Phosphatidylinositol mannosides: Synthesis and adjuvant properties of phosphatidylinositol di- and tetramannosides

Article abstract of DOI:10.1016/j.bmc.2006.07.003

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 (PIM2, 1) and phos

Full text of DOI:10.1016/j.bmc.2006.07.003

Bioorganic & Medicinal Chemistry 14 (2006) 7615–7624
Phosphatidylinositol mannosides: Synthesis and adjuvant properties
of phosphatidylinositol di- and tetramannosides
Gary D. Ainge,a,c Natalie A. Parlane,b Michel Denis,b Colin M. Hayman,a
David S. Larsenc and Gavin F. Paintera,*
aCarbohydrate Chemistry Team, Industrial Research Limited, PO Box 31-310, Lower Hutt, New Zealand
bAgResearch, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand
cUniversity of Otago, PO Box 56, Dunedin, New Zealand
Received 19 May 2006; revised 26 June 2006; accepted 1 July 2006
Available online 27 July 2006
Abstract—Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of glyco-
lipids with significant immune modulating properties. We present here the syntheses of phosphatidylinositol dimannoside (PIM2, 1)
and phosphatidylinositol tetramannoside (PIM4, 2) and evaluate their adjuvant properties in a transgenic mouse model. The key
step in the synthetic methodology for the synthesis of 2 relies on the selective glycosylation of diol 3 with mannosyl donor 11. Both
synthetic PIMs were effective at enhancing IFN-c when given as adjuvants with a model antigen, with PIM2 being the more active.
These data suggest that in this assay the PIM core structure is responsible for the observed biological activity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
The mycobacterial vaccine Bacille Calmette–Guerin
(BCG) has been used to protect humans and animals
against tuberculosis. In addition, it is now known that
it can also prevent the development of atopic disorders
in humans and experimental animals.3,4 The precise
mechanism as to how these mycobacterial products con-
fer protection against atopy is not well understood but is
thought to involve the production of TH1 cytokines that
inhibit TH2 cytokine release that are crucial in mediating
atopy.5 This effect has been, at least in part, attributed to
the presence of certain cell surface glycolipids in the
BCG vaccine such as: lipoarabinomannan (LAM), lipo-
mannans (LM) and phosphatidylinositol manno-
oligosaccharides (PIMs).6 We and others have
demonstrated that the smaller PIM molecules, by them-
selves, retain immunomodulatory activities.7–10 In par-
ticular, they stimulate cytokine production via key
cells of the immune system and generate an inflammatory
response.7,9,11 Thus, we reasoned that PIM compounds,
besides their potential as antagonists of the development
of atopy, may have potential applications as adjuvants
to enhance the activity of sub-unit vaccines.
Vaccines that consist of chemically defined components,
such as sub-unit vaccines, have significant advantages
over live attenuated vaccines in terms of their reproduc-
ibility, safety and hence ease of registration.1 However,
relatively poor immunogenicity is a major drawback in
their use. In order to elicit a strong and robust cell-
mediated immune response, the co-administration of
an adjuvant is required2 and there is at present a paucity
of safe, defined and effective adjuvants. Currently avail-
able immune modulators used in experimental animals,
such as Complete Freund’s Adjuvant (CFA) and Chol-
era Toxin (CT), are based on complex bacterial cell
components and are crude products which are not suit-
able for use in humans owing to their considerable tox-
icities. Therefore, there is a need for the development of
novel immune modulators that are safe, potent, selective
and consist of chemically defined entities.
One obstacle hampering the elucidation of this glycolipid
based immunomodulatory effect is the lack of discrete
molecules of known structure that can be used for
testing. The oligosaccharide backbone of the PIM
Keywords: Phosphatidylinositol mannosides; Adjuvant; Synthesis;
Immunomodulation.
*
Corresponding author. Tel.: +64 4 931 3103; fax: +64 4 931 3055;
e-mail: g.painter@irl.cri.nz
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.003

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G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 7615–7624
molecules has been rigorously investigated,12,13 however,
the precise acylation state still remains an area of
uncertainty. Differently acylated PIM molecules are dif-
ficult to separate from each other when isolated from
natural sources13,14 and it is likely that subtle changes
in the acylation pattern affect their bioactivity.
previously used allyl diol 324 which can be doubly
glycosylated with mannosyl donor 4 in dichloromethane
to afford the a,a-pseudo-trisaccharide 5 in 57% yield.8
We now find the reaction applied on a larger scale
affords the a,b-pseudo-trisaccharide 6 (22%) as by-prod-
uct together with the desired product 5 (59%, Scheme 1).
The anomeric configurations of the mannopyranose res-
idues of 6 were established from the glycosidic C-1
13C–1H coupling constants of 176 and 161 Hz which
are consistent with a- and b-linkages, respectively.26,27
A correlation between H-2 of the inositol and the
anomeric carbon atom of the b-linked mannopyranosyl
residue was apparent in the heteronuclear multiple bond
coherence (HMBC) spectrum, establishing that the
b-mannopyranosyl residue was attached to O-2 of the
inositol moiety and therefore O-6 carried the a-linked
sugar. The anomeric selectivity was increased by
changing the synthesis solvent to diethyl ether which
has been reported28 to favour the formation of a-glyco-
sidic bonds. Consistent with this finding, when the gly-
cosidation reaction was repeated in this solvent the
isolated yield of 5 was increased to 70% and that of 6
decreased to 10%.
In recent work, a bacterial antigen that induces interfer-
on-c (IFN-c) release in a CD1d restricted manner was
identified as a tetra-mannosylated-dipalmitoyl phospha-
tidyl inositol mannoside (PIM4).15 Also, the first crystal
structure of a CD1d–PIM2 complex, where the acyl
groups were palmitoyl residues, has recently been deter-
mined16 and revealed that the mannopyranosyl residue
bonded to O-6 of the inositol moiety projects away from
the surface of the protein implying that CD1d could
accommodate higher molecular weight ligands such as
PIM4 and PIM6. Their tri- and penta-manno-oligosac-
charide chains are linked to the O-6 position and would
also project away from the CD1d hydrophobic binding
groove. This work also revealed that the F0 binding
pocket, of the CD1d protein, accommodates the sn-2
palmitoyl group of the PIM2 molecule and that this
group was an optimal fit.
Routine protecting group manipulation as described
previously8 afforded alcohol 7 that was subsequently
coupled with phosphoramidite 8 to give the protected
PIM2 9 in good yield (Scheme 2). Hydrogenolytic deb-
enzylation of this material using reaction solvent mix-
tures that contained some water led to a small amount
of the deacylation product 10. This product was slightly
more polar than the desired PIM2 product so could be
separated by column chromatography. This side reac-
tion was overcome by using anhydrous solvent mixtures
and trapping the free acid as the triethylammonium salt.
In our ongoing programme to elucidate the biological
mechanism by which PIM compounds act, we have syn-
thesized and investigated the biological properties of dis-
crete PIM molecules.8 Here, we present an improved
synthesis of dipalmitoyl PIM2 (1), and, to our knowl-
edge, the first chemical synthesis of dipalmitoyl PIM4
(2). We have also investigated the adjuvant properties
of these molecules in an in vivo ovalbumin (OVA)
specific transgenic mouse model.
2. Results and discussion
2.1. Synthesis of PIM2
2.2. Synthesis of PIM4
The protected inositol 3 was also used as the starting
material for our synthesis of PIM4 2. The strategy
requires either a chemo-selective O-6 glycosylation of
acceptor 3 with an orthogonally protected mannosyl
donor or temporary protection of one of the hydroxyl
groups of 3 to establish the required unsymmetrical gly-
cosylation pattern. As we had previously demonstrated8
that 3 could be selectively glycosylated we chose the
former approach as the latter would necessitate both
protection and subsequent deprotection steps (Scheme
3). Glycosylation of the C-6 hydroxyl group of inositol
A number of approaches for the synthesis of PIM1 and
PIM2 compounds have been reported8,17–24 and, very
recently, the first synthesis of PIM6 has been pub-
lished.25 The synthetic methodologies used to date to
obtain a suitable unsymmetrical myo-inositol derivative
as starting material rely on its synthesis from glucose by
application of the Ferrier rearrangement or the derivati-
zation and desymmetrization of myo-inositol. Our
approach utilizes the former methodology to give the
BnO
OH
OH
OAc
O
BnO
OAc
BnO
BnO
BnO
BnO
O
BnO
BnO
OAllyl
(i) or (ii)
O
O
BnO
BnO
O
3
BnO
+
BnO
OAllyl
+
BnO
BnO
OAllyl
BnO
BnO
BnO
OAc
O
O
OBn
OBn
O
OBn
OBn
O
BnO
OBn
OAc
OC(NH)CCl3
OBn
OAc
6
5
4
˚
Scheme 1. Reagents: (i) TMSOTf, MS-4 A, CH2Cl2 (59% for 5 and 22% for 6); (ii) TMSOTf, MS-4 A, Et2O (70% for 5 and 10% for 6).
˚

G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 7615–7624
7617
RO
OR
BnO
OBn
O
O
RO
BnO
RO
BnO
O
O
O
RO
BnO
(i)
OR2
OR1
RO
BnO
O
O
OH
RO
BnO
P
O
O
OCOC15H31
OR
OR
OBn
OBn
O
O
OR
OBn
OR
OBn
7
OCOC15H31
9 R = R1 = Bn, R2 = COC15H31
1 R = H, R1 = Et3NH, R2 = COC15H31
10 R = R1 = R2 = H
(ii)
BnO
O
P
NiPr2
OCOC15H31
8
Scheme 2. Reagents: (i) 1H-tetrazole, 8, CH2Cl2 then m-CPBA (67%); (ii) Pd/C, MeOH, THF, H2 then Et3N (98%).
BnO
OAc
OH
OH
BnO
BnO
O
BnO
OAc
O
BnO
BnO
BnO
BnO
OR
O
BnO
BnO
BnO
OAllyl
(i)
O
O
BnO
(iii) or (iv)
O
OAllyl
BnO
BnO
+
3
BnO
BnO
OAllyl
OAllyl
BnO
BnO
+
OTBDPS
OBn
O
O
TBDPSO
OTBDPS
OBn
OR
OBn
OAc
O
OBn
O
O
OAc
BnO
BnO
OBn
OBn
OAc
OAc
12 R = H
13 R = Bz
(ii)
14
OC(NH)CCl3
11
15 R = TBDPS
16 R = H
(v)
˚
Scheme 3. Reagents: (i) TMSOTf, MS-4 A, CH2Cl2 (73%); (ii) Py, BzCl, DMAP (44%); (iii) 4, TMSOTf, MS-4 A, CH2Cl2 (54% for 14); (iv) 4,
˚
˚
TMSOTf, MS-4 A, Et2O (80% for 15); (v) TBAF, THF (47%).
acceptor 3 with orthogonally protected donor 11 affor-
ded the mono-mannoside 12 in good yield (Scheme 3).
The glycosylation site was established by benzoylation
of 12 to give the 2-O-benzoyl-inositol 13. The expected
downfield shift (4.18 ppm for 12 to 5.98 ppm for 13)
for the characteristic broad triplet of the inositol H-2
acid-labile triisopropylsilyl group as this group was used
successfully in the synthesis of PIM6.25
The synthesis of disaccharide donor 17 from 4 and thio-
glycoside 18,29 through the intermediacy of 19 proceed-
ed smoothly. Glycosylation of the pseudo-trisaccharide
16 with trichloroacetimidate 17 using standard condi-
tions gave pseudo-pentasaccharide 20 in 80% yield.
The newly formed glycosidic bond was confirmed as
having a-stereochemistry as all of the four one-bond
C–H coupling constants of the anomeric carbon signals
were in the range of 172–175 Hz. Routine manipulation
of the protecting groups of 20 by deacetylation to 21,
subsequent benzylation to 22 and deallylation gave the
protected PIM4 headgroup 23 in 63% overall yield for
the sequence Scheme 4.
1
proton was evident in the H NMR spectra. The a-ano-
meric configuration of glycoside 12 was established from
the one-bond 13C–1H coupling constant of 177 Hz for
the C-10 resonance in the coupled 13C NMR spectrum.
Surprisingly, glycosylation of pseudo-disaccharide 12
with 4 in dichloromethane afforded the a,b-bis-manno-
side 14 as the major product. Although we observed
some formation of the b-glycoside in the preparation
of PIM2 (vide infra), we did not expect that b-glycosyl-
ation would dominate in this reaction. Gratifyingly,
the use of diethyl ether as the reaction solvent reversed
the stereoselectivity of the mannosylation such that the
a,a-bis-mannoside 15 was obtained in high yield. The
anomeric carbons of 15 resonated at 99.0 and
98.6 ppm in the 13C NMR spectrum with the one-bond
13C–1H coupling constants of 173 Hz each confirming
the assignment of anomeric stereochemistry. The next
step in the sequence involved unblocking the 60-hydroxyl
group for glycosylation. Removal of the TBDPS ether
group to give alcohol 16 proved to be problematic.
A number of reaction conditions were investigated but
the yield could not be improved above 50%. Forcing
reaction conditions lead to the formation of deacylation
products. We are currently investigating the use of the
The phosphodiester linkage was introduced by coupling
excess phosphoramidite 8 (4 equiv) with 23 (Scheme 5)
to give protected PIM4 24 in 52% yield as a 1:1.5 mix-
ture of stereoisomers about the phosphorus centre.
The final step involved hydrogenolytic debenzylation
under anhydrous conditions to furnish dipalmitoyl
PIM4 2 in 89% yield.
2.3. Adjuvant properties of PIM2 and PIM4
Adjuvants normally generate relatively subtle quantita-
tive changes in the immune response to an antigen.
For this reason, we used an ovalbumin (OVA)-specific

7618
G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 7615–7624
BnO
OAc
O
BnO
OAc
O
BnO
BnO
BnO
HO
BnO
BnO
OAc
O
(i)
(ii), (iii)
O
OAc
O
O
OAc
O
BnO
BnO
BnO
BnO
SPh
BnO
SPh
OC(NH)CCl3
19
17
18
BnO
BnO
BnO
BnO
OAc
O
OR
O
BnO
BnO
20 R = Ac, R1 = Allyl
21 R = H, R1 = Allyl
22 R = Bn, R1 = Allyl
23 R = Bn, R1 = H
(v)
(vi)
(vii)
O
O
BnO
BnO
BnO
BnO
BnO
BnO
(iv)
OAllyl
OR1
O
O
O
OBn
OH
O
O
O
OBn
OBn
O
OBn
OBn
OBn
16
OBn
OBn
O
OAc
OR
OBn
OR
OR
˚
Scheme 4. Reagents: (i) 4, TMSOTf, MS-4 A, CH2Cl2 (67%); (ii) NIS, TfOH, H2O, Me2CO (62%); (iii) DBU, Cl3CCN, CH2Cl2 (87%); (iv) 17,
TMSOTf, MS-4 A, Et2O (80%); (v) NaOMe, MeOH (85%); (vi) NaH, BnBr, DMF (80%); (vii) (Ph2MeP)2(COD)Ir+ PF6ꢀ, THF then AcCl, CH2Cl2,
˚
MeOH (92%).
RO
30
25
20
15
10
5
OR
O
RO
RO
O
RO
OCOC15H31
OCOC15H31
OR
(i)
RO
23
O
O
RO
P
O
O
O
O
O
OR
OR
O
24 R = Bn
R = H
OR
OR
OR
(ii)
O
0
OR
2
OR
OR
OVA alone
OVA+CFA
OVA +
OVA +
OR
OR
synPIM2
synPIM4
Scheme 5. Reagents: (i) 1H-tetrazole, 8, CH2Cl2 then m-CPBA (52%);
(ii) Pd(OH)2/C, MeOH, THF, H2 (89%).
Figure 1. Effects of subcutaneous administration of PIM2 and PIM4
with the model antigen, OVA, for the development of an immune
response in C57BL/6 mice which had been transferred with OT-II
spleen cells. Mice were given 100 lg of OVA emulsified in CFA, or
with 36 lg of PIM2 or PIM4. Axillary lymph node cells were isolated 4
days following injection. Cells were cultured for 4 days with 1 lM of
OVA peptide, after which IFN-c levels were measured in supernatants.
Results are means of triplicates, repeated twice with similar results.
Standard errors of the means were in the range of 5%.
MHC class II restricted ab T cell receptor (TCR) trans-
genic mouse model (OT-II) for assessing the adjuvant
properties of PIM2 and PIM4. Expression of the T cell
receptors Va2 and Vb5.1 is increased to 20–40% of total
T cells in these mice, compared to the wild-type C57BL/6,
where expression is less than 1%.30 This increased
expression leads to a robust immune response versus
OVA (used here as a model antigen), and increases the
opportunities for detecting changes when testing adju-
vants, that otherwise would not be apparent.
OVA alone (P < 0.01), although PIM2 was more effi-
cient than PIM4 (P < 0.01). It was observed that injec-
tion of OVA and CFA induced the formation of
substantial abscesses and fibrosis in subcutaneous tis-
sues, whereas OVA and PIM2 and PIM4 induced only
a slight reddening of tissues, with no abscesses nor fibro-
sis. Data were analyzed for significance using a Fischer’s
unprotected test with pairwise comparisons.
Mice were administered 100 lg of OVA by the subcuta-
neous route with the synthetic PIMs (36 lg) as an adju-
vant, or OVA emulsified in CFA (0.1 mL) given as a
positive control adjuvant. The ability of lymph node
cells from the axillary lymph nodes to release IFN-c
in vitro in response to OVA was then evaluated, as a
measure of the magnitude of the induction of a TH1
response. Figure 1 shows that administration of OVA with
synthetic PIM2 generated significant IFN-c release
(about 40% of that compared with CFA). In compari-
son, PIM4 was also able to generate significant IFN-c
release but only at about 20% of that compared with
CFA. Both PIM2 and PIM4 induced significant
enhancements of IFN-c release, compared to mice given
3. Conclusions
We have prepared synthetic samples of PIM2 1 and
PIM4 2 and evaluated their adjuvant activities in an
OVA-specific mouse model. Both synthetic molecules
given as adjuvants with a model antigen resulted in sig-
nificantly higher quantities of IFN-c released by cells of

G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 7615–7624
7619
immunized mice, with PIM2 being more potent than
PIM4. Significantly, in contrast to CFA, neither com-
pound caused any local edema nor visible lesions follow-
ing administration. We are currently undertaking
further studies to fully evaluate the potential of this class
of molecule to act as adjuvants, both when given paren-
terally and by the mucosal route.
139.1, 138.7, 138.6, 138.3, 138.0, 137.8, 135.1, 128.6,
128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7,
127.6, 127.5, 127.3, 127.2, 117.5, 98.6, 98.2, 81.5, 81.3,
81.1, 80.4, 78.6, 78.2, 75.8, 75.6, 75.5, 75.2 (2· CH2),
74.9, 74.7, 74.3, 73.5, 73.4, 73.2, 71.7, 71.5, 71.4, 70.1,
69.8, 68.9, 68.4, 67.9, 21.2. Gated decoupled 13C NMR
1
0
0
(75 MHz, CDCl3) selected data,
d
98.6, JC1 ꢀH1
161 Hz, d 98.2, JC1 ꢀH1 176 Hz. HRMS-ESI [M+Na]+
calcd for C88H94O18Na: 1461.6338. Found 1461.6371.
Further elution afforded 5 (1.77 g, 1.23 mmol, 59%).
1
00
00
4. Experimental procedures for synthesis of
PIM analogues
4.1.2. 1-O-Allyl-2,6-di-O-(2-O-acetyl-3,4,6-tri-O-benzyl-
a-D-mannopyranosyl)-3,4,5-tri-O-benzyl-D-myo-inositol
(5).8 TMSOTf (24 lL, 0.13 mmol) was added drop-wise
to a stirred mixture of 3 (217 mg, 0.442 mmol), 2-O-ac-
etyl-3,4,6-tri-O-benzyl-a-D-mannopyranosyl trichloro-
4.1. General
Specific optical rotations, given in 10ꢀ1 deg cm2 gꢀ1
,
were measured at ambient temperature using a Perkin-
Elmer 241 polarimeter with a cell of path length
1.0 dm. H NMR spectra were obtained at either 300
˚
acetimidate (4) (844 mg, 1.33 mmol) and 4 A molecular
1
sieves (100 mg) in Et2O (20 mL) cooled to ꢀ30 ꢁC. The
reaction mixture was allowed to warm to 0 ꢁC over
90 min when Et3N (2.0 mL) was added. The mixture
was filtered through Celiteꢂ and the filter cake washed
with further Et2O (2 · 100 mL). The solvent was
removed and the residue purified by column chromato-
graphy on silica gel. Elution with EtOAc/light petroleum
(1:9 to 3:7) afforded 6 (63 mg, 0.044 mmol, 10%)
or 500 MHz and referenced to either the residual solvent
peak or tetramethylsilane (0.0 ppm). 13C NMR spectra
were recorded at 75 or 125 MHz and referenced to the
residual solvent peak (77.08). 31P NMR spectra were
recorded at 121.5 or 202.5 MHz. Chemical shifts are
reported using the d scale, and coupling constants (J)
are reported in Hz. High resolution electro-spray ioniza-
tion (ESI) mass spectra were recorded on a Waters
Micromass Q-Tof Premier mass spectrometer. Elemen-
tal analyses were carried out by the Campbell Microan-
alytical Laboratory, University of Otago, Dunedin, New
Zealand. Thin-layer chromatography (TLC) was per-
formed on Merck silica gel 60 F254 plates and was visu-
alized under UV light and/or with a cerium sulfate–
ammonium molybdate solution and subsequent heating.
Flash column chromatography was carried out using
Scharlau silica gel 60 (0.04–0.06 mm, 230–400 mesh).
All chromatography solvents were of reagent grade.
Dry solvents were purchased from Aldrich.
1
followed by 5 (445 mg, 0.309 mmol, 70%) as an oil. H
and 13C NMR spectra were consistent with the previous-
ly reported data. Anal. (C88H94O18) C, H.
4.1.3. 3,4,5-Tri-O-benzyl-2,6-di-O-2,3,4,6-tetra-O-ben-
zyl-a-D-mannopyranosyl-1-O-(1,2-di-O-hexadecanoyl-sn-
glycero-3-benzylphosphoryl)-D-myo-inositol (9). 1H-Tet-
razole (41 mg, 0.59 mmol) was added to a stirred solu-
tion of 3,4,5-tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-
benzyl-a-D-mannopyranosyl)-D-myo-inositol (7) and
phosphoramidite 8 (476 mg, 0.590 mmol) in dry CH2Cl2
(8 mL) cooled to 0 ꢁC under an argon atmosphere. After
stirring at rt for 2 h the reaction mixture was cooled to
ꢀ40 ꢁC and a solution of m-CPBA (50%, 204 mg,
0.591 mmol) in CH2Cl2 (10 mL) was transferred by can-
nular into the reaction mixture. After warming to rt over
2 h, the reaction was quenched with the addition of aq
Na2SO3 (10%, 50 mL) and the combined mixture
extracted with Et2O (100 mL). The ethereal extract
was washed with aq NaHCO3 (satd, 3· 50 mL) and
dried (MgSO4). After filtration, the solvent was removed
in vacuo and the residue purified by column chromatog-
raphy on silica gel. Elution with EtOAc/light petroleum
(1:9 to 1:4) afforded the title compound 9 (175 mg,
0.079 mmol, 67%) as an oil. 1H NMR (300 MHz,
CDCl3) d 7.39–7.01 (m, 60H), 5.49 (br s, 1H), 5.37–
5.29 (m, 1H), 5.12–4.40 (m, 24H), 4.30–3.75 (m, 17H),
3.52–3.20 (m, 6H), 2.23–2.09 (m, 4H), 1.58–1.41 (m,
4H), 1.31–1.17 (m, 48H), 0.89–0.82 (m, 6H). 31P NMR
4.1.1. 1-O-Allyl-2-O-(2-O-acetyl-3,4,6-tri-O-benzyl-b-D-
mannopyranosyl)-6-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-
D-mannopyranosyl)-3,4,5-tri-O-benzyl-D-myo-inositol (6)
and 1-O-Allyl-2,6-di-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-D-
mannopyranosyl)-3,4,5-tri-O-benzyl-D-myo-inositol (5)8.
TMSOTf (200 lL, 1.11 mmol) was added drop-wise to
a stirred mixture of 1-O-allyl-3,4,5-tri-O-benzyl-D-myo-
inositol (3) (1.02 g, 2.08 mmol), 2-O-acetyl-3,4,6-tri-O-
benzyl-a-D-mannopyranosyl trichloroacetimidate (4)
˚
(2.83 g, 4.44 mmol) and 4A molecular sieves (400 mg) in
CH2Cl2 (60 mL) cooled to ꢀ40 ꢁC. The reaction mixture
was allowed to warm to 0 ꢁC over 90 min when Et3N
(5.0 mL) was added. The mixture was filtered through
Celiteꢂ and the filter cake washed with further CH2Cl2
(2· 200 mL). The solvent was removed and the residue
purified by column chromatography on silica gel. Elution
with EtOAc/light petroleum (1:9 to 3:7) afforded the a,b-
(121.5 MHz, CDCl3)
d
1.16, 0.94. HRMS-ESI
diglycoside 6 (650 mg, 0.451 mmol, 22%) as an oil.
[M+Na]+ calcd for C137H171O23NaP: 2238.1847. Found
2238.1833. Anal. (C137H171O23P) C, H, P.
20
D
½aꢁ ꢀ 18:4 (c 2.72, CH2Cl2). 1H NMR (300 MHz,
CDCl3) d 7.37–7.01 (m, 45H), 6.01–5.88 (m, 1H), 5.70
(d, J = 3.1 Hz, 1H), 5.45 (s, 2H), 5.22–5.06 (m, 2H),
4.93–4.38 (m, 19H), 4.29 (d, J = 12.0 Hz, 1H), 4.23–4.17
(m, 1H), 4.00–3.90 (m, 6H), 3.79–3.66 (m, 3H), 3.53
(dd, J = 9.2, 3.3 Hz, 1H), 3.43–3.20 (m, 6H), 2.17 (s,
3H), 2.10 (s, 3H). 13C NMR (75 MHz) d 171.0, 170.4,
4.1.4. 2,6-(Di-O-a-D-mannopyranosyl)-1-O-(1-O-hexa-
decanoyl-sn-glycero-3-phosphoryl)-D-myo-inositol (10).
Pd(OH)2/C (20%, 31 mg) was added to a stirred solution
of 7 (30 mg, 0.014 mmol) in CHCl3/MeOH/H2O
(1:1:0.1, 2.1 mL). The mixture was stirred under a

7620
G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 7615–7624
hydrogen atmosphere for 10 h at rt when the hydrogen
was replaced with argon and the mixture was filtered
through Celiteꢂ and concentrated in vacuo. The residue
was purified on silica gel eluting with CHCl3 then
CHCl3/MeOH/H2O (70:40:1 to 70:40:4) to afford
PIM2 (8.0 mg, 0.0070 mmol, 50%) followed by the title
compound (3.0 mg, 0.0033 mmol, 24%). 1H NMR
(300 MHz, CDCl3/CD3OD/D2O, 70:40:6) d 5.18 (br s,
1H), 5.11 (br s, 1H), 4.34 (br s, 1H), 4.18–3.93 (m,
7H), 3.87–3.58 (m, 10H), 3.59–3.42 (m, 1H), 3.31–3.25
(m, 1H), 2.36 (t, J = 7.5 Hz, 2H), 1.67–1.55 (m, 2H),
1.67–1.55 (m, 2H), 1.35–1.21 (m, 24H), 0.88 (t,
J = 6.6 Hz, 3H). 31P NMR (121.5 MHz, CDCl3/
CD3OD/D2O, 70:40:6) d 1.21. HRMS-ESI [MꢀH]ꢀ
calcd for C37H68O22P: 895.3940. Found 895.3950.
136.1, 135.6, 134.5, 134.3, 133.5, 129.5, 129.4, 128.6,
128.4, 128.3, 128.1, 128.1, 128.0. 127.7, 127.6, 127.5,
127.3, 127.2, 118.1, 98.2, 81.4, 81.2, 80.6, 79.8, 78.1,
75.9, 75.5, 75.3, 75.1, 74.2, 72.8, 72.4, 71.8, 71.4, 69.2,
66.9, 62.4, 26.8, 21.0, 19.4. Gated decoupled 13C NMR
1
0
0
(75 MHz, CDCl3) selected data, d 98.2, JC1 ꢀH1
176 Hz. HRMS-ESI [M+Na]+ calcd for C68H76O12Na-
Si: 1135.5004. Found 1135.5034.
4.1.7.
1-O-Allyl-6-O-(2-O-acetyl-3,4-di-O-benzyl-6-O-
tert-butyldiphenylsilyl-a-D-mannopyranosyl)-2-O-benzo-
yl-3,4,5-tri-O-benzyl-D-myo-inositol (13). Benzoyl chlo-
ride (22 lL, 0.19 mmol) was added drop-wise to a
stirred solution of alcohol 12 (69 mg, 0.062 mmol) and
DMAP (1.0 mg, 0.008 mmol) in pyridine (5 mL) cooled
to 0 ꢁC. After being stirred for 10 h at rt, the solvent was
removed in vacuo and the residue partitioned with H2O
(50 mL) and Et2O (100 mL). The ethereal extract was
washed with aq HCl (0.5 M, 50 mL), aq NaHCO3 (satd,
50 mL) and brine (50 mL). After drying (MgSO4) and
filtration the solvent was removed in vacuo and the res-
idue purified by column chromatography on silica gel.
Elution with EtOAc/light petroleum (1:4 to 3:7) afforded
the title compound 13 as an oil (33 mg, 0.027 mmol,
44%). 1H NMR (300 MHz, CDCl3) d 8.07–7.99 (m,
2H), 7.66–7.52 (m, 5H), 7.40–7.10 (m, 30H), 7.06–6.98
(m, 1H), 6.90–6.84 (m, 2H), 6.04–5.90 (m, 2H), 5.44–
5.42 (m, 2H), 5.31–5.24 (m, 2H), 4.96–4.54 (m, 10H),
4.25–3.88 (m, 7H), 3.65–3.61 (2H, m), 3.56 (dd,
J = 9.9, 2.7 Hz, 1H), 3.47 (dd, J = 9.6, 2.7 Hz, 1H),
3.38 (t, J = 9.6 Hz, 1H), 2.07 (s, 3H), 1.02 (s, 9H). 13C
NMR (75 MHz, CDCl3) d 170.4, 166.0, 139.2, 138.6,
138.3, 137.8, 137.7, 136.0, 135.6, 134.4, 134.3, 133.4,
133.1, 129.9, 129.5, 128.6, 128.4, 128.3, 128.2, 127.9,
127.8 127.7, 127.6, 127.4, 127.3, 118.3, 98.2, 81.8, 81.2,
78.7, 78.4, 78.1, 76.2, 75.8, 75.3, 74.2, 72.5, 72.2, 71.8,
71.1, 69.0, 66.9, 62.4, 26.8, 21.1, 19.4. HRMS-ESI
[M+Na]+ calcd for C75H80O13NaSi: 1239.5266. Found
1239.5304.
4.1.5. Triethyl ammonium 2,6-(di-O-a-D-mannopyrano-
syl)-1-O-(1,2-di-O-hexadecanoyl-sn-glycero-3-phosphor-
yl)-D-myo-inositol (1). Pd(OH)2/C (20%, 40 mg) was
added to stirred solution of 7 (112 mg, 0.0505 mmol)
in THF/MeOH (2:3, 5 mL). The mixture was stirred un-
der a hydrogen atmosphere for 2.5 h at rt when the
hydrogen was replaced with argon and triethylamine
(1.0 mL) was added. The mixture was filtered through
Celiteꢂ and concentrated in vacuo. The residue was
lyophilized to afford 1 as its tetrahydrate triethylammo-
nium salt (62 mg, 0.050 mmol, 98%) as a white powder.
20
½aꢁ þ 38 (c 0.19, CHCl3/CH3OH/H2O, 70:40:6). 1H
D
NMR (300 MHz, CDCl3/CD3OD/D2O, 70:40:6)
d
5.31–5.22 (m, 1H), 5.13 (br s, 1H), 5.10 (br s, 1H),
4.45–4.40 (m, 1H), 4.30 (br s, 1H), 4.08–3.92 (m, 7H),
3.87–3.58 (m, 10H), 3.50–3.42 (m, 1H), 3.31–3.25 (m,
1H), 3.10 (q, J= 7.5 Hz, 6H), 2.40–2.27 (m, 4H), 1.67–
1.55 (m, 4H), 1.33–1.21 (m, 57H), 0.89 (t, J = 6.9 Hz,
6H). 31P NMR (121.5 MHz, CDCl3/CD3OD/D2O,
70:40:6) d ꢀ3.7. HRMS-ESI [MꢀEt3NH]ꢀ calcd for
C53H98O23P: 1133.6237. Found 1133.6232. Anal.
(C59H114O23NP. 4H2O) C, H, N.
4.1.6.
1-O-Allyl-6-O-(2-O-acetyl-3,4-di-O-benzyl-6-O-
tert-butyldiphenylsilyl-a-D-mannopyranosyl)-3,4,5-tri-O-
benzyl-D-myo-inositol (12). TMSOTf (40 lL, 0.22 mmol)
was added drop-wise to a stirred mixture of 3 (1.08 g,
2.20 mmol), 2-O-acetyl-3,4-di-O-benzyl-6-O-tert-buty-
ldiphenylsilyl-a-D-mannopyranosyl trichloroacetimidate
4.1.8. 1-O-Allyl-2-O-(2-O-acetyl-3,4,6-tri-O-benzyl-b-D-
mannopyranosyl)-6-O-(2-O-acetyl-3,4-di-O-benzyl-6-O-
tert-butyldiphenylsilyl-a-D-mannopyranosyl)-3,4,5-tri-O-
benzyl-D-myo-inositol
(14).
TMSOTf
(7.0 lL,
0.04 mmol) was added drop-wise to a stirred mixture
of 12 (208 mg, 0.187 mmol), trichloroacetimidate 4
˚
(237 mg, 0.373 mmol) and 4 A molecular sieves
(400 mg) in CH2Cl2 (15 mL) at ꢀ30 ꢁC. After 10 min,
Et3N (0.5 mL) was added and the mixture was filtered
through Celiteꢂ. The solvent was removed in vacuo
and the residue purified by column chromatography
on silica gel. Elution with acetone/toluene (1:49 to
˚
(11) (2.07 g, 2.64 mmol) and 4 A molecular sieves
(120 mg) in CH2Cl2 (30 mL) at ꢀ30 ꢁC. After 10 min
Et3N (2.5 mL) was added. The mixture was filtered
through Celiteꢂ and the filter cake washed with further
CH2Cl2 (2· 100 mL). The solvent was removed in vacuo
and the residue purified by column chromatography
on silica gel. Elution with EtOAc/light petroleum (1:9
to 1:4) afforded the title compound 12 (1.78 g,
1:19) afforded title compound 14 (160 mg, 0.101 mmol,
20
20
D
1.60 mmol, 73%) as an oil. ½aꢁ þ 17 (c 0.68, CHCl3).
54%) as an oil. ½aꢁ þ 5:0 (c 0.80, CHCl3). 1H
D
1H NMR (300 MHz, CDCl3) d 7.69 (d, J = 6.5 Hz,
2H), 7.58 (d, J = 6.5 Hz, 2H), 7.42–7.07 (m, 28H),
7.02–6.96 (m, 1H), 6.89–6.81 (m, 2H), 6.04–5.91 (m,
1H), 5.42–5.40 (m, 2H), 5.32–5.5.20 (m, 2H), 4.92–4.66
(m, 7H), 4.61–4.56 (m, 3H), 4.18 (t, J = 2.6 Hz, 1H),
4.15–3.83 (m, 7H), 3.57–3.49 (m, 2H), 3.38 (dd,
J = 9.6, 2.4 Hz, 1H), 3.30–3.22 (m, 2H), 2.38 (br s,
1H), 2.10 (s, 3H), 1.03 (s, 9H). 13C NMR (75 MHz,
CDCl3) d 170.4, 139.2, 138.6, 138.3, 138.1, 138.0,
NMR (500 MHz, CDCl3) d 7.71–7.68 (m, 2H), 7.60–
7.57 (m, 2H), 7.40–7.10 (m, 43H), 7.01 (t, J = 7.4 Hz,
1H), 6.91 (t, J = 7.7 Hz, 2H), 6.07–5.98 (m, 1H), 5.74
(d, J = 3.4 Hz, 1H), 5.48–5.46 (m, 1H), 5.44–5.42 (m,
1H), 5.22 (dd, J = 17.2, 1.7 Hz, 1H), 5.16–5.12 (m,
1H), 4.93–4.75 (m, 8H), 4.70 (d, J = 11.2 Hz, 1H),
4.61–3.51 (m, 7H), 4.40 (d, J = 11.2 Hz, 1H), 4.36 (t,
J = 2.3 Hz, 1H), 4.26–4.20 (m, 2H), 4.05–3.90 (m, 4H),
3.83–3.66 (m, 4H), 3.55–3.41 (m, 4H), 3.36 (dd,

G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 7615–7624
7621
J = 9.8, 2.1 Hz, 1H), 3.28 (t, J = 9.4 Hz, 1H), 3.23 (dd,
J = 9.7, 2.6 Hz, 1H), 2.13 (s, 3H), 2.05 (s, 3H), 1.06 (s,
9H). 13C NMR (125 MHz, CDCl3) d 170.9, 170.3,
139.2, 138.7, 138.4, 138.2, 138.2, 138.1, 137.8, 136.0,
135.6, 135.3, 134.3, 133.4, 129.5, 129.4, 128.6, 128.5,
128.4, 128.4, 128.4, 128.3, 128.2, 128.1, 128.0. 128.0,
127.9, 127.9, 127.8, 127.7, 127.6, 127.6, 127.5, 127.5,
127.2, 127.2, 127.0, 117.7, 99.2, 98.4, 81.4, 81.2, 81.0,
80.5, 78.5, 78.3, 76.2, 75.8, 75.5, 75.3, 75.0, 74.5, 73.9,
73.6, 73.4, 72.7, 72.3, 71.7, 71.4, 70.3, 69.9, 69.0, 67.9,
washed with HCl (0.5 M, 50 mL), aq NaHCO3 (satd,
50 mL) and brine (50 mL). After drying (MgSO4) and
filtration, the solvent was removed in vacuo and the res-
idue purified by column chromatography on silica gel.
Elution with EtOAc/light petroleum (3:7 to 1:1 to 3:1)
afforded the starting material 15 (88 mg, 0.055 mmol,
19%) followed by the title compound 16 (180 mg,
20
D
0.133 mmol, 47%) as an oil. ½aꢁ þ 99 (c 0.88,
1
CHCl3). H NMR (300 MHz, CDCl3) d 7.41–7.09 (m,
40H), 5.94–5.82 (m, 1H), 5.49–5.41 (m, 3H), 5.25–5.13
(m, 3H), 4.92–4.52 (m, 14H), 4.42 (d, J = 10.8 Hz,
1H),4.34–4.29 (m, 2H), 4.14–3.79 (m, 10H), 3.50–3.23
(m, 7H), 2.17 (s, 3H), 2.16 (s, 3H). 13C NMR
(75 MHz, CDCl3) d 170.1, 138.8, 138.7, 138.4, 138.2,
138.1, 137.9, 137.9, 133.9, 128.4, 128.4, 128.3, 128.3,
128.2, 128.3, 127.9, 127.9, 127.9, 127.8, 127.7, 127.6,
127.5, 127.5, 127.4, 127.3, 117.6, 98.8, 98.2, 81.4, 81.1,
78.8, 77.9, 77.5, 76.1, 75.7, 75.6, 75.1, 75.0, 74.1, 73.4,
72.5, 71.8, 71.6, 71.5, 71.5, 71.1, 68.7, 68.6, 68.6, 61.5,
21.1, 21.0. HRMS-ESI [M+Na]+ calcd for
C81H88O18Na: 1371.5868. Found 1371.5873.
62.3, 26.9, 21.1, 21.1, 19.5. Gated decoupled 13C NMR
1
0
0
(125 MHz, CDCl3) selected data, d 99.2, JC1 ꢀH1
1
00
00
160 Hz,
d
99.2,
JC1 ꢀH1
176 Hz. HRMS-ESI
[M+Na]+ calcd for C97H106O18NaSi: 1609.7046. Found
1609.7087. Further elution afforded the di-a mannoside
15 (119 mg, 0.0749 mmol, 40%), characterized below.
4.1.9. 1-O-Allyl-2-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-D-
mannopyranosyl)-6-O-(2-O-acetyl-3,4-di-O-benzyl-6-O-
tert-butyldiphenylsilyl-a-D-mannopyranosyl)-3,4,5-tri-O-
benzyl-D-myo-inositol (15). TMSOTf (30 lL, 0.17 mmol)
was added drop-wise to a stirred mixture of 12 (920 mg,
0.828 mmol), trichloroacetimidate 4 (1.05 g, 1.65 mmol)
4.1.11. Phenyl 2-O-acetyl-6-O-(2-O-acetyl-3,4,6-tri-O-
benzyl-a-D-mannopyranosyl)-3,4-di-O-benzyl-1-thio-a-D-
mannopyranoside (19). TMSOTf (55 lL, 0.30 mmol) was
added drop-wise to a stirred mixture of 18 (1.15 g,
2.33 mmol), trichloroacetimidate 4 (1.92 g, 3.02 mmol)
˚
and 4 A molecular sieves (400 mg) in Et2O (20 mL) at
ꢀ30 ꢁC. The reaction mixture was allowed to warm to
0 ꢁC over 30 min when Et3N (2.5 mL) was added. The
mixture was filtered through Celiteꢂ and the filter cake
washed with further CH2Cl2 (2· 100 mL). The solvent
was removed in vacuo and the residue purified by col-
umn chromatography on silica gel. Elution with ace-
˚
and 4 A molecular sieves (150 mg) in CH2Cl2 (35 mL)
cooled to ꢀ30 ꢁC. The reaction mixture was allowed
to warm to 0 ꢁC over 90 min when Et3N (2.0 mL) was
added. The mixture was filtered through Celiteꢂ and
the filter cake washed with further CH2Cl2
(2· 100 mL). The solvent was removed in vacuo and
the residue purified by column chromatography on silica
gel. Elution with EtOAc/light petroleum (1:9 to 1:4 to
tone/toluene (1:49 to 3:97) afforded the title compound
20
D
15 (1.05 g, 0.661 mmol, 80%) as an oil. ½aꢁ þ 42:0
(c 1.06, CHCl3). 1H NMR (500 MHz, CDCl3)
d
7.70–7.67 (m, 2H), 7.58–7.55 (m, 2H), 7.41–7.12 (m,
43H), 7.03 (t, J = 7.4 Hz, 1H), 6.92 (t, J = 7.7 Hz, 2H),
6.01–5.92 (m, 1H), 5.50–5.41 (m, 3H), 5.29–5.19 (m,
3H), , 4.95–4.70 (m, 8H), 4.64–4.57 (m, 5H), 4.53 (d,
J = 11.2 Hz, 1H), 4.41 (d, J = 10.7 Hz, 1H), 4.33 (t,
J = 2.3 Hz, 1H), 4.30 (d, J = 12.0 Hz, 1H), 4.25–3.93
(m, 8H), 3.88 (d, J = 9.8 Hz, 1H), 3.79 (t, J = 9.6 Hz,
1H), 3.56–3.47 (m, 3H), 3.31–3.22 (m, 4H), 2.19 (s,
3H), 2.15 (s, 3H), 1.04 (s, 9H). 13C NMR (125 MHz,
CDCl3) d 170.4, 170.0, 139.2, 138.8, 138.4, 138.3,
138.2, 138.0, 137.9, 137.9, 136.0, 135.6, 134.2, 134.1,
133.2, 129.5, 129.4, 128.6, 128.5, 128.4, 128.4, 128.3,
128.2, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.7,
127.6, 127.6, 127.5, 127.4, 127.3, 117.8, 99.0, 98.6,
81.5, 81.2, 80.9, 78.7, 78.4, 76.7, 75.7, 75.7, 75.1, 75.0,
74.1, 74.0, 73.4, 72.6, 72.4, 71.7, 71.7, 71.6, 71.4, 68.8,
68.6, 68.5, 62.3, 26.9, 21.3, 21.2, 19.4. Gated decoupled
13C NMR (125 MHz, CDCl3) selected data, d 99.0,
1:3) afforded the title compound 19 (1.52 g, 1.56 mmol,
20
D
67%) as an oil. ½aꢁ þ 60 (c 1.1, CHCl3). 1H NMR
(300 MHz, CDCl3) d 7.43–7.09 (m, 30H), 5.61–5.58
(m, 1H), 5.45–5.42 (m, 2H), 4.95–4.83 (m, 3H), 4.74–
4.41 (m, 8H), 4.31–4.25 (m, 1H), 3.97–3.58 (m, 9H),
2.13 (s, 3H), 2.12 (s, 3H). 13C NMR (75 MHz,
CDCl3) d 170.4, 170.3, 138.6, 138.3, 137.9, 137.6,
133.9, 131.5, 129.3, 128.6, 128.5, 128.4, 128.3, 128.2,
128.0, 127.9, 127.8, 127.7, 127.6, 98.1, 86.3, 78.6, 78.0,
75.2, 74.4, 74.3, 73.4, 72.3, 71.9, 71.7, 71.6, 70.3, 68.8,
68.5, 66.2, 21.2, 21.0. Gated decoupled 13C NMR
1
0
0
(125 MHz, CDCl3) selected data, d 98.1, JC1 –H1
172 Hz, d 86.3, JC1–H1 169 Hz. HRMS-ESI [M+Na]+
1
calcd for C57H60O12NaS: 991.3703. Found 991.3664.
4.1.12. 2-O-Acetyl-6-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-
D-mannopyranosyl)-3,4-di-O-benzyl-a-D-mannopyranosyl
trichloroacetimidate (17). N-Iodosuccinimide (414 mg,
1.84 mmol) followed by triflic acid (5.0 lL, 0.06 mmol)
was added to a stirred solution of 19 (595 mg,
0.614 mmol) in acetone/H2O (9:1, 20 mL). After stirring
at rt for 12 h, the reaction mixture was diluted with
CH2Cl2 (100 mL). The organic phase was washed with
aq Na2S2O3 (10%, 50 mL), aq NaHCO3 (satd, 50 mL),
brine (50 mL) and dried (MgSO4). After filtration, the
solvent was removed in vacuo and the residue purified
by column chromatography on silica gel. Elution
with EtOAc/light petroleum (3:7 to 1:1) afforded the
1
1
0
0
00
00
JC1 ꢀH1 173 Hz, d 98.6, JC1 ꢀH1 173 Hz. HRMS-ESI
[M+Na]+ calcd for C97H106O18NaSi: 1609.7046. Found
1609.7014.
4.1.10. 1-O-Allyl-2-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-D-
mannopyranosyl)-6-O-(2-O-acetyl-3,4-di-O-benzyl-a-D-
mannopyranosyl)-3,4,5-tri-O-benzyl-D-myo-inositol (16).
Tetrabutylammonium fluoride (1 M in THF, 2.00 mL,
2.00 mmol) was added to stirred solution of the silyl
ether 15 (450 mg, 0.283 mmol) and acetic acid (17 lL,
0.29 mmol) in THF (10 mL) heated to 40 ꢁC. After
5 d, the mixture was diluted with EtOAc (100 mL) and

7622
G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 7615–7624
intermediate hemiacetal (332 mg, 0.379 mmol, 62%) that
was dissolved in CH2Cl2 (15 mL) and cooled to 0 ꢁC.
Trichloroacetonitrile (380 lL, 3.79 mmol) and DBU
(6 lL, 0.04 mmol) were then added. After 2 h, the sol-
vent was removed in vacuo and the residue purified by
column chromatography on silica gel. Elution with
EtOAc/light petroleum (1:5 to 3:7) afforded the title
4.1.14. 1-O-Allyl-2-O-(3,4,6-tri-O-benzyl-a-D-mannopyr-
anosyl)-6-O-[(3,4,6-tri-O-benzyl-a-D-mannopyranosyl)-
(1 ! 6)-(3,4-di-O-benzyl-a-D-mannopyranosyl)-(1 ! 6)-
(3,4-di-O-benzyl-a-D-mannopyranosyl)]-3,4,5-tri-O-ben-
zyl-D-myo-inositol (21). Sodium methoxide in MeOH
(30%, approx 0.05 mL) was added drop-wise to a stirred
solution of tetraacetate 20 (251 mg, 0.114 mmol) in
CH2Cl2/MeOH (1:9, 15 mL). After being stirred for
24 h, the reaction mixture was diluted with aq NH4Cl
(satd, 50 mL). The aqueous phase was extracted with
CHCl3 (3· 40 mL) and the combined organic extracts
were washed with H2O (100 mL). After drying (MgSO4)
and filtration, the solvent was removed in vacuo and the
residue purified by column chromatography on silica
gel. Elution with EtOAc/light petroleum (1:1 to 3:1)
1
compound 17 (336 mg, 0.329 mmol, 87%) as an oil. H
NMR (300 MHz, CDCl3) d 8.68 (s, 1H), 7.34–7.12
(m, 25H), 6.21 (br s, 1H), 5.50–5.42 (m, 2H), 4.96–4.43
(m, 11H), 3.92–3.62 (m, 10H), 2.18 (s, 3H), 2.12 (s,
3H). 13C NMR (75 MHz, CDCl3) d 170.3, 170.1,
159.6, 138.6, 138.4, 138.0, 137.9, 137.5, 128.5, 128.4,
128.3, 128.2, 128.2, 128.1, 128.0, 127.9, 127.8, 127.5,
97.8, 95.0, 90.8, 77.9, 75.3, 75.1, 74.2, 73.9, 73.5, 73.4,
72.1, 71.6, 71.5, 68.8, 68.4, 67.4, 65.5, 21.1, 20.9. Gated
decoupled 13C NMR (125 MHz, CDCl3) selected data, d
afforded title compound 21 (197 mg, 0.097 mmol, 85%)
20
D
as an oil. ½aꢁ þ 70 (c 0.80, CHCl3). 1H NMR
1
1
0
0
97.8, JC1 ꢀH1 172 Hz, d 95.0, JC1–H1 179 Hz. HRMS-
ESI [M+Na]+ calcd for C53H56O13Cl3NNa: 1042.2715.
Found 1042.2715.
(500 MHz, CDCl3) d 7.43–7.14 (m, 65H), 5.94–5.86
(m, 1H), 5.41 (s, 1H), 5.32–5.20 (m, 3H), 5.01 (d,
J = 11.0 Hz, 1H), 4.95–4.55 (m, 21H), 4.49–4.39 (m,
6H), 4.36 (d, J = 12.0 Hz, 1H), 4.19–4.06 (m, 6H),
4.00–3.73 (m, 12H), 3.64–3.54 (m, 3H), 3.51–3.46 (m,
2H), 3.41–3.28 (m, 6H), 3.23–3.17 (m, 2H), 2.22 (br s,
4H). 13C NMR (125 MHz, CDCl3) d 138.7, 138.7,
138.6, 138.4, 138.3, 138.3, 138.1, 138.0, 138.0, 138.0,
137.9, 137.8, 133.8, 128.7, 128.6, 128.5, 128.5, 128.4,
128.4, 128.3, 128.3, 128.3, 128.3, 128.2, 128.2, 128.2,
128.0, 128.0, 128.0, 128.0, 127.9, 127.8, 127.8, 127.7,
127.6, 127.5, 127.5, 127.5, 127.4, 127.4, 127.3, 127.1,
117.9, 100.5, 100.1, 100.1, 99.8, 81.5, 81.5, 81.3, 80.5,
79.8, 79.6, 79.3, 78.9, 75.9, 75.8, 75.1, 75.0, 74.7, 74.2,
73.8, 73.6, 73.4, 73.4, 72.4, 72.0, 71.9, 71.7, 71.6, 71.3,
71.2, 71.1, 70.6, 70.6, 70.5, 68.7, 68.4, 68.0, 67.8, 65.8.
HRMS-ESI [M+Na]+ calcd for C124H134O26Na:
2061.9061. Found 2061.9070.
4.1.13. 2-O-(2-O-Acetyl-3,4,6-tri-O-benzyl-a-D-manno-
pyranosyl)-6-O-[(2-O-Acetyl-3,4,6-tri-O-benzyl-a-D-
mannopyranosyl)-(1 ! 6)-(2-O-acetyl-3,4-di-O-benzyl-a-
D-mannopyranosyl)-(1 ! 6)-(2-O-acetyl-3,4-di-O-benzyl-
a-D-mannopyranosyl)]-1-O-allyl-3,4,5-tri-O-benzyl-D-
myo-inositol (20). TMSOTf (5 lL, 0.03 mmol) was
added drop-wise to a stirred mixture of 16 (194 mg,
0.144 mmol),
trichloroacetimidate
17
(257 mg,
˚
0.252 mmol) and 4 A molecular sieves (150 mg) in
Et2O (15 mL) cooled to ꢀ30 ꢁC. After 10 min, Et3N
(1.5 mL) was added, the mixture filtered through
Celiteꢂ and the filter cake washed with further CH2Cl2
(2· 100 mL). The solvent was removed in vacuo and
the residue purified by column chromatography on sil-
ica gel. Elution with EtOAc/light petroleum (1:9 to 1:4
to 3:7) afforded the title compound 20 (253 mg,
4.1.15. 1-O-Allyl-2-O-(2,3,4,6-tetra-O-benzyl-a-D-manno-
pyranosyl)-6-O-[(2,3,4,6-tetra-O-benzyl-a-D-mannopyr-
anosyl)-(1 ! 6)-(2,3,4-tri- O-benzyl-a-D-mannopyranosyl)-
(1 ! 6)-(2,3,4-tri- O-benzyl-a-D-mannopyranosyl)]-3,4,5-
tri-O-benzyl-D-myo-inositol (22). Sodium hydride (60%
dispersion in mineral oil, 30 mg, 0.75 mmol) was added
to a stirred solution of tetraol 21 (188 mg, 0.092 mmol)
in DMF (15 mL) cooled to 0 ꢁC. After 20 min benzyl,
bromide (66 lL, 0.55 mmol) was added and the reaction
mixture stirred at rt for 14 h when aq NH4Cl (satd,
50 mL) was added. The mixture was extracted with ether
(2· 50 mL) and the combined ethereal extracts were
washed with H2O (2· 50 mL). After drying (MgSO4)
and filtration, the solvent was removed in vacuo and
the residue purified by column chromatography on silica
gel. Elution with EtOAc/light petroleum (1:9 to 1:4 to
20
D
0.115 mmol, 80%) as an oil. ½aꢁ þ 47 (c 1.4,
1
CHCl3). H NMR (500 MHz, CDCl3) d 7.43–7.05 (m,
65 H), 5.97–5.88 (m, 1H), 5.54–5.40 (m, 5H), 5.26–
5.16 (m, 3H), 5.05 (d, J = 11.0 Hz, 1H), 4.95–4.58 (m,
19H), 4.54 (d, J = 10.7 Hz, 1H), 4.47 (d, J = 11.3 Hz,
1H), 4.44–4.40 (m, 4H), 4.35–4.29 (m, 4H), 4.19 (d,
J = 8.5 Hz, 1H), 4.12 (dd, J = 12.5, 5.6 Hz, 1H), 4.05
(dd, J = 12.5, 5.7 Hz, 1H), 4.02–3.84 (m, 11H), 3.79
(t, J = 9.6 Hz, 1H), 3.60–3.43 (m, 5H), 3.34–3.23 (m,
7H), 3.16 (d, J = 11.0 Hz, 1H), 2.17 (s, 3H), 2.14 (s,
6H), 2.13 (s, 3H). 13C NMR (125 MHz, CDCl3) d
170.3, 170.1, 170.0, 169.9, 138.9, 138.8, 138.7, 138.6,
138.3, 138.2, 138.1, 138.0, 138.0, 137.7, 137.7, 134.0,
128.7, 128.6, 128.5, 128.4, 128.4, 128.3, 128.3, 128.2,
128.2, 128.2, 128.1, 128.1, 128.1, 128.0, 127.9, 127.9,
127.8, 127.8, 127.6, 127.6, 127.5, 127.5, 127.4, 127.3,
127.2, 127.1, 126.8, 117.8, 99.0, 98.8, 98.2, 98.0, 81.3,
81.2, 80.7, 78.7, 77.7, 77.7, 76.9, 75.8, 75.8, 75.1,
75.0, 74.8, 74.5, 74.1, 74.1, 73.7, 73.4, 73.4, 72.6,
71.7, 71.6, 71.6, 71.5, 71.4, 71.3, 71.2, 70.9, 70.7,
68.6, 68.3, 68.1, 67.8, 65.2, 65.1, 21.1, 21.0. Gated
decoupled 13C NMR (125 MHz, CDCl3) selected data,
1:3) afforded the title compound 22 as an oil (178 mg,
20
D
0.074 mmol, 80%). ½aꢁ þ 40 (c 0.71, CHCl3). 1H
NMR (500 MHz, CDCl3) d 7.40–7.09 (m, 85H), 5.78–
5.70 (m, 1H), 5.44 (s, 1H), 5.24–5.19 (m, 2H), 5.11–5.03
(m, 3H), 4.96–4.77 (m, 8H), 4.70–4.34 (m, 27H), 4.18–
3.82 (m, 18H), 3.63–3.50 (m, 5H), 3.40–3.25 (m, 6H),
3.19–3.14 (m, 2H). 13C NMR (125 MHz, CDCl3)
d 139.0, 138.9, 138.9, 138.9, 138.6, 138.6, 138.6, 138.5,
138.4, 138.4, 138.4, 138.3, 138.3, 138.1, 134.0, 128.6,
128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.2, 128.2,
128.2, 128.2, 128.1, 128.1, 128.1, 128.1, 128.0, 128.0,
1
1
0
0
00
00
d 99.0, JC1 ꢀH1 174 Hz, d 98.8, JC1 –H1 175 Hz,
1
1JC1
173 Hz, JC1
172 Hz. HRMS-ESI
000–H1000
0000–H10000
[M+Na]+ calcd for C132H142O30Na: 2229.9484. Found
2229.9512.

G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 7615–7624
7623
127.9, 127.8, 127.8, 127.7, 127.7, 127.6, 127.5,
127.5, 127.4, 127.4, 127.4, 127.3, 127.3, 127.2, 127.2,
127.1, 127.0, 117.7, 99.1, 98.8, 98.4, 98.3, 81.6, 81.4,
81.2, 80.7, 79.6, 79.2, 79.1, 78.8, 75.8, 75.7, 75.3,
75.0, 75.0, 74.9, 74.7, 74.7, 74.3, 73.8, 73.4, 73.3, 72.7,
72.6, 72.6, 72.3, 72.2, 72.2, 72.0, 71.9, 71.9, 71.6, 71.3,
71.2, 71.0, 70.4, 69.1, 69.0, 65.6, 65.5. HRMS-ESI
[M+Na]+ calcd for C152H158O26Na: 2422.0939. Found
2422.0933.
(5 mL) was transferred by cannular into the reaction
mixture. After warming to rt over 2 h, the reaction
was quenched with the addition of aq Na2SO3 (10%,
50 mL) and the combined mixture was extracted with
CH2Cl2 (2· 30 mL). The combined organic extract
was washed with aq NaHCO3 (satd, 3· 50 mL) and
dried (Na2SO4). After filtration, the solvent was re-
moved in vacuo and the residue purified by column
chromatography on silica gel. Elution with EtOAc/light
petroleum (1:9 to 1:4) followed by a further purification
with fresh silica gel eluting with EtOAc/CH2Cl2 (1:49 to
4.1.16. 2-O-(2,3,4,6-Tetra-O-benzyl-a-D-mannopyrano-
syl)-6-O-[(2,3,4,6-tetra-O-benzyl-a-D-mannopyranosyl)-
(1 ! 6)-(2,3,4-tri- O-benzyl-a-D-mannopyranosyl)-(1 ! 6)-
(2,3,4-tri- O-benzyl-a-D-mannopyranosyl)]-3,4,5-tri-O-
1:24) afforded the title compound 24 (61 mg,
20
D
0.020 mmol, 52%) as an oil. ½aꢁ þ 27:0 (c 1.22,
1
CHCl3). H NMR (500 MHz, CDCl3), mixture of iso-
mers, d 7.38–7.05 (m, 80 H), 5.42–5.5.40 (1H, m),
5.31–5.27 (m, 1H), 5.18–4.26 (m, 40H), 4.23–3.78 (m,
21H), 3.61–3.42 (m, 6H), 3.34–3.24 (m, 5H), 3.13–3.08
(m, 1H), 2.24–2.13 (m, 4H), 1.58–1.48 (m, 4H), 1.34–
1.18 (m, 48H), 0.89 (t, J = 6.5 Hz, 6H). 13C NMR
(125 MHz, CDCl3), mixture of isomers, d 173.1, 173.0,
172.8, 172.6, 139.1, 139.0, 139.0, 138.9, 138.8, 138.6,
138.6, 138.5, 138.4, 138.4, 138.4, 138.2, 138.0, 137.9,
137.8, 135.2, 135.2, 135.1, 135.0, 129.1, 128.9, 128.8,
128.6, 128.4, 128.4, 128.4, 128.3, 128.3, 128.2, 128.2,
128.2, 128.2, 128.1, 128.1, 128.1, 128.0, 128.0, 127.9,
127.8, 127.8, 127.7, 127.6, 127.6, 127.5, 127.5, 127.4,
127.4, 127.4, 127.3, 127.3, 127.2, 127.2, 127.1, 126.9,
99.6, 99.1, 99.0, 98.4, 98.2, 98.2, 81.0, 80.7, 80.6, 80.3,
80.2, 79.6, 79.2, 79.0, 78.7, 78.5, 76.0, 75.8, 75.7, 75.6,
75.0, 74.9, 74.7, 74.7, 74.6, 74.1, 73.9, 73.8, 73.7, 73.3,
72.9, 72.6, 72.6, 72.4, 72.3, 72.2, 71.9, 71.7, 71.6, 71.3,
71.3, 71.2, 70.2, 70.2, 69.5, 69.4, 69.1, 68.8, 66.1, 66.1,
65.5, 61.5, 34.1, 34.1, 34.0, 33.9, 32.0, 29.8, 29.7, 29.6,
29.4, 29.4, 29.2, 29.2, 24.8, 22.8, 14.2. 31P NMR
benzyl-D-myo-inositol
(23).
(1,5-Cyclooctadiene)-
bis(methyldiphenylphosphine)iridium(I) hexafluorophos-
phate (10 mg, 0.01 mmol) was added to a stirred solu-
tion of allyl ether 22 (68 mg, 0.028 mmol) in THF
(5 mL) under argon. This atmosphere was replaced with
hydrogen for ca 1 min and then, in turn, the hydrogen
was replaced with argon. The mixture was stirred at
20 ꢁC for 70 min, the solvent was removed in vacuo
and the residue dissolved with stirring in CH2Cl2/MeOH
(2:1, 9 mL). Acetyl chloride (100 lL, 0.26 mmol) was
added to this solution and stirring was continued for
12 h when solid NaHCO3 (200 mg, 2.38 mmol) was add-
ed. The mixture was stirred for an additional 5 min
when H2O (50 mL) was added. This mixture was
extracted with CHCl3 (2· 60 mL), and after drying
(MgSO4) and filtration the solvent was removed in vac-
uo and the residue purified by column chromatography
on silica gel. Elution with EtOAc/light petroleum (1:4 to
3:7) afforded the title compound 23 as an oil (61 mg,
20
D
0.026 mmol, 92%). ½aꢁ þ 37:5 (c 1.22, CHCl3). 1H
NMR (500 MHz, CDCl3) d 7.39–7.12 (m, 85H), 5.46
(s, 1H), 5.23 (s, 1H), 5.15 (s, 1H), 4.93–4.85 (m, 7H),
4.76–4.42 (m, 27H), 4.30–4.27 (m, 2H), 4.12–3.78 (m,
17H), 3.70–3.51 (m, 8H), 3.39–3.31 (m, 3H), 3.23 (t,
J = 9.5 Hz, 1H). 13C NMR (125 MHz, CDCl3) d 138.8,
138.7, 138.6, 138.5, 138.5, 138.5, 138.4, 138.4, 138.3,
138.2, 138.1, 138.0, 128.5, 128.5, 128.4, 128.4, 128.3,
128.3, 128.3, 128.3, 128.3, 128.2, 128.2, 128.2, 128.1,
128.1, 128.1, 128.0, 127.9, 127.8, 127.8, 127.8, 127.7,
127.7, 127.7, 127.6, 127.6, 127.5, 127.5, 127.5, 127.5,
127.4, 127.4, 127.4, 127.3, 99.0, 98.5, 98.3, 96.6 (br),
81.3, 80.6, 80.2, 79.2, 78.9, 78.7, 75.6, 75.4, 75.0, 75.0,
74.9, 74.8, 74.7, 74.1, 73.4, 73.3, 73.0, 72.5, 72.3, 72.3,
72.1, 72.1, 72.0, 71.9, 71.8, 71.8, 71.7, 71.7, 71.3, 69.2,
69.1, 66.4, 65.6. HRMS-ESI [M+Na]+ calcd for
C149H154O26Na: 2382.0626. Found 2382.0732.
(121.5 MHz, CDCl3)
d
1.11, 0.80. HRMS-ESI
[M+Na]+ calcd for C191H227O33NaP: 3012.5720. Found
3012.5745.
4.1.18. 1-O-(1,2-di-O-Hexadecanoyl-sn-glycero-3-phos-
phoryl)-2-O-(a-D-mannopyranosyl)-6-O-[(a-D-manno-
pyranosyl)-(1 ! 6)- (a-D-mannopyranosyl)-(1 ! 6)-(a-D-
mannopyranosyl)]-D-myo-inositol (2). Pd(OH)2/C (20%,
25 mg) was added to a stirred solution of 24 (59 mg,
0.019 mmol) in THF/MeOH (2:3, 5 mL). The mixture
was stirred under hydrogen atmosphere for 4 h at rt
when the hydrogen was replaced with argon. The mix-
ture was filtered through Celiteꢂ and concentrated in
vacuo. The residue was lyophilized to afford 2 (25 mg,
20
D
0.017 mmol, 89%) as a white powder. ½aꢁ þ 37 (c
1
0.30, D2O). H NMR (500 MHz, CDCl3/CD3OD/D2O,
1.0:1.5:0.5), d 5.32–5.26 (m, 1H), 5.15 (br s, 1H), 5.11
(br s, 1H), 4.88 (br s, 1H), 4.86 (br s, 1H), 4.40–3.48
(m, 36H), 2.40–2.31 (m, 4H), 1.67–1.57 (m, 4H), 1.35–
1.20 (m, 48H), 0.89 (t, J = 6.9 Hz, 6H). 31P NMR
(202.5 MHz, CDCl3/CD3OD/D2O, 1.0:1.5:0.5) d 0.5.
HRMS-ESI [MꢀH]ꢀ calcd for C65H118O33P:
1457.7293. Found 1457.7284.
4.1.17.
2-O-(2,3,4,6-tetra-O-benzyl-a-D-mannopyrano-
syl)-6-O-[(2,3,4,6-tetra-O-benzyl-a-D-mannopyranosyl)-
(1 ! 6)-(2,3,4-tri- O-benzyl-a-D-mannopyranosyl)-(1 ! 6)-
(2,3,4-tri- O-benzyl-a-D-mannopyranosyl)]-3,4,5-tri-O-
benzyl-1-O-(1,2-di-O-hexadecanoyl-sn-glycero-3-benzyl-
phosphoryl)-D-myo-inositol (24). 1H-Tetrazole (13 mg,
0.19 mmol) was added to a stirred solution of 23
(90 mg, 0.038 mmol) and phosphoramidite 8 (154 mg,
0.191 mmol) in CH2Cl2 (5 mL) cooled to 0 ꢁC under
an argon atmosphere. After stirring at rt for 2 h, the
reaction mixture was cooled to ꢀ40 ꢁC and a solution
of m-CPBA (50%, 66 mg, 0.191 mmol) in CH2Cl2
4.2. Experimental Procedures for the Adjuvant activity of
PIM2 and PIM4
Female C57BL/6 mice were purchased from the Hercus
Taieri Resource Unit, Dunedin, New Zealand. Female

7624
G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 7615–7624
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The authors thank the New Zealand Foundation for
Research and Technology (C08X0209) and the Marsden
Fund (Royal Society of New Zealand, IRL0401 to GFP)
for financial support. We thank Rosemarie Meo for help
with animal husbandry and Lilian Morrison for statisti-
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Stewart for recording high field NMR spectra. We
thank Professor Robin Ferrier for assisting in the prep-
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29. The thioglycoside 18 was prepared from phenyl 3-O-
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the sequence of acetylation and regioselective reduction of
the benzylidene acetal with BH3ÆTHF-Bu2BOTf.
30. Barnden, M. J.; Allison, J.; Heath, W. R.; Carbone, F. R.
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References and notes
1. Lussow, A. R.; Aguado, M. T.; Del Giudice, G.; Lambert,
P. H. Immunol. Lett 1990, 25, 255.
2. Marciani, D. J. Drug Discovery Today 2003, 8, 934.