Phosphatidylinositol mannoside ether analogues: Syntheses and interleukin-12-inducing properties

Article abstract of DOI:10.1021/jo070639m

(Chemical Equation Presented) Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of glycolipids with significant immune modulating properties. We present here the syntheses of phosphatidylinositol

Full text of DOI:10.1021/jo070639m

Phosphatidylinositol Mannoside Ether Analogues: Syntheses and
Interleukin-12-Inducing Properties
Gary D. Ainge,†,§ Natalie A. Parlane, Michel Denis, Blake S. Dyer, Andrea H a¨ rer,
‡
‡
§
†
†
§
,†
Colin M. Hayman, David S. Larsen, and Gavin F. Painter*
Carbohydrate Chemistry Team, Industrial Research Limited, P.O. Box 31-310, Lower Hutt, New Zealand,
AgResearch, Hopkirk Research Institute, PriVate Bag 11 222, Palmerston North, New Zealand, and
UniVersity of Otago, P.O. Box 56, Dunedin, New Zealand
g.painter@irl.cri.nz
ReceiVed April 5, 2007
Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important
class of glycolipids with significant immune modulating properties. We present here the syntheses of
phosphatidylinositol dimannoside ether analogues 2 and 3 and evaluate their interleukin-12 (IL-12)-
inducing properties along with dipalmitoyl PIM2 (1) in an in Vitro bovine dendritic cell assay. Both
synthetic PIM analogues and synthetic dipalmitoyl PIM2 (1) were effective at enhancing IL-12 production
by immature bovine dendritic cells. Unexpectedly, ether analogue 2 was significantly more active than
dipalmitoyl PIM2 (1) which indicates that modified PIM compounds can be strongly immunoactive and
may have significant adjuvant activities.
Introduction
products act as adjuvants; their structures, the cell types on
which they act, and their modes of action cover wide spectra.4
Adjuvants are particularly important in the case of vaccines that
consist of chemically defined components, such as subunit
vaccines, which have significant advantages over live attenuated
vaccines in terms of their reproducibility, safety, and concomi-
Mycobacteria and their products have strong inherent immu-
nomodulatory properties. The bulk of the stimulatory activity
resides in the cell walls of mycobacteria, with various products
such as lipoarabinomannan (LAM), phosphatidylinositol man-
nosides (PIMs), and a plethora of other mycobacterial com-
1
5
tant ease of registration. However, discrete antigens often have
2
pounds being able to modulate the immune response. Conse-
poor immunogenicity compared to live attenuated vaccines, and
this is a major drawback to their use. In order to elicit a strong
and robust cell-mediated immune response, the coadministration
quently, mycobacterial products have been exploited to produce
formulations to modulate the immune response.3
6
Adjuvants stimulate the immune system to respond to an
antigen or a set of antigens and are used extensively to enhance
the efficacy of vaccines. Numerous natural and synthetic
of an adjuvant is required, and there is at present a scarcity of
effective adjuvants which are safe and reliable. A canonical
adjuvant based on mycobacterial products is Complete Freund’s
Adjuvant (CFA), which is a formulation of heat-killed myco-
7
bacteria in an oil emulsion that has been used extensively for
*
Author to whom correspondence should be addressed. Phone: 64 4 931
3
103. Fax: 64 4 931 3055.
†
Industrial Research Limited.
AgResearch.
University of Otago.
‡
(4) Petrovsky, N.; Aguilar, J. C. Immunol. Cell Biol. 2004, 82, 488-96.
(5) Francis, J. N.; Larche, M. Curr. Opin. Allergy Clin. Immunol. 2005,
5, 537-43.
§
(
1) Goren, M. B. Am. ReV. Respir. Dis. 1982, 125, 50-69.
(2) Briken, V.; Porcelli, S. A.; Besra, G. S.; Kremer, L. Mol. Microbiol.
(6) Clark, T. G.; Cassidy-Hanley, D. DeV. Biol. (Basel) 2005, 121, 153-
2
004, 53, 391-403.
3) Ivanyi, J.; Sharp, K.; Jackett, P.; Bothamley, G. Springer Semin.
Immunopathol. 1988, 10, 279-300.
63.
(
(7) Claassen, E.; de Leeuw, W.; de Greeve, P.; Hendriksen, C.; Boersma,
W. Res. Immunol. 1992, 143, 478-83.
1
0.1021/jo070639m CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/09/2007
J. Org. Chem. 2007, 72, 5291-5296
5291

Ainge et al.
SCHEME 1. Synthesis of Monoether 2
experimental purposes for many decades. CFA, however, has
toxic side effects which preclude its use in humans, and there
is an urgent need for the development of novel immune
modulators that are safe, potent, and based on chemically defined
entities.
genolytic debenzylation of the perbenzylated compound 6 using
solvent mixtures that contained some water led to a small
amount of the monodeacylated product 7. The chemical instabil-
ity of the sn-2 acyl linkage of phospholipids is known; indeed,
specific loss of this group is used as a means of structural
characterization by mass spectral techniques. Inasmuch as acyl
ester groups are also subject to enzymatic removal, we
postulated that PIM analogues containing more stable linkages
may be better immune modulators than natural PIMs.
15
A major challenge in this field is therefore to isolate or
synthesize compounds that have adjuvant activities (i.e., stimu-
4
late a pro-inflammatory response), with limited side effects,
and are chemically well defined. Phosphatidylinositol manno-
sides (PIMs) are glycolipids, embedded in the cell walls of
mycobacteria, that anchor an array of more complex glycolipids
We present here the synthesis of the PIM ether analogues 2
and 3 and assess their ability to induce bovine dendritic cells
8
16
to the cell membrane. Native PIMs generally occur with two
(DC) to secrete interleukin-12 (IL-12).
(PIM2) or six mannose (PIM6) residues attached, singly or as
oligosaccharides, to their inositol component. We and others
have demonstrated that the smaller natural PIM compounds,
9
-12
by themselves, retain immunomodulatory activities
synthetic PIM samples also retain these activities.
particular, they stimulate cytokine production via key cells of
and that
1
3,14
In
1
0
the immune system and generate an inflammatory response.
The precise acylation state of the PIM molecule appears
1
0
crucial in the type and magnitude of the observed bioactivity,
and we have already demonstrated14 that synthetic PIM2 (1) is
sensitive to degradation by deacylation. In particular, hydro-
(
9.
8) Stadelmaier, A.; Schmidt, R. R. Carbohydr. Res. 2003, 338, 2557-
6
(
9) Sprott, G. D.; Dicaire, C. J.; Gurnani, K.; Sad, S.; Krishnan, L. Infect.
Discussion
Immun. 2004, 72, 5235-46.
(10) Gilleron, M.; Quesniaux, V. F.; Puzo, G. J. Biol. Chem. 2003, 278,
The phosphoramidite 14 was prepared from (R)-benzyl
glycidol (8) which was obtained by hydrokinetic resolution of
2
9880-9.
(
11) Torrelles, J. B.; Azad, A. K.; Schlesinger, L. S. J. Immunol. 2006,
1
77, 1805-16.
the racemate using the methodology of Jacobsen et al. (Scheme
(12) Sayers, I.; Severn, W.; Scanga, C. B.; Hudson, J.; le Gros, G.;
17
1
). The ee of 8 was estimated as greater than 95% from the
Harper, J. L. J. Allergy Clin. Immunol. 2004, 114, 302-309.
13) Ainge, G. D.; Hudson, J.; Larsen, D. S.; Painter, G. F.; Gill, G. S.;
Harper, J. L. Bioorg. Med. Chem. 2006, 14, 5632-42.
14) Ainge, G. D.; Parlane, N. A.; Denis, M.; Hayman, C. M.; Larsen,
D. S.; Painter, G. F. Bioorg. Med. Chem. 2006, 14, 7615-7624.
(
(15) Hsu, F. F.; Turk, J. J. Am. Soc. Mass Spectrom. 2000, 11, 986-99.
(16) Boyaka, P. N.; Lillard, J. W., Jr.; McGhee, J. Immunol. Res. 1999,
20, 207-17.
(
5292 J. Org. Chem., Vol. 72, No. 14, 2007

Phosphatidylinositol Mannoside Ether Analogues
SCHEME 2. Synthesis of Diether 3
The Th1-inducing activities of PIM compounds 1, 2, and 3
were tested in an in Vitro bovine dendritic cell (DC) assay, and
the results were compared with responses induced by li-
popolysaccharide (LPS), a known potent IL-12 inducer (Figure
1
). Products which direct the immune responses toward a Th1
profile are IL-12 inducers, and specialized antigen presenting
cells such as DCs are excellent bioprobes for the detection of
factors likely to direct an immune response toward a Th1 profile,
via their high potential for IL-12 release.
Interestingly, the three synthetic compounds 1, 2, and 3 all
induced significant levels of IL-12 from bovine DCs, suggesting
that these compounds are good adjuvant candidates and, in
particular, have potential for stimulating a Th1 cell-mediated
immune response, which is important for protection against
intracellular pathogens. The diether 3 and dipalmitoyl PIM2 (1)
induced IL-12 production to a similar level, indicating that the
acyl linkages in PIM2 are not required for the observed activity.
On the other hand, the mono-ether 2 was significantly more
active than either the diacyl 1 or diether 3 compounds. This
would indicate that the acyl linkage attached at the sn-1 carbon
center together with a stable linkage at the sn-2 carbon center
are both important for inducing higher levels of IL-12.
The lipid antigen-presenting molecule CD1d has been im-
plicated in the observed activity of PIM compounds in ViVo,25
and the crystal structure of a PIM2-CD1d complex has been
1
H and 19F NMR spectra of a corresponding Mosher ester
derivative.18 Here, Lewis acid-promoted opening of epoxide 8
with allyl alcohol provided allyl ether 9 which had an optical
rotation consistent with that reported by Mikkilineni et al. The
sn-2 hydroxyl group in compound 9 was alkylated with
1
19
-bromohexadecane without incident, affording ether 10 in good
yield. Removal of the allyl group was effected by treatment
with a catalytic amount of tetrakis(triphenylphosphine)palladium-
2
6
27
resolved. However, it has recently been discovered that the
bovine CD1 family contains group 1 CD1 proteins but no
functional CD1d, suggesting that cattle have no functional NKT
cells. In addition, synthetic dipalmitoyl PIM2 (1) does not
stimulate mouse NKT cells in the same way, if at all, as do
2
0
(
0) and N,N′-dimethylbarbituric acid to give alcohol 11 in
excellent yield. The specific optical rotation was equal, but
opposite in sign, to that reported for the enantiomer by Duclos.
Esterification of compound 11 with palmitoyl chloride afforded
monoester 12 in good yield. The benzyl ether group was
removed with Pd(OH)2/C in the presence of acid to give alcohol
21
28
lipid products such as R-galactosylceramide. Therefore, recep-
tors of the innate immune system, such as Toll receptors10 or
perhaps the group 1 CD1 proteins (CD1a, CD1b, and CD1c),
may be more relevant here, when purified DCs are used as a
readout system. It is emphasized that PIMs have been reported
to have multiple effects on a variety of cell types, including
binding to integrins present on CD 4 + T cells, which is likely
1
3, and the phosphoramidite 14 was prepared from it by use of
benzyloxy-bis(diisopropylamino)phosphine activated by 1H-
tetrazole. Coupling of compound 14 with the pseudo-trisaccha-
ride 15
afforded the monoether 2 in good yield. The H NMR spectrum
showed all the expected signals in the appropriate ratios. In
particular, the two anomeric protons were evident as broad
singlets at δ 5.14 and 5.11, and the sn-2 proton now resonated
at higher field as expected (for PIM2 1, the sn-2 proton appears
at δ 5.3). The 31P NMR displayed a single peak at δ -3.5.
1
4,22
gave the fully substituted 16, and deprotection
1
29
to activate these cells. It therefore is unclear which mechanism-
s) of action is responsible for the complete immunomodulatory
(
actions of PIMs in ViVo, and the possibility that multiple
mechanisms are implicated (binding to integrins on T cells,
engagement to TLR receptors on antigen-presenting cells,
activation of NKT cells) remains a possibility.
2
3,24
The phosphoramidite 19 was prepared from alcohol 18
Scheme 2) by phosphitylation with benzyloxy-bis(diisopropy-
(
We have prepared synthetic samples of PIM2 ether analogues
lamino)phosphine. Coupling with the pseudo-trisaccharide 15
afforded the benzyl-protected compound 20 from which the
required diether 3 was obtained after deprotection. The H NMR
spectrum showed all the expected signals in the appropriate
ratios. In particular, the two anomeric protons were evident at
δ 5.16 and 5.11, and as expected the sn-2 proton was absent
from this region.
2
and 3 and together with dipalmitoyl PIM2 (1) evaluated their
relative abilities to induce IL-12 in bovine DCs as a model for
Th1-inducing activity. The three synthetic compounds are good
IL-12 inducers with mono-ether 2 being significantly more
potent than either the diacylated (1) or diether (3) counterparts.
Hence, non-natural PIMs can be at least as active as natural
PIMs at inducing IL-12 from DCs in Vitro, and may identify a
1
(
17) 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.
002, 124, 1307-1315.
18) Dyer, B. S.; Jones, J. D.; Ainge, G. D.; Denis, M.; Larsen, D. S.;
Painter, G. F. J. Org. Chem. 2007, 72, 3282-3288.
19) Mikkilineni, A. B.; Kumar, P.; Abushanab, E. J. Org. Chem. 1988,
(25) Fischer, K.; Scotet, E.; Niemeyer, M.; Koebernick, H.; Zerrahn, J.;
Maillet, S.; Hurwitz, R.; Kursar, M.; Bonneville, M.; Kaufmann, S. H.;
Schaible, U. E. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10685-10690.
(26) Zajonc, D. M.; Ainge, G. D.; Painter, G. F.; Severn, W. B.; Wilson,
I. A. J. Immunol. 2006, 177, 4577-4583.
2
(
(
(27) Rhijn, I. V.; Koets, A. P.; Im, J. S.; Piebes, D.; Reddington, F.;
Besra, G. S.; Porcelli, S. A.; van Eden, W.; Rutten, V. P. M. G. J. Immunol.
2006, 176, 48884893.
5
3, 6005-6009.
(20) Tsukamoto, H.; Kondo, Y. Synlett 2003, 7, 1061-1063.
(21) Duclos, R., Jr. I. Chem. Phys. Lipids 1993, 66, 161-170.
(22) Elie, C. J. J.; Verduyn, R.; Dreef, C. E.; Van der Marel, G. A.; Van
(28) Kinjo, Y.; Tupin, E.; Wu, D.; Fujio, M.; Garcia-Navarro, R.; Zajonc,
D. M.; Ben-Menache, G.; Ainge, G. D.; Painter, G. F.; Khurana, A.; Hoebe,
K.; Behar, S. M.; Beutler, B.; Wilson, I. A.; Tsuji, M.; Sellati, T. J.; Wong,
C. H.; Kronenberg, M. Nat. Immunol. 2006, 7, 978-986.
(29) Rojas, R. E.; Thomas, J. J.; Gehring, A. J.; Hill, P. J.; Belisle, J. T.;
Harding, C. V.; Boom, W. H. J. Immunol. 2006, 177, 2959-68.
Boom, J. H. J. Carbohydr. Chem. 1992, 11, 715-39.
(
23) Bhattacharya, S.; De, S. Chem. Eur. J. 1999, 5, 2335-2347.
(24) Browne, J. E.; Freeman, R. T.; Russel, J. C.; Sammes, P. G. J. Chem.
Soc., Perkin Trans. 1 2000, 645-652.
J. Org. Chem, Vol. 72, No. 14, 2007 5293

Ainge et al.
FIGURE 1. Induction of IL-12 by synthetic PIM compounds, 1, 2, and 3 on bovine dendritic cells (50 µg of each compound) compared with saline
control (Control). Supernatants were collected 48 h after stimulation and IL-12 levels then measured. Columns with different letters are significantly
different from each other (P < 0.05).
novel avenue for creating more stable and potent adjuvants. We
are currently pursuing this possibility.
Elution with EtOAc/light petroleum (1:9) afforded the title com-
28
pound 11 (342 mg, 0.84 mmol, 93%) as an oil. [R]
D
) +10.8 (c
21
1
.23, CH
glycerol [R]
CDCl ) δ 7.38-7.22 (m, 5H), 4.54 (m, 2H), 3.77-3.42 (m, 7H),
.13 (t, J ) 5.7 Hz, 1H), 1.61-1.52 (m, 2H), 1.39-1.19 (m, 26H),
2 2
Cl ). {lit. for ent-11, 1-O-benzyl-2-O-hexadecyl-sn-
28
1
D
2 2
) -10.2 (c 5.00, CH Cl )}. H NMR (300 MHz,
Experimental Section
3
2
0
1
2
1
-O-Allyl-3-O-benzyl-sn-glycerol (9). BF
mmol) was added to a stirred solution of (R)-benzyl glycidol (8)
260.8 mg, 1.6 mmol) and allyl alcohol (1.1 mL, 16 mmol) in dry
CH Cl
3
2
‚OEt (50 µL, 0.4
.92-0.82 (m, 3H). 13C NMR (75 MHz, CDCl
) δ 138.1, 128.5,
3
27.8, 127.7, 78.6, 73.6, 70.5, 70.1, 63.0, 32.0, 30.2, 29.8, 29.7,
(
+
9.5, 29.4, 26.2, 22.8, 14.2. HRMS-ESI [M + Na] calcd for
2
2
(10 mL). After 1h, the solvent was removed in Vacuo and
26 46 3
C H O Na: 429.3345. Found 429.3351.
the residue purified by column chromatography on silica gel. Elution
3-O-Benzyl-1-O-hexadecanoyl-2-O-hexadecyl-sn-glycerol (12).
with EtOAc/light petroleum (1:9) afforded the title compound 9
2
8
Palmitoyl chloride (1.10 mL, 3.64 mmol) was added dropwise to
a stirred solution of alcohol 11 (1.34 g, 3.31 mmol) and pyridine
(
269 mg, 1.20 mmol, 76%) as a clear oil. [R]
D
1
) +0.76 (c 1.00,
19
EtOH). {lit. [R]
CDCl
1
4
4
1
D
) +0.74 (c 3.53, EtOH)}. H NMR (300 MHz,
(
1.34 mL, 16.6 mmol) in CH
being stirred for 12 h at rt, the reaction mixture was quenched with
O (100 mL). The mixture was extracted with Et
O (2 × 150
mL) and the ethereal extract washed with a 0.5M HCl solution
100 mL) and saturated NaHCO solution (100 mL) and dried
2 2
Cl (20 mL) cooled to 0 °C. After
3
) δ 7.40-7.26 (m, 5H), 5.97 (ddt, J ) 17.3, 10.4, 5.7 Hz,
H), 5.27 (dq, J ) 17.2, 1.6 Hz, 1H), 5.19 (dq, J ) 10.4, 1.4, 1H),
.57 (s, 2H), 4.39-4.23 (m, 3H), 3.60-3.45 (m, 4H), 2.52 (d, J )
H
2
2
.4 Hz, 1H). 1 C NMR (75 MHz, CDCl
3
3
) δ 138.0, 134.5, 128.5,
(
3
27.8, 127.8, 117.3, 73.5, 72.4, 71.4, 71.3, 69.6. HRMS-ESI [M +
+
(MgSO ). After filtration, the solvent was removed in Vacuo and
Na] calcd for C13
H O
18 3
Na: 245.1154. Found 245.1163.
4
the residue purified by column chromatography on silica gel. Elution
1-O-Allyl-3-O-benzyl-2-O-hexadecyl-sn-glycerol (10). 1-Bro-
with EtOAc/light petroleum (0:1 to 1:9) afforded the title compound
mohexadecane (685 µL, 2.2 mmol) was added to a stirred
suspension of 9 (248 mg, 1.1 mmol) and sodium hydride (60%
dispersion in mineral oil, 150 mg, 3.8 mmol) in dry DMF (10 mL)
under nitrogen. After being stirred for 16 h, the reaction was
quenched by addition of 1 M HCl (100 mL). The mixture was
2
0
1
2 (2.04 g, 3.18 mmol, 96%) as an oil. [R]
D
) +5.3 (c 0.92,
) δ 7.37-7.22 (m, 5H), 4.54
m, 2H), 4.27-4.10 (m, 2H), 3.66 (quintet, J ) 5.2 Hz, 1H), 3.57-
1
3 3
CHCl ). H NMR (300 MHz, CDCl
(
3
1
1
3
.52 (m, 4H), 2.29 (t, J ) 7.4 Hz, 2H), 1.67-1.50 (m, 4H), 1.38-
.17 (m, 50H), 0.92-0.85 (m, 6H). 13C NMR (75 MHz, CDCl
) δ
extracted with CH
solvent was removed in Vacuo and the residue purified by column
chromatography on silica gel. Elution with Et O/light petroleum
0:1 to 1:19) afforded the title compound 10 (426 mg, 0.95 mmol,
2
Cl
2
(2 × 100 mL) and dried (MgSO
4
). The
3
73.7, 138.2, 128.4, 127.7, 76.7, 73.5, 70.7, 69.7, 63.7, 34.3, 32.0,
0.1, 29.8, 29.7, 29.6, 29.4, 29.3, 29.2, 26.1, 25.0, 22.8, 14.2.
2
+
HRMS-ESI [M + Na] calcd for C42
667.5632.
76 4
H O Na: 667.5641. Found
(
8
2
8
1
5%) as an oil.[R]
D
) +0.60 (c 1.00, EtOH). H NMR (300 MHz,
CDCl
3
) δ 7.36-7.24 (m, 5H), 5.89 (ddt, J ) 17.3, 10.2, 5.6 Hz,
1-O-Hexadecanoyl-2-O-hexadecyl-sn-glycerol (13). A mixture
of the benzyl ether 12 (1.00 g, 1.55 mmol) and Pd(OH) /C (20%,
1
1
7
H), 5.26 (dq, J ) 17.2, 1.6 Hz, 1H), 5.17 (dq, J ) 10.4, 1.5 Hz,
2
H), 4.56 (s, 2H), 4.00 (dt, J ) 5.6, 1.5 Hz, 2H), 3.67-3.48 (m,
H), 1.63-1.52 (m, 2H), 1.39-1.20 (m, 26H), 0.88 (t, J ) 6.8
300 mg) in EtOH (100 mL)/HOAc (10 mL) was stirred under
hydrogen for 14 h. The hydrogen was removed and the mixture
filtered through Celite. The filtrate was concentrated in Vacuo and
the residue purified by column chromatography on silica gel. Elution
13
Hz, 3H). C NMR (75 MHz, CDCl
1
2
3
) δ 138.5, 134.9, 128.4, 127.64,
27.57, 116.9, 78.0, 73.4, 72.4, 70.7, 70.3, 70.2, 32.0, 30.2, 29.8,
+
9.73, 29.70, 29.6, 29.4, 26.2, 22.8, 14.2. HRMS-ESI [M + Na]
with EtOAc/CH
2 2
Cl (1:24) afforded the title compound 13 (850
20
calcd for C29
50
H O
3
Na: 469.3658. Found 469.3651.
mg, 1.54 mmol, 99%) as an oil. [R]
D
) -4.8 (c 1.20, CHCl
3
).
1
H NMR (300 MHz, CDCl
3
) δ 4.18-4.15(m, 2H), 3.65-3.43 (m,
3-O-Benzyl-2-O-hexadecyl-sn-glycerol (11). A mixture of the
5
4
H), 2.32 (d, J ) 7.4 Hz, 2H), 2.09-2.02 (m, 1H), 1.65-1.52 (m,
ether 10 (403 mg, 0.90 mmol), N,N′-dimethylbarbituric acid (373
H), 1.36-1.21 (m, 50H), 0.92-0.85 (m, 6H). 13C NMR (75 MHz,
mg, 2.4 mmol), and tertakis(triphenylphosphine)palladium (60.8 mg,
.05 mmol)20 in dry THF (4 mL) was heated at 90 °C under nitrogen
CDCl ) δ 173.8, 77.8, 70.6, 62.8, 62.8, 34.3, 32.0, 30.1, 29.8, 29.7,
0
3
2
9.6, 29.4, 29.3, 29.2, 26.1, 25.0, 22.8, 14.2. HRMS-ESI [M +
in a sealed tube. After being stirred at the same temperature for 40
h, the reaction mixture was cooled to rt and poured into a saturated
+
70 4
Na] calcd for C35H O Na: 577.5172. Found 577.5172.
NaHCO
organic layer was dried (MgSO
residue was purified by column chromatography on silica gel.
3
solution and extracted with CH
2
Cl
2
(2 × 100 mL). The
Benzyl (1-O-Hexadecanoyl-2-O-hexadecyl-sn-glycero)-diiso-
propylphosphoramidite (14). 1H-Tetrazole (55 mg, 0.79 mmol)
was added to a stirred solution of alcohol 13 (395 mg, 0.714 mmol)
4
) and concentrated in Vacuo. The
5294 J. Org. Chem., Vol. 72, No. 14, 2007

Phosphatidylinositol Mannoside Ether Analogues
and benzyloxy-bis(diisopropylamino)phosphine (482 mg, 1.43
3,4,5-Tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-benzyl-r-D-man-
nopyranosyl)-1-O-(1,2-di-O-hexadecyl-sn-glycero-3-benzylphos-
phoryl)-D-myo-inositol (20). 1H-Tetrazole (16 mg, 0.23 mmol) was
added to a stirred solution of pseudo-trisaccharide 15 (90 mg, 0.06
mmol) and phosphoramidite 19 (181 mg, 0.23 mmol) in CH Cl
mmol) in dry CH
removed in Vacuo and the residue purified by column chromatog-
raphy on silica gel. Elution with Et N/EtOAc/light petroleum (1:
:16) afforded the title compound 14 (544 mg, 0.689 mmol, 96%)
2 2
Cl (10 mL). After 1 h at rt, the solvent was
3
3
2
2
1
as an oil. H NMR (300 MHz, CDCl
3
) δ 7.37-7.23 (m, 5H), 4.79-
(9 mL) cooled to -10 °C. The ice bath was removed after 15 min
and after being stirred at rt for 2 h under argon the reaction mixture
was cooled to -40 °C and a solution of predried m-CPBA (50%,
55.0 mg, 0.32 mmol) in CH Cl (5 mL) was transferred by cannula
4
(
(
1
.62 (m, 2H), 4.30-4.20 (m, 1H), 4.15-4.05 (m, 1H), 3.70-3.48
m, 7H), 2.29 (t, J ) 7.3 Hz, 2H), 1.65-1.50 (m, 4H), 1.30-1.16
m, 62H), 0.90-0.83 (m, 6H). 31P NMR (121.5 MHz, CDCl
) δ
NaP:
3
2
2
+
49.4, 149.2. HRMS-ESI, [M + Na] calcd for C48
H90NO
5
into the reaction mixture. After being warmed to rt over 3 h, the
reaction was quenched by the addition of a 10% Na SO solution
8
14.6454. Found 814.6469.
2
3
3
,4,5-Tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-benzyl-r-D-man-
(50 mL) and the mixture was extracted with Et O (2 × 30 mL).
2
nopyranosyl)-1-O-(1-O-Hexadecanoyl-2-O-hexadecyl-sn-glycero-
The combined organic extracts were washed with a saturated
3-benzylphosphoryl)-D-myo-inositol (16). 1H-Tetrazole (19 mg,
0.27 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-mannopyranosyl)-D-myo-inosi-
NaHCO solution (3 × 50 mL) and brine (50 mL). After drying
3
(MgSO ) and filtration, the solvent was removed in Vacuo and the
4
residue purified by column chromatography on silica gel. Elution
tol (15) (81 mg, 0.054 mmol) and phosphoramidite 14 (214 mg,
.271 mmol) in dry CH Cl (8 mL) cooled to 0 °C under argon.
After being stirred at rt for 2 h, the reaction mixture was cooled to
40 °C and a solution of m-CPBA (50%, 93 mg, 0.27 mmol) in
CH Cl (10 mL) was transferred by cannula into the reaction
mixture. After being stirred at rt over 2 h, the reaction was quenched
by addition of a 10% Na SO solution (50 mL) and the combined
mixture extracted with Et O (100 mL). The ethereal extract was
washed with a saturated NaHCO
solution (3 × 50 mL) and dried
MgSO
with EtOAc/light petroleum (2:8 to 2.5:7.5) followed by two further
purifications on fresh silica gel eluted with Et O/CH Cl (0.25:
0
2
2
2
2
2
9
.75) afforded the title compound 20 (20 mg, 0.0091 mmol, 15%)
-
1
as an oil. H NMR (300 MHz, CDCl
3
) δ 7.30-6.95 (m, 60H), 5.44
2
2
(
2
0
d, J ) 7.3 Hz, 1H), 5.28 (d, J ) 9.7 Hz, 1H), 4.99 (t, J ) 7.9 Hz,
H), 4.86-3.16 (m, 49H), 1.49-1.31 (m, 4H), 1.30-1.10 (m, 52H),
31
2
3
.83-0.79 (m, 6H). P NMR (121.5 MHz, CDCl
) δ 1.29, 1.25.
175
H O21PNa: 2210.2364.
3
+
2
HRMS-ESI [M + Na] calcd for C137
Found: 2210.2314.
3
(
4
). After filtration, the solvent was removed in Vacuo and
2
,6-Di-O-(r-D-mannopyranosyl)-1-O-(1,2-di-O-hexadecyl-sn-
the residue purified by column chromatography on silica gel. Elution
glycero-3-phosphoryl)-D-myo-inositol (3). Pd(OH) /C (20%, 7.0
2
with EtOAc/light petroleum (1:9) afforded the title compound 16
mg) was added to a stirred solution of the fully substituted 20 (20
mg, 0.0091 mmol) in THF/MeOH (2:3, 2.5 mL), and stirring was
continued under hydrogen for 3 h at rt after which Et N (1 mL)
3
1
(
38 mg, 0.017 mmol, 32%) as an oil. H NMR (300 MHz, CDCl
3
)
δ 7.38-7.01 (m, 60H), 5.53-5.47 (m, 1H), 5.37-5.31 (m, 1H),
5
1
1
.04 (ap t, J ) 7.2 Hz, 2H), 4.92-4.37 (m, 21H), 4.30-3.75 (m,
was added. The mixture was filtered through Celite and the filter
cake washed with further THF/MeOH (2:3, 10 mL). The filtrate
and washings were concentrated in Vacuo, and the residue was
7H), 3.54-3.20 (m, 9H), 2.21-2.10 (m, 2H), 1.58-1.41 (m, 4H),
13
.31-1.15 (m, 50H), 0.89-0.82 (m, 6H). C (75 MHz, CDCl
3
)
)
31
selected signals δ 173.3, 99.6, 98.6. P NMR (121.5 MHz, CDCl
3
purified by column chromatography on silica gel. Elution with H
MeOH/CHCl (0:2:7 to 0:4:7 to 0.2:4:7 to 0.8:4:7) and evaporation
of the solvent afforded the title compound 3 (4.6 mg, 0.0042 mmol,
2
O/
+
δ 1.17, 1.13. HRMS-ESI [M + Na] calcd for C137
224.2054. Found 2224.2051.
,6-(Di-O-R-D-mannopyranosyl)-1-O-(1-O-hexadecanoyl-2-O-
hexadecyl-sn-glycero-3-phosphoryl)-D-myo-inositol (2). Pd-
OH) /C (20%, 25 mg) was added to a stirred solution of the fully
173
H O22NaP:
3
2
2
2
0
4
6%) as a white powder. [R]
D
) +32 (c 0.23, D
2
O/CD
3
OD/
1
CDCl
3
, 0.6:4:7). H NMR (300 MHz, D O/CD OD/CDCl
2
3
3
, 0.5:4:
(
2
7
3
) δ 5.16 (br s, 1H), 5.11 (br s, 1H), 4.05-3.43 (m, 28H), 3.36-
substituted 16 (38 mg, 0.017 mmol) in THF/MeOH (2:3, 5 mL).
The mixture was stirred under hydrogen for 2.5 h at rt and the
hydrogen was replaced with argon. The mixture was filtered through
Celite and the filtrate concentrated in Vacuo. The residue was
lyophilized to afford 2 (19 mg, 0.16 mmol, 94%) as a white powder.
.19 (m, 12H), 1.58 (br s, 5H), 1.27 (br s, 51H), 0.89 (t, J ) 6.5
31
Hz, 6H). P NMR (121.5 MHz, D
2
O/CD
3
OD/CDCl
21PNa: 1129.6627.
21P·4H O: C, 53.97;
3
, 0.5:4:7) δ
+
0
.74. HRMS-ESI [M + Na] calcd for C53
103
H O
Found: 1129.6664. Anal. Calcd for C53
H, 9.49. Found: C, 53.71; H, 9.21.
103
H O
2
2
0
1
[
R]
MHz, CDCl
H), 4.50-3.20 (m, 25H), 2.35 (t, J ) 7.2 Hz, 2H), 1.65-1.55
D
) +34 (c 0.20, CHCl
3 3 2
/CH OH/H O, 70:40:6). H NMR (300
Isolation of Bovine Dendritic Cells. Peripheral blood mono-
3
/CD OD/D O, 70:40:6) δ 5.14 (br s, 1H), 5.11 (br s,
3
2
nuclear cells (PBMCs) were isolated from heparinized blood of
1
7
31
cattle by density-gradient centrifugation. Cells were adjusted to 10 /
(
(
[
m, 4H), 1.33-1.22 (m, 50H), 0.86 (t, J ) 6.9 Hz, 6H). P NMR
121.5 MHz, CDCl /CD OD/D O, 70:40:6) δ -3.5. HRMS-ESI
M - H] calcd for C53 22P: 1119.6444. Found 1119.6453.
22P·4H O: C, 53.34; H, 9.21. Found: C,
mL in RPMI-1640 medium containing 10% heat-inactivated FCS,
3
3
2
-
5
-
10 mM HEPES, antibiotics, and 5 × 10 M 2-mercaptoethanol
101
H O
Anal. Calcd for C53
3.30; H, 8.92.
Benzyl (1,2-Di-O-hexadecyl-sn-glycero)-diisopropylphosphor-
amidite (19). Benzyloxy-bis(diisopropylamino)phosphine (250 mg,
.74 mmol) in dry CH Cl (10 mL) was transferred by cannula
into a stirred mixture of alcohol 18 (200 mg, 0.37 mmol) and 1H-
tetrazole (26.0 mg, 70.1 mmol) in dry CH Cl (20 mL) at 0 °C.
101
H O
2
(all obtained from InVitrogen, Auckland, New Zealand). Cells were
7
5
seeded at 10 cells/well of a 24-well plastic tissue dish. Nonadherent
cells were removed after 3 h by washing with warm phosphate-
buffered saline (PBS). Adherent cells were then incubated in
complete medium with 0.2 U/mL of recombinant bovine GM-CSF
and 200 U/mL of recombinant bovine IL-4 (obtained from Serotec,
Oxford, UK). After 3-4 days, fresh media and cytokines were
added to cells. Cells were harvested after 7-10 days of culture,
washed three times, and adjusted to give the desired cell numbers.
When harvested, cells were more than 90% DCs based on the
following: DCs lacked canonical B and T cells markers and had
high expression of MHC class 2 and class 1 (Serotec), and had
low expression of CD11a (Serotec) and CD14 (Serotec). DCs after
10 days of culture also expressed CD80 and CD86 (Veterinary
Medical Research and Development, Inc. (VMRD, Pullman, WA)).
In addition, DCs had the characteristic veiled morphology and
functional phenotype of DCs. DCs were used immediately by
0
2
2
2
2
The ice-bath was removed after 15 min, and after the solution had
been stirred for 2 h at rt under argon the solvent was removed in
Vacuo and the residue purified by column chromatography on silica
gel. Elution with Et
the title compound 19 (233 mg, 0.30 mmol, 81%) as an oil. H
NMR (300 MHz, CDCl ) δ 7.37-7.25 (m, 5H), 4.79-4.63 (m,
H), 3.68-3.40 (m, 11H), 1.57-1.53 (m, 4H), 1.25-1.17 (m, 64H),
3
N/EtOAc/light petroleum (0.3:1:9) afforded
1
3
2
0
1
4
1
3
.88 (t, 6.43, 6H). C NMR (75 MHz, CDCl
27.2, 127.0, 78.6, 71.7, 71.0, 70.7, 65.5, 65.2, 63.3, 63.1, 43.2,
3.0, 32.0, 30.2, 29.8, 29.6, 29.4, 26.2, 24.7, 24.7, 22.8, 14.2. 31
3
) δ 139.7, 128.3,
P
+
5
NMR (121.5 MHz, CDCl
calcd for C48 NPNa: 800.6662. Found: 800.6664.
3
) δ 148.9, 148.8. HRMS-ESI [M + Na]
transferring to a 96-well microtiter plate at 2 × 10 cells per 200
H
92
O
4
uL of complete medium. The cells were cultured in RPMI-1640
J. Org. Chem, Vol. 72, No. 14, 2007 5295

Ainge et al.
with 10% FCS, 10 mM HEPES, and 4 mM L-glutamine, with the
indicated concentrations of reagents for 48 h prior to measurements
of IL-12.
Acknowledgment. The authors wish to thank the New
Zealand Foundation for Research and Technology (C08 × 0209)
and the Marsden Fund (Royal Society of New Zealand,
IRL0401) for financial support (to G.P.). We thank Lilian
Morrison for statistical analyses and Professor Robin Ferrier
for assisting in the preparation of the manuscript.
IL-12 ELISA. Plates (Maxisorp, Nunc, Denmark) were coated
with 8 µg/mL anti-IL-12 (Serotec) and incubated overnight at rt.
The plates were washed in washing buffer, and blocking buffer
was added for 1 h. Following a further washing step, samples were
added for 1 h. Dilutions of the samples were added to the plates
for 1 h. Following washing, biotin-labeled anti-IL-12 (Serotec: 8
µg/mL in blocking buffer) was added for 1 h, followed by washing
and addition of SA-HRP for 45 min. Following the final washing
step TMB substrate was added, the reaction was stopped by the
Supporting Information Available: General experimental
remarks. NMR spectra for all compounds reported in the Experi-
mental Section. This material is available free of charge via the
Internet http://pubs.acs.org.
addition of H SO
2 4
and the absorbance values read at 450 nm.
JO070639M
5296 J. Org. Chem., Vol. 72, No. 14, 2007