A PIM2 analogue suppresses allergic airway disease

Article abstract of DOI:10.1016/j.bmc.2010.11.058

Two approaches for the synthesis of a phosphatidylinositol dimannoside (PIM₂) analogue 4 that mimics the suppressive activity of natural PIMs and also synthetic PIM₂ have been developed. This analogue, where the inositol core was rep

Full text of DOI:10.1016/j.bmc.2010.11.058

Bioorganic & Medicinal Chemistry 19 (2011) 917–925
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A PIM2 analogue suppresses allergic airway disease
Jacquie L. Harper a, Colin M. Hayman b, David S. Larsen c, , Gavin F. Painter b, Gurmit Singh-Gill c
⇑
a The Malaghan Institute of Medical Research, Wellington, New Zealand
b Carbohydrate Chemistry Team, Industrial Research Limited, PO Box 31310, Lower Hutt, New Zealand
c University of Otago, PO Box 56, Dunedin, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Two approaches for the synthesis of a phosphatidylinositol dimannoside (PIM2) analogue 4 that mimics
the suppressive activity of natural PIMs and also synthetic PIM2 have been developed. This analogue,
where the inositol core was replaced by glycerol, was tested for its ability to suppress cellular inflamma-
tion in a mouse model of allergic asthma and shown to be effective in suppressing airway eosinophilia.
Suppression of all inflammatory cells monitored was observed, indicating a general blockade of cellular
activity. These data indicate that the inositol core is not essential for this suppressive activity.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 29 September 2010
Revised 23 November 2010
Accepted 25 November 2010
Available online 2 December 2010
Keywords:
Phosphatidylinositol mannans
PIMs
PIM analogues
Asthma
Inflammation
1. Introduction
(2) and PIM2 (3) indicates that suppressive activity requires the
presence of fatty acid residues and at least one mannopyranosyl
The increasing prevalence of allergic disease, including asthma,
in industrialised nations is commonly linked with decreased child-
hood exposure to infectious agents including mycobacteria.1–8
Mycobacterial products and components have been shown to in-
duce a wide range of immune responses including stimulatory
and suppressive immune cell function.9–13 Previously, we have
shown that the cell wall components lipoarabinomannan (LAM),
and phosphatidylinositol mannanoside (PIM) isolated from
Mycobacterium bovis exert a suppressive phenotype in a model of
moiety where the mannosylation site can be at either O-2 or O-6
of the inositol moiety.14,15
The heterogeneity of the native PIMs makes their separation
technically challenging. As a result a rational approach to progress-
ing structure activity relationship studies on PIMs is to synthesise
discrete PIM molecules for testing. However, it is important to note
that the myo-inositol core complicates the synthetic route to PIMs.
Enantiomerically pure myo-inositol intermediates for PIM
syntheses are available either by low yielding desymmetrization
OH
HO
HO
HO
HO
HO
OH
OR
HO
HO
OH
O
OCOC17H35
OCOC17H35
O
P
O
R =
HO
O
O
O
O
OH
OH
O
OH
O
OR
O
HO
HO
OR
OH
HO
HO
OH
OH
PIM1
OH
OH
OH
2
O
OH
OH
OH
PIM1
1
PIM2
3
Compounds 1 to 3
allergic airway inflammation.14 Initial work using native lipogly-
cans and three discrete synthetic PIM structures: PIM1 (1), PIM1
processes16,17 or via a lengthy multi-step strategy from methyl
-glucopyranoside.18,19 We felt that we could circumvent these
a-D
problems and evaluate whether the inositol moiety of PIMs was
essential for the suppressive activity by elimination of the C-3, -4
⇑
Corresponding author. Tel.: +64 3 479 7816; fax: +64 4 479 7906.
E-mail address: dlarsen@alkali.otago.ac.nz (D.S. Larsen).
and -5 fragment from the central inositol unit to give
a
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.058

918
J. L. Harper et al. / Bioorg. Med. Chem. 19 (2011) 917–925
Figure 1. Design of the cutaway PIM analogue.
symmetrical molecule containing a glycerol core (Fig. 1). The
advantage of this is that the core structure would contain no asym-
metric centres thus simplifying its preparation. Furthermore, the
64 and 5% respectively. The one-bond coupling constants (1JC–H
)
for each of the anomeric carbon signals of 10 which resonated at
d 98.55 and 98.58 ppm in the 13C NMR spectrum were both
170 Hz confirming the anomeric configuration of each mannose
incorporation of two
a-D-mannopyrannosyl residues would en-
hance the water solubility of these PIM-like compounds. We refer
here to this simplified PIM2 structure as a ‘cutaway’ PIM2 analogue.
This paper reports our strategy for the synthesis of a cutaway
PIM2 (4) analogue that suppresses infiltration of inflammatory cells
into the lung in an in vivo allergic airway disease model in detail.20
residue as a 24
.
With the dimannoside 10 in hand attention turned to attaching
the phosphatidyl group. We initially utilised the H-phosphonate
method for forming the phosphate ester. Reaction of 10 and
H-phosphonate 1225–27 gave, after oxidation with iodine, the tri-
ethylammonium salt of the protected PIM analogue 13 in 76% yield
(Scheme 2). The ꢀve ion mass spectrum showed a peak consistent
with the anion of 13 and the 31P NMR spectrum showed a peak at d
0.15 ppm. The remaining spectral data were also consistent with
13. The final step in the synthesis was the global hydrogenolysis
of the benzyl groups by treatment with hydrogen over 10% Pd on
carbon using a 2:1:1:1 mixture of EtOAc, THF, EtOH and H2O to
give the target cutaway PIM2 analogue 4 as the triethylammonium
salt.
Although the chemistry approach above provided the target
PIM analogue 4, problems with synthetic efficiency needed to be
addressed. The use of armed donor 8 resulted in an inseparable
mixture of bis-mannosides 9; the separation of debenzoylated gly-
cosides 10 and 11 was inefficient; and hydrogenolysis of benzyl
groups of the phosphodiester 13 was unpredictable due to the tri-
ethylammonium phosphate moiety. A bis-mannosylation of 2-O-p-
methoxybenzylglycerol 14 with a donor possessing a participating
group at O-2 (i.e., a disarmed donor) and phosphorylation utilising
2. Results and discussion
2.1. Chemistry
Our initial approach to the synthesis of the cutaway PIM ana-
logue started with the conversion of benzylated allyl mannoside
5
21 into a 1:1 diastereoisomeric mixture of the glyceryl mannoside
6 by dihydroxylation using the Upjohn process (Scheme 1 and 88%
yield). Selective protection of the secondary glyceryl hydroxyl
group was achieved by sequential reaction with trityl chloride then
benzoyl chloride in pyridine followed by treatment of the crude
product with acidified methanol to give the 2-O-benzoyl glyceryl
glycoside 7 in 77% yield. Mannosylation of 7 with mannosyl
phosphite 822,23 gave a 4:1 inseparable mixture of the
,b-dimannosylated glycerols 9 in 71% yield. Fortunately, after
a,a- and
a
hydrolysis of the benzoyl group under Zémplen conditions, the
glyceryl dimannosides 10 and 11 could be separated in yields of
Scheme 1. Reagents and conditions: (i) 1% aq OsO4, 9:1 acetone–H2O (88%); (ii) (a) TrCl, py, 100 °C, (b) BzCl, py, (c) pTSA, 7:3 CH2Cl2–MeOH (77%); (iii) NIS, Et2O (71%); (iv)
1 M NaOMe, MeOH (64% for 10, 5% for 11).

J. L. Harper et al. / Bioorg. Med. Chem. 19 (2011) 917–925
919
An alternative approach to the target PIM analogue 4 was devel-
oped from 14 (Scheme 5). Reaction of 14 with disarmed trichloro-
acetimidate donor 21 possessing a 2-O-methoxyacetyl group was
attempted under the conditions optimised for donor 17. In this
case the use of 5 mol % TMSOTf to promote this reaction resulted
in the formation of orthoester products. Fortunately, increasing
the amount of promoter to 20 mol % gave the desired bis-manno-
side 22 in 79% yield. The reaction time was kept as short as
possible (15 min) to minimise uncontrolled in situ loss of the
PMB group. Removal of the PMB protecting group with DDQ
to 23 (81%) and subsequent 4,5-dicyanoimidazole-promoted phos-
phitylation with phosphoramidite 2432,33 and subsequent mCPBA
oxidation resulted in the fully protected PIM2 analogue 25.
Hydrogenolysis of the benzyl groups gave 26 in 89% yield and
followed by selective hydrolysis of the methoxyacetate groups
effected by treatment with 0.33 mM sodium methoxide in
methanol–CH2Cl2 gave the target cutaway PIM2 analogue 4 iso-
lated as its sodium salt in 84% yield (96% pure by HPLC analysis).
Modifying the work-up of this process gave 4 as its triethylamine
salt. The spectroscopic data of the triethylamine salt of 4 produced
by this route were identical to those of 4 produced by the strategy
shown in Scheme 2.
Scheme 2. Reagents and conditions: (i) PivCl, py then I2 in 9:1 Py–H2O (76%); (ii)
H2, 10% Pd/C, 2:1:1:1 EtOAc–THF–EtOH–H2O (69%).
phosphoramidite coupling methodology was used to overcome
these issues. Known glycerol derivative 14 was synthesised
(Scheme 3) using a modification of the strategy reported by Chong
et al.28
Commercially available 1,3-di-O-benzylglycerol (15) was alkyl-
ated with p-methoxybenzyl chloride under standard conditions to
give 16 in 79% yield. Hydrogenolytic removal of the benzyl groups
using palladium on carbon as described28 proved unreliable. In-
stead, selective hydrogenolysis of the benzyl groups was effected
using Raney nickel29 providing the target glycerol 14 in 77% yield.
Bis-glycosylation of 14 with disarmed trichloroacetimidate
(TCA) donor 1730 promoted by TMSOTf in a mixture of toluene
and CH2Cl2 was investigated31 (Scheme 4). Although the reaction
2.2. Suppression of airway eosinophilia
As in earlier studies,14,15 the cutaway PIM2 analogue 4 was also
assayed in a mouse model of ovalbumin (OVA)-induced allergic
airway inflammation using infiltration of eosinophils as a key read-
out of allergic airways inflammation. As shown in Figure 2A, mol-
ecule 4 suppressed the recruitment of eosinophils into the lung of
OVA challenged mice. A similar decrease in both macrophage and
lymphocyte cell numbers was also observed. As shown in previous
studies, the percentage of eosinophils, macrophages and lympho-
cytes did not change significantly (Fig. 2B). Therefore, like the
PIM extracts and synthetic PIMs, the suppression of lung eosino-
philia by the cutaway PIM mimic appeared to result from overall
suppression of cell infiltration rather than a blockade of a specific
cell type.
using 20 mol % of the promoter provided the desired a,a-bisman-
noside 18 in 60% yield, a significant amount of material lacking
the p-methoxybenzyl group was also recovered. Optimal reaction
conditions were obtained by lowering the amount of promoter to
5 mol % to give 18 in 86% yield. This sequence converged with that
shown in Scheme 1 by conversion of 18 into the corresponding
benzylated bis-mannoside 10 via a sequence of deacetylation to
19, benzylation to 20 and then oxidative removal of the
p-methoxybenzyl group with DDQ. Compound 10 produced by this
strategy was identical to that synthesised using the route shown in
Scheme 1.
Previous work had shown that deacylation of native LAM and
PIM from M. bovis, and the absence of mannose caps in LAM from
M. smegmatis, results in loss of lipoglycan-dependent suppression
of OVA-induced airway eosinophilia.14,15 The current research
now indicates that the inositol core of PIMs is not necessary for
suppressive activity in vivo. Similar structure activity requirements
have also been observed in relation to the suppressive activity of a
closely related compound in the context of LPS-induced macro-
phage activation in vitro and neutrophil infiltration in vivo.34 To-
gether with our data, it appears that PIM molecules have the
ability to suppress aspects of both adaptive and innate inflamma-
tion, and that these immunosuppressive activities require the pres-
Scheme 3. Reagents and conditions: (i) PMBCl, NaH, DMF (79%); (ii) Raney Ni, H2,
EtOH (77%).
ence of at least one a-D-mannopyranosyl residue, acyl chains, and
the phosphodiester moiety. However, the inositol core of PIMs ap-
pears to act solely as a scaffold for the pharmacophore and is not
essential for the anti-inflammatory activity.
3. Conclusion
In conclusion, we have developed two approaches for the syn-
thesis of cutaway PIM2 analogues that mimic the suppressive
activity of natural PIMs and also synthetic PIM2 and added to our
understanding of the structural requirements for suppression of
allergic airway inflammation by PIMs. As this molecule serves as
a lead compound for the development of an immunotherapeutic
agent the synthesis of further PIM analogues and investigation of
their immunosuppressant activity is underway.
Scheme 4. Reagents and condition: (i) 5 mol % TMSOTf, 2:5 CH2Cl2–toluene, 0 °C
(86%); (ii) NaOMe, CH2Cl2–MeOH (90%); (iii) BnBr, NaH, DMF (88%); (iv) DDQ,
CH2Cl2–H2O (84%).

920
J. L. Harper et al. / Bioorg. Med. Chem. 19 (2011) 917–925
Scheme 5. Reagents and conditions: (i) 20 mol % TMSOTf, 2:5 CH2Cl2–toluene, 0 °C (79%); (ii) DDQ, CH2Cl2–H2O (81%); (iii) 4,5-dicyanoimidazole, CH2Cl2, then mCPBA (82%);
(iv) H2, 20% Pd(OH)2/C, THF–MeOH (89%); (v) NaOMe, CH2Cl2–MeOH (84%).
A
B
300
200
100
0
80
60
40
20
0
Total Cells
Eosinophils
Macrophages
Lymphocytes
OVA Control
µg/ml
20
OVA Control
µg/ml
20
Figure 2. Intra-nasal administration of cutaway PIM2 mimic 4 suppresses cell infiltration in a mouse model of allergic airway disease (AAD). (A) Cells per mL and (B) cell
percentages in the brochoalveolar lavage fluid of OVA challenged mice treated with compound 4. Graph is representative of two experiments (n = 5 per group, SEM).
4.2. Experimental procedures for synthesis of PIM analogues
4. Experimental section
Specific optical rotations, given in 10ꢀ1 deg cm2 gꢀ1, were mea-
sured at ambient temperature using either a Jasco DIP-1000 polar-
imeter or a Perkin Elmer 241 polarimeter each with a cell of path
length 1.0 dm. 1H NMR spectra were obtained at 500 MHz and ref-
erenced to tetramethylsilane (TMS) (0.0 ppm) or the residual sol-
vent peak (CHCl3 7.26 ppm). Chemical shifts are reported as parts
per million (ppm) using the d scale and coupling constants (J)
and separations are reported to the nearest 0.5 Hz. 13C NMR spec-
tra were recorded at 125 MHz and referenced either to TMS
(0.0 ppm) or internal solvent (CDCl3 77.0 ppm). Chemical shifts
are reported to 1 decimal place with the chemical shifts for signals
that are similar reported to two decimal places. 31P NMR spectra
were recorded at 202 MHz and are reported to 1 decimal place
with H3PO4 (0.0 ppm) as the external reference. Electro-spray ion-
ization (ESI) mass spectra were recorded either on a PerSpective
Biosystems Mariner time-of-flight mass spectrometer or a Q-TOF
Premier mass spectrometer. Elemental analyses were carried out
at the Campbell Microanalytical Laboratory, University of Otago,
Dunedin, New Zealand. Thin layer chromatography (TLC) was per-
formed on Merck silica gel DC Alurolle Kieselgel 60F254 plates and
were visualised under an UV lamp and/or with a spray consisting
of 5% w/v dodecamolybdophosphoric acid in ethanol with subse-
quent heating. Flash column chromatography was carried out
using Merck Kieselgel 60 (230–400 mesh). All chromatography sol-
vents were reagent grade. Petroleum ether used was bp 60–80°
range. Anhydrous solvents were either sourced from Aldrich and
used without further treatment or reagent grade solvents distiled
fresh prior to use (THF was distiled from sodium-benzophenone
ketyl under nitrogen and dichloromethane was distilled from
phosphorus pentoxide). Powdered molecular sieves were flame
4.1. Airway inflammation model
C57BL/6 male mice were bred and housed in a conventional ani-
mal facility at the Wellington School of Medicine. All animals used
for the experiments were aged between 8 and 12 weeks. Experi-
mental procedures were approved by the animal ethics committee
in accordance with Victoria University of Wellington (Wellington,
NZ) guidelines for the care of animals.
4.1.1. Immunisation and airway challenge
Mice were injected intraperitoneally (ip) with 2
(Sigma–Aldrich, St. Louis, MO) in 200 L of alum adjuvant (Serva,
Heidelberg, Germany) on day 0 and 14 (n = 5 mice/group). On
day 28 mice were anaesthetised by an ip injection of a mixture
of Ketamine and Xylazine (Sigma–Aldrich) and intranasally (in)
lg of OVA
l
challenged with 50 lL of PBS containing 100 lg OVA.
4.1.2. Treatment with PIM analogue 4
One week (day 21) prior to OVA challenge mice were anaesthe-
tised and analogue 4 was administered intranasally (50 L, 20 g/
mL, in). OVA control mice were given 50 L of PBS intranasally.
l
l
l
4.1.3. Detection of cell types in the bronchoalveolar lavage fluid
Four days (day 32) post OVA challenge (day 28) the mice were
sacrificed and bronchoalveolar lavage (BAL) was performed (three
washes with 1 mL PBS). Total BAL cell numbers were counted, cells
were fixed onto slides using a cytospin and stained with the Diff-
Quick Staining Kit (Dade Behring, Newark, USA). The percentages
of different cell types were determined microscopically using stan-
dard histological criteria.

J. L. Harper et al. / Bioorg. Med. Chem. 19 (2011) 917–925
921
dried under vacuum immediately prior to use. All compounds were
4.2.3. (2R⁄)-2-O-Benzoyl-1,3-bis-O-(2,3,4,6-tetra-O-benzyl-
D-
isolated after silica gel column chromatography and fractions col-
lected were one spot by TLC. HPLC analyses were performed on
an Agilent 1100 quaternary pump system using an ESA Corona
Charged Aerosol Detector (Filter = none). The column used was a
mannopyranosyl)glycerol 9
Powdered 4 Å molecular sieves (50 mg) were added to a solu-
tion of glycerol 7 (730 mg, 1.02 mmol), phosphite 8 (771 mg,
1.22 mmol) and N-iodosuccinimide (NIS) (275 mg, 1.22 mmol) in
dry diethyl ether (20 mL) under an atmosphere of nitrogen. The
reaction was stirred at room temperature for 15 min, trifluoro-
Phenomenex Synergi 4
l
m Fusion-RP (80 Å, 4.6 ꢁ 250 mm). HPLC
elution gradient: solvent A H2O; solvent B 100 mM NH4OAc;
solvent C Methanol; Program 0–10 min 5–0% A, 5% B, 85–95% C,
10–28 min hold (0% A, 5% B, 95% C) and operated at a flow rate
methanesulfonic acid (110 lL, 1.24 mmol) was added and the stir-
ring was continued for 2 h. The reaction was diluted with more
diethyl ether (50 mL) and the organic layer was washed with 10%
sodium thiosulfate solution (30 mL), water (2 ꢁ 20 mL) and dried
over magnesium sulfate. The solvent was removed and the residue
was purified over silica (9:1 hexanes–diethyl ether as eluent) to
of 1.0 mL minꢀ1
.
4.2.1. (2R⁄)-1-O-(2,3,4,6-Tetra-O-benzyl-
a
-
D
-mannopyranosyl)-
(1.00 g,
glycerol 6
Allyl 2,3,4,6-tetra-O-benzyl-
a
-D
-mannopyranoside
5
give 9 (900 mg, 71%, 4:1 mixture of aa to ab diastereoisomers)
1.72 mmol) and N-methylmorpholine-1-oxide (302 mg, 2.58 mmol)
were dissolved in a mixture of 9:1 acetone–water (40 mL) and a 1%
aqueous osmium tetraoxide solution (1.5 mL) was added. The reac-
tion mixture was stirred overnight at room temperature, then
poured into 10% sodium thiosulfate solution (20 mL) and extracted
with CH2Cl2 (40 mL). The organic layer was washed with water
and dried over magnesium sulfate. The solvent was removed and
the residue was purified over silica (1:2 hexanes–diethyl ether as
eluent) to give the title compound 6 (927 mg, 88%, 1:1 mixture of
epimers) as a colourless syrup; Rf 0.4 (1:2 hexanes–EtOAc); (found:
as a colourless syrup; Rf 0.6 (2:3 hexanes–diethyl ether); mmax
cmꢀ1/(CHCl3) 1716.7; 1H NMR (500 MHz, CDCl3) d inter alia
3.50–4.13 (m, 16H), 4.42–4.52 (m, 4H), 4.55–4.65 (m, 4H), 4.69
(d, 2H, J 12.5 Hz, PhCH2), 4.72 (d, 2H, J 12.5 Hz, PhCH2), 4.82 (d,
2H, J 11 Hz, PhCH2), 4.86 (d, 2H, J 11 Hz, PhCH2), 4.91 (d, 1H, J
2 Hz), 4.96 (d, 1H, J 2 Hz), 5.33–5.38 and 5.41–5.53 (2 ꢁ m, 1H,
H-20), 7.08–7.38 (m, 42H, Ph-H), 7.55 (t, 1H, J 7.7 Hz), 8.00 (d, 2H,
J 7.7); 13C NMR (125 MHz, CDCl3) d inter alia 65.5, 65.9, 69.1,
69.2, 71.4, 72.2, 72.3, 72.4, 72.65, 72.68, 73.38, 73.42, 74.7, 74.8,
74.9, 75.0, 75.1, 79.9, 80.0, 97.7, 98.4, 127.52, 127.60, 127.63,
127.73, 127.76, 127.78, 127.81, 127.87, 127.92, 128.03, 128.29,
128.33, 128.35, 128.41, 128.5, 129.8, 130.0, 133.2, 138.35 138.42,
138.43, 138.5, 138.5, 138.6, 165.9; HRMS-ESI (+ve) found: m/z
1241.5595 [M+H]+; C78H81O14 calcd m/z 1241.5626.
C, 71.69; H, 6.92. C37H42O8ꢂ0.5H2O requires C, 71.25; H, 6.95); mmax
/
cmꢀ1 3439 (OH); 1H NMR (500 MHz, CDCl3) d 3.46–3.94 (m, 11H),
4.48–4.64 (m, 5H), 4.71–4.87 (m, 2H), 4.82–4.88 (m, 2H), 7.18–
7.21 (m, 2H, Ph-H) and 7.24–7.36 (m, 18H, Ph-H);13C NMR
(125 MHz, CDCl3) d 63.5, 63.6, 69.4, 69.5, 69.9, 70.6, 70.8, 70.9,
72.2, 72.3, 72.37, 72.42, 72.85, 72.9, 73.5, 73.6, 74.9, 75.02, 75.04,
75.10, 75.12, 79.9, 99.1, 127.70, 127.72, 127.76, 127.90, 127.95,
127.99, 128.06, 128.10, 128.40, 128.43, 128.45, 138.0, 138.1,
138.23, 138.24, 138.3, 138.4; LRMS-ESI (+ve) m/z (%) 638 [MNa]+
(24), 91 (100).
4.2.4. 1,3-Bis-O-(2,3,4,6-tetra-O-benzyl-a-D-mannopyranosyl)-
glycerol 10
Bis-mannoside 9 (800 mg, 0.65 mmol) was dissolved in 1 M so-
dium methoxide in methanol (80 mL) and the reaction was stirred
overnight at room temperature. The solvent was removed and the
residue was dissolved in CH2Cl2, washed with 2 M HCl (2 ꢁ 50 mL)
and water (50 mL), and dried over magnesium sulfate. Removal of
the solvent gave a residue (10 to 11 4:1, 615 mg) which was puri-
fied over silica (75:25–65:35 hexanes–diethyl ether gradient elu-
tion) to afford the title compound 10 (470 mg, 64%), a mixture of
4.2.2. (2R⁄)-2-(O-Benzoyl-3-O-(2,3,4,6-tetra-O-benzyl-
a-D-
mannopyranosyl)glycerol 7
Glycerol 6 (900 mg, 1.47 mmol) and trityl chloride (490 mg,
1.76 mmol) were dissolved in dry pyridine (60 mL) and heated at
100 °C. The disappearance of 6 was monitored by TLC and after
90 min the reaction was cooled to 0 °C and a solution of benzoyl
chloride (1.0 g, 7.0 mmol) in dry CH2Cl2 (10 mL) was added. The
reaction was warmed to room temperature and stirring was
continued for 2 h. The solvent was removed and the residue was
dissolved in chloroform (50 mL), washed with 2 M HCl
(2 ꢁ 20 mL), saturated sodium bicarbonate solution (2 ꢁ 20 mL),
water (25 mL) and dried over magnesium sulfate. The solvent
was removed, the residue was dissolved in a 7:3 CH2Cl2–MeOH
(50 mL) and p-TSA (75 mg) was added. The reaction was stirred
at room temperature overnight and solvent was removed. The
residue was purified over silica (8:2–7:3 hexanes–diethyl ether
as eluent) to afford the title compound 7 (816 mg, 77%) as a pale
syrup; Rf 0.4 (2:1 diethyl ether–hexanes); 1H NMR (500 MHz,
CDCl3) d 3.62–4.05 (m, 10H); 4.46–4.77 (m, 7H), 4.83 and 4.85
(2 ꢁ d, each 1H, J 10.5 Hz, PhCH2), 4.89 and 4.94 (2 ꢁ d, each 1H,
J 2 Hz, H-1), 5.20–5.30(m, 1H, H-20), 7.10–7.12 and 7.20–7.41 (m,
20 H, Ph-H), 7.51 (t, 1H, J 7.5 Hz), 8.05 (br d, 2H, J 7.5 Hz); 13C
NMR (125 MHz, CDCl3) d 57.9, 61.2, 65.25, 65.3, 65.6, 68.9, 69.0,
71.9, 72.0, 72.4, 73.1, 73.2, 73.4, 73.8, 74.6, 74.7, 74.8, 79.6, 79.7,
97.5, 98.0, 127.32, 127.36, 127.38, 127.41, 127.56, 127.59, 127.62,
127.65, 127.74, 127.80, 127.83, 128.07, 128.10, 128.13, 128.2,
128.2, 128.3, 129.5, 129.6, 129.7, 129.8, 132.96, 133.0, 138.05,
10 and 11 (90 mg, 12%), and (2R⁄)-1-O-(2,3,4,6-tetra-O-benzyl-
a-
D
-mannopyranosyl)-3-O-(2,3,4,6-tetra-O-benzyl-b- -mannopyran-
D
osyl)-glycerol 11 (40 mg, 5%, 1:1 mixture of diastereoisomers).
Data for 10: Rf 0.55 (2:3 hexanes–diethyl ether); ½a D20
ꢃ
+27 (c
0.89, CH2Cl2); 1H NMR (500 MHz, CDCl3) d 3.45 (dd, 1H, J = 7.0,
10.6 Hz), 3.52 (dd, 1H, J = 4.1, 10.8 Hz), 3.56 (dd, 1H, J = 5.9,
10.8 Hz), 3.61 (dd, 1H, J = 4.3, 10.6 Hz), 3.67–3.81 (m, 8H), 3.83–
3.92 (m, 3H), 3.94 (d, 1H, J = 9.7 Hz), 3.98 (d, 1H, J = 9.4 Hz),
4.46–4.52 (m, 4 H), 4.57–4.65 (m, 6H), 4.67–4.76 (m, 4H) 4.82–
4.88 (m, 4H), 7.19–7.19 (m, 4H), 7.20–7.38 (m, 36H); 13C NMR
69.26, 69.29, 69.4, 69.6, 70.0, 72.27, 72.29, 72.33, 72.4, 72.8,
73.41, 73.42, 74.9, 75.00, 75.02, 75.05, 75.08, 80.0, 80.1, 98.8 (1JC–
170 Hz), 98.9 (1JC–H 170 Hz), 127.52, 127.55, 127.61, 127.62,
H
127.64, 127.68, 127.70, 127.8, 128.0, 128.33, 128.34, 128.37,
128.39, 138.3, 138.4, 138.45, 138.47, 138.52; HRMS-ESI (+ve)
found: m/z 1159.5186 [M+Na]+; C71H76O13Na+ calcd m/z
1159.5184.
Data for 11: Rf 0.5 (2:3 hexanes–diethyl ether); 1H NMR
(500 MHz, CDCl3)
d 3.42–.58 (m, 1H), 3.64–4.00 (m, 16H),
4.44–4.76 (m, 15H), 4.83–4.92 (m, 2H), 5.04 (d, 1H, J 2 Hz, H-1),
7.16–7.34 (m, 40H, Ph-H), 13C NMR (125 MHz, CDCl3) d 61.69,
67.03, 69.29, 72.23, 72.36, 72.38, 72.40, 72.71, 72.78, 73.46,
74.88, 74.92, 74.96, 75.11, 75.17, 79.95, 80.14, 98.6 and 97.7 (both
C-1), 127.55, 127.59, 127.66, 127.69, 127.76, 127.84, 127.92,
127.97, 128.06, 128.08, 128.14, 128.18, 128.22, 128.37, 128.40,
138.23, 138.26, 138.30, 138.36, 138.42, 138.50, 138.53; LRMS-ESI
138.1, 138.2, 138.3, 166.0, 166.1;
m
max/cmꢀ1 (CHCl3) 1716;
HRMS-ESI (+ve) found: m/z 719.3220 [M+H]+; C44H47O9 calcd m/z
719.3220.

922
J. L. Harper et al. / Bioorg. Med. Chem. 19 (2011) 917–925
(+ve) m/z 1176 [M+K]+ (100), 1160 [M+Na]+ (89); (found: C, 74.87;
H, 6.86; C71H76O13 requires C, 74.98; H, 6.74).
CDCl3–CD3OD–D2O)
d
ꢀ0.9; HRMS-ESI (ꢀve) found m/z
1101.6699 [MꢀH]ꢀ; C54H102O20 Pꢀ calcd 1101.6702;
4.2.5. Triethylammonium 2-O-(1,2-di-O-stearoyl-sn-glycero-3-
phosphoryl)-1,3-bis-O-(2,3,4,6-tetra-O-benzyl-a-D-mannopyr-
anosyl)glycerol 13
4.2.7. 2-O-(4-Methoxybenzyl)glycerol 14
A solution of 1629 (2.19 g, 5.58 mmol) in ethanol (35 mL) was
stirred with RaneyÒ-Ni (50% in water, 4.5 g) under an H2 atmo-
sphere for 24 h, filtered and concentrated. Flash chromatography
on silica gel (deactivated with 8% w/w water) using 4:5–9:10
EtOAc–petroleum ether gave 14 (917 mg, 77%) as a colourless so-
lid. Rf 0.13 (2:1 EtOAc–petroleum ether); 1H NMR (500 MHz,
CDCl3) d 2.35–2.41 (m, 2H), 3.55 (quintet, 1H, J = 4.7 Hz, 2-H),
3.64–3.77 (m, 4H), 3.79 (s, 3H, CH3), 4.56 (s, 2H, ArCH2), 6.86–
6.90 (m, 2H), 7.25–7.29 (m, 2H); 13C NMR (500 MHz, CDCl3) d
55.3, 62.3, 71.7, 78.9, 114.0, 129.5, 130.2, 159.5; HRMS-ESI (+ve)
found: m/z 235.0947 [M+Na]+; C11H16O4Na+ calcd 235.0946.
A mixture of salt 12, prepared from (200 mg, 0.32 mmol), salicyl
chlorophosphite (107 mg, 0.53 mmol), dimannoside 10 (200 mg,
0.176 mmol)and 1,2-di-O-stearoyl-sn-glycerol (200 mg, 0.176
mmol) was dried by co-evaporation with pyridine (2 ꢁ 20 mL) and
was then dissolved in pyridine (8 mL). Pivaloyl chloride (200 lL,
1.62 mmol) was added and the resulting solution was stirred for
1 h at room temperature. After this time a solution of iodine
(120 mg, 0.47 mmol) in 9:1 pyridine–water (30 mL) was added
and stirring was continued for 45 min. The reaction mixture was di-
luted with CH2Cl2 (50 mL) and stirred for 15 min, washed with 10%
sodium thiosulfate solution (20 mL), and with 1 M tetraethylammo-
nium bromide (2 ꢁ 20 mL) and water (100 mL). The organic layer
was dried over magnesium sulfate and the solvent was removed.
The residue was purified over silica (98:1:1 CH2Cl2–MeOH–NEt3 as
eluent) to give the title compound 13 (252 mg, 76%) as a clear glass;
4.2.8. 1,3-Bis-(2-O-acetyl-3,4,5-tri-O-benzyl-
anosyl)-2-O-(4-methoxybenzyl)glycerol 18
a-D-mannopyr-
A solution of 14 (48 mg, 0.22 mmol) and donor 17 (357 mg,
0.56 mmol) in CH2Cl2 (10 mL) was concentrated and dried under
high vacuum for 30 min. The mixture was re-dissolved in dry
CH2Cl2 (2 mL) and dry toluene (5 mL). To the homogeneous solu-
tion was added powdered 4 Å molecular sieves (approx 1 g) and
the mixture stirred for 1 h at ambient temperature before being
cooled in an ice-water bath. Trimethylsilyl trifluoromethanesulfo-
Rf 0.45 (5:1:0.1 CH2Cl2–MeOH–NEt3); ½a D22
ꢃ
+11.7 (c 1.2, CH2Cl2); 1H
NMR (500 MHz, CDCl3) d 0.88 (t, 6H, J 7.5 Hz, 2 ꢁ CH3), 1.15 (t, 9H,
J 7.5 Hz, 3 ꢁ NCH2CH3), 1.20–1.32(m, 48H), 1.44–1.58 (br m, 4H,
2 ꢁ COCH2CH2), 2.21 (t, 4H, J 7.5 Hz, 2 ꢁ COCH2), 2.80–2.90 (br m,
6H, 3 ꢁ NCH2), 3.62–3.90 (m, 14H), 3.92–4.06 (m, 5H), 4.10–4.15
(m, 1H), 4.25–4.74 (m, 15H, PhCH2), 4.80–4.86 (m, 2H, PhCH2),
4.96 (br s, 1H, H-1), 5.03 (d, 1H, J 6 Hz, H-1), 5.18–5.22 (m, 1H, H-
100), 7.11–7.38 (m, 40H, Ph-H); 13C NMR (125 MHz, CDCl3) d 8.71,
14.16, 22.73, 24.89, 24.96, 29.20, 29.37, 29.40, 29.57, 29.70, 29.75,
31.96, 34.11, 34.29, 45.43, 62.95, 63.58, 66.97, 69.21, 69.32, 70.52,
72.02, 72.08, 72.54, 72.83, 73.33, 73.37, 74.75, 74.80, 75.04, 75.08,
80.39, 80.47, 98.29 (C-10), 127.38, 127.45, 127.58, 127.60, 127.65,
127.67, 127.71, 127.74, 127.80, 128.03, 128.04, 128.22, 128.24,
nate (2
tored for completion by TLC (3:7 EtOAc–petroleum ether). After
1 h at 0 °C NEt3 (25 L, 0.18 mmol) was added and the crude reac-
lL, 0.011 mmol) was added and the reaction was moni-
l
tion mixture was filtered, concentrated and purified by silica gel
chromatography (3:7 EtOAc–petroleum ether) to provide 18
(225 mg, 86%). Rf 0.15 (1:2 EtOAc–petroleum ether); ½a D20
ꢃ
+33 (c
d 2.13 (s, 6H,
0.92, CH2Cl2); 1H NMR (500 MHz, CDCl3)
2 ꢁ C(O)CH3), 3.45–3.55 (m, 2H), 3.63–3.82 (m, 12H), 3.85–3.97
(m, 4H), 4.42–4.54 (m, 8H), 4.62–4.72 (m, 4H), 4.81–4.88 (m,
4H), 5.34–5.38 (m, 2H, 2-H0 and 2-H00), 6.74–6.79 (m, 2H, Ph-H),
7.12–7.35 (m, 32H, Ph-H); 13C NMR (125 MHz, CDCl3) d 21.1,
55.2, 67.2, 67.4, 68.6, 68.7, 68.8, 71.5, 71.7, 71.8, 71.9, 72.3,
73.37, 73.45, 74.3, 75.1, 76.1, 78.1, 97.8 (1JC–H = 171 Hz), 98.2
(1JC–H = 171 Hz), 113.8, 127.5, 127.76, 127.83, 128.1, 128.3, 128.4,
129.6, 130.2, 138.0, 138.3, 138.5, 138.6, 159.3, 170.4; HRMS-ESI
(+ve) m/z found 1183.5016 [M+Na]+; C69H76O16Na+ calcd
1183.5031.
128.27, 128.35, 138.58, 138.66, 138.74, 138.76, 172.99, 173.40; 31
P
NMR (121 MHz, CDCl3) d 0.15 ppm; HRMS-ESI (ꢀve) found: m/z
1822.0492 [MꢀNHEt3]ꢀ C110H150O20Pꢀ calcd m/z 1822.0458.
4.2.6. Triethylammonium 2-O-(1,2-O-distearoyl-sn-glycero-3-
phoshoryl)-1,3-bis-O-(a-D-mannopyranosyl)glycerol 4
Phosphate 13 (100 mg, 0.055 mmol) was dissolved in 2:1:1:1
EtOAc–THF–EtOH–H2O (30 mL). 10% Pd/C (200 mg) was added
and the reaction was stirred under the atmosphere of hydrogen
for 18 h. The mixture was filtered through Celite, the filter pad
was washed with THF (5 mL), methanol (5 mL) and CH2Cl2
(2 ꢁ 5 mL), and the solvent from the combined filtrates was re-
moved in vacuo. The water was removed by azeotropic distillation
with toluene (5 ꢁ 4 mL). The residue was purified by silica gel pre-
parative plate chromatography (94:5:1 CH2Cl2–MeOH–NEt3 as elu-
ent). The baseline region of the plate was cut and the silica washed
with warm methanol (20 mL) and CH2Cl2 (20 mL). The solvent was
removed to give a residue which was lypophilised from a methanol
and water mixture to give the title compound 4 (42 mg, 69%) as a
4.2.9. 1,3-Bis-(3,4,5-tri-O-benzyl-a-D-mannopyranosyl)-2-O-(4-
methoxybenzyl)glycerol 19
To a solution of 18 (173 mg, 0.149 mmol) in 2:3 CH2Cl2–MeOH
(5 mL) was added sodium methoxide (30%, 25 L) and the reaction
l
mixture was stirred at ambient temperature. The reaction was
monitored by TLC (1:1 EtOAc–petroleum ether) and at 4.5 h the
reaction was quenched by the addition of DowexWX8-100 (H+) re-
sin. Flash chromatography on silica gel, using 3:2 EtOAc–petro-
leum ether gave the title compound 19 (145 mg, 90%) as a
colourless foam. Rf 0.22 (3:2 EtOAc–petroleum ether); ½a D20
ꢃ
+55 (c
white solid; Rf 0.2 (2:1:0.17 CHCl3–MeOH–H2O); ½a D20
ꢃ
+38 (c 0.6,
0.88, CH2Cl2); 1H NMR (500 MHz, CDCl3) d 2.45 (br s, 2H), 3.45–
3.55 (m, 2H), 3.61–3.80 (m, 12H), 3.80–3.88 (m, 4H), 3.97–4.01
(m, 2H, 2-H0 and 2-H00), 4.44–4.53 (m, 6H), 4.57–4.70 (m, 6H),
4.79–4.83 (m, 2H), 4.86 (d, 1H, J = 1.5 Hz, 1-H0), 4.90 (d, 1H,
J = 1.5 Hz, 1-H00), 6.75–6.79 (m, 2H), 7.13–7.36 (m, 32H); 13C NMR
125 MHz, CDCl3) d 55.2, 67.0, 67.4, 68.3, 68.4, 68.90, 68.94, 71.2,
71.3, 72.0, 72.1, 73.4, 73.5, 74.2, 74.3, 75.06, 75.07, 76.1, 80.09,
80.11, 99.4, 99.6, 113.8, 127.52, 127.55, 127.59, 127.62, 127.82,
127.84, 127.86, 127.87, 127.89, 127.91, 127.93, 128.0, 128.30,
128.31, 128.51, 128.52, 129.5, 130.3, 137.92, 137.93, 138.3, 138.4,
138.5, 159.2; HRMS-ESI (+ve) found m/z 1099.4824 [M+Na]+;
2:1:0.17 CHCl3–MeOH–H2O); 1H NMR (500 MHz, 2:1:0.17 CDCl3–
CD3OD–D2O) d 0.85–0.92 (m, 6H), 1.20–1.37 (m, 65H), 1.55–1.67
(m, 4H), 2.28–2.38 (m, 4H), 3.13 (q, 6H, J = 7.3 Hz), 3.58–3.69 (m,
6H), 3.71–3.78 (m, 4H), 3.82–3.91 (m, 6H), 36.94–4.02 (m, 2H),
4.17–4.22 (obs m, 1H), 4.35–4.45 (obs m, 2H), 4.82 (d, 1H,
J = 1.6 Hz), 4.84 (d, 1H, J = 1.6 Hz), 5.21–5.27 (m, 1H); 13C NMR
(125 MHZ, 2:1:0.17 CDCl3–CD3OD–D2O) d 8.0, 13.4, 22.2, 24.4,
24.5, 28.66, 28.69, 28.8, 28.9, 29.06, 29.09, 29.14, 29.19, 29.22,
31.4, 33.6, 33.8, 46.0, 47.5, 47.7, 47.8, 48.0, 48.2, 48.3, 48.5,
61.00, 61.03, 62.5, 63.0, 66.0, 66.4, 66.88, 66.93, 70.00, 70.04,
70.1, 70.2, 70.7, 72.5, 72.6, 76.7, 77.0, 77.3, 99.8 (1JC–H = 170),
99.9 (1JC–H = 169), 173.4, 173.8; 31P NMR (202 MHz, 2:1:0.17
C
65H72O14Na+ calcd 1099.4820.

J. L. Harper et al. / Bioorg. Med. Chem. 19 (2011) 917–925
923
4.2.10. 1,3-Bis-(2,3,4,5-tetra-O-benzyl-
O-(4-methoxybenzyl)glycerol 20
a
-
D
-mannopyranosyl)-2-
C
33H38O10Na+ calcd 617.2363. Data for the b-anomer: Rf 0.15 (1:2
EtOAc–petroleum ether); ½a D20
ꢃ
ꢀ16 (c 0.50, CH2Cl2); 1H NMR
To a solution of 19 (137 mg, 0.127 mmol) and benzyl bromide
(76 L, 0.636 mmol) in DMF (1 mL) stirred in an ice-water bath
(500 MHz, CDCl3) d 3.44 (s, 6H, 2 ꢁ CH3), 3.59 (ddd, 1H, J = 2.1,
4.1, 9.7 Hz, 5-H), 3.72–3.80 (m, 3H), 3.86 (dd, 1H, J = 9.5, 9.5 Hz,
4-H), 4.02 (d, 1H, J = 16.9 Hz), 4.08 (d, 1H, J = 16.9 Hz), 4.15 (d,
1H, J = 16.7 Hz), 4.21 (d, 1H, J = 16.7 Hz), 4.47–4.55 (m, 3H), 4.64
(d, 1H, J = 12.1 Hz), 4.71 (d, 1H, J = 11.2 Hz), 4.84 (d, 1H,
J = 10.8 Hz), 5.66 (dd, 1H, J = 0.8, 3.1 Hz, 2-H), 5.85 (d, 1H,
J = 0.8 Hz, 1-H), 7.13–7.17 (m, 2H), 7.23–7.34 (m, 13H); 13C NMR
(125 MHz, CDCl3) d 59.3, 59.4, 67.9, 68.4, 69.3, 69.5, 71.9, 73.5,
73.6, 75.2, 76.3, 79.6, 91.3 (1JCH = 162 Hz), 127.7, 127.8, 127.9,
128.0, 128.1, 128.4, 128.5, 137.2, 138.0, 168.3, 170.1; HRMS-ESI
(+ve) found m/z 617.2363 [M+Na]+; C33H38O10Na+ calcd 617.2363.
l
was added sodium hydride (60%, 20 mg, 0.50 mmol). The cooling
bath was removed and after 4 h the reaction was complete (TLC;
1:2 EtOAc–petroleum ether). The reaction mixture was diluted
with diethyl ether (50 mL) and washed with water (3 ꢁ 30 mL),
dried (MgSO4) and concentrated. Flash chromatography on silica
gel using 1:3 EtOAc–petroleum ether gave the title compound 20
(140 mg, 88%) as a colourless foam. Rf 0.36 (1:2 EtOAc–petroleum
ether); ½a 2D0
ꢃ
+27 (c 1.4, CH2Cl2); 1H NMR (500 MHz, CDCl3) d
3.41–3.50 (m, 2H), 3.59–3.80 (m, 14H) 3.85–3.90 (m, 2H), 3.97–
4.04 (m, 2H), 4.42–4.54 (m, 6H), 4.57–4.74 (m, 10H), 4.84–4.91
(m, 4H), 6.71–6.76 (m, 2H), 7.13–7.38 (m, 42H); 13C NMR
(125 MHz, CDCl3) d 55.1, 66.8, 67.5, 69.1, 69.2, 71.9, 72.0, 72.09,
72.12, 72.2, 72.5, 72.6, 73.2, 73.3, 74.82, 74.84, 74.88, 74.94, 75.0,
76.4, 80.01, 80.02, 98.0, 98.3, 113.7, 127.31, 127.34, 127.40,
127.44, 127.5, 127.56, 127.61, 127.62, 127.64, 127.8, 127.9,
128.16, 128.18, 128.20, 128.22, 128.24, 128.3, 129.3, 130.3, 138.3,
138.36, 138.43, 138.46, 138.48, 138.6, 159.1; HRMS-ESI (+ve)
found m/z 1279.5748 [M+Na]+; C79H84O14Na+ calcd 1279.5759.
4.2.12.2. 3,4,5-Tri-O-benzyl-2-O-methoxyacetyl-
nose. To a solution of 3,4,5-tri-O-benzyl-1,2-di-O-methoxyacetyl-
-mannopyranoside (1.22 g, 2.11 mmol) in THF (12 mL) was
D-mannopyra-
a-D
added methanolic ammonia (7 M, 0.84 mL, 5.9 mmol). The reaction
was monitored by TLC (1:1 EtOAc–petroleum ether) and at 80 min
was diluted with toluene (20 mL) and concentrated under reduced
pressure. Flash chromatography on silica gel using 1:2–2:3 EtOAc–
petroleum ether gave 3,4,5-tri-O-benzyl-2-O-methoxyacetyl-
mannopyranose (0.967 g, 88%) as a 9:1 mixture of - and b-anomers
as a colourless oil. Rf -anomer) 0.35, Rf (b-anomer) 0.24 (1:1
D-
a
4.2.11. 1,3-Bis-(2,3,4,5-tetra-O-benzyl-
a
-
D-mannopyranosyl)-
(a
glycerol 10
EtOAc–petroleum ether); 1H NMR (500 MHz, CDCl3) d 3.41 (s,
2.7H), 3.42 (s, 0.3H), 3.44–3.49 (m, 0.1H), 3.53 (d, 0.9H, J = 3.9 Hz,
OH), 3.60 (dd, 0.1H, J = 3.1, 9.2 Hz), 3.63–3.74 (m, 3H), 3.96 (d,
0.1H, J = 7.8 Hz, OH) 4.01–4.08 (m, 1.8H), 4.09–4.11 (m, 1.8H), 4.13
(d, 0.1H, J = 16.6 Hz), 4.20 (d, 0.1H, J = 16.6 Hz), 4.44–4.61 (m, 4H),
4.67–4.73 (m, 1H), 4.73–4.76 (m, 0.1H, b-1-H), 4.80–4.86 (m, 1H),
To a rapidly stirred mixture of 20 (117 mg, 93
(3.6 mL) and water (0.4 mL) was added DDQ (25 mg, 0.11
lmol) in CH2Cl2
l
mol)
at ambient T. After 1.5 h trace 20 remained (TLC; 1:2 EtOAc–petro-
leum ether) and the mixture was diluted with diethyl ether
(50 mL) and washed with NaHCO3 (4 ꢁ 40 mL) until the aqueous
phase was only a pale yellow colour and the combined organic por-
tions were dried (MgSO4) and concentrated. Flash chromatography
on silica gel using 7:20 EtOAc–petroleum ether as eluant gave 10
(89 mg, 84%) as a colourless foam. Rf 0.16 (1:2 EtOAc–petroleum
ether); Spectroscopic data for 10 prepared in this manner matched
well the data for 10 prepared by the alternative route described
above.
5.21 (dd, 0.9H, J = 1.8, 3.8 Hz,
a-1-H), 5.44 (dd, 0.9H, J = 1.9, 3.2 Hz,
a
-2-H), 5.52 (dd, 0.1H, J = 1.0, 3.1 Hz, b-2-H), 7.13–7.17 (m, 2H),
7.24–7.35 (m, 13H); 13C NMR (125 MHz, CDCl3) d 59.3, 69.0, 69.3,
69.5, 69.6, 69.7, 71.2, 71.8, 71.9, 73.5, 73.6, 73.9, 74.5, 75.1, 75.2,
77.3, 77.6, 80.2, 92.3, 93.0, 127.69, 127.75, 127.8, 127.91, 127.92,
127.95, 128.00, 128.04, 128.1, 128.2, 128.3, 128.39, 128.41, 128.5,
137.5, 137.86, 137.89, 138.1, 138.2, 169.9, 170.3; HRMS-ESI (+ve)
found m/z 545.2144 [M+Na]+; C30H34O8Na+ calcd 545.2151.
4.2.12. 3,4,5-Tri-O-benzyl-2-O-methoxyacetyl-a-D-mannopyr-
anosoyl trichloroacetimidate 21
4.2.12.1. 3,4,5-Tri-O-benzyl-1,2-di-O-methoxyacetyl-b-
nopyranoside. To a solution of 3,4,5-tri-O-benzyl- -mannopy-
4.2.12.3. 3,4,5-Tri-O-benzyl-2-di-O-methoxyacetyl-
pyranosyl trichloroacetimidate 21. To solution of 3,4,5-
tri-O-benzyl-2-O-methoxyacetyl- -mannopyranose (897 mg,
1.72 mmol) and trichloroacetonitrile (0.86 mL, 8.6 mmol) in CH2
Cl2 (5 mL) being stirred at 0 °C was added DBU (26 L, 0.17 mmol).
The reaction was monitored for loss of the starting pyranose by
TLC (1:2 EtOAc–petroleum ether) and after 15 min the reaction mix-
ture was applied directly to a flash chromatography silica column
and eluted with 2:8 EtOAc–petroleum ether to give 21 (1.08 g,
a-D-manno-
D
-man-
a
a
-
D
D
ranose (3.29 g, 7.29 mmol) in CH2Cl2 (11 mL) cooled in an ice/
water bath was added pyridine (2.0 mL, 25 mmol) followed by
careful addition of methoxyacetyl chloride (1.67 mL, 18 mmol).
The reaction was allowed to warm to ambient temperature over
18 h. The mixture was diluted with CH2Cl2 (100 mL) and washed
with dil. HCl (3 ꢁ 50 mL), satd. NaHCO3 (50 mL) and brine
(50 mL). The organic phase was dried (MgSO4) and concentrated.
Flash chromatography on silica gel using 1:2–2:3 EtOAc–petro-
leum ether as eluant gave clean samples of each 3,4,5-tri-O-ben-
l
94%) as approx 95:5 mixture of a-/b-anomers as a colourless oil. Rf
0.18 (1:4 EtOAc–petroleum ether); 1H NMR (500 MHz, CDCl3) d inter
alia 3.42 (s, 3H), 3.69–3.73 (m, 1H), 3.81–3.85 (m, 1H), 3.96–4.07 (m,
3H), 4.15 (s, 2H), 4.50 (d, 1H, J = 12.0 Hz), 4.54 (d, 1H, J = 10.7 Hz),
4.59 (d, 1H, J = 11.3 Hz), 4.65 (d, 1H, J = 12.1 Hz), 4.73 (d, 1H, d,
J = 11.3 Hz), 4.86 (d, 1H, J = 10.7 Hz), 5.58 (dd, 1H, J = 2.1, 3.0 Hz, 2-
H), 6.31 (d, 1H, J = 2.0 Hz, 1-H), 7.17–7.20 (m, 2H), 7.24–7.35 (m,
13H), 8.70 (s, 1H); 13C NMR (125 MHz, CDCl3) d 59.3, 67.7, 68.3,
69.5, 72.1, 73.4, 73.6, 74.3, 75.4, 90.7, 95.1, 127.6, 127.7, 127.8,
127.9, 128.0, 128.27, 128.32, 128.4, 128.5, 137.3, 138.0, 138.1,
159.9, 169.5; HRMS-ESI (+ve) found m/z 688.1253 [M+Na]+;
C32H34NO8Cl3Na+ calcd 688.1248.
zyl-1,2-di-O-methoxyacetyl-
and 3,4,5-tri-O-benzyl-1,2-di-O-methoxyacetyl-b-
oside (0.39 g, 9%) and a sample as a mixture of anomers (0.93 g,
a
-D
-mannopyranoside (2.75 g, 63%)
D
-mannopyran-
21%) all as colourless oils. Data for the
a
-anomer: Rf 0.23 (1:2
EtOAc–petroleum ether); ½a D20
ꢃ
+33 (c 1.12, CH2Cl2); 1H NMR
(500 MHz, CDCl3) d 3.41 (s, 3H, CH3), 3.42 (s, 3H, CH3), 3.68 (dd,
1H, J = 1.6, 10.9 Hz), 3.77–3.85 (m, 2H), 3.91–3.99 (m, 2H), 4.02
(s, 2H, CH2(OCH3)), 4.13 (s, 2H, CH2(OCH3)), 4.48–4.59 (m, 3H),
4.65 (d, 1H, J = 12.1 Hz), 4.73 (d, 1H, J = 11.1 Hz), 4.84 (d, 1H,
J = 10.6 Hz), 5.45 (dd, 1H, J = 2.4, 2.4 Hz, 2-H), 6.22 (d, 1H,
J = 1.9 Hz, 1-H), 7.15–7.19 (m, 2H), 7.23–7.35 (m, 13 H); 13C NMR
(125 MHz, CDCl3) d 59.4, 59.5, 67.9, 68.4, 69.4, 69.5, 72.1, 73.56,
73.60, 74.1, 75.4, 77.4, 91.5 (1JCH = 177 Hz), 127.7, 127.79, 127.82,
127.95, 127.98, 128.1, 128.36, 128.40, 128.5, 137.5, 138.0, 138.1,
167.9, 169.6; HRMS-ESI (+ve) found m/z 617.2360 [M+Na]+;
4.2.13. 1,3-Bis-(3,4,5-tri-O-benzyl-2-O-methoxyacetyl-
nopyranosyl)-2-O-(4-methoxybenzyl)glycerol 22
a-D-man-
A mixture of glycerol 14 (21 mg, 0.099 mmol), trichloroacetim-
idate donor 21 (165 mg, 0.247 mmol) and powdered 4 Å molecular
sieves (approx 0.5 g) in dry CH2Cl2 (1 mL) and dry toluene (2.5 mL)

924
J. L. Harper et al. / Bioorg. Med. Chem. 19 (2011) 917–925
was stirred for 1 h at ambient temperature before being cooled in
an ice-water bath. Trimethylsilyl trifluoromethanesulfonate
(1 ꢁ 30 mL), dried (MgSO4) and concentrated. Purification using
flash silica gel chromatography (35:65–55:45 EtOAc–petroleum
ether) afforded 25 (194 mg, 82%). Rf 0.36 (1:3 EtOAc–petroleum
ether); 1H NMR (500 MHz, CDCl3) d 0.82–0.82 (m, 6H), 1.09–1.42
(m, 56H), 1.49–1.61 (m, 4H), 2.18–2.27 (m, 4H), 3.37–3.39 (m,
6H), 3.57–3.71 (m, 4H), 3.72–3.90 (m, 8H), 3.90–3.98 (m, 2H),
4.00–4.16 (m, 6H), 4.16–4.27 (m, 2H), 4.39–4.54 (m, 6H), 4.57–
4.66 (m, 4H), 4.66–4.71 (m, 1H), 4.77–4.84 (m, 2H), 4.84–4.92
(m, 2H), 5.02–5.09 (m, 2H), 5.09–5.18 (m, 1H), 5.40–5.49 (m, 2H),
7.05–7.17 (m, 4H), 7.18–7.37 (m, 31H); 13C NMR (125 MHz, CDCl3)
d 14.0, 22.6, 24.7, 29.98, 29.02, 29.20, 29.24, 29.4, 29.6, 31.8, 33.9,
34.0, 59.2, 61.6, 65.5, 66.4, 66.6, 68.6, 68.7, 69.25, 69.32, 69.4, 71.8,
71.9, 73.3, 73.9, 75.1, 75.4, 78.0, 97.8, 98.1, 127.5, 127.6, 127.7,
127.97, 128.01, 128.15, 128.20, 128.5, 135.5, 137.7, 138.1, 138.2,
169.7, 172.6, 173.0; 31P NMR (202 MHz, CDCl3) d ꢀ1.5, ꢀ1.4;
HRMS-ESI (+ve) found m/z 1900.0389 [M+Na]+; C109H153O24PNa+
calcd 1900.0387.
(3.6
for completion by TLC (3:7 EtOAc–petroleum ether). After 15 min
at 0 °C NEt3 (50 L, 0.36 mmol) was added and the crude reaction
lL, 0.020 mmol) was added and the reaction was monitored
l
mixture was filtered, concentrated and purified by silica gel chro-
matography (1:3–45:55 EtOAc–petroleum ether) to provide the ti-
tle compound 22 (95 mg, 79%). Rf 0.26 (1:2 EtOAc–petroleum
ether); ½a 2D0
ꢃ
+31 (c 0.91, CH2Cl2); 1H NMR (500 MHz, CDCl3) d
3.395 and 3.398 (2 ꢁ unresolved s, 2 ꢁ 3H, 2 ꢁ CH2OCH3), 3.47–
3.56 (m, 2H), 3.62–3.89 (m, 14H), 3.93–3.98 (m, 2H), 4.10 (s, 4H,
2 ꢁ C(O)CH2), 4.41–4.56 (m, 8H, 8 ꢁ ArCH), 4.61–4.65 (m, 2H,
2 ꢁ ArCH), 4.68–4.72 (m, 2H, 2 ꢁ ArCH), 4.81–4.86 (m, 3H, 1-H0
and 2 ꢁ ArCH), 4.88 (d, 1H, J = 1.7 Hz, 1-H00), 5.44–5.47 (m, 2H, 2-
H0 and 2-H00), 6.78–6.80 (m, 2H, Ar), 7.12–7.35 (m, 32H, Ar). 13C
NMR (125 MHz, CDCl3) d 55.1, 59.2, 67.1, 67.2, 68.6, 68.8, 68.9,
69.5, 71.4, 71.5, 71.8, 71.9, 72.1, 73.26, 73.33, 74.1, 75.0, 75.9,
77.9, 97.6 (1JC–H = 171 Hz), 97.9 (1JC–H = 171 Hz), 113.7, 127.5,
127.6, 127.7, 128.0, 128.2, 128.3, 129.5, 130.0, 137.7, 138.1,
138.26, 138.34, 159.2, 169.7; HRMS-ESI (+ve) found m/z
1243.5236 [M+Na]+; C71H80O18Na+ calcd 1243.5242.
4.2.16. 2-O-(1,2-Di-O-stearoyl-sn-glycero-3-phosphoryl)-1,3-
bis-(2-O-methoxycetyl-
a-
D-mannopyranosyl)glycerol 26
A mixture of 25 (185 mg, 98
lmol) and Pd(OH)2 (20% on C,
125 mg) in 2:3 THF–MeOH (10 mL) was stirred under an
atmosphere of H2 at ambient temperature for 4 h, filtered and con-
centrated. Flash chromatography on silica gel, using 1:9–2:3
MeOH–CHCl3 gave 26 as a colourless powder (109 mg, 89%). Rf
4.2.14. 1,3-Bis-(3,4,5-tri-O-benzyl-2-O-methoxycetyl-a-D-man-
nopyranosyl)glycerol 23
To a stirred mixture of 22 (188 mg, 0.154 mmol) in CH2Cl2
(3.6 mL) and water (0.4 mL) was added DDQ (52 mg, 0.231 mmol).
After 2.25 h TLC (2:3 EtOAc–petroleum ether) indicated the reac-
tion was complete. The mixture was diluted with diethyl ether
(100 mL) and washed NaHCO3 (4 ꢁ 20 mL) until the aqueous phase
was almost colourless. The organic phase was dried (MgSO4) and
concentrated and the residue purified by silica gel chromatography
(2:3 to 1:1 EtOAc–petroleum ether) to afford the title compound
0.44 (2:1:0.17 CHCl3–MeOH–H2O); ½a D20
ꢃ
+24 (c 0.6, 2:1 CHCl3–
MeOH); 1H NMR (500 MHz, 2:1 CDCl3–CD3OD) d 0.86–0.92 (m,
6H), 1.22–1.37 (m, 56H), 1.55–1.67 (m, 4H), 2.28–2.368 (m, 4H),
3.46 (s, 6H, 2 ꢁ OCH3), 3.52–3.60 (m, 2H), 3.62–3.68 (m, 2H),
3.68–3.80 (m, 4H), 3.86–4.05 (m, 8H), 4.08–4.23 (m, 5H), 4.40–
4.47 (m, 2H), 4.84 (br s, 1H, 1-H0), 4.86 (d, 1H, J = 1.2 Hz, 1-H00),
5.12–5.18 (m, 2H), 5.22–5.29 (m, 1H, 2-H); 13C NMR 125 MHz,
2:1 CDCl3–CD3OD) d 13.8, 22.5, 24.76, 24.82, 29.0, 29.2, 29.5,
29.6, 31.8, 34.0, 34.1, 59.0, 61.9, 62.1, 62.7, 63.4, 66.8, 66.9, 68.1,
68.3, 69.3, 70.5, 72.6, 73.0, 73.1, 97.3, 170.3, 170.4, 173.7, 174.0;
31P NMR (202 MHz, 2:1 CDCl3–CD3OD) d ꢀ0.8; HRMS-ESI (ꢀve)
found m/z 1245.7139 [MꢀH]ꢀ; C60H110O24Pꢀ calcd 1245.7125.
23 (138 mg, 81%). Rf 0.26 (1:1 EtOAc–petroleum ether); ½a D20
ꢃ
+27
(c 1.4, CH2Cl2); 1H NMR (500 MHz, CDCl3) d 3.40 (s, 6H, 2 ꢁ CH3),
3.52 (dd, 1H, J = 6.6, 10.6 Hz), 3.57 (dd, 1H, J = 4.3, 10.7 Hz), 3.63
(dd, 1H J = 6.1, 10.7 Hz), 3.65–3.71 (m, 3H), 3.71–3.77 (m, 2H),
3.77–3.85 (m, 4H), 3.95–3.98 (m, 3H), 4.11 (s, 4H, 2 ꢁ C(O)CH2),
4.45–4.50 (m, 4H, 4 ꢁ ArCH), 4.51–4.56 (m, 2H, 2 ꢁ ArCH), 4.61–
4.65 (m, 2H, 2 ꢁ ArCH), 4.68–4.72 (m, 2H, 2 ꢁ ArCH), 4.80–4.85
(m, 2H, 2 ꢁ ArCH), 4.86–4.89 (m, 2H, 1-H0 and 1-H00), 5.45–5.48
(m, 2H, 2-H0 and 2-H00), 7.12–7.17 (m, 4H, Ar), 7.21–7.34 (m, 26H,
Ar); 13C NMR d 59.3, 68.8, 69.01, 69.04, 69.2, 69.6, 69.7, 69.9,
71.8, 72.03, 72.05, 73.5, 74.2, 75.2, 78.0, 98.2 (1JCH = 171 Hz),
98.3 (1JCH = 171 Hz), 127.7, 127.8, 127.9, 128.1, 128.3, 128.4,
137.8, 138.1, 138.2, 169.8; HRMS-ESI (+ve) found m/z 1123.4664
[M+Na]+; C63H72O17Na+ calcd 1123.4667.
4.2.17. Sodium salt of 2-O-(1,2-di-O-stearoyl-sn-glycero-3-
phosphoryl)-1,3-bis-(
To a solution of 26 (37 mg, 30.9
(6 mL) was added sodium methoxide (0.5 M, 4
a
-
D
-mannopyranosyl)glycerol 4
mol) in 1:5 CH2Cl2–MeOH
L, 2 mol) and
l
l
l
the reaction mixture was stirred at ambient T. The reaction was
monitored by TLC (2:1:0.17 CHCl3–MeOH–H2O) and at 2 h a further
portion of sodium methoxide was added (0.5 M, 4 lL, 2 lmol). After
a further 45 min the reaction was quenched by the addition of
DowexWX8-100 (H+) resin (approx 0.5 g), filtered and subjected
to two sequential treatments with DowexWX8-100 (Na+) resin
(2 ꢁ approx 0.5 g). Flash chromatography on silica gel, using
2:1:0–2:1:0.05–2:1:0.1 CHCl3–MeOH–H2O gave 4 as a colourless
4.2.15. 2-O-(Benzyloxy-1,2-di-O-stearoyl-sn-glycero-3-phos-
phoryl)-1,3-bis-(3,4,5-tri-O-benzyl-2-O-methoxycetyl-a-D-
mannopyranosyl)glycerol 25
After being dried separately under high vacuum for 2 h alcohol
23 (138 mg, 0.125 mmol) and phosphoramidite 2433 (189 mg, s
0.219 mmol) were dissolved together in dry CH2Cl2 (6 mL) and
cooled in an ice-water bath before adding 4,5-dicyanoimidazole
(31 mg, 0.263 mmol). The cooling bath was removed and the initial
suspension became homogenous over 15 min. The reaction was
monitored for loss of 23 by TLC (2:3 EtOAc–petroleum ether) and
after 2 h mCPBA (60%, 90 mg as a solution in CH2Cl2 and dried over
MgSO4, 0.31 mmol) was added over a period of 1 min. After stirring
for 30 min the mixture was concentrated under vacuum to half
volume, diluted with diethyl ether (150 mL) and washed with
10% sodium thiosulfate solution (100 mL). The aqueous phase
was back-extracted with diethyl ether (50 mL) and the combined
organic portions were washed with NaHCO3 (3 ꢁ 100 mL), brine
powder (28 mg, 84%). Rf 0.30 (2:1:0.17 CHCl3–MeOH–H2O); ½a D20
ꢃ
+37 (c 1.4, 2:1:0.17 CHCl3–MeOH–H2O); 1H NMR (500 MHz,
2:1:0.17 CDCl3–CD3OD–D2O) d 0.83–0.96 (m, 6H), 1.11–1.44 (m,
56H), 1.54–1.68 (m, 4H), 2.26–2.40 (m, 4H), 3.54–3.71 (m, 6H),
3.71–3.80 (m, 4H), 3.80–3.93 (m, 6H), 3.94–4.02 (m, 2H), 4.17–
4.23 (obs m, 1H), 4.36–4.45 (obs m, 2H), 4.84 (s, 1H, 1-H0), 4.87 (s,
1H, 1-H00), 5.22–5.28 (m, 1H, 2-H); 13C NMR (125 MHz, 2:1:0.17
CDCl3–CD3OD–D2O) d 14.2, 23.0, 25.2, 25.3, 29.50, 29.54, 29.68,
29.70, 29.8, 29.91, 29.95, 29.98, 30.03, 30.1, 32.3, 34.5, 34.6, 61.6,
61.7, 63.3, 63.8, 63.9, 66.9, 67.2, 67.55, 67.63, 70.76, 70.81, 70.9,
71.0, 71.38, 71.40, 73.3, 73.4, 73.5, 100.57, 100.64, 174.4, 174.7;
31P NMR (202 MHz, 2:1:0.17 CDCl3–CD3OD–D2O) d ꢀ0.8; HRMS-
ESI (ꢀve) found m/z 1101.6700 [MꢀH]ꢀ; C54H102O20Pꢀ calcd
1101.6702; HPLC purity 96.0.

J. L. Harper et al. / Bioorg. Med. Chem. 19 (2011) 917–925
925
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The authors wish to thank ComOne Ltd and the New Zealand
Foundation of Research Science and Technology (Contract C08X
0808) for financial support.
15. Ainge, G. D.; Hudson, J.; Larsen, D. S.; Painter, G. F.; Gill, G. S.; Harper, J. L.
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Supplementary data
Supplementary data (1H and 13C NMR spectra for compounds 6,
7, 10, 13, 18, 19, 20, 21, 23 and 25 as well as those of the sodium
and triethylammonium salt of the final compound 4 are provided.
The HPLC chromatogram of 4 is also provided) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmc.2010.11.058. These data include MOL files and InChiKeys of
the most important compounds described in this article.
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