-6-en-1-yl Triisopropylsilyl (TIPS) Ether
((S)-Citronellyl Triisopropylsilyl Ether, 16). Triisopropylchlorosilane
(7.06 mL, 33.3 mmol) was added slowly at 0 °C to a solution of (S)-
citronellol (15, 4.956 g, 31.71 mmol) and imidazole (4.32 g, 63.45
mmol) in CH2Cl2 (75 mL). The mixture was allowed to warm to room
temperature (rt), stirred for 1 h, and treated with a saturated aqueous
(aq) NH4Cl solution (20 mL). The organic phase was separated, washed
with H2O (3 × 20 mL) and brine (20 mL), and dried over Na2SO4.
Evaporation gave 16 (10.134 g, quantitative), which was used for the
next reaction without further purification. TLC: Rf (hexane/Et2O 1:1)
0.84. 1H NMR (300 MHz, CDCl3): δ 5.13-5.07 (m, 1H), 3.74-3.67
(m, 2H), 2.03-1.96 (m, 2H), 1.70-0.98 (m, 32H), 0.88 (d, J ) 6.9,
Me).
Methyl (2E,4RS,5Z,9Z,4′S)-4-(Benzenesulfonyl)-11-hydroxy-
6,10-dimethyl-2-[4′-methyl-6′-(triisopropylsilyloxy)hexylidene]undeca-
5,9-dienoate (22). Under Ar atmosphere at -78 °C, n-butyllithium
(1.3 m in hexane, 15.2 mL, 19.8 mmol) was added dropwise to a
solution of 21 (2.325 g, 7.90 mmol) in THF/1,3-dimethyl-3,4,5,6-
tetrahydro-2(1H)-pyrimidinone (49 mL/12 mL), leading to an orange
precipitate. The mixture was warmed to -10 °C, kept at this
temperature for 10 min until phase separation, cooled to -78 °C, and
treated dropwise within 70 min with 20 (3.274 g, 7.90 mmol) in THF
(21 mL). The mixture was stirred and warmed to -30 °C (2 h), leading
to a homogeneous pale yellow solution. The mixture was poured into
saturated aq NH4Cl (60 mL). The aqueous phase was extracted at 0
°C with hexane/Et2O 1:1 (4 × 20 mL). The combined organic layers
were washed with brine (30 mL) and dried over Na2SO4. Flash column
chromatography (hexane/EtOAc 3:1) of the residue (5.85 g) gave 22
(3.544 g, 69% yield) as a colorless oil. TLC: Rf (hexane/Et2O 1:1)
0.08. IR (CHCl3): 3523w, 3007m, 2945s, 2867s, 1772w, 1706s,
1646m, 1586w, 1462s, 1448s, 1438s, 1380m, 1305s, 1146s, 1085s,
(4S)-4-Methyl-6-(triisopropylsilyloxy)hexanal (17). Ozone was
bubbled through a solution of 16 (8.983 g, 28.74 mmol) in CH2Cl2 (74
mL) at -78 °C for 4.5 h. After Me2S (4.8 mL) was added, the solution
was allowed to warm to 25 °C and stirred overnight. Evaporation and
flash column chromatography (hexane/EtOAc 15:1) gave 17 (6.812 g,
83% yield) as a colorless oil. TLC: Rf (hexane/CH2Cl2 2:1) 0.18. IR
(CHCl3): 2944s, 2867s, 2727m, 1722s, 1603w, 1463s, 1410w, 1382m,
1367m, 1248m, 1100s, 1070s, 1013s, 996s, 919m, 883s, 838w, 657s,
998s, 943w, 883s, 836w, 657m, 601m, 549m, 512m cm-1 1H NMR
.
560w, 543w, 530w, 514w, 502w cm-1 1H NMR (200 MHz, CDCl3):
.
(200 MHz, CDCl3): δ 7.89-7.84 (m, 2H), 7.63-7.49 (m, 3H), 6.83
(t, J ) 7.3, 1H), 5.07-4.98 (m, 2H), 4.15 (dt, J ) 3.7, 10.5, 1H), 4.01
(s, 2H), 3.74-3.64 (m, 2H), 3.65 (s, OMe), 2.92 (dd, J ) 3.7, 13.2,
1H), 2.67 (dd, J ) 10.5, 13.2, 1H), 2.25-2.04 (m, 2H), 1.88-0.98
(m, 37H), 0.90 (d, J ) 6.3, Me). 13C NMR (75 MHz, CDCl3, 1:1
mixture): δ 167.8 (s), 146.8 (d), 145.6 (s), 138.1 (s), 135.4 (s), 133.5
δ 9.77 (t, J ) 1.8, 1H), 3.72-3.65 (m, 2H), 2.48-2.40 (m, 2H), 1.80-
1.20 (m, 5H), 1.20-0.96 (m, 21H), 0.91 (d, J ) 6.4, Me). 13C NMR
(75 MHz, CDCl3): δ 203.93 (d), 61.33 (t), 41.73 (t), 39.69 (t), 29.23
(55) Elmberger, P. G.; Eggens, I.; Dallner, G. Biomed. Chromatogr. 1989,
3, 20-28.

A PhotoactiVatable Dolichol Analogue
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10997
(d), 129.1 (2d), 128.8 (2d), 126.8 (d), 126.7 (s), 116.6 (d), 63.2 (d),
61.4 (t), 61.3 (t), 51.9 (q), 40.0/39.9 (2t), 36.0 (t), 32.4 (t), 29.3 (d),
26.6/26.5 (2t), 26.1 (t), 25.8 (t), 23.5 (q), 21.3 (q), 19.4 (q), 18.1 (6q),
12.0 (3d). FAB-MS (NOBA): m/z 650 (18), 649 (38, M+), 507 (16),
491 (14), 490 (41), 489 (97), 475 (15), 463 (17), 457 (16), 454 (12),
431 (13), 413 (16), 379 (12), 283 (20), 273 (12), 267 (10), 257 (10),
256 (17), 255 (69), 241 (13), 229 (12), 227 (16), 215 (17), 213 (14),
201 (17), 199 (17), 191 (15), 189 (17), 187 (31), 185 (20), 183 (11),
179 (11), 177 (11), 175 (15). Anal. Calcd for C36H60O6SSi (649.02):
C, 66.62; H, 9.32; S, 4.94. Found: C, 66.46; H, 9.38; S, 4.71.
23.7 (q), 23.5 (q), 23.4 (q), 21.4 (q), 19.4 (q), 18.1 (6q), 12.0 (3d).
FAB-MS (NOBA): m/z 1057 (11), 927 (6), 926 (10, M+), 768 (17),
767 (39), 766 (70), 642 (17), 626 (19), 625 (50), 624 (100), 621 (11),
609 (17), 591 (15), 501 (16), 475 (11), 255 (23), 201 (21), 199 (11),
187 (11), 175 (24), 173 (16), 171 (10), 165 (12), 161 (15), 159 (27),
157 (34), 154 (26), 151 (14). Anal. Calcd for C52H80O8S2Si
(925.40): C, 67.49; H, 8.71; S, 6.93. Found: C, 67.32; H, 8.83; S,
7.13.
Methyl (2E,4RS,5Z,9Z,12RS,13Z,17Z,4′S)-4,12-Di(benzenesulfo-
nyl)-19-chloro-6,10,14,18-tetramethyl-2-[4′-methyl-6′-(triisopropyl-
silyloxy)hexylidene]nonadeca-5,9,13,17-tetraenoate (25). Mesyl chlo-
ride (0.8 mL, 10.2 mmol) was added dropwise to a solution of 24 (3.476
g, contaminated with sym-collidine, approximately 3.44 mmol), LiCl
(0.432 g, 10.2 mmol) and sym-collidine (2.25 mL, 17 mmol) in DMF
(36 mL) at -3 °C. The mixture was allowed to warm to 3 °C within
75 min, leading to the formation of a colorless precipitate after 15 min.
The mixture was poured into saturated aq NaHCO3 solution (30 mL)
and extracted with hexane/Et2O 1:1 (3 × 20 mL). The combined
organic phases were washed with aq NH4Cl (20 mL) and brine (20
mL) and dried over Na2SO4. Evaporation followed by flash column
chromatography (hexane/EtOAc 3:1) gave 25 (2.963 g, 92% yield) as
a colorless oil. TLC: Rf (hexane/EtOAc 1:1) 0.82. IR (CHCl3): 2944s,
2867s, 1708s, 1646w, 1462m, 1447s, 1379m, 1304s, 1248m, 1145s,
Methyl (2E,4RS,5Z,9Z,4′S)-4-(Benzenesulfonyl)-11-chloro-6,10-
dimethyl-2-[4′-methyl-6′-(triisopropylsilyloxy)hexylidene]undeca-
5,9-dienoate (23). Mesyl chloride (1.6 mL, 20 mmol) was added
dropwise to a solution of 22 (4.440 g, 6.65 mmol), LiCl (0.808 g, 19.1
mmol), and sym-collidine (4.46 g, 36.8 mmol) in N,N-dimethylforma-
mide (DMF, 46 mL) at -3 °C. The mixture was allowed to warm to
3 °C within 1 h leading to the formation of a colorless precipitate after
15 min. The mixture was poured into saturated aq NaHCO3 (30 mL).
The solution was extracted with hexane/Et2O 1:1 (3 × 30 mL), and
the combined organic phases were dried over Na2SO4 and evaporated.
Crude 23 (5.917 g) was used for the next step without further
purification. TLC: Rf (hexane/EtOAc 5:1) 0.16. IR (CHCl3): 2945s,
2867s, 1707s, 1646w, 1462m, 1447s, 1438m, 1380m, 1305s, 1281s,
1146s, 1085s, 1037w, 1014w, 998m, 918w, 883m, 837w, 657w, 604w,
1085s, 1014w, 998w, 883m, 658w, 601m, 548w, 536w cm-1 1H NMR
.
548w, 516w, 502w cm-1
.
1H NMR (200 MHz, CDCl3): δ 7.91-7.88
(200 MHz, CDCl3): δ 7.93-7.82 (m, 4H), 7.73-7.49 (m, 6H), 6.90-
6.78 (m, 1H), 5.23-5.13 (m, 1H), 5.07-4.92 (m, 3H), 4.24-4.10 (m,
1H), 3.97 (s, CH2Cl), 3.92-3.69 (m, 3H), 3.66 (s, OMe), 3.02-2.9
(m, 1H), 2.80-2.58 (m, 2H), 2.50-2.30 (m, 1H), 2.28-2.10 (m, 2H),
1.94-0.98 (m, 46H), 0.92 (d, J ) 6.6, Me). 13C NMR (75 MHz,
CDCl3, mixture): δ 167.7 (s), 146.7 (d), 145.6/145.5 (2s), 144.7 (s),
138.3 (s), 138.1 (s), 133.8 (d), 133.8 (d), 132.4 (s), 130.8/130.7 (2s),
130.1 (d), 129.4 (2d), 129.3 (2d), 129.1 (2d), 129.0 (2d), 128.1 (d),
126.9 (s), 118.3 (d), 116.9 (d), 63.3 (2d), 61.5 (t), 51.8 (q), 43.4 (t),
40.0/39.9 (2t), 36.1 (t), 31.8 (several t), 30.2 (t), 29.4/29.3 (2d), 26.6/
26.5 (2t), 25.9-25.8 (several t), 23.5 (q), 23.4 (q), 23.3 (q), 21.6 (q),
19.4 (q), 18.1 (6q), 12.0 (3d). FAB-MS (NOBA): m/z 946 (3), 945
(7), 944 (7), 943 (11, M+), 803 (16), 802 (16), 801 (29), 662 (19), 661
(46), 660 (48), 659 (100), 657 (12), 627 (14), 501 (17), 255 (10), 211
(11), 157 (12).
(m, 2H), 7.65-7.52 (m, 3H), 6.83 (t, J ) 7.3, 1H), 5.16 (t, J ) 6.8,
1H), 5.03 (d, J ) 10.6, 1H), 4.14 (dt, J ) 3.5, 10.8, 1H), 4.02-3.93
(m, CH2Cl), 3.75-3.67 (m, 2H), 3.66 (s, OMe), 2.94 (dd, J ) 3.4,
13.4, 1H), 2.72 (dd, J ) 10.8, 13.4, 1H), 2.22-2.05 (m, 2H), 1.90-
1.50 (m, 10H), 1.50-0.95 (m, 26H), 0.90 (d, J ) 6.4, Me). 13C NMR
(75 MHz, CDCl3, 1:1 mixture): δ 167.8 (s), 146.8 (d), 145.4 (s), 138.3
(s), 133.8 (d), 132.3 (s), 130.1 (d), 129.3 (2d), 129.1 (2d), 127.0/126.9
(2s), 117.2 (d), 63.4 (d), 61.5 (t), 51.9 (q), 43.5 (t), 40.0/39.9 (2t), 36.1
(t), 31.8 (t), 29.4/29.3 (2d), 26.6/26.5 (2t), 26.0 (t), 25.9 (t), 23.5 (q),
21.5 (q), 19.4 (q), 18.1 (6q), 12,0 (3d). FAB-MS (NOBA): m/z 670
(12), 669 (30), 668 (28), 667 (59, M+), 625 (11), 623 (22), 528 (17),
527 (49), 526 (43), 525 (100), 493 (14), 489 (15), 483 (16), 482 (11),
481 (31), 449 (14), 379 (13), 291 (15), 256 (11), 255 (45), 187 (12),
185 (11), 173 (10), 171 (12), 169 (12), 165 (14), 161 (12), 159 (18),
157 (44), 155 (13), 154 (21), 151 (11).
Methyl (2E,4RS,5Z,9Z,12RS,13Z,17Z,20RS,21Z,25Z,4′S)-4,12,20-
Tri(benzenesulfonyl)-6,10,14,18,22,26-hexamethyl-27-hydroxy-2-[4′-
methyl-6′-(triisopropylsilyloxy)hexylidene]heptacosa-5,9,13,17,21,25-
hexaenoate (26). Under an Ar atmosphere at -78 °C, n-butyllithium
(4.6 mL, 1.6 m in hexane, 7.37 mmol) was added dropwise to a solution
of 21 (943.7 mg, 3.21 mmol) in THF/1,3-dimethyl-3,4,5,6-tetrahydro-
2(1H)-pyrimidinone (37 mL/9 mL), leading to an orange solution, which
was warmed to -20 °C, kept at this temperature for 20 min, cooled to
-78 °C, and treated dropwise with 25 (2.924 g, 3.1 mmol) in THF (21
mL) within 20 min. This mixture was allowed to warm to -40 °C
within 120 min, leading to a yellow solution that was poured into
saturated aq NH4Cl (50 mL). The aqueous phase was extracted at 0
°C with hexane/Et2O 1:1 (3 × 20 mL). The combined organic phases
were washed with brine (20 mL) and dried over Na2SO4. Evaporation
followed by flash column chromatography (hexane/EtOAc 3:1) gave
26 (2.824 g, 76% yield) as a colorless oil. TLC: Rf (hexane/EtOAc
1:1) 0.33. IR (CHCl3): 3536 w, 2944s, 2867m, 1708m , 1660w,
1447m, 1378m, 1304s, 1248m, 1145s, 1085s, 999w, 883w, 658w,
Methyl (2E,4RS,5Z,9Z,12RS,13Z,17Z,4′S)-4,12-Di(benzenesulfo-
nyl)-19-hydroxy-6,10,14,18-tetramethyl-2-[4′-methyl-6′-(triisopro-
pylsilyloxy)hexylidene]nonadeca-5,9,13,17-tetraenoate (24). Under
an Ar atmosphere at -78 °C, n-butyllithium (9.33 mL, 1.6 m in hexane,
14.9 mmol) was added dropwise to a solution of 21 (1.759 g, 5.98
mmol) in THF/1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (69
mL/17 mL), leading to an orange solution. It was warmed to -15 °C,
kept at this temperature for 25 min, cooled to -78 °C, and treated
dropwise with crude 23 (5.917 g) in THF (21 mL) within 30 min. The
solution was allowed to warm to -40 °C within 80 min, leading to a
yellow solution. The mixture was poured into saturated aq NH4Cl (80
mL). The aqueous phase was extracted at 0 °C with hexane/Et2O 1:1
(3 × 30 mL). The organic phases were combined, washed with brine
(30 mL), and dried over Na2SO4. Evaporation followed by flash column
chromatography (hexane/EtOAc 3:1) gave a mixture of 24 and sym-
collidine from the preceding deoxyhalogenation step (3.604 g, 1:1
mixture), containing approximately 3.190 g of 24 (52% yield from 22).
A sample of 24 was purified by dissolving it in CH2Cl2 and washing
twice with 0.1 M HCl at 0 °C. TLC: Rf (hexane/EtOAc 1:1) 0.32. IR
(CHCl3): 3528w, 3008w, 2944s, 2867s, 1706s, 1646w, 1458m, 1447s,
1438m, 1379w, 1304s, 1145s, 1085s, 998m, 909m, 883m, 657w, 598m,
596m, 550w, 506w cm-1 1H NMR (200 MHz, CDCl3): δ 7.91-7.78
.
(m, 6H), 7.70-7.44 (m, 9H), 6.81 (t, J ) 7.4, 1H), 5.10-4.82 (m,
6H), 4.23-4.05 (m, 1H), 4.05-3.95 (m, 2H), 3.90-3.64 (m, 4H), 3.63
(s, OMe), 2.98-2.85 (m, 1H), 2.78-2.52 (m, 3H), 2.50-1.98 (m, 4H),
1.98-1.00 (m, 57H), 0.89 (d, J ) 6.5, Me). 13C NMR (75 MHz,
CDCl3, mixture): δ 167.8 (s), 146.9 (d), 145.7 (s), 145.2 (s), 145.0
(s), 138.3 (s), 138.0 (s), 135.5 (s), 133.8 (several d), 130.7 (several s),
129.4-129.1 (several d), 128.0 (d), 127.1 (d), 126.9 (s), 118.0 (2d),
116.8 (d), 63.3 (several d), 61.5 (2t), 51.80 (q), 39.9 (t), 36.1 (t), 32.3-
32.0 (several t), 30.2 (t), 29.3 (d), 26.1-25.6 (several t), 23.5 (several
q), 21.4 (q), 19.4 (q), 18.1 (6q), 12.0 (3d). FAB-MS (NOBA): m/z
1223.6 (8, [M + Na]+), 1043 (16), 1042 (22), 919 (14), 918 (22), 916
(11), 902 (26), 901 (53), 900 (80), 868 (12), 778 (11), 777 (21), 776
(35), 774 (10), 760 (24), 759 (57), 758 (93), 756 (11), 751 (15), 745
(10), 744 (17), 727 (10), 726 (16), 610 (11), 609 (20), 502 (13), 501
549w, 536w, 528w, 502w cm-1
.
1H NMR (300 MHz, CDCl3): δ
7.88-7.81 (m, 4H), 7.65-7.48 (m, 6H), 6.85-6.78 (t, J ) 7.7, 1H),
5.10-4.88 (m, 4H), 4.20-4.09 (m, 1H), 4.09-3.95 (m, 2H), 3.88-
3.65 (m, 3H), 3.64 (s, OMe), 2.91 (dd, J ) 3.5, 13.3, 1H), 2.74-2.58
(m, 2H), 2.43-2.28 (m, 1H), 2.22-2.04 (m, 2H), 1.90-1.00 (m, 47H),
0.89 (d, J ) 6.5, Me). 13C NMR (75 MHz, CDCl3): δ 167.8 (s), 146.9
(d), 145.7 (s), 145.2 (s), 138.2 (s), 138.0 (s), 135.5 (s), 133.9 (d), 133.8
(d), 130.9/130.7 (2s), 129.4 (2d), 129.3 (2d), 129.1 (2d), 129.0 (2d),
128.0 (d), 127.1 (d), 126.9 (s), 118.0 (d), 116.8 (d), 63.4 (d), 63.3 (d),
61.5 (t), 61.4 (t), 51.9 (q), 40.0/39.9 (2t), 36.1 (t), 32.4 (t), 32.0 (t),
31.8 (t), 30.3 (t), 29.4/29.3 (2d), 26.6/26.5 (2t), 26.0/25.9 (2t), 25.7 (t),

10998 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Grassi et al.
(30), 476 (13), 475 (31), 379 (10), 267 (17), 255 (31), 229 (10), 227
(11), 213 (11), 201 (26), 199 (16), 187 (18), 185 (13), 175 (19), 173
(20), 171 (16), 169 (11), 167 (10), 165 (16), 163 (12), 161 (23), 159
(42), 158 (11), 157 (38), 155 (20), 154 (40), 152 (13), 151 (13). Anal.
Calcd for C68H100O10S3Si (1201.80): C, 67.96; H, 8.39; S, 8.00.
Found: C, 67.79; H, 8.63; S, 7.95.
614 (16), 611 (12), 610 (23), 514 (10), 501 (24), 475 (23), 471 (13),
405 (11), 379 (11), 337 (21), 271 (19), 269 (10), 255 (29), 229 (11),
227 (14), 215 (14), 213 (19), 211 (12), 203 (20), 201 (26), 199 (23),
197 (12), 191 (10), 189 (17), 187 (38), 185 (22), 183 (12), 177 (11),
175 (25), 173 (29), 171 (20), 169 (12), 165 (15), 163 (14), 161 (43),
160 (12). Anal. Calcd for C89H128O11S4Si (1530.42): C, 69.85; H,
8.43; S, 8.38. Found: C, 70.03; H, 8.70; S, 8.26.
Methyl (2E,4RS,5Z,9Z,12RS,13Z,17Z,20RS,21Z,25Z,4′S)-4,12,20-
Tri(benzenesulfonyl)-27-chloro-6,10,14,18,22,26-hexamethyl-2-[4′-
methyl-6′-(triisopropylsilyloxy)hexylidene]heptacosa-5,9,13,17,21,25-
hexaenoate (27). Mesyl chloride (0.42 mL, 5.4 mmol) was added
dropwise to a solution of 26 (2.159 g, 1.80 mmol), LiCl (0.229 g, 5.4
mmol) and sym-collidine (1.2 mL, 9.0 mmol) in DMF (36 mL) at -3
°C. The mixture was stirred at -3 °C for 135 min, leading to the
formation of a colorless precipitate after 15 min. The mixture was
poured into saturated aq. NaHCO3 (20 mL) and extracted with hexane/
Et2O 1:1 (3 × 15 mL). The combined organic phases were dried over
Na2SO4 and evaporated. Purification by flash column chromatography
(hexane/EtOAc 3:1) gave 27 (1.953 g, 89% yield) as a colorless oil.
TLC: Rf (hexane/EtOAc 3:1) 0.16. IR (CHCl3): 2944s, 2867s, 2361w,
2341w, 1772w, 1706m, 1684w, 1670w, 1662w, 1654w, 1647w, 1636w,
1612w, 1586w, 1570w, 1558w, 1540w, 1522w, 1508w, 1498w, 1489w,
1458m, 1447s, 1379m, 1304s, 1145s, 1085s, 1014w, 998w, 883w,
(2E,4RS,5Z,9Z,12RS,13Z,17Z,20RS,21Z,25Z,28RS,29E,33E,4′S)-
4,12,20,28-Tetra(benzenesulfonyl)-6,10,14,18,22,26,30,34,38-nonam-
ethyl-2-[4′-methyl-6′-(triisopropylsilyloxy)hexylidene]nonatriaconta-
5,9,13,17,21,25,29,33,37-nonaen-1-ol (30). Under an Ar atmosphere
at -75 °C, a solution of 29 (1.338 g, 0.907 mmol) in CH2Cl2 (43 mL)
was treated with diisobutylaluminium hydride (3.63 mL, 1 m in hexane)
over 20 min. The solution was allowed to warm to -30 °C within 3
h, diluted with CH2Cl2 (20 mL), and poured into saturated aq NH4Cl
(20 mL). The aqueous phase was extracted with hexane/Et2O 1:1 (3
× 10 mL), and the extracts were washed with brine (30 mL) and
evaporated to give 1.542 g of crude 30. Flash column chromatography
(hexane/EtOAc 4:1) afforded 30 (1.061 g, 78% yield) as a colorless
oil. TLC: Rf (hexane/EtOAc 2:1) 0.11. IR (CHCl3): 2942m, 2866m,
1447m, 1379w, 1303s, 1178w, 1145s, 1084s, 999w, 883w, 595m, 551w,
538m, 514w, 507w cm-1 1H NMR (200 MHz, CDCl3): δ 7.92-7.80
.
843w, 658w, 596m, 550w, 537w, 526w, 517w cm-1 1H NMR (200
.
(m, 8H), 7.72-7.43 (m, 12H), 5.58-5.44 (m, 1H), 5.15-4.83 (m, 9H),
4.22-4.04 (m, 1H), 4.04-3.97 (m, 2H), 3.97-3.62 (m, 5H), 2.93-
2.30 (m, 8H), 2.18-0.97 (m, 79H), 0.89 (d, J ) 6.2, Me). 13C NMR
(75 MHz, CDCl3, mixture): δ 145.5-144.9 (several s), 138.1 (s), 138.0
(s), 136.0 (s), 133.9-131.2 (several d), 130.9-130.7 (several s), 129.4-
127.7 (several d), 124.4 (d), 123.5 (d), 118.0 (several d), 117.3 (d),
67.6 (t), 63.5-63.2 (4d), 61.7 (t), 40.2 (t), 40.1 (t), 39.8 (t), 37.1 (t),
32.0-31.9 (several t), 29.9 (several t), 29.4 (d), 27.1-26.2 (several t),
25.8 (q), 25.4 (t), 23.5-23.3 (several q), 19.6 (q), 18.1 (6q), 17.8 (q),
16.2 (q), 16.0 (q), 12.0 (3d). FAB-MS (NOBA): m/z 1343 (12, [M -
TIPS]+), 1342 (15), 1202 (24), 1201 (42), 1200 (519), 1061 (19), 1060
(23), 1059 (76), 1057 (100), 1056 (11), 917 (30), 915 (35), 884 (42),
742 (14), 705 (14), 255 (17), 201 (10), 187 (16), 173 (12), 161 (18),
159 (24).
MHz, CDCl3): δ 7.93-7.78 (m, 6H), 7.70-7.43 (m, 9H), 6.82 (t, J )
7.5, 1H), 5.20-5.08 (m,1H), 5.03-4.83 (m, 5H), 4.15 (dt, J ) 3.7,
10.8, 1H), 3.93 (s, CH2Cl), 3.90-3.65 (m, 4H), 3.64 (s, OMe), 2.93
(dd, J ) 3.7, 10.8, 1H), 2.79-2.55 (m, 3H), 2.53-2.03 (m, 4H), 1.98-
1.00 (m, 56H), 0.89 (d, J ) 6.2, Me). 13C NMR (75 MHz, CDCl3,
mixture): δ 167.5 (s), 146.5 (d), 145.4 (s), 144.6 (s), 144.4 (s), 138.1
(s), 137.9 (s), 133.6 (several d), 132.2 (s), 130.7 (s), 130.5 (s), 130.3
(s), 129.9-127.8 (several d), 126.7 (s), 118.1 (d), 117.9 (d), 116.7 (d),
63.2-63.1 (3d), 61.4 (t), 51.7 (q), 43.4 (t), 40.0 (t), 39.9 (t), 36.0 (t),
31.9-30.1 (several t), 29.4 (d), 26.5-25.6 (several t), 23.5-23.3
(several q), 21.6 (q), 19.4 (q), 18.1 (6q), 12.0 (3d). FAB-MS
(NOBA): m/z 1241.5 (9, [M + Na]+, 1078 (12), 939 (19), 938 (40),
937 (44), 936 (68), 904 (10), 797 (25), 796 (54), 795 (56), 794 (100),
792 (10), 761 (16), 752 (10), 751 (18), 610 (13), 609 (23), 502 (12),
501 (30), 475 (22), 303 (13), 255 (16), 237 (20), 213 (10), 211 (15),
201 (11).
(2E,5Z,9Z,13Z,17Z,21Z,25Z,29E,33E,4′S)-6,10,14,18,22,26,30,34,-
38-nonamethyl-2-[4′-methyl-6′-(triisopropylsilyloxy)hexylidene]-
nonatriaconta-5,9,13,17,21,25,29,33,37-nonaen-1-ol (31). Under an
Ar atmosphere, 30 (509.7 mg, 0.339 mmol) in THF (8.8 mL) was added
to a mixture of [PdCl2(dppp)] (61 mg, 0.1 mmol) in THF (2.5 mL).
The mixture was cooled to 0 °C, and a solution of LiEt3BH (3.4 mL,
1 M in THF, 3.4 mmol) in THF was added dropwise over 3.5 h. The
mixture was stirred for additional 2.5 h at 0 °C and diluted with Et2O
(35 mL). The color of the mixture changed from orange (during the
addition of LiEt3BH) to black. The mixture was washed with NaCN
(10 mL, 1 M in H2O) and brine (10 mL); the organic phase was dried
over Na2SO4 and evaporated. Flash chromatography (hexane/EtOAc
40:1) afforded 31 (163.9 mg, 51% yield) as a colorless oil. TLC: Rf
(hexane/EtOAc 10:1) 0.13. IR (CHCl3): 3599w, 2930s, 2865s, 2729w,
1662w, 1602w, 1456s, 1378m, 1317w, 1099m, 1070m, 996m, 918w,
Methyl (2E,4RS,5Z,9Z,12RS,13Z,17Z,20RS,21Z,25Z,28RS,29E,-
33E,4′S)-4,12,20,28-Tetra(benzenesulfonyl)-6,10,14,18,22,26,30,34,-
38-nonamethyl-2-[4′-methyl-6′-(triisopropylsilyloxy)hexylidene]-
nonatriaconta-5,9,13,17,21,25,29,33,37-nonaenoate (29). Under an
Ar atmosphere at -78 °C, n-butyl lithium (1.75 mL, 1.5 m in hexane,
2.63 mmol) was added dropwise to a solution of 28 (720.8 mg, 2.08
mmol) in THF/1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (25
mL/6 mL). The resulting orange solution was warmed to -20 °C,
kept at this temperature for 25 min, cooled to -78 °C, and treated
dropwise with 27 (1.953 g, 1.6 mmol) in THF (14 mL). The mixture
was allowed to warm to -30 °C within 130 min, leading to a yellow
solution that was poured into saturated aq. NH4Cl (30 mL). The
aqueous phase was extracted at 0 °C with hexane/Et2O 1:1 (3 × 10
mL). The combined organic phases were washed with brine (20 mL)
and dried over Na2SO4. Evaporation followed by flash column
chromatography (hexane/EtOAc 3:1) gave 29 (1.612 g, 66% yield) as
a colorless oil. TLC: Rf (hexane/EtOAc 2:1) 0.19. IR (CHCl3):
3038w, 2943m, 2866m, 1706m, 1661w, 1602w, 1447m, 1379w, 1304s,
884m, 841w, 657w, 570w, 529w, 520w, 514w, 5005m cm-1 1H NMR
.
(200 MHz, CDCl3): δ 5.44 (t, J ) 7.3, 1H), 5.22-5.08 (m, 9H), 4.05
(d, J ) 5.8, 2H), 3.78-3.68 (m, 2H), 2.19-1.92 (m, 38H), 1.71 (s,
7Me), 1.62 (s, 3Me), 1.52-1.00 (m, 27H), 0.92 (d, J ) 6.6, Me). 13C
NMR (75 MHz, CDCl3): δ 138.8 (s), 135.8-135.2 (several s), 131.5
(s), 127.9 (d), 125.3-124.4 (several d), 67.5 (t), 61.7 (t), 40.2-39.8
(several t), 37.3 (t), 32.3-32.1 (several t), 29.4 (d), 28.5 (t), 26.9-
26.5 (several t), 25.8 (q), 25.1 (t), 23.5 (several q), 19.7 (q), 18.1 (6q),
17.7 (q), 16.0 (several q), 12.1 (3d). Anal. Calcd for C64H112O2Si
(941.68): C, 81.63; H, 11.99. Found: C, 81.46; H, 11.91.
(2E,5Z,9Z,13Z,17Z,21Z,25Z,29E,33E,4′S)-4-(1-Azi-2,2,2-trifluoro-
ethyl)-1-{[6,10,14,18,22,26,30,34,38-nonamethyl-2-[4′-methyl-6′-(tri-
isopropylsilyloxy)hexylidene]nonatriaconta-5,9,13,17,21,25,29,33,37-
nonaen-1-oxy]methyl}-2-iodobenzene (34). Aqueous NaOH (0.25 mL
50%) was added to a solution of 31 (40 mg, 42.5 µmol), 32 (50.9 mg,
112.6 µmol), and tetrabutylammonium iodide (12 mg, 32 µmol) in
benzene (0.25 mL). The emulsion was stirred in the dark at 25 °C for
23 h, treated with benzene (0.2 mL), and stirred for 49 h at 25 °C. Ice
water (3mL) and hexane (2 mL) were added, and the aqueous phase
was extracted with hexane/Et2O 10:1 (2 × 2 mL). The combined
organic phases were dried over Na2SO4 and evaporated to give 76 mg
1178w, 1145s, 1085s, 999w, 884w, 595m, 552w, 539w cm-1 1H NMR
.
(300 MHz, CDCl3): δ 7.91-7.78 (m, 8H), 7.70-7.47 (m, 12H), 6.87-
6.79 (m, 1H), 5.12-4.85 (m, 9H), 4.16 (dt, J ) 3.7, 10.7, 1H), 3.91-
3.77 (m, 3H), 3.77-3.67 (m, 2H), 3.65 (s, OMe), 2.99-2.89 (m, 1H),
2.80-2.55 (m, 4H), 2.52-2.32 (m, 3H), 2.29-1.00 (m, 78H), 0.91 (d,
J ) 6.3, Me). 13C NMR (75 MHz, CDCl3, mixture): δ 167.7 (s), 146.7
(d), 145.4-144.8 (several s), 138.1 (several s), 133.8 (several d), 131.6-
130.6 (several s), 129.4-127.6 (several d), 126.9 (s), 124.3 (d), 123.4
(d), 118.0 (d), 117.3 (d), 116.8 (d), 63.5-63.2 (4d), 61.8 (t), 51.8 (q),
39.7 (several t), 36.1 (t), 31.9 (several t), 30.0 (several t), 29.3 (d),
26.7-25.7 (several t), 23.3 (several q), 19.4 (q), 18.1 (6q), 17.7 (q),
16.2 (q), 16.0 (q), 12.0 (3d). FAB-MS (NOBA): m/z 1552.4 (14, [M
+ Na]+), 1248 (11), 1247 (28), 1246 (37), 1107 (12), 1106 (32), 1105
(1), 1104 (100), 1102 (17), 1072 (12), 964 (27), 963 (63), 961 (91),
960 (14), 929 (14), 886 (13), 752 (13), 752 (24), 744 (11), 656 (10),

A PhotoactiVatable Dolichol Analogue
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10999
of crude extract. Flash column chromatography gave 32 (elution with
hexane), 34 (49.5 mg, elution with hexane/Et2O 10:1) containing an
unknown contamination, and 31 (6.2 mg, 6.6 µmol, elution with hexane/
Et2O 1:1). TLC: Rf (hexane/Et2O 10:1) 0.74. 1H NMR (200 MHz,
CDCl3): δ 7.63-7.47 (m, 2H), 7.32-7.20 (m, 1H), 5.49 (t, J ) 7.2,
1H), 5.20-5.05 (m, 9H), 4.63 (s, 2H, contamination), 4.41 (s, CH2-
Ar), 4.02 (s, CH2OBn), 3.79-3.66 (m, 2H), 2.19-1.90 (m, 38H), 1.70
(s, 7Me), 1.62 (s, 3Me), 1.50-0.99 (m, 26H), 0.92 (d, J ) 6.7, Me).
(3S,6E,10Z,14Z,18Z,22Z,26Z,30Z,34E,38E,42E)-7-{[4-(1-Azi-2,2,2-
trifluoroethyl)-2-iodophenyl]methoxymethyl}-3,11,15,19,23,27,31,-
35,39,43-decamethyltetratetraconta-6,10,14,18,22,26,30,34,38,42-
decaen-1-ol (6). Tetrabutylammonium fluoride trihydrate (24 mg, 68
µmol) was added in the dark to a solution of slightly impure 34 (49.5
mg) in THF (0.8 mL) at rt. The mixture changed from colorless to
black. After 4.5 h, ice water (2 mL) and hexane (3 mL) were added.
The aqueous phase was extracted with hexane/Et2O 10:1 (3 × 3 mL),
and the combined organic phases were washed with brine (3 mL), dried
over Na2SO4, and evaporated. Flash column chromatography (hexane/
EtOAc 40:1) afforded pure 6 (26.6 mg) in 56% yield from 31. TLC:
Rf (hexane/Et2O 1:1) 0.47. IR (CHCl3): 3005m, 2963s, 2930s, 2856m,
2090w, 1720w, 1451m, 1378m, 1348w, 1322w, 1260m, 1162s, 1112s,
Cl) was removed by filtration and washed with absolute toluene. The
filtrate was evaporated to give crude phosphate 7 (16 mg) which was
dissolved in chloroform/methanol (2:1, 30 mL) and applied to a DEAE
sepharose column (11.5 × 1.2 cm). Increasing concentrations of
ammonium acetate in CHCl3/MeOH 2:1 were used for elution (30 mL
0.01 M, 30 mL 0.02 M, 30 mL 0.04 M). The fractions containing 7
(monitored by TLC, CHCl3/MeOH/H2O 65:25:4) were combined and
evaporated to give 8.8 mg of a mixture of 7 and ammonium acetate.
The mixture was dissolved in CHCl3/MeOH 2:1 (0.5 mL) and
ammonium acetate was removed by gel filtration (21.5 × 1 cm, CHCl3/
MeOH 2:1). The progress of the filtration was followed by TLC
(CHCl3/MeOH/H2O 65:25:4); fractions containing 7 were combined
and evaporated to give 7 (6.7 mg, 68% yield) as a colorless solid.
TLC: Rf (CHCl3/MeOH/H2O 65:25:4) 0.58. IR (CHCl3): 2961s, 2929s,
2855s, 1617w, 1585w, 1452m, 1377m, 1340w, 1262m, 1161s, 1097s,
1050s, 1030s, 1012s, 952m. cm-1 1H NMR (500 MHz, CDCl3): δ
.
7.54 (s, 1H), 7.47-7.46 (m, 1H), 7.30-7.19 (m, 1H), 5.45-5.40 (m,
1H), 5.13-5.08 (m, 9H), 4.37 (s, 2H), 4.00-3.90 (m, 4H), 2.61 (s br,
8H, NH4), 2.24-1.94 (m, 38H), 1.67-1.65 (m, 7 Me), 1.60 (s, Me),
1.59 (s, 2 Me), 1.50-1.12 (m, 5H), 0.89 (d, J ) 6.5, Me), two additional
signals were observed.3.88 (s br, 1H), 3.22 (s br, 1H). 13C NMR (125
MHz, CDCl3): δ 143.1 (s), 136.8 (d), 135.5-134.9 (several s), 131.2
(s), 130.0 (d), 129.7 (s), 128.4 (d), 126.4 (d), 125.0-124.2 (several d),
97.2 (s), 75.4 (t), 75.0 (t), 39.8 (several t), 37.2 (t), 32.2-32.0 (several
t), 29.7 (t), 29.7 (d), 28.6 (t), 26.8-26.4 (several t), 25.7 (s), 25.0 (t),
23.4 (several q), 19.1 (q), 17.7 (q), 16.0 (q), (multiplets of CF3, CN2,
CH2OP, missing). 31P NMR (121MHz, CDCl3): 1.25. ESI-MS: m/z
(MeOH) 1188 (M-).
1034w, 999w cm-1 UV (EtOH): λmax 355 nm (ꢀ 314). 1H NMR
.
(500 MHz, CDCl3): δ 7.56 (d, J ) 1.85, 1H), 7.48 (d, J ) 8.1, 1H),
7.22 (d, J ) 8.1, 1H), 5.46 (t, J ) 7.2, 1H), 5.14-5.08 (m, 9H), 4.39
(s, CH2Ar), 4.00 (s, CH2OBn), 3.71-3.65 (m, 2H), 2.16-1.96 (m, 38H),
1.68 (s, 7Me), 1.62-1.60 (m, 10H), 1.47-1.36 (m, 2H), 1.27-1.20
(m, 2H), 1.15-1.13 (m, 1H), 0.92 (d, J ) 6.6, Me). 13C NMR (125
MHz, CDCl3): δ 143.1 (s), 136.8 (d), 135.6-135.0 (several s), 131.3
(s), 130.0 (d), 129.7 (s), 128.5 (d), 126.5 (d), 125.0-124.2 (several d),
4-(1-Azi-2,2,2-trifluoroethyl)-2-iodo-1-(iodomethyl)benzene (32).
Methyltriphenoxyphosphonium iodide (132 mg, 302 µmol) was added
to a solution of 2-iodo-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl
alcohol (33, 35.6 mg, 109 µmol) in acetonitrile (0.2 mL). After 23 h
in the dark, the solution was diluted with Et2O (5mL) and extracted
with aqueous NaOH (1 m, 2 mL). The organic phase was washed
with water and dried over MgSO4. Flash column chromatography
(hexane/Et2O 3:1) gave 32 (39.9 mg, 84% yield) as a yellowish solid.
TLC: Rf (hexane/Et2O 1:1) 0.77. IR (CHCl3): 1616w, 1600w, 1547w,
1
121.9 (q, JCF ) 275), 97.2 (s), 75.3 (t), 74.9 (t), 61.1 (t), 39.9-39.7
(several t), 37.1 (t), 32.3-32.0 (several t), 29.3 (d), 28.6 (t), 27.6 (q,
2JCF ) 41), 26.8-26.4 (several t), 25.7 (q), 25.1 (t), 23.5-23.4 (several
q), 19.5 (q), 17.7 (q), 16.0 (q). FAB-MS (NOBA): m/z 1131.5 (11,
[M + Na]+), 768 (11), 189 (12). Anal. Calcd for C64H96F3IN2O2
(1109.54): C, 69.29; H, 8.72; I, 11.44; N, 2.53. Found: C, 69.20; H,
8.93; I, 11.25; N, 2.23.
Diammonium (3S,6E,10Z,14Z,18Z,22Z,26Z,30Z,34E,38E,42E)-7-
{[4-(1-Azi-2,2,2-trifluoroethyl)-2-iodophenyl]methoxymethyl}-3,11,-
15,19,23,27,31,35,39,43-decamethyltetratetraconta-6,10,14,18,22,-
26,30,34,38,42-decaen-1-yl Phosphate (7). Under an Ar atmosphere,
triethylamine (184 µL, absolute) was added dropwise to a solution of
POCl3 (123 µL) in hexane (6.15 mL). The solution was stirred for 15
min at 25 °C, while a colorless precipitate formed. An aliquot (0.65
mL) of this mixture was transferred into another flask, where 6 (8.9
mg, 8.02 µmol) in hexane (0.41 mL, 15 min) was added under an Ar
atmosphere. The mixture was stirred for 20 min at 25 °C and poured
into a mixture of acetone, H2O, and NEt3 (88:10:2, 6 mL). After 20 h,
the solution was concentrated, forming a milky liquid. The evaporation
was repeated after addition of 2-propanol (2 mL). The residue was
treated with toluene (3 mL) and evaporated. Evaporation with toluene
was repeated (2 × 3 mL). The residue was dissolved in dry chloroform
(1 mL) and treated with toluene (3 mL), leading to the precipitation of
a colorless solid. The suspension was concentrated to a volume of 3
mL and was left at -20 °C for 1 h. The crystalline precipitate (NH4-
1491w, 1339m, 1160s, 1035w, 953w, 830w cm-1 1H NMR (200 MHz,
.
CDCl3): δ 7.55-7.46 (m, 2H), 7.20-7.16 (m, 1H), 4.52 (s, 2H). 13C
NMR (50 MHz): δ 143.6 (s), 138.0 (d), 130.3 (s), 129.7 (d), 127.1
(d), 99.7 (s), 10.2 (t), (multiplets of CF3 and CN2 missing). FAB-MS
(NOBA): m/z 557 (12), 543 (11), 499 (11), 478 (11), 463 (20), 462
(19), 448 (11), 447 (31), 425 (11), 424 (15), 413 (12), 393 (11), 392
(28), 391 (100), 389 (12), 372 (11), 371 (32), 369 (12), 355 (10), 342
(15), 341 (13), 339 (12), 329 (11), 327 (18), 325 (92 [M]+), 315 (10),
313 (16), 311 (12), 309 (12), 308 (13), 307 (37).
Acknowledgment. This work was made possible by a grant
from the Swiss Federal Institute of Technology, Zu¨rich. We
thank Dr. S. Vonhoff and Dr. B. Bernet for stimulating
discussions and R. K. Mu¨ller, F. Hoffmann-La Roche AG,
Basel, for the generous gift of (E,E)-farnesol and (S)-citronellol.
JA9721677