Synthesis and structure of phosphatidylinositol dimannoside

Article abstract of DOI:10.1021/jo0625599

(Chemical Equation Presented) (R)-Tuberculostearic acid (2) was synthesized in seven steps from (S)-citronellol (5). The carbon chain of 2 was assembled by copper-catalyzed cross coupling of (5)-citronellol tosylate (6) and hexylmagnesium bromide; subsequent ozonolysis and reaction with 6-benzyloxyhexylmagnesium bromide furnished alcohol 10. Functional group manipulation afforded (R)-2 in 49% overall yield from 5. DCC coupling of (R)-2 with 3-O-benzyl-1-O-palmitoyl-sn-glycerol (16), followed by hydrogenolytic removal of the benzyl group and treatment with benzyl bis(diisopropyl) phosphoramidite, afforded phosphoramidite 20. Tetrazole-mediated coupling of 20 with PIM1 head group 21 gave 22, and subsequent debenzylation afforded phosphatidylinositol mono-mannoside, PIM1 (23). Similarly, coupling of 20 and 24 and removal of the benzyl protecting groups gave PIM2 (1c). Both 23 and 1c have a clearly defined acylation pattern, which was confirmed by mass spectrometry, with sn-1 palmitoyl and sn-2 tuberculostearoyl groups on the glycerol moiety. Both 23 and 1c were shown to modulate the release of the pro-inflammatory cytokine, IL-12, in a dendritic cell assay.

Full text of DOI:10.1021/jo0625599

Synthesis and Structure of Phosphatidylinositol Dimannoside
Blake S. Dyer,† Jeremy D. Jones,† Gary D. Ainge,†,‡ Michel Denis,§ David S. Larsen,*,† and
Gavin F. Painter‡
UniVersity of Otago, P.O. Box 56, Dunedin, New Zealand, Carbohydrate Chemistry Team, Industrial
Research Limited, P.O. Box 31-310, Lower Hutt, New Zealand, and AgResearch Limited, Hopkirk
Research Institute, Grasslands Research Centre, PriVate Bag 11008, Palmerston North, New Zealand
dlarsen@alkali.otago.ac.nz
ReceiVed December 13, 2006
(R)-Tuberculostearic acid (2) was synthesized in seven steps from (S)-citronellol (5). The carbon chain
of 2 was assembled by copper-catalyzed cross coupling of (S)-citronellol tosylate (6) and hexylmagnesium
bromide; subsequent ozonolysis and reaction with 6-benzyloxyhexylmagnesium bromide furnished alcohol
10. Functional group manipulation afforded (R)-2 in 49% overall yield from 5. DCC coupling of (R)-2
with 3-O-benzyl-1-O-palmitoyl-sn-glycerol (16), followed by hydrogenolytic removal of the benzyl group
and treatment with benzyl bis(diisopropyl)phosphoramidite, afforded phosphoramidite 20. Tetrazole-
mediated coupling of 20 with PIM1 head group 21 gave 22, and subsequent debenzylation afforded
phosphatidylinositol mono-mannoside, PIM1 (23). Similarly, coupling of 20 and 24 and removal of the
benzyl protecting groups gave PIM2 (1c). Both 23 and 1c have a clearly defined acylation pattern, which
was confirmed by mass spectrometry, with sn-1 palmitoyl and sn-2 tuberculostearoyl groups on the glycerol
moiety. Both 23 and 1c were shown to modulate the release of the pro-inflammatory cytokine, IL-12, in
a dendritic cell assay.
Introduction
ongoing program investigating the potential of PIMs for
modulating immune responses we recently reported the syn-
theses of diacylated phosphatidylinositol mannosides, PIM1,
PIM2, and PIM4.6,7 Mixtures of PIMs isolated from Mycobac-
terium boVis and synthetic PIM1 and PIM2 1a, which contain
the stearoyl acyl residue, were shown to suppress the recruitment
of eosinophils in a mouse model of allergic asthma.6,8 Synthetic
PIM2 1b and PIM4, containing palmitoyl residues, were shown
Phosphatidylinositol mannosides (PIMs), present in the cell
walls of mycobacteria, are attracting a great deal of attention
as they elicit a range of immune responses and constitute the
biosynthetic precursors of lipomannan and lipoarabinomannan.
PIMs act as agonists of Toll-like receptor 2 (TLR2), which is
involved with innate immunity.1,2 They are also known to recruit
natural killer T-cells1,3 and cause T-cell expansion via binding
to the lipid presentation molecule, CD1b.1,4,5 As part of an
(4) Ernst, W. A.; Maher, J.; Cho, S.; Niazi, K. R.; Chatterjee, D.; Moody,
D. B.; Besra, G. S.; Watanabe, Y.; Jensen, P. E.; Porcelli, S. A.; Kronenberg,
M.; Modlin, R. L. Immunity 1998, 8, 331-340.
† University of Otago.
‡ Industrial Research Limited.
(5) Sieling, P. A.; Chatterjee, D.; Porcelli, S. A.; Prigozy, T. I.;
Mazzaccaro, R. J.; Soriano, T.; Bloom, B. R.; Brenner, M. B.; Kronenberg,
M.; Brennan, P. J. Science 1995, 269, 227-230.
§ AgResearch Limited.
(1) Gilleron, M.; Ronet, C.; Mempel, M.; Monsarrat, B.; Gachelin, G.;
Puzo, G. J. Biol. Chem. 2001, 276, 34896-34904.
(2) Gilleron, M.; Quesniaux, V. F. J.; Puzo, G. J. Biol. Chem. 2003,
278, 29880-29889.
(3) Fischer, K.; Scotet, E.; Niemeyer, M.; Koebernick, H.; Zerrahn, J.;
Maillet, S.; Hurwitz, R.; Kursar, M.; Bonneville, M.; Kaufmann, S. H. E.;
Schaible, U. E. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10685-10690.
(6) Ainge, G. D.; Hudson, J.; Larsen, D. S.; Painter, G. F.; Gill, G. S.;
Harper, J. L. Bioorg. Med. Chem. 2006, 14, 5632-5642.
(7) Ainge, G. D.; Parlane, N. A.; Denis, M.; Hayman, C. M.; Larsen, D.
S.; Painter, G. F. Bioorg. Med. Chem. 2006, 14, 7615-7624.
(8) Sayers, I.; Severn, W.; Scanga, C. B.; Hudson, J.; le Gros, G.; Harper,
J. L. J. Allergy Clin. Immunol. 2004, 114, 302-309.
10.1021/jo0625599 CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/27/2007
3282
J. Org. Chem. 2007, 72, 3282-3288

Synthesis Phosphatidylinositol Dimannoside
to be effective in inducing IFN-γ production when given as an
adjuvant with a model antigen in an in vivo system.7 Structure-
activity relationships of PIMs are complicated by the fact that
these molecules exist in nature in multiacylated forms, and
obtaining single entities from cell wall material is challenging.
Although structures of the pseudo-oligosaccharide moieties of
PIMs have been well established,9-11 those of the diacyl-glyceryl
component are still unclear. Gilleron et al.1 carried out a
comprehensive study of this unit of PIMs from Mycobacterium
boVis Bacilluss Calmette Gue´rin using negative-ion ESI-MS/
MS mass spectrometry to elucidate the acylation pattern. The
specific location of these acyl groups was deduced on the basis
of work by Hsu and Turk,12 who showed that loss of a fatty
acid at the sn-2 position occurs more readily than that at the
sn-1 position of deprotonated molecular ions of phosphatidyli-
nositols. Gilleron et al.1 concluded from their mass spectral data
that PIM2 existed as a 62:38 mixture of two compounds with
differing acylation patterns. The major form 1c contained a
palmitate residue at the sn-1 position of the glycerol unit and
an (R)-tuberculostearate (TBSA) residue at the sn-2 position.
The minor form 1b possessed palmitate residues at both of these
sites. The glyceryl moieties of tri- and tetra-acylated PIM2
species were shown to have palmitate at the sn-1 and tubercu-
lostearate at the sn-2 positions only. In a later paper, Gilleron
et al.2 depicted a structure of the hexa-mannoside, AcPIM6. In
this case they showed the tuberculostearate residue at the sn-1
and palmitate at the sn-2 positions of the glycerol unit.
Liu et al.13 recently reported the total synthesis of “native”
AcPIM2 and AcPIM6 based upon the later structural assignment
of Gilleron et al.2 They synthesized (R)-tuberculostearic acid
(TBSA, 2) from the Roche ester 3 and prepared differentially
acylated diacylglycerol 4. Glycerol 4 was coupled to protected
PIM head groups to furnish AcPIM2 1d and AcPIM6. In this
paper, we report an alternative synthesis of (R)-TBSA (2) from
(S)-citronellol (5), the regioisomer of 4 and PIM2 1c, where
the acylation pattern is consistent with the 2001 findings of
Gilleron et al.1 We aim to gain insight into the structures of
these natural products.
Discussion
Several syntheses of racemic and two of enantiopure (R)-
tuberculostearic acid (2) have been reported to date.13-17 The
elegant synthesis of (R)-2, by Liu et al.,13 relied on building up
the carbon chain from a derivative of Roche ester 3 by copper-
catalyzed cross-coupling reactions. Our approach is somewhat
similar and builds upon the work of Djerassi and co-workers,18
who reported the synthesis of 10-methylhexadecanoic acid from
citronellol. In our synthesis (Scheme 1) commercially available
(S)-citronellol (5) was tosylated to give 6,19 which was then
coupled with hexylmagnesium bromide catalyzed by dilithium
copper tetrachloride to give hydrocarbon 7 (Scheme 1). Due to
difficulties in separating 7 from dodecane produced from the
Grignard homocoupling reaction the mixture was used as is for
subsequent reaction. Ozonolysis of the alkene furnished alde-
hyde 8 in 88% from tosylate 6. Attempts to extend the carbon
chain of 8 using Wittig chemistry proved low yielding, and cross
coupling with Grignard reagent 9 after reduction and subsequent
tosylation of 8 failed. Thus, aldehyde 8 was directly reacted
with Grignard reagent 9 to give the secondary alcohol 10 as a
mixture of epimers in 78% yield. Tosylation of 10 gave 11,
and subsequent elimination on alumina furnished a mixture of
alkenes 12. Simultaneous removal of the alkene and benzyl
group was achieved by treatment with hydrogen to give the
known alcohol 1313 in 58% yield from 10. Oxidation of 13 with
acidified potassium permanganate under phase-transfer condi-
tions afforded (R)-tuberculostearic acid (2) in 98% yield.
With tuberculostearic acid in hand our attention turned to
synthesis of the diacylated glycerol derivative. We felt that the
most efficient method to prepare a protected diacyl glycerol
derivative was via nucleophilic opening of benzyl glycidol by
a carboxylate salt.20 This was achieved from (R)-benzyl glycidol
(14), which is readily available by hydrokinetic resolution of
(rac)-1421,22 using Jacobsen’s methodology.23 The resolution of
14, using catalyst (S,S)-15 (Schaus et al.23 reported that the
(14) Wallace, P. A.; Minnikin, D. E.; McCrudden, K.; Pizzarello, A.
Chem. Phys. Lipids 1994, 71, 145-162.
(15) Prout, F. S.; Cason, J.; Ingersoll, A. W. J. Am. Chem. Soc. 1947,
69, 1233.
(16) Schmidt, G. A.; Shirley, D. A. J. Am. Chem. Soc. 1949, 71, 3804-
3864.
(9) Severn, W. B.; Furneaux, R. H.; Falshaw, R.; Atkinson, P. H.
Carbohydr. Res. 1998, 308, 397-408.
(10) Hill, D. L.; Ballou, C. E. J. Biol. Chem. 1966, 241, 895-902.
(11) Brennan, P.; Ballou, C. E. J. Biol. Chem. 1967, 242, 3046-3056.
(12) Hsu, F. F.; Turk, J. J. Am. Soc. Mass Spectrom. 2000, 11, 986-
999.
(17) Linstead, R. P.; Lunt, J. C.; Weedon, B. C. L. J. Chem. Soc. 1950,
3331-3333.
(18) Raederstorff, D.; Shu, A. Y. L.; Thompson, J. E.; Djerassi, C. J.
Org. Chem. 1987, 52, 2337-2346.
(19) Santangelo, E. M.; Correa, A. G.; Zarbin, P. H. G. Tetrahedron
Lett. 2006, 47, 5135-5137.
(13) Liu, X.; Stocker, B. L.; Seeberger, P. H. J. Am. Chem. Soc. 2006,
128, 3638-3648.
(20) Lindberg, J.; Svensson, S. C. T.; Pahlsson, P.; Konradsson, P.
Tetrahedron 2002, 58, 5109-5117.
J. Org. Chem, Vol. 72, No. 9, 2007 3283

Dyer et al.
SCHEME 1. Synthesis of (R)-Tuberculostearic acid (2)
SCHEME 2. Synthesis of Phosphoramidite 20
hydrokinetic resolution of 14 with (R,R)-15 gave (R)-benzyl
glycidol), afforded (R)-14, which had an optical rotation
consistent with that reported by Mikkilineni et al.24 and also
that of a commercial sample (Aldrich). Reaction of (R)-14 with
palmitic acid and tetraethylammonium bromide in a melt25
yielded, after careful column chromatography, mono-acyl-
glycerol 16 in 64% yield. The ee of 16 was estimated as greater
than 95% from the 1H and 19F NMR spectra of the correspond-
ing (R)-MTP-ester 17. Carbodimide coupling of 16 and TBSA
(R)-2 gave the corresponding differentially acylated diacyl
glycerol 18 in 97% yield. Removal of the benzyl protecting
group was achieved without significant acyl migration under
the hydrogenolytic conditions as reported for debenzylation of
the corresponding regioisomer by Liu et al.13 to afford 19 in
93% yield.
The next step in the synthesis was the tetrazole-mediated
coupling of phosphoramidite 20 with PIM1 head group 216
(Scheme 3). After an in situ oxidation, the reaction furnished
protected PIM1 22 as a mixture of isomers about the phosphorus
atom in a 51% yield. Hydrogenolysis of the benzyl groups of
22 gave PIM1 (23) in 86% yield as a white amorphous solid
after lyopholization. Of note in the 1H NMR spectrum recorded
in a mixture of CDCl3, CD3OD and D2O was a multiplet at δ
5.29-5.34 due to sn-2 proton and a broad one-proton singlet
at δ 5.14 attributed to the anomeric proton, H-1′.
SCHEME 3. Synthesis of PIM1 (23)
PIM2 (1c) was synthesized in a similar manner (Scheme 4).
Tetrazole-mediated coupling of phosphoramidite 20 with PIM2
head group 247 gave, after an in situ oxidation, protected PIM2
25 as a mixture of isomers about the phosphorus atom in a 48%
yield. Hydrogenolytic removal of the benzyl groups of 22 gave
PIM2 (1c) in 86% yield as a white amorphous solid after
(21) Brugat, N.; Duran, J.; Polo, A.; Real, J.; Alvarez-Larena, A.; Piniella,
J. F. Tetrahedron: Asymmetry 2002, 13, 569-577.
(22) Borah, J. C.; Gogoi, S.; Boruwa, J.; Kalita, B.; Barua, N. C.
Tetrahedron Lett. 2004, 45, 3689-3691.
1
lyopholization. The H NMR spectrum recorded in a mixture
of CDCl3, CD3OD, and D2O showed a multiplet at δ 5.22-
5.33 due to sn-2 proton and two broad one-proton singlets at δ
5.10 and 5.13 attributed to the two anomeric protons, H-1′ and
H-1′′.
The negative-ion ESI MS/MS CID spectrum of 1c showed
two fragments at m/z 877.4 and 920.4. The former was the more
(23) Schaus, S. E.; Brandes, B. D.; Larrow, J. F.; Tokunaga, M.; Hansen,
K. B.; Gould, A. E.; Furrow, M. E.; Jacobsen, E. N. J. Am. Chem. Soc.
2002, 124, 1307-1315.
(24) Mikkilineni, A. B.; Kumar, P.; Abushanab, E. J. Org. Chem. 1988,
53, 6005-6009.
(25) Lok, C. M. Chem. Phys. Lipids 1978, 22, 323-337.
3284 J. Org. Chem., Vol. 72, No. 9, 2007

Synthesis Phosphatidylinositol Dimannoside
SCHEME 4. Synthesis of PIM2 (1c)
significant peak that arose by elimination of the tuberculostearate
(C19) residue from the sn-2 position of the glyceryl moiety of
the molecular ion. The latter less significant peak was due to
loss of the sn-1 palmitate (C16) residue. This result is consistent
with the acylation pattern of 1c and the findings of Hsu and
Turk,12 who have shown that the acid on the sn-2 position of a
glyceryl phosphate is more readily lost. Very recently, Gilleron
et al. revisited their structural characterization of natural PIMs.26
In this work they have used negative-ion ES-IRMPD and
MALDI-TOF/TOF CID mass spectrometry on tetra-acylated
PIM2 (Ac2PIM2) from Mycobacterium boVis Bacilluss Calmette
Gue´rin to establish the acylation pattern. In this study they
proposed that the C19 fatty acid is located on the sn-1 and the
C16 acid at is on the sn-2 position of the glycerol contrary to
their earlier assignment. The basis for this new assignment was
the presence of a peak at m/z 433.3 that was assigned to fragment
26 consistent with loss of the C16 fatty acid from the sn-2
position. In the current study, prominent in the ESI MS/MS
CID spectrum of 1c is a peak at m/z 391.2268 attributed to ion
27, clearly establishing the presence of the C16 fatty acid at the
sn-1 position. A much smaller peak at m/z 433.2733 was
attributed to 28. A similar pattern of peaks due to ions 27 and
28 were apparent in a negative-ion MALDI-TOF/TOF CID
spectrum of PIM1 (23).
FIGURE 1. Bone marrow cells from BALB/c mice were cultured in
the presence of 100 U of granulocyte-macrophage stimulating factor
(GM-CSF) and interleukin-4 (IL-4) for 10 days. Cells (95% DCs) were
stimulated with 50 µg of indicated products or diluent (control). Levels
of interleukin-12 (IL-12) released by DCs were measured by a
commercial ELISA kit. Columns identified by different letters are
significantly different from each other (ANOVA test). Each data point
represents results from four mice cell cultures. Bars indicate standard
errors of the means.
recently been published.28 The sn-2 palmitate inserts into the
hydrophobic F′ pocket, making an optimal fit, whereas the sn-1
palmitate only partially fills the larger hydrophobic A′ pocket.
In the current work, if binding of CD1d to PIM is important, it
would follow that for tight binding the larger tuberculostearic
acid would need to insert into the larger A′ pocket, and this
would only occur if it was attached to the sn-1 position of the
glycerol moiety.
In summary, we developed simple syntheses of (R)-tubercu-
lostearic acid (2) from commercially available (S)-citronellol
and differentially acylated glycerol 19 incorporating 2 from (R)-
benzyl glycidol. The latter was then utilized in the total syntheses
of the PIM1 (23) and PIM2 (1c), both of which possess a defined
acylation pattern. Our MS/MS studies on 1c and 23 unambigu-
ously confirm the findings of Turk12 and Gilleron26 that loss of
the sn-2 fatty acid residue is more facile than that of the sn-1
fatty acid. The fatty acid composition and distribution on the
glycerol moiety of natural PIM molecules can now be validated
using this method. Although PIM1 (23) and PIM2 (1c), both
of which possess an ‘unnatural’ acylation pattern of the glycerol
moiety, were immunologically active in a dendritic cell assay
which measured the release of the Th1 cytokine IL-12; they
were significantly less active than PIM2 (1b). We plan to
synthesize a library of PIM variants, including those with
tuberculostearoyl and palmitoyl residues at the sn-1 and sn-2
positions of the glyceryl unit, to establish a structure-activity
relationship. Careful study of these newly synthesized variants
may provide some clues in the mechanisms of immunomodu-
lation of PIMs.
Given the potential of PIMs to modulate immune responsive-
ness,1,2 we elected to test the obtained products to ascertain if
they could modulate the release of pro-inflammatory cytokines.
To that aim, dendritic cells (DCs) from mice were exposed to
50 µg of synthesized compounds and the release of the key
Th1 cytokine interleukin-12 (IL-12)27 was assessed (Figure 1).
It was found that PIM2 (1b),7 possessing palmitoyl residues at
the sn-1 and sn-2 positions, was significantly more active than
PIM1 (23) and PIM2 (1c) at enhancing IL-12, suggesting that
the modified products, although immunologically active, are less
potent than 1b at stimulating DCs. This result confirms that
subtle changes in the composition of the acyl residues in the
PIM structure affect the bioactivity of these compounds, at least
in this in vitro system. The crystal structure of the lipid antigen
presenting molecule, CD1d, with a PIM2 (1b) ligand has
Experimental Section
(R)-2,6-Dimethyl-tetradec-2-ene (7). LiCl (0.525 g, 12.4 mmol)
and CuCl2 (0.915 g, 6.81 mmol) were stirred in THF (100 mL)
under a N2 atmosphere for 5 min; then tosylate 619 (9.72 g, 31.3
mmol) was added. The solution was cooled to 0 °C, and freshly
(26) Gilleron, M.; Lindner, B.; Puzo, G. Anal. Chem., 2006, 78, 8543-
8548.
(27) Trinchieri, G. Nat. ReV. Immunol. 2003, 3, 133-146.
(28) Zajonc, D. M.; Ainge, G. D.; Painter, G. F.; Severn, W. B.; Wilson,
I. A. J. Immunol. 2006, 177, 4577-4583.
J. Org. Chem, Vol. 72, No. 9, 2007 3285

Dyer et al.
prepared hexylmagnesium bromide, from 1-bromohexane (17.8 g,
108 mmol) and magnesium (3.59 g, 148 mmol) in THF (150 mL),
was added dropwise to the mixture, which was stirred at 0 °C for
a further 1 h. The solution was then stirred 24 h at room
temperature. Saturated NH4Cl solution (200 mL) was added, and
the mixture was filtered though Celite, diluted with ether (400 mL),
washed with water (150 mL), brine (150 mL), and dried (MgSO4).
Removal of the solvent and purification of the residue by silica
gel column chromatography (hexanes as eluent) gave an inseparable
mixture of the title compound 7 and dodecane as a colorless oil
29.2, 29.4, 29.70, 29.74, 29.8, 30.0, 30.1, 31.68, 31.72, 31.8, 31.9,
32.0, 32.58, 32.61, 34.1, 34.2, 36.8, 36.9, 70.4, 73.0, 85.0, 127.6,
127.7, 127.8, 128.4, 129.6, 129.7, 134.9, 138.7, 144.4. Anal. Calcd
for C33H52O4S: C, 72.75; H, 9.62; S, 5.89. Found: C, 72.49; H,
9.55; S, 5.55.
(R)-1-Benzyloxy-10-methyloctadec-6- and -7-ene (12). Diethyl
ether (50 mL) was added to freshly activated neutral alumina (28
g), and the mixture was cooled to room temperature. Tosylate 11
(2.10 g, 3.83 mmol) in ether (25 mL) was added, and the resultant
slurry was stirred for 17 h under nitrogen. The mixture was filtered,
and the alumina was washed with diethyl ether (100 mL).
Concentration of the combined filtrates and purification of the
residue by silica gel chromatography (light petroleum/Et2O ) 9:1)
gave the title compounds 12 (1.08 g, 75%) as a colorless oil; υmax
(film), 2923, 2854, 1455, 1112 cm-1; 1H NMR (500 MHz, CDCl3)
δ 0.82-0.90 (m, 6H), 1.05-1.19 (m, 2H), 1.20-1.44 (m, 20H),
1.58-1.66 (m, 2H), 1.94-2.07 (m, 2H), 3.47 (dt, J ) 1.0, 7.0 Hz,
2H), 5.31-5.39 (m, 2H), 7.26-7.36 (m, 5H); 13C NMR (125 MHz,
CDCl3) δ 14.2, 19.57, 19.64, 19.66, 19.69, 22.8, 24.9, 25.8, 26.0,
26.1, 26.16, 26.18, 27.09, 27.12, 27.17, 27.23, 27.3, 27.4, 29.0,
29.3, 29.4, 29.5, 29.6, 29.7, 29.75, 29.82, 30.0, 30.1, 30.2, 32.0,
32.3, 32.48, 32.6, 32.7, 33.3, 33.51, 34.6, 36.7, 36.8, 37.0, 37.1,
40.2, 70.4, 70.5, 70.6, 72.92, 72.94, 127.5, 127.7, 128.4, 128.6,
129.0, 129.5, 130.0, 130.3, 130.6, 130.8, 131.5, 138.76, 138.78.
Anal. Calcd for C26H43O: C, 84.03; H, 11.66. Found: C, 84.05;
H, 11.89.
1
(9.76 g). H NMR (500 MHz, CDCl3) δ inter alia 0.87-0.91 (m,
6H), 1.08-1.20 (m, 2H), 1.30-1.46 (m, 17H), 1.62 (s, 3H), 1.70
(s, 3H), 1.89-2.08 (m, 2H), 5.10-5.14 (m, 1H); 13C NMR (125
MHz, CDCl3) δ 14.2, 17.7, 19.7, 22.8, 25.7, 25.8, 27.2, 29.5, 29.8,
30.2, 32.1, 32.6, 37.1, 37.3, 125.2, 131.0. Anal. Calcd for C16H32:
C, 85.63; H, 14.37. Found: C, 85.66; H, 14.16.
(R)-4-Methyl-dodecanal (8). Ozone was bubbled though a
solution of 7 and dodecane (9.76 g, 31.3 mmol) from the previous
reaction in CH2Cl2 (400 mL) at -78 °C until the solution turned
blue. Oxygen was bubbled through the solution to remove excess
ozone; then dimethyl sulfide (4.6 mL, 63 mmol) was added. The
stirred solution was warmed to room temperature over 2 h, then
diluted with dichloromethane (200 mL), and washed with water
(300 mL) and brine (300 mL). The solution was dried (MgSO4),
and the solvent was removed. Purification of the residue by silica
gel column chromatography (light petroleum to light petroleum/
CH2Cl2 ) 3:1) gave the title compound 8 (5.85 g, 88% from 6) as
a colorless oil. [R]D18 +1.1 (c ) 0.44, CH2Cl2); υmax (film), 2925,
2855, 2713, 1728 cm-1; 1H NMR (500 MHz, CDCl3) δ 0.86-0.90
(m, 6H), 1.08-1.16 (m, 2H), 1.21-1.34 (m, 12H), 1.39-1.48 (m,
2H), 1.62-1.65 (m, 1H), 2.36-2.48 (m, 2H), 9.77 (t, J ) 2 Hz,
1H); 13C NMR (125 MHz, CDCl3) δ 14.2, 19.4, 22.7, 27.0, 29.0,
29.4, 29.7, 30.0, 32.0, 32.5, 36.7, 41.8, 203.1. Anal. Calcd for
C13H26O: C, 78.72; H, 13.21. Found: C, 78.57; H, 13.12.
(R)-(-)-10-Methyl-octadecan-1-ol (13). A mixture of 10%
palladium on carbon (0.305 g), 12 (2.46 g, 6.37 mmol), and hexanes
(100 mL) was stirred under an atmosphere of hydrogen for 72 h.
The reaction mixture was filtered though Celite, and the solvent
was removed. Purification of the residue by silica gel chromatog-
raphy (light petroleum/Et2O ) 9:1) gave the title compound 13
(1.84 g, 96%) as a colorless oil. [R]D29 -0.03 (c ) 2.75, CH2Cl2)
{Lit.13 [R]D -0.02 (c ) 8.0, CHCl3)}; υmax (film), 3336, 2922, 1459,
1370, 1057 cm-1; 1H NMR (500 MHz, CDCl3) δ 0.83 (d, J ) 6.6
Hz, 3H), 0.88 (t, J ) 6.6 Hz, 3H), 1.02-1.14 (m, 1H), 1.18-1.44
(m, 29H), 1.50-1.65 (2H, m), 3.64 (t, J ) 6.6 Hz, 2H); 13C NMR
(125 MHz, CDCl3) δ 14.2, 19.8, 22.8, 24.7, 27.1, 27.2, 29.1, 29.3,
29.4, 29.5, 26.8, 30.0, 30.1, 32.0, 32.8, 34.1, 37.1, 37.2, 63.3. Anal.
Calcd for C19H40O: C, 80.21; H, 14.17. Found: C, 80.56; H, 14.06.
(R)-10-Methyloctadecanoic acid [(R)-(2)]. Alcohol 13 (0.255
g, 0.898 mmol), tetrabutylammonium bromide (0.157 g, 0.487
mmol), and potassium permanganate (0.443 g, 2.80 mmol) were
refluxed in a mixture of CH2Cl2 (50 mL), acetic acid (2 mL), and
water (30 mL) for 4 h. The solution was cooled, and 1 M HCl (50
mL) was added. Sodium sulfite (0.5 g) was added, and the aqueous
phase was extracted with CH2Cl2 (50 mL). The organic extracts
were dried (MgSO4), and the solvent was removed. Purification of
the residue by silica gel column chromatography (light petroleum/
Et2O ) 4:1) gave the title compound (R)-2 (0.263 g, 98%) as a
(7R*,10R)-1-Benzyloxy-10-methyloctadecan-7-ol (10). A solu-
tion of aldehyde 8 (2.38 g, 11.2 mmol) in THF (20 mL) was added
to 6-benzyloxylhexylmagnesium bromide (9), prepared from 6-ben-
zyloxy-1-bromohexane (7.0 g, 26 mmol) and magnesium turnings
in THF (60 mL), dropwise at room temperature. The reaction was
stirred at room temperature for 16 h; then saturated NH4Cl solution
(100 mL) was added. The mixture was diluted with CH2Cl2 (100
mL), washed with 1 M HCl (50 mL), and dried (MgSO4). Removal
of the solvent and purification of the residue by silica gel column
chromatography (light petroleum/Et2O ) 4:1) gave the title
compound 10 (3.291 g, 75%, mixture of diastereoisomers) as a
1
colorless oil. υmax (film), 3364, 2926, 2854, 1454 cm-1; H NMR
(500 MHz, CDCl3) δ 0.86 (dd, J ) 3.3, 6.6 Hz, 3H), 0.89 (d, J )
7.0 Hz, 3H), 1.06-1.14 (m, 2H), 1.19-1.52 (m, 26H), 1.58-1.66
(m, 2H), 3.47 (t, J ) 6.6 Hz, 2H), 3.52-3.58 (m, 1H), 4.50 (s,
2H) 7.25-7.35 (m, 5H); 13C NMR (125 MHz, CDCl3) δ 14.2, 19.7,
19.8, 22.8, 25.6, 25.7, 26.3, 27.1, 27.2, 29.4, 29.6, 29.7, 29.8, 30.1,
32.0, 32.9, 32.9, 33.0, 35.0, 35.1, 37.0, 37.2, 37.4, 37.5, 70.5, 72.4,
72.5, 72.9, 127.5, 127.7, 128.4, 138.8. Anal. Calcd for C26H46O2:
C, 79.94; H, 11.87. Found: C, 79.67; H, 11.89.
30
colorless oil. [R]D -0.03 (c ) 4.75, CHCl3) {Lit.13 [R]D -0.02
(c ) 10.5, CHCl3)}; υmax (film), 3016, 2923, 1705, 1459, 1057
1
cm-1; H NMR (500 MHz, CDCl3) δ 0.83 (d, J ) 6.7 Hz, 3H),
0.88 (t, J ) 6.9 Hz, 3H), 1.05-1.10 (m, 1H), 1.18-1.39 (m, 29H),
1.60-1.66 (2H, m), 2.35 (t, J ) 7.5 Hz, 2H); 13C NMR (125 MHz,
CDCl3) δ 14.2, 19.8, 22.8, 24.8, 27.1, 27.2, 29.1, 29.3, 29.4, 29.5,
26.8, 30.0, 30.1, 32.0, 32.8, 34.1, 37.1, 37.2, 180.2. Anal. Calcd
for C19H38O2: C, 76.45, H, 12.83. Found: C, 76.09; H, 12.93.
(R)-Benzyl Glycidyl Ether (-)-(14). A mixture of (S,S)-15 (76.0
mg, 126 µmol), (()-benzyl glycidyl ether (14) (4.15 g, 25.3 mmol),
AcOH (28 µL, 0.49 mmol), and THF (0.25 mL) was cooled to 0
°C; then H2O (240 µL, 13.3 mmol) was added with stirring. The
mixture was warmed to rt, and after 16 h, (R)-benzyl glycidyl ether
(-)-(14) (2.05 g, 49%) was isolated by vacuum distillation (bpt,
120 °C 0.7 mm/Hg) followed by further purification by silica gel
column chromatography (light petroleum/Et2O ) 9:1); [R]D20 -5.7
(c ) 5, C6H5CH3); Aldrich: (-)-Benzyl (R)-glycidyl ether, cat.
(7R*,10R)-1-Benzyloxy-10-methyl-7-tosyloxyoctadecane (11).
Tosyl chloride (2.3 g, 13 mmol) was added to a solution of 10
(3.29 g, 8.43 mmol) in a 1:1 mixture of pyridine and CH2Cl2 (20
mL) at 0 °C. The mixture was maintained at 0 °C for 3 days. The
reaction mixture was concentrated in vacuo, diluted with CH2Cl2
(100 mL), washed with 1 M HCl (50 mL), and dried (MgSO4).
Removal of the solvent and purification of the residue by silica
gel column chromatography (light petroleum/CH2Cl2 ) 1:1, then
light petroleum/EtOAc ) 4:1) gave the title compound 11 (4.30 g,
93%) as a colorless oil; υmax (film), 2926, 1454, 1599, 1454 cm-1
;
1H NMR (500 MHz, CDCl3) δ 0.76 (d, J ) 6.6 Hz, 3H), 0.89 (d,
J ) 7.0 Hz, 3H), 0.95-1.24 (m, 2H), 1.10-1.35 (m, 27H), 1.50-
1.61 (m, 2H), 2.42 (s, 3H), 3.44 (t, J ) 6.6 Hz, 2H), 4.50 (s, 2H),
4.50-4.56 (m, 1H), 7.26-7.35 (m, 7H), 7.76-7.80 (m 2H); 13C
NMR (125 MHz, CDCl3) δ 14.2, 19.4, 19.5, 21.7, 22.8, 24.7, 24.8,
20
1
No. 363529 [R]D -5.4 (c ) 5, C6H5CH3); H NMR (500 MHz,
CDCl3) δ 2.60-2.64 (m, 1H); 2.80 (1H, t, J ) 4.6 Hz); 3.07-3.12
3286 J. Org. Chem., Vol. 72, No. 9, 2007

Synthesis Phosphatidylinositol Dimannoside
1
(m, 1H); 3.44 (dd, J ) 6.2, 5.3 Hz, 1H); 3.77 (dd, J ) 8.4, 3.0 Hz,
1H); 4.59 (dt, J ) 15.8, 11.7 Hz, 2H), 7.26-7.40 (m, 5H); 13C
NMR (125 MHz, CDCl3) δ 44.3, 50.9, 70.8, 73.3, 127.8, 128.4,
137.9.
-2.6 (c ) 0.40, CHCl3); H NMR (500 MHz, CDCl3) δ 0.83 (d,
J ) 6.5 Hz, 3H), 0.88 (t, J ) 7.0 Hz, 6H), 1.10-1.13 (m, 1H),
1.17-1.40 (m, 48H), 1.57-1.66 (m, 4H), 2.30-2.36 (m, 6H),
3.69-3.76 (m, 2H), 4.23 (dd, J ) 6.0, 12.1 Hz, 1H), 4.32 (dd, J )
5.5, 11.8 Hz, 1H), 5.06-5.11 (m, 1H); 13C NMR (125 MHz, CDCl3)
δ 14.2, 19.8, 22.8, 24.9, 25.0, 27.1, 29.2, 29.3, 29.36, 29.43, 29.5,
29.6, 29.67, 29.72, 29.8, 30.0, 30.1, 32.0, 32.8, 34.2, 34.4, 37.2,
61.6, 62.1, 72.2, 173.5, 173.9; HRMS-ESI: [M + Na]+ calcd for
C38H74O5Na, 633.5434; found, 633.5462. Anal. Calcd for C38H74O5:
C, 74.70; H, 12.21. Found: C, 74.87; H, 11.99.
3-O-Benzyl-1-O-palmitoyl-sn-glycerol29 (16). A melt of (R)-
benzyl glycidol (R)-14 (1.06 g, 6.46 mmol), palmitic acid (1.68,
6.53 mmol), and tetraethylammonium bromide (30 mg, 0.14 mmol)
at 100 °C was stirred for 3 h. The reaction mixture was purified by
silica gel chromatography (toluene to toluene/EtOAc ) 95:5) to
15
give the title compound 16 (1.75 g, 64%) as a white wax. [R]D
+0.6 (c ) 3.00, CH2Cl2); υmax (film), 3494, 2917, 2852, 1715 cm-1
;
Benzyl (2-O-(R)-10-Methyloctadecanoyl-1-O-hexadecanoyl-
sn-glycero)-diisopropylphosphoramidite (20). 1H-Tetrazole (15.0
mg, 0.214 mmol) was added to a stirred solution of alcohol 19
(100 mg, 0.164 mmol) and benzyl bis(diisopropyl)phosphoramidite
(124 mg, 0.367 mmol) in dry CH2Cl2 (7 mL) cooled to 0 °C under
an argon atmosphere. After stirring at rt for 1.5 h, the solvent was
removed in vacuo and the residue purified by silica gel column
chromatography (Et3N/EtOAc/light petroleum ) 3:10:90), affording
1H NMR (500 MHz, CDCl3) δ 0.88 (t, J ) 6.9 Hz, 3H), 1.20-
1.34 (m, 24H), 1.57-1.67 (m, 2H), 2.32 (t, J ) 7.5 Hz, 2H), 2.51
(bs, 1H), 3.49 (dd, J ) 6.2, 9.6 Hz, 1H), 3.56 (dd, J ) 4.4, 9.6 Hz,
1H), 4.00-4.06 (m, 1H), 4.14 (dd, J ) 6.1, 11.5 Hz, 1H), 4.19
(dd, J ) 4.4, 11.5 Hz, 1H), 4.56 (s, 2H), 7.27-7.38 (m, 5H); 13C
NMR (125 MHz, CDCl3) δ 14.1, 22.7, 24.9, 29.1, 29.3, 29.4, 29.5,
29.6, 29.65, 29.69, 31.9, 34.1, 65.3, 68.9, 70.9, 73.5, 127.7, 127.8,
128.4, 137.7, 173.9.
20
the title compound 20 (128 mg, 0.151 mmol, 92%) as an oil. [R]D
5.4 (c ) 1.7, CHCl3); H NMR (300 MHz, CDCl3) δ 0.81-0.91
1
3-O-Benzyl-2-O-((R)-R,R,R-methoxytrifluoromethylphenyl-
acetyl)-1-O-palmitoyl-sn-glycerol (17). A mixture of 16 (20 mg,
48 µmol), DCC (20 mg, 97 µmol), (R)-MTPA (26 mg, 111 µmol),
and DMAP (7.0 mg, 57 µmol) was dissolved in CH2Cl2 (5 mL) at
room temperature and stirred overnight. The reaction mixture was
filtered through Celite and concentrated. The residue was purified
by silica chromatography (light petroleum/Et2O ) 9:1) to give
(m, 9H), 1.02-1.38 (m, 63H), 1.52-1.62 (m, 4H), 2.21-2.31 (m,
4H), 3.58-3.80 (m, 4H), 4.12-4.19 (m, 1H), 4.30-4.38 (m, 1H),
4.61-4.77 (m, 2H), 5.17-5.24 (m, 1H), 7.19-7.37 (m, 5H); 13C
NMR (75 MHz) δ 14.2, 19.8, 22.7, 24.6, 24.7, 24.8, 25.0, 27.1,
29.2, 29.4, 29.5, 29.6, 29.7, 30.1, 32.0, 32.8, 34.2, 34.4, 37.2, 43.1,
43.3, 61.5, 61.7, 61.9, 62.6, 65.3, 65.6, 70.9, 127.0, 127.3, 128.3,
139.4, 173.4, 173.4; 31P NMR (121.5 MHz, CDCl3) δ 150.0, 150.2;
HRMS-ESI: [M + H]+ calcd for C51H95O6NP, 848.6897; found,
848.6885.
26
MTPA-ester 17 (27 mg, 89%) as a white wax. [R]D 31.0 (c )
1.40, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 0.88 (t, J ) 7.0 Hz,
3H), 1.20-1.40 (m, 24H), 1.50-1.60 (m, 2H), 2.21 (dt, J ) 2.4,
5.2 Hz, 2H), 3.54 (d, J ) 1.0 Hz, 3H), 3.69 (d, J ) 5.5 Hz, 2H),
4.14 (dd, J ) 6.8, 12.3 Hz, 1H), 4.37 (dd, J ) 3.53, 12.0 Hz, 1H),
4.55 (dd, J ) 11.8, 17.9 Hz, 2H), 5.50-5.54 (m, 1H), 7.28-7.40
(m, 8H), 7.56 (d, J ) 7.5 Hz, 2H); 13C NMR (125 MHz, CDCl3)
δ 14.2, 22.8, 24.8, 29.2, 29.3, 29.4, 29.5, 29.67, 29.71, 29.8, 32.0,
34.0, 55.5, 62.3, 68.3, 72.7, 73.4, 127.5, 127.7, 127.9, 128.4, 128.5,
129.6, 132.2, 137.4, 166.1, 173.3; 19F NMR (470 MHz, CDCl3) δ
-72.5. HRMS-ESI: [M + Na]+ calcd for C36H51F3O6Na, 659.3535;
found, 659.3530.
3,4,5,6-Tetra-O-benzyl-2-O-(2,3,4,6-tetra-O-benzyl-R-D-man-
nopyranosyl-1-O-(2-O-(R)-10-methyloctadecanoyl-1-O-hexade-
canoyl-sn-glycero-3-benzylphosphoryl)-D-myo-inositol (22). 1H-
Tetrazole (16 mg, 0.23 mmol) was added to a stirred solution of
3,4,5,6-tri-O-benzyl-2-O-(2,3,4,6-tetra-O-benzyl-R-D-mannopyra-
nosyl)-D-myo-inositol (21) (94 mg, 0.088 mmol) and phosphora-
midite 20 (149 mg, 0.176 mmol) in dry CH2Cl2 (6 mL) cooled to
0 °C under an argon atmosphere. After stirring at rt for 1.5 h, the
reaction mixture was cooled to -40 °C and a solution of m-CPBA
(50%, 61 mg, 0.18 mmol) in CH2Cl2 (4 mL) was added dropwise
to the reaction mixture. After warming to rt over 2 h the reaction
was quenched with addition of 10% aqueous Na2SO3 solution (50
mL) and the combined mixture extracted with Et2O (100 mL). The
ethereal extract was washed with saturated NaHCO3 solution (3 ×
50 mL) and dried (MgSO4). After filtration the solvent was removed
in vacuo and the residue purified by silica gel column chromatog-
raphy (EtOAc/light petroleum ) 1:9 to 1:4), followed by a second
column (CH2Cl2 to EtOAc/CH2Cl2 ) 1:25), giving the title
compound 21 (83 mg, 51%) as an oil. 1H NMR (500 MHz, CDCl3)
δ 0.83 (d, J ) 6.3 Hz, 3H), 0.89 (t, J ) 6.9 Hz, 6H), 1.03-1.11
(m, 2H), 1.15-1.35 (m, 49H), 1.45-1.59 (m, 4H), 2.17-2.25 (m,
4H), 3.21-3.55 (m, 4H) 3.66-4.34 (m, 12H), 4.44-5.07 (m, 18H),
3-O-Benzyl-2-O-((R)-10-methyloctadecanoyl)-1-O-palmitoyl-
sn-glycerol (18). A mixture of 16 (344 mg, 817 µmol), DCC (343
mg, 1.66 mmol), (R)-2 (268 mg, 898 µmol), and DMAP (5.9 mg,
48 µmol) was dissolved in CH2Cl2 (15 mL) at rt and stirred
overnight. The reaction mixture was filtered through Celite and
concentrated. The residue was purified by silica gel column
chromatography (light petroleum/Et2O ) 19:1) to give the title
compound 18 (556 mg, 97%) as a white wax. [R]D20 5.2 (c ) 3.00,
CHCl3); 1H NMR: (500 MHz, CDCl3) δ 0.83 (d, J ) 6.4 Hz, 3H),
0.88 (t, J ) 6.9 Hz, 6H), 1.02-1.12 (m, 1H), 1.19-1.40 (m, 48H),
1.55-1.65 (m, 4H), 2.28 (t, J ) 7.6 Hz, 3H), 2.32 (t, J ) 7.6 Hz,
3H), 3.56-3.62 (m, 2H), 4.19 (dd, J ) 6.4, 12.1 Hz, 1H), 4.34
(dd, J ) 3.7, 12.0 Hz, 1H), 4.52 (d, J ) 12.1 Hz, 1H), 4.56 (d, J
) 12.1 Hz, 1H), 5.22-5.27 (m, 1H), 7.26-7.36 (m, 5H); 13C NMR
(125 MHz, CDCl3) δ 14.2, 19.8, 22.8, 24.9, 25.0, 27.1, 27.2, 29.18,
29.20, 29.36, 29.39, 29.43, 29.44, 29.56, 29.61, 29.70, 29.73, 29.74,
29.8, 30.0, 30.1, 32.0, 32.8, 34.2, 34.4, 37.2, 62.7, 68.3, 70.1, 73.4,
127.7, 127.8, 128.5, 137.8, 173.2, 173.5. HRMS-ESI: [M + Na]+
calcd for C45H80O5Na, 723.5903; found: 723.5931. Anal. Calcd
for C45H80O5: C, 77.09; H, 11.50. Found: C, 76.72; H, 11.29.
2-O-((R)-10-methyloctadecanoyl)-1-O-palmitoyl-sn-glycerol (19).
A mixture of glycerol 18 (200 mg, 285 µmol) and 10% Pd/C (30
mg, 28 µmol) in ethanol (15 mL) and AcOH (0.15 mL) was stirred
at room temperature for 1.5 h under an atmosphere of H2. The
mixture was filtered through Celite, and the solvent was removed.
Purification of the residue by silica gel column chromatography
(CH2Cl2/light petroleum 1:1 to CH2Cl2 to CH2Cl2/EtOAc ) 19:1)
5.07-5.13 (m, 1H), 5.34-5.38 (m, 1H), 7.14-7.44 (m, 45H); 13
C
NMR (125 MHz, CDCl3) δ 13.8, 14.2, 19.8, 22.8, 24.8, 24.85,
24.89, 27.2, 29.1, 29.17, 29.21, 29.38, 29.40, 29.42, 29.43, 29.58,
29.64, 29.7, 29.8, 30.09, 30.12, 31.3, 32.0, 32.9, 34.0, 34.07, 34.11,
37.2, 38.2, 59.6, 61.58, 61.60, 65.6, 65.78, 65.82, 66.0, 67.5, 68.7,
68.8, 69.4, 69.5, 69.70, 69.74, 69.8, 71.8, 71.9, 72.1, 72.15, 72.19,
72.24, 73.4, 73.7, 74.0, 74.4, 74.5, 74.6, 74.7, 75.1, 75.4, 75.5, 75.7,
75.8, 76.2, 77.3, 78.5, 78.7, 79.0, 79.1, 79.3, 79.34, 79.6, 79.7, 80.7,
80.8, 82.98, 83.01, 98.9, 127.3, 127.40, 127.42, 127.48, 127.51,
127.6, 127.66, 127.70, 127.77, 127.83, 127.9, 128.0, 128.06, 128.07,
128.07, 128.11, 128.15, 128.23, 128.26, 128.31, 128.33, 128.38,
128.42, 128.44, 128.49, 128.53, 128.57, 128.63, 128.68, 128.72,
128.8, 130.96, 130.97, 131.2, 135.5, 135.6, 138.0, 138.1, 138.31,
138.34, 138.39, 138.43, 138.43, 138.45, 138.53, 138.54, 138.87,
138.88, 172.8, 173.09, 173.13; 31P NMR (202 MHz, CDCl3) δ
-0.64 (0.7P) and -0.34 (0.3P). HRMS-ESI: [M + Na]+ calcd
for C113H149NaO18P, 1849.0413; found, 1849.0452. Anal. Calcd for
20
gave the title compound 19 (163 mg, 93%) as a colorless oil. [R]D
(29) Martin, S. F.; Josey, J. A.; Wong, Y.-L.; Dean, D. W. J. Org. Chem.
1994, 59, 4805-4820.
C
133H149O18P: C, 74.31; H, 8.22. Found: C, 74.23; H, 8.32.
J. Org. Chem, Vol. 72, No. 9, 2007 3287

Dyer et al.
2-(O-R-D-Mannopyranosyl)-1-O-(2-O-(R)-10-methyloctade-
canoyl-1-O-hexadecanoyl-sn-glycero-3-phosphoryl)-D-myo-inosi-
tol (23). Pd(OH)2/C (20% , 19 mg) was added to a stirred solution
of 22 (51 mg, 0.028 mmol) in a 3:2 mixture of MeOH and THF (5
mL). The mixture was stirred under a hydrogen atmosphere for 4
h at rt and filtered through Celite, washed with further 3:2 MeOH
and THF (30 mL), and concentrated in vacuo. The residue was
purified on silica gel (CHCl3/MeOH/H2O ) 90:10:0 to 70:40:6) to
5.36 (m, 1H), 5.49 (brs, 1H), 6.98-7.39 (m, 60H), 13C NMR (75
MHz) δ 14.2, 19.8, 22.8, 24.9, 27.2, 29.2, 29.4, 29.8, 30.1, 32.0,
32.9, 33.9, 34.1, 37.2, 61.4-81.1 (CH and CH2), 98.5, 99.6, 127.2-
128.9 (CH-Aryl),137.8-139.2 (Cq-Aryl), 172.6, 173.0; 31P NMR
(121.5 MHz, CDCl3) δ 1.16, 0.94. HRMS-ESI: [M + Na]+ calcd
for C140H177O23NaP, 2280.2316; found, 2280.2317.
2,6-(Di-O-R-D-mannopyranosyl)-1-O-(2-O-(R)-10-methylocta-
decanoyl-1-O-hexadecanoyl-sn-glycero-3-phosphoryl)-D-myo-
inositol (1c). Pd(OH)2/C (20%, 37 mg) was added to a stirred
solution of 25 (32 mg, 0.014 mmol) in a 3:2 mixture of MeOH
and THF (5 mL). The mixture was stirred under a hydrogen
atmosphere for 3 h at rt when the hydrogen was replaced with argon,
and the mixture was filtered through Celite, washed with 3:2 MeOH
and THF (30 mL), and concentrated in vacuo. The residue was
purified on silica gel (CHCl3/MeOH/H2O ) 70:40:1 to 70:40:6) to
1
afford the title compound 23 (26 mg, 91%). H NMR (500 MHz,
CDCl3:MeOD:D2O 70:40:6) δ 0.85 (d, J ) 6.6 Hz, 3H), 0.89 (t, J
) 6.9 Hz, 6H), 1.05-1.12 (m, 2H), 1.17-1.40 (m, 49H), 1.65-
1.55 (m, 4H), 2.31 (t, J ) 7.6, Hz, 2H), 2.34 (t, J ) 7.5, Hz, 2H),
3.27 (t, J ) 9.1 Hz, 1H), 3.47-3.52 (m, 1H), 3.57-3.67 (m, 2H),
3.62-3.84 (m, 4H), 3.84-3.68 (m, 1H), 3.93-4.10 (m, 5H), 4.27-
4.35 (m, 1H), 4.31 (bs, 1H), 4.44 (dd, J ) 12.0, 2.5 Hz, 2H), 5.14
(s, 1H), 5.29-5.23 (m, 1H); 13C NMR (125 MHz, CDCl3:
MeOD: D2O 70: 40: 6) δ 16.7, 22.4, 25.4, 27.7, 27.8, 29.87, 29.92,
32.0, 32.1, 32.2, 32.4, 32.5, 32.6, 32.8, 32.9, 34.7, 35.6, 36.9, 37.1,
39.91, 39.93, 57.3, 58.3, 62.9, 63.0, 64.0, 65.9, 66.5, 69.8, 71.4,
73.1, 73.3, 73.35, 73.42, 73.45, 75.3, 75.7, 76.9, 77.4, 78.2, 78.9,
79.3, 81.5, 90.9, 104.3, 176.9, 177.2; 31P NMR (121 MHz, CDCl3:
MeOD: D2O 70: 40: 6) δ -0.06. HRMS-ESI: [M - H]- calcd
for C50H94O18P, 1013.6183; found, 1013.6172.
3,4,5-Tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-benzyl-R-D-man-
nopyranosyl-1-O-(2-O-(R)-10-methyloctadecanoyl-1-O-hexade-
canoyl-sn-glycero-3-benzylphosphoryl)-D-myo-inositol (25). 1H-
Tetrazole (18 mg, 0.26 mmol) was added to a stirred solution of
3,4,5-tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-benzyl-R-D-mannopy-
ranosyl)-D-myo-inositol (24) and phosphoramidite 20 (215 mg,
0.253 mmol) in dry CH2Cl2 (6 mL) cooled to 0 °C under an argon
atmosphere. After stirring at rt for 1.5 h the reaction mixture was
cooled to -40 °C and a solution of m-CPBA (50%, 88 mg, 0.25
mmol) in CH2Cl2 (4 mL) was transferred by cannula into the
reaction mixture. After warming to rt over 2 h the reaction was
quenched with addition of 10% aqueous Na2SO3 solution (50 mL)
and the combined mixture extracted with Et2O (100 mL). The
ethereal extract was washed with saturated NaHCO3 solution (3 ×
50 mL) and dried (MgSO4). After filtration the solvent was removed
in vacuo and the residue purified by silica gel column chromatog-
raphy (EtOAc/light petroleum ) 1:9 to 1:4) followed by a second
column (CH2Cl2 to EtOAc/CH2Cl2 ) 1:25) to give the title
compound 21 (69 mg, 0.031 mmol, 48%) as an oil. 1H NMR (300
MHz, CDCl3) δ 0.81-0.91 (m, 9H), 1.01-1.13 (m, 2H), 1.14-
1.34 (m, 49H), 1.42-1.58 (m, 4H), 2.09-2.24 (m, 4H), 3.21-
3.52 (m, 6H), 3.75-4.26 (m, 17H), 4.40-5.15 (m, 24H), 5.27-
1
afford the title compound 5 (14 mg, 86%). H NMR (300 MHz,
CDCl3/CD3OD/D2O, 70:40:6) δ 0.82-0.90 (m, 9H), 1.05-1.13 (m,
2H), 1.55-1.66 (m, 4H), 1.21-1.34 (m, 49H), 2.27-2.40 (m, 4H),
3.25-3.31(m, 1H), 3.43-3.50 (m, 1H), 3.57-3.86 (m, 10H), 3.91-
4.09 (m, 7H), 4.30 (br s, 1H), 4.40-4.43 (m, 1H), 5.10 (br s, 1H),
5.13 (br s, 1H), 5.22-5.32 (m, 1H); 31P NMR (121.5 MHz, CDCl3/
CD3OD/D2O, 70:40:6) δ -3.7. HRMS-ESI: [M - H]- calcd for
C56H104O23P, 1175.6706; found, 1175.6725. Anal. Calcd for
C56H105O23P.1.5SiO2: C, 53.07; H, 8.35. Found: C, 53.11; H, 8.27.
Acknowledgment. We are grateful to the University of
Otago for financial support for Blake Dyer, and the Marsden
fund for Gary Ainge (grant no. IRL0401). We also thank Ian
Stewart and Dr. Mervyn Thomas for help recording NMR
spectra and Yinrong Lu, Glycosyn, Industrial Research Limited,
and Dr. Torsten Kleffmann, Department of Biochemistry,
University of Otago, for recording mass spectra. We would like
to thank Professor Robin Smith, University of Otago, and
Professor Martine Gilleron, Centre National de la Recherche
Scientific, France, for helpful discussions and the latter for a
preprint of ref 26.
Supporting Information Available: General experimental
remarks, experimental details for the synthesis of 6, NMR spectra
for all compounds reported in the Experimental Section, and mass
spectra for 1c and 23. This material is available free of charge via
the Internet at http://pubs.acs.org.
JO0625599
3288 J. Org. Chem., Vol. 72, No. 9, 2007