The synthesis of phosphorylated disaccharide components of the extracellular phosphomannan of Pichia (Hansenula) holstii NRRL Y-2448

Article abstract of DOI:10.1016/j.bmc.2004.09.005

Methods for the stereoselective synthesis of α-(1→2)- and α-(1→3)-linked 6^II-O-phosphomannobiosides were developed. Two strategies were successfully employed: a d-mannosyl acceptor was coupled with a phosphorylated d-mannosyl trichloroacetimida

Full text of DOI:10.1016/j.bmc.2004.09.005

Bioorganic & Medicinal Chemistry 12 (2004) 6063–6075
The synthesis of phosphorylated disaccharide components of
the extracellular phosphomannan of Pichia (Hansenula) holstii
NRRL Y-2448
Jon K. Fairweather, Tomislav Karoli and Vito Ferro*
Drug Design Group, Progen Industries Ltd, 2806 Ipswich Road, Darra, Qld 4076, Australia
Received 26 August 2004;accepted 8 September 2004
Available online 29 September 2004
Abstract—Methods for the stereoselective synthesis of a-(1!2)- and a-(1!3)-linked 6II-O-phosphomannobiosides were developed.
Two strategies were successfully employed: a D-mannosyl acceptor was coupled with a phosphorylated D-mannosyl trichloroacetim-
idate donor, or alternatively with a differentially 6-O-protected D-mannosyl trichloroacetimidate donor which, after glycosylation,
was selectively deprotected and phosphorylated. Two target phosphomannobiosides intended for use in SAR studies of the antian-
giogenic drug candidate PI-88, 2-O-(6-O-phospho-a-D-mannopyranosyl)-D-mannopyranose and methyl 3-O-(6-O-phospho-a-D-
mannopyranosyl)-a-D-mannopyranoside, were synthesized. The former is a minor component of the side-chain repeating unit of
the extracellular phosphomannan of Pichia (Hansenula) holstii NRRL Y-2448, whilst the latter represents a nonreducing end frag-
ment of the phosphomannan.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
and tetrasaccharide phosphates
3
(ꢀ60%) and
4
(ꢀ30%), respectively, whilst the remaining 10% com-
prises di-, tri- and hexasaccharide phosphates (5–7)
and a tetrasaccharylamine (not shown).13,14 These oligo-
saccharides represent the repeating units of the side
chains attached to the core of the phosphomannan.
The individual components of 2 have not previously
been isolated primarily due to an inability to effectively
fractionate the mixture.ꢀ Consequently, structure–activ-
ity relationship (SAR) studies of PI-88 have been re-
stricted to sulfonating the more readily available
nonphosphorylated derivatives of oligosaccharides 3–
6,3,10 or using partially purified fractions obtained by
size exclusion chromatography of PI-88 itself (Fig. 1).11
The phosphosulfomannan agent PI-88 (1)1 has been
identified as a promising inhibitor of tumour growth
and metastasis and is currently undergoing Phase II clin-
ical trials in patients with advanced malignancies.2 PI-88
inhibits angiogenesis by antagonizing the interactions of
angiogenic growth factors (principally FGF-1, FGF-2
and VEGF) and their receptors with heparan sulfate.1,3
PI-88 is also a potent inhibitor of heparanase, an endo-
glucuronidase that cleaves heparan sulfate and plays a
key role in metastasis and angiogenesis.4,5 In addition
to its anticancer effects, PI-88 inhibits the blood coagu-
lation cascade,6–8 blocks vascular smooth muscle cell
proliferation and intimal thickening9 and inhibits herpes
simplex virus (HSV) infection of cells and cell-to-cell
spread of HSV-1 and HSV-2.10
To address the need for quantities of pure, phosphoryl-
ated PI-88 oligosaccharides for SAR studies, the synthe-
sis of the individual phosphomannans present in 2 (and
their subsequent sulfonation) is required. We therefore
sought to develop methods for the synthesis of phospho-
mannans containing a-(1!2) and a-(1!3) linkages. The
initial synthetic targets were the phosphomannobiosides
5 and 8. The a-(1!2)-linked phosphomannobioside 5 is
the smallest component present in 2 and is thus the first
target, whilst the a-(1!3)-linked 8 is a nonreducing end
PI-88 is prepared by exhaustive sulfonation11 of the
oligosaccharide phosphate fraction (2) obtained by mild,
acid-catalyzed hydrolysis of the extracellular phospho-
mannan of the yeast Pichia (Hansenula) holstii NRRL
Y-2448.12,13 The major components of 2 are the penta-
Keywords: Phosphomannobiosides;Synthesis; Pichia;PI-88.
*
Corresponding author. Tel.: +61 7 3273 9150;fax: +61 7 3375
6746;e-mail: vito.ferro@progen.com.au
ꢀ This has been attributed to the presence of the phophate group.13
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.005

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J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
2. Results and discussion
2.1. Synthesis via phosphorylated D-mannopyranosyl
donors
Many of the known procedures for synthesising 6-O-
phosphomannobiosides rely on the condensation of a
phosphorylated D-mannopyranosyl donor with a suita-
bly protected alcohol acceptor15–19 with good yields
being reported for both primary (6-OH) and secondary
(2-OH or 3-OH) glycosyl acceptors, though not with
universally successful outcomes. With precedent,18 the
bromide 9 was condensed with the alcohol 1020 in a
silver triflate (AgOTf)-promoted reaction incorporating
1,1,3,3-tetramethylurea (TMU) as a free-acid scavenger.
However,18 in our hands this procedure resulted in
exclusive formation of the orthoester 11 (65%) (Scheme
1). While the potential for rearrangement21 to the a-gly-
coside under acidic conditions exists, it was subse-
quently found that omission of TMU from the
reaction gave the desired (1!2)-linked disaccharide 12
in good yield (70%) (Scheme 1).
Figure 1.
fragment of these phosphomannans, with the methyl
glycoside representing the fixed a-linkage to succeeding
sugars. Two strategies were explored for the synthesis
of these disaccharides. Firstly, the glycosylation of a
suitable acceptor with a 6-O-phosphorylated D-manno-
pyranosyl donor, or alternatively, glycosylation with a
differentially 6-O-protected D-mannopyranosyl donor,
followed by removal of the protecting group and subse-
quent phosphorylation.
The alcohol 1322 was then targeted for glycosylation for
the preparation of a (1!3)-linked disaccharide, but
under these same conditions the reaction was not suc-
cessful. Similar observations were noted by Matta
et al. in attempts to glycosylate some secondary alcohol
acceptors.15 An alternative, more reactive glycosyl
donor was thus sought to effect the desired glycosylation
(Fig. 2).
Scheme 1. (a) Literature18 conditions, 65%;(b) modified conditions, 70%.
Figure 2.

J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
6065
From the multitude of available methods for activating
monosaccharides for glycosylation, trichloroacetimi-
dates were selected for further investigation. A classical
route to preparing an imidate such as 14 from the read-
ily available23 tetraacetate 15 necessitates selective
(anomeric) deacylation prior to condensation with tri-
chloroacetonitrile. Treatment of 15 with benzylamine
or hydrazine acetate, however, led to unwanted reac-
tions at the phosphate ester. The documented versatility
of the bis(trichloroethyl) phosphate protecting group in
disaccharide synthesis17 lead us to prepare compound 16
from the alcohol 1724 as an alternative synthon. Disap-
pointingly, this group proved equally prone to nucleo-
philic attack during manipulation of the anomeric
centre. A new strategy for preparing a suitable glycosyl
donor was therefore required.
upon treatment with trichloroacetonitrile and DBU was
successfully converted into the trichloroacetimidate 24.
A trimethylsilyl triflate (TMSOTf) promoted glycosyla-
tion of the alcohol 10 with the imidate 24 resulted in
an unexpected outcome. In addition to a chromato-
graphically indistinguishable mixture (9:1) of the phos-
phomannobiosides 25 and 26 (55%), the amide 27,
presumably resulting from an acid catalyzed rearrange-
ment of the imidate,27 was obtained (31%) (Scheme 3).
Imidate rearrangements of this type have been previ-
ously noted in attempted glycosylations of poor nucleo-
philes.28–30
In order to improve selectivity in glycosylation, a 2-O-
acyl (participating) protecting group was desired. The
tribenzoylated mannoside 28 was prepared from the tri-
tyl ether 19 in a similar procedure to the one previously
outlined (Scheme 2). In contrast to the tribenzylether 22,
treatment of the tribenzoate 28 with CAN under similar
conditions yielded an equal mixture of the hemiacetal 29
and an oxidatively-coupled by-product (30) (Scheme 4).
The occurrence of this by-product was effectively dimin-
ished, however, when the reaction was performed at
slightly elevated temperatures (30ꢁC).22 Upon treatment
with trichloroacetonitrile and potassium carbonate, the
hemiacetal 29 gave the imidate 31 in good yield
(85%).
The p-methoxyphenyl a-D-mannoside 1825 was con-
verted by way of the trityl ether 19 to the tribenzyl ether
20. Acid-catalyzed hydrolysis of the trityl ether was
rapid, yielding the alcohol 21 as the sole product. This
in turn was treated with diphenyl phosphorochloridate
and triethylamine to yield the diphenyl phosphate 22
in good yield (72%) (Scheme 2). Oxidative cleavage of
the p-methoxyphenyl protecting group with cerium(IV)
ammonium nitrate (CAN) at low temperature (0ꢁC)26
afforded a product, presumably the hemiacetal 23, which
Scheme 2.
Scheme 3.
Scheme 4.

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J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
Scheme 5.
TMSOTf was once again employed for glycosylation of
the alcohol 10 with the imidate 31 and resulted in the
exclusive formation of the a-(1!2)-phosphomannobio-
side 32 (79%) (Scheme 5). The identity of the phospho-
mannobioside was confirmed through a variety of
spectroscopic techniques including 1H, 31P and 13C
Though necessarily lengthy, these conditions did not
lead to either complete or partial hydrolysis of the 6II-
O-diphenylphosphate group. Sabesan and Neira31 have
observed that conversion of benzoyl to cyclohexylcarb-
onyl esters may occur under the forcing conditions
required for hydrogenation of sugar diphenylphosphates
(PtO2/H2/100psi). While such groups may well have
been formed during the hydrogenation of 36, all esters
were susceptible to methoxide-induced transesterifica-
tion and the target a-(1!2)-phosphomannobioside 5
was obtained as the sole product over the three steps
(30%). This sample was isolated as the disodium salt fol-
lowing purification by size exclusion chromatography
(Bio-Gel P-2) and characterized both spectroscopically
(1H and 31P NMR) and by capillary electrophoresis
(CE, tm = 18.8min). In an improved protocol, the hexa-
benzoate 35 was first subjected to base-catalyzed hydro-
lysis (2M KOH/THF/18-crown-6), during which the
diphenyl phosphate was partially deprotected, and then
rapid (<1h each) hydrogenolyses of the benzyl ether and
remaining phenyl phosphate. The product of these reac-
tions was acidified (AGꢂ 50W-X8, H+) and then neu-
tralized with Na2CO3 (1M) prior to size exclusion
chromatography (Bio-Gel P-2) to yield the phospho-
mannobioside 5 (49%), identical in all respects to the
sample previously prepared.
13
NMR with characteristic C–31P coupling constants19
3
observed (2JC6,P = 5.7Hz and JC5,P = 7.3Hz).
A published protocol16 for deprotection of the newly
synthesized 6II-O-phosphomannobioside 32 was then
followed. This involved Pd/C-catalyzed hydrogenolysis
of the benzyl ethers, PtO2-catalyzed hydrogenation of
the phosphate phenyl groups (100psi) and transesterifi-
cation of the benzoate esters. Thus, phosphomanno-
bioside 32 was submitted to the reaction sequence but
gave irreproducible results—the reaction was generally
plagued by an incomplete and indiscriminate hydro-
genolysis of the benzyl ether protecting groups even over
extended periods of time. In one outcome, following an
exhaustive hydrogenolysis with Pd/C and then Pearl-
manꢁs catalyst [Pd(OH)2/C], the crude reaction mixture
was successively hydrogenated with PtO2 and transeste-
rified but yielded only the 1-O-methylcyclohexyl manno-
bioside 33 in 23% yield (Scheme 5).
In an effort to monitor the removal of the benzyl ether
protecting group more closely, the alcohol 34 [derived
from 3,4,6-tri-O-acetyl-1,2-O-(benzyloxyethylidene)-b-
D-mannopyranose20 through the processes of transeste-
rification, benzoylation, orthoester rearrangement and
acid-catalyzed deacetylation] was glycosylated with the
imidate 31 to form the hexabenzoate 35 in good yield
(84%). Hydrogenolysis of the 1I-O-benzyl ether of 35
over Pearlmanꢁs catalyst was slow (72h at 100psi)
although the hemiacetal product 36 was confirmed by
Attention was then turned to investigating the outcomes
of glycosylation of the alcohol 13 with the two imidates
at hand for the preparation of a (1!3)-linked disaccha-
ride. Firstly, when the tribenzyl ether 24 was used as
glycosyl donor, a mixture (2:1) of the disaccharides 37
and 38 (72%) was obtained and once again the amide
1
an absence of benzylic-CH2 protons in the H NMR
spectrum (d 4.61, 4.79 AB quartet) (Scheme 6).
Scheme 6.
Scheme 7.

J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
6067
Scheme 8.
by-product 27 was formed (31% on 24). In contrast,
complete selectivity in glycosylation was observed when
the tribenzoylated imidate 31 was used as glycosyl donor
and the a-(1!3)-phosphomannobioside 39 was isolated
as the sole product (78%). The deprotection of the phos-
phomannobioside 39 was less challenging than for 35 as
a consequence of having only two types of protecting
groups in place. Slight modifications to the two previ-
ously established protocols were investigated: hydrogen-
ation of the diphenyl phosphate (PtO2/H2/100psi) and
then transesterification (NaOMe) of the benzoyl esters,
or base catalyzed ester hydrolysis (2M KOH/THF/18-
crown-6) followed by hydrogenation of the partially
deprotected diphenyl phosphate. In both cases, the reac-
tion mixtures were acidified upon completion (AGꢂ
50W-X8, H+) and then neutralized with Na2CO3 (1M)
before passage through a size exclusion column (Bio-
Gel P-2). An identical product, confirmed (1H and 31P
NMR spectroscopy) to be the a-1,3-phosphomannobio-
side disodium salt 8, was obtained from both strategies
(Scheme 7).
ation and base catalyzed condensation with trichloroac-
etonitrile (Scheme 8).
While a TMSOTf-catalyzed glycosylation of the alcohol
10 with 41 proceeded in near quantitative yield to give
the disaccharide 44, the attempted desilylation of 44
with tetrabutylammonium fluoride (TBAF), even when
buffered with acetic acid, resulted in quantitative acyl
migration to give 45. The tribenzoyl disaccharide 46,
prepared via glycosylation with the corresponding ben-
zoylated donor 42, was more amenable to deprotection
with TBAF but still susceptible to some acyl migration.
Kong and co-workers32 have demonstrated the utility of
the 6-O-acetyl imidate 43 in generating branched oligo-
mannans since this D-mannopyranosyl moiety may be
deacetylated without benzoyl ester migration. Following
these procedures, the imidate 43 was used in the glycosyl-
ation of the alcohol 10 furnishing the disaccharide 47 in
good yield (80%, Scheme 8). The acetyl protecting group
was easily removed thereby liberating the alcohol 48.
Reaction of the alcohol 48 with diphenyl phosphoro-
chloridate at ambient temperature resulted in the slow
formation of diphenyl phosphate 32.ꢂ The reaction
was greatly accelerated upon heating, as described for
the preparation of inositol phosphates,33 with the de-
sired product 32 obtained in a relatively short period
(3h).§ The phosphomannobioside 32 was identical in
all respects to that prepared by the previous method
(Scheme 9).
2.2. Synthesis via differentially protected D-mannopyrano-
syl imidates
An alternative approach to the target phosphomanno-
biosides was also investigated. The strategy was to gly-
cosylate the acceptor alcohols (such as 10 and 13) with
a glycosyl donor lacking a 6-O-phosphodiester and then
to introduce the phospho group after assembly of the
disaccharide. This necessitated the use of a differentially
protected D-mannopyranosyl donor to provide access to
an alcohol at C-6II, the site of phosphorylation, after
assembly of the disaccharide. Such an approach would
also allow for the synthesis of C-6II-analogues of the
disaccharides, if required. The initial glycosyl donor
chosen was the imidate 41, which was readily obtained
from the alcohol 17 via silylation, anomeric de-O-acetyl-
In accordance with the above procedure, the imidate
43 was condensed with the alcohol 13 to yield the
ꢂ The phosphorylation reaction was also very difficult to follow by
TLC because the alcohol 48 and product 32 co-migrate under many
solvent systems.
§ In repeated syntheses, this reaction was conveniently run over a
longer period of time (o/n).

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J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
detection by indirect UV absorbance at 214nm. Com-
1
pound homogeneity was determined by H and/or 13C
NMR spectroscopy and, where appropriate, by capillary
electrophoresis.
3.2. 3,4-Di-O-acetyl-6-O-diphenylphosphoryl-a-D-manno-
pyranose-1,2-(benzyl 3,4,6-tri-O-benzyl-a-D-mannopyr-
anosid-2-yl)orthoacetate (11)
Scheme 9.
A combined mixture of the freshly prepared bromide 918
(38mg, 63lmol), the alcohol 1020 (35mg, 64lmol) and
TMU (15lL, 133lmol) in CH2Cl2 (2mL) was stirred
disaccharide 49 in good yield (73%). Deacetylation
furnished the alcohol 50 which was, in turn, phosphoryl-
ated, thereby affording the a-(1!3)-phosphomannobio-
side 39. Once again, the product was identical in all
respects to that prepared by the previous method.
˚
in the presence of mol sieves (200mg of 3A powder)
(ꢁ40ꢁC, 20min). AgOTf (17mg, 66lmol) was added
and stirring was continued (45min). The mixture was
neutralized with Et3N (50lL), filtered and concentrated
and the residue was subjected to FC (25–40% EtOAc/
hexanes) to yield the orthoester 11 as a colourless oil
(45mg, 65%, a 93:7 mixture of diastereomers). 1H
NMR (major diastereoisomer: 400MHz, CDCl3) d
1.67 (s, 3H, ortho-Me),34 1.98, 1.99 (2s, 2 · 3H,
2 · Ac), 3.66–3.83 (m, 5H, H4I,5I,6aI,6bI,5II), 3.88 (dd,
1H, J2,3 3.3, J3,4 8.8Hz, H3I), 4.06 (dd, 1H, J1,2 1.7Hz,
H2I), 4.28–4.55, 4.62–4.81 (2m, 11H, H2II, 6aII, 6bII,
4 · CH2Ph), 4.56 (dd, 1H, J1,2 2.9, J2,3 4.0Hz, H2II),
4.95 (d, 1H, H1I), 5.07 (dd, 1H, J3,4 9.7Hz, H3II), 5.18
(dd, 1H, J3,4ꢀ4,5 9.4Hz, H4II), 5.37 (d, 1H, H1II),
7.09–7.35 (m, 30H, Ph);ESMS: m/z 1061.3 [M+H]+.
The successful synthesis of these disaccharides demon-
strates the utility of the imidates 31 and 43 as useful
glycosyl donors for the stereoselective assembly of a-
linked 6II-O-phosphomannobiosides such as 35 and 39.
In addition, we have employed reproducible protocols
for the deprotection of such phosphomannobiosides to
yield the target compounds 5 and 8, respectively. Efforts
are now directed towards employing these strategies for
the synthesis of the higher phosphorylated oligosacchar-
ides from the Pichia (Hansenula) holstii extracellular
phosphomannan.
3. Experimental
3.1. General
3.3. Benzyl 2-O-(2,3,4-tri-O-acetyl-6-O-diphenylphos-
phoryl-a-D-mannopyranosyl)-3,4,6-tri-O-benzyl-a-D-
mannopyranoside (12)
Nuclear magnetic resonance (NMR) spectra were re-
1
corded at 400 or 200MHz for H, 100MHz for 13C or
AgOTf (17mg, 66lmol) was added to the bromide 9
(38mg, 63lmol) and alcohol 10 (35mg, 64lmol) as de-
scribed for 11 except for the omission of TMU, to yield
the mannobioside 12 as a colourless oil (48mg, 70%). 1H
NMR (400MHz, CDCl3) d 1.96, 2.00, 2.04 (3s, 3 · 3H,
3 · Ac), 3.76–3.86 (ABX, 2H, H6aI,6bI), 3.80–3.95,
4.20–4.40 (2m, 6H, H3I,4I,5I,6aI,6bI,5II), 3.98 (dd, 1H,
J1,2ꢀ2,3 2.2Hz, H2I), 4.38–4.83 (m, 8H, PhCH2), 4.96
(d, 1H, J1,2 1.8Hz, H1I), 5.01 (d, 1H, J1,2 1.8Hz,
H1II), 5.28 (dd, 1H, J3,4ꢀ4,5 9.7Hz, H4II), 5.43 (dd,
1H, J2,3 3.5, J3,4 9.7Hz, H3II), 5.54 (dd, 1H, H2II),
7.10–7.40 (m, 30H, Ph);HRMS calcd for C 58H62O17P
[M+H]+ 1061.3719, found 1061.3794.
162MHz for 31P, either in deuteriochloroform (CDCl3)
with residual CHCl3 (1H, d 7.26) or deuterium oxide
(D2O), employing residual HOD (1H, d 4.78) as internal
standard, at ambient temperatures (298K), unless spec-
ified otherwise. Where appropriate, analysis of 1H NMR
spectra was aided by gCOSY experiments. Flash
chromatography (FC) or rapid silica filtration
(RSF) was performed on silica gel (40–63lm) under a
positive pressure with the specified eluants. Size
exclusion chromatography was performed on Bio-Gel
P-2;50 · 1000mm;0.1M NH 4HCO3;flow rate
2.8mLminꢁ1;collecting fractions each 2.8min. All sol-
vents used were of analytical grade. The progress of
the reactions was monitored by thin layer chromatogra-
phy (TLC) using commercially prepared silica gel 60
F254 aluminium-backed plates. Compounds were visual-
ized by charring with 5% sulfuric acid in methanol and/
or by visualization under ultraviolet light. The term
ꢃworkupꢁ refers to dilution with water, extraction into
an organic solvent, sequential washing of the organic ex-
tract with aqueous hydrochloric acid (1M, where appro-
priate), saturated aqueous sodium bicarbonate and
brine, followed by drying over anhydrous magnesium
sulfate, filtration and evaporation of the solvent by
means of a rotary evaporator at reduced pressure and
where appropriate, extensive drying of the residue at
<1mmHg. Capillary electrophoresis was performed as
previously described14 with the use of 6mM potassium
sorbate (pH10.3) as the background electrolyte and
3.4. 1,2,3,4-Tetra-O-acetyl-6-O-bis(trichloroethyl)phos-
phoryl-b-D-mannopyranose (16)
A mixture of the alcohol 1724 (377mg, 1.08mmol),
bis(2,2,2-trichloroethyl)phosphorochloridate
(1.23g,
3.24mmol) and Et3N (820lL, 11.1mmol) in 1,2-DCE
(25mL) was stirred (rt, 3h). The mixture was concen-
trated, filtered and the filtrate washed (H2O and then
satd NaCl). The solvent was evaporated and the residue
subjected to FC (35–50% EtOAc/hexanes) to yield the
1
tetraacetate 16 as a pale yellow oil (550mg, 87%). H
NMR (200MHz, CDCl3) d 2.01, 2.08, 2.10, 2.21 (4s,
4 · 3H), 3.82–3.88 (m, 1H), 4.27–4.37 (m, 2H), 4.64 (s,
2H), 4.67 (s, 2H), 5.14 (dd, 1H, J 3.2, 9.9Hz), 5.32 (t,
1H, J 9.9Hz), 5.49 (dd, 1H, J 1.1, 3.2Hz), 5.87 (d, 1H,
J 1.1Hz);ESMS: m/z 688.9 [M+H]+.

J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
6069
3.5. p-Methoxyphenyl 3,4,6-tri-O-benzyl-a-D-mannopyr-
anoside (21)
CDCl3) d 55.8, 67.9 (d, JC,P 5.7Hz), 71.6 (d, JC,P
8.2Hz), 72.5, 73.2, 75.4, 74.2, 74.9, 80.0, 97.4,
114.8, 117.8, 120.4(0), 120.4(3), 120.4(6), 120.4(8),
125.4(3), 125.4(4), 125.4(6), 125.4(8), 127.8(9), 127.9(2),
127.9(6), 128.0(0), 128.0(3), 128.2, 128.6(1), 128.6(4),
128.7, 138.2(8), 138.2(9), 138.5, 150.4, 155.2; 31P NMR
(162MHz, CDCl3) d ꢁ11.4 (PO(OPh)2);ESMS: m/z
789.2 [M+H]+.
(A) Trityl chloride (3.06g, 11.0mmol) was added to a
solution of the mannoside 1825 (2.82g, 9.86mmol) in
pyridine (20mL) and the combined mixture stirred
(0ꢁC!rt, o/n). The mixture was subjected to workup
(CH2Cl2) and FC (10–35% EtOAc/CH2Cl2) to yield,
presumably, the trityl ether 19 as a colourless foam
(4.39g). This sample was further dried under high vac-
uum (18h) and used in the following reaction without
further purification or characterization. (B) The trityl
ether 19 (2.22g, 4.20mmol, max.) in DMF (10mL)
was added dropwise to a cooled (0ꢁC), stirred suspen-
sion of pre-washed (hexane) NaH (1.21g of 50% oil sus-
pension, 25.2mmol) in DMF (30mL). Once the addition
was complete, stirring was maintained (0ꢁC!rt,
20min). The mixture was cooled (0ꢁC, 5min) and BnBr
(1.94mL, 16.4mmol) was introduced dropwise with con-
tinued stirring (0ꢁC!rt, 2h). The mixture was cooled
once again (0ꢁC) and MeOH (5mL) was introduced
with continued stirring (5min). The solvent was evapo-
rated and the residue was subjected to workup (CH2Cl2)
and evaporation to yield, presumably, the tribenzylether
20 as a pale yellow oil. This residue was co-evaporated
(2 · 100mL MeCN) and used in the next reaction with-
out further purification or characterization. (C) p-TsOHÆ
H2O (150mg) was added to the crude mixture from (B)
in MeOH (30mL) and MeCN (30mL) and the combined
mixture stirred (rt, o/n). The mixture was treated with
Et3N (1mL), the solvents were evaporated and the resi-
due subjected to workup (EtOAc) and FC (0–35%
EtOAc/hexane) to yield the alcohol 21 as a pale yellow
oil (2.06g, 88%, three steps). 1H NMR (400MHz,
CDCl3) d 3.73–3.78 (m, 6H, H5,6a,6b,OMe), 3.94 (dd,
1H, J1,2ꢀ2,3 2.3Hz, H2), 4.03–4.11 (m, 2H, H3,4), 4.66,
4.94 (AB quartet, JA,B 10.9Hz, PhCH2a), 4.68, 4.74
(AB quartet, JA,B 11.6Hz, PhCH2b), 4.71, 4.81 (AB
quartet, JA,B 12.4Hz, PhCH2c), 5.39 (d, 1H, H1),
6.75–6.88, 7.23–7.39 (2m, 19H, ArH); 13C NMR
(100MHz, CDCl3) d 55.8, 62.3, 73.1, 74.8, 75.0, 72.7,
73.3, 75.5, 80.1, 97.6, 114.8, 117.9, 127.8(7), 127.8(8),
128.0, 128.1, 128.3, 128.6(2), 128.6(3), 128.6(4), 128.7,
138.3, 138.5, 138.6, 150.2, 155.2.
3.7. 3,4,6-Tri-O-benzyl-6-O-diphenylphosphoryl-a-D-
mannopyranosyl trichloroacetimidate (24)
(A) CAN (3.35g, 6.09mmol) was added portionwise to a
solution of the mannoside 22 (1.60g, 2.03mmol) in 4:1
MeCN–H2O (60mL) and the combined mixture stirred
(0ꢁC, 10min). The mixture was then diluted (H2O),
and extracted (EtOAc). The organic extracts were suc-
cessively washed (satd aq NaHCO3, Na2S2O3, NaCl),
dried (MgSO4), filtered and evaporated. The residue
was subjected to FC (20–50% EtOAc/hexane) to yield
an orange oil. This residue was co-evaporated
(2 · 30mL MeCN) and used in the next reaction without
further purification or characterization. (B) DBU
(50lL) was added to a solution of the product from
(A) and trichloroacetonitrile (355lL, 3.55mmol) in
1,2-DCE (10mL) and the combined mixture stirred
(0ꢁC, 10min). The mixture was concentrated and the
residue subjected to FC (10–20% EtOAc/hexane) to
yield the imidate 24 as a pale yellow oil (728mg, 43%,
two steps). 1H NMR (400MHz, CDCl3) d 3.83 (dd,
1H, J1,2 2.0, J2,3 3.0Hz, H2), 3.90 (dd, 1H, J3,4 9.4Hz,
H3), 3.94–3.98 (m, 1H, H5), 4.05 (dd, 1H, J3,4ꢀ4,5
9.6Hz, H4), 4.45, 4.84 (AB quartet, JA,B 10.6Hz,
PhCH2a), 4.46–4.48 (m, 2H, H6a,6b), 4.56, 4.69 (AB
quartet, JA,B 11.8Hz, PhCH2b), 4.72 (s, 2H, PhCH2c),
6.31 (d, 1H, H1), 7.08–7.39 (m, 25H, Ph), 8.51 (br s,
1H, NH); 13C NMR (100MHz, CDCl3) d 67.5 (d, JC,P
5.4Hz), 72.4, 73.0, 73.5, 73.7(0), 73.7(1) (d, JC,P
8.0Hz), 75.6, 79.0, 96.0, 120.4(1), 120.4(4), 120.4(6),
120.4(9), 125.4(6), 125.4(7), 125.4(9), 125.5(0), 128.0,
128.0(8), 128.0(9), 128.1(4), 128.4, 128.6(1), 128.6(3),
128.7, 129.8(9), 129.9(0), 129.9(1), 129.9(2), 138.0,
138.0(8), 138.0(9), 160.5; 31P NMR (162MHz,
CDCl3)
d
ꢁ11.4 (PO(OPh)2);HRMS calcd for
C39H38O8P [M+HꢁCCl3CONH2]+ 665.2304, found
3.6. p-Methoxyphenyl 3,4,6-tri-O-benzyl-6-O-diphenyl-
phosphoryl-a-D-mannopyranoside (22)
665.2143.
Diphenyl phosphorochloridate (1.9mL, 9.0mmol) was
added to a solution of the alcohol 21 (2.0g, 3.6mmol)
and Et3N (2.5mL, 18mmol) in 1,2-DCE (20mL) and
the combined mixture stirred (0ꢁC!rt, 4h). The
mixture was subjected to workup (CHCl3) and FC
(10–40% EtOAc/hexane) to yield the phosphate 22 as a
3.8. Benzyl 2-O-(2,3,4-tri-O-benzyl-6-O-diphenylphos-
phoryl-D-mannopyranosyl)-3,4,6-tri-O-benzyl-a-D-man-
nopyranoside (25/26)
A mixture of the imidate 24 (430mg, 0.52mmol) and the
alcohol 10 (192mg, 0.36mmol) in 1,2-DCE (8mL) was
˚
stirred in the presence of mol sieves (400mg of 3A pow-
1
colourless oil (2.0g, 72%). H NMR (400MHz, CDCl3)
d 3.71 (s, 3H, OMe), 3.88–3.93 (m, 1H, H5), 3.94 (dd,
1H, J1,2 2.0, J2,3 3.0Hz, H2), 4.02 (dd, 1H, J3,4ꢀ4,5
9.6Hz, H4), 4.09 (dd, 1H, J3,4 9.2Hz, H3), 4.43–4.46
(m, 2H, H6a,6b), 4.48, 4.83 (AB quartet, JA,B 10.4Hz,
PhCH2a), 4.68 (s, 2H, PhCH2b), 4.73, 4.77 (AB quartet,
JA,B 12.6Hz, PhCH2c), 5.40 (d, 1H, H1), 6.76–6.84,
7.26–7.39 (2m, 29H, ArH); 13C NMR (100MHz,
der) under an atmosphere of argon (30min). The mix-
ture was cooled (0ꢁC) with continued stirring (10min)
prior to the addition of TMSOTf (94lL, 0.52mmol).
After 10min, Et3N (100lL) was introduced and the mix-
ture was filtered. The solvent was evaporated and the
residue subjected to FC (10–30% EtOAc/hexane) to
yield two fractions. Firstly, an anomeric mixture (9:1)

6070
J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
of the disaccharides 25/26 was produced as a colourless
oil (242mg, 55%). Partial 1H NMR (400MHz, CDCl3) d
4.88 (d, 1H, J1,2 1.9Hz, H1I-major), 4.89 (d, 1H, J1,2
2.0Hz, H1I-minor), 5.01 (d, 1H, J1,2 1.8Hz, H1II-major);
31P NMR (162MHz, CDCl3) d ꢁ11.4 (PO(OPh)2-min-
or), ꢁ11.3 (PO(OPh)2-major). ESMS: m/z 1205.4
[M+H]+. Next, the amide 27 was obtained as a colour-
less oil (136mg, 31%). 1H NMR (major anomer:
400MHz, CDCl3) d 3.55–3.59 (m, 1H, H5), 3.71 (dd,
1H, J2,3 2.4, J4,5 9.2Hz, H3), 3.93 (dd, 1H, J1,2 1.6Hz,
H2), 3.96 (dd, 1H, J4,5 9.6Hz, H4), 4.37–4.60 (m, 3H,
H6a,6b,PhCH2a), 4.65, 5.04 (AB quartet, JA,B 11.6Hz,
PhCH2b), 4.78 (s, 2H, PhCH2c), 4.80 (B of AB quartet,
JA,B 10.8Hz, PhCH2a), 5.17 (dd, 1H, J1,NH 8.8Hz, H1),
7.10–7.36 (m, 25H, Ph), 7.52 (d, 1H, NH); 13C NMR
(major anomer: 100MHz, CDCl3) d 67.5 (d, JC,P
4.2Hz), 73.4, 73.6, 75.1, 75.2, 75.3, 75.9 (d, JC,P
8.0Hz), 78.7, 83.1, 120.3(8), 120.4(2), 125.5, 127.9,
128.1, 128.2, 128.3, 128.5, 128.6, 128.7, 128.8, 128.9,
129.9, 137.7, 137.8, 137.9, 150.6, 150.7, 161.3; 31P
NMR (162MHz, CDCl3) d ꢁ11.7(5) (PO(OPh)2-minor),
ꢁ11.6(6) (PO(OPh)2-major). ESMS: m/z 826.3 (100%),
828.3 (97%) [M+H]+.
3H, H5,6a,6b), 5.59 (d, 1H, J1,2 1.9Hz, H1), 5.82 (dd,
1H, J2,3 3.2Hz, H2), 5.98 (dd, 1H, J3,4 10.0, J4,5
9.5Hz, H4), 6.05 (dd, 1H, H3), 6.77–7.58, 7.82–8.09
(m, 24H, ArH); 13C NMR (100MHz, CDCl3) d 55.8,
66.7, 67.4 (d, JC,P 5.5Hz), 70.0, 70.2 (d, JC,P 8.5Hz),
70.6, 97.1, 114.9, 118.2, 120.1(8), 120.2(3), 120.2(6),
120.3(1), 125.4, 125.4(6), 125.5(1), 125.5(2), 128.5,
128.7, 128.9, 129.0, 129.2, 129.3, 129.8(2), 129.8(3),
129.8(8), 129.8(9), 129.9(6), 130.0(4), 130.1, 150.3,
155.7, 165.6, 165.6(7), 165.6(9); 31P NMR (CDCl3) d
ꢁ11.5 (PO(OPh)2). ESMS: m/z 831.2 [M+H]+.
3.10. 2,3,4-Tri-O-benzoyl-6-O-diphenylphosphoryl-a-D-
mannopyranose (29)
(A) CAN (1.66g, 3.03mmol) was added to the
mannoside 28 (841mg, 1.01mmol) as described for the
preparation of 24 to yield two fractions. Firstly, 4-meth-
oxy-3-[1,4]benzoquinonyl-1-phenyl 2,3,4-tri-O-benzoyl-
6-O-diphenylphopshoryl-a-D-mannopyranoside (30) was
obtained as an orange oil (183mg, 19%). 1H NMR
(400MHz, CDCl3) d 3.69 (s, 3H, OMe), 4.43–4.49 (m,
3H, H5,6a,6b), 5.63 (d, 1 H, J1,2 1.9Hz, H1), 5.82 (dd,
1H, J2,3 3.0Hz, H2), 5.99 (dd, 1H, J3,4 10.0, J4,5
9.3Hz, H4), 6.05 (dd, 1H, H3), 6.72–6.82, 7.03–7.58,
7.81–8.09 (3m, 31H, ArH); 13C NMR (100MHz,
CDCl3) d 56.4, 66.6, 67.4 (d, JC,P 5.5Hz), 69.9, 70.3(9)
(d, JC,P 8.8Hz), 70.4(3), 97.2, 112.5, 119.3, 119.8,
120.2(2), 120.2(4), 120.2(6), 120.3(1), 123.6, 125.4(8),
125.4(9), 125.5(3), 125.5(4), 128.6, 128.7, 128.9(0),
128.9(4), 129.1, 129.2, 129.8, 129.9, 130.0, 130.1, 130.2,
133.5, 133.8, 133.9, 134.8, 136.3, 137.2, 145.1, 149.9,
153.3, 165.5(6), 165.6(4), 165.7, 185.6, 187.5; 31P NMR
(162MHz, CDCl3) d ꢁ11.4 (PO(OPh)2);ESMS: m/z
937.2 [M+H]+. Next, the hemiacetal 29 was obtained
3.9. p-Methoxyphenyl 2,3,4-tri-O-benzoyl-6-O-diphenyl-
phosphoryl-a-D-mannopyranoside (28)
(A) Benzoyl chloride (1.19mL, 10.3mmol) was added
dropwise to a cooled (0ꢁC) solution of the trityl ether
19 (1.21g, 2.30mmol) in 1,2-DCE (10mL) and the com-
bined mixture was stirred (0ꢁC!rt, o/n). After this
time, the mixture was cooled (0ꢁC) and MeOH (5mL)
was introduced with continued stirring (5min). The sol-
vent was evaporated and the residue was subjected to
workup (EtOAc) and evaporation, and the residual oil
subjected to RSF (10–35% EtOAc/hexanes) to yield a
yellow oil. This residue was co-evaporated (2 · 50mL
MeCN) and used in the next reaction without further
purification or characterization. (B) p-TsOHÆH2O
(100mg) was added to the crude product from (A) in
MeOH (10mL) and MeCN (10mL) and the combined
mixture heated (70ꢁC, 10min). Et3N (1mL), was added,
the solvents were evaporated and the residue subjected
to FC (10–40% EtOAc/hexane) to yield p-methoxy-
phenyl 2,3,4-tri-O-benzoyl-a-D-mannopyranoside (51)
1
as a colourless oil (353mg, 48%). H NMR (400MHz,
CDCl3) d 4.42 (dd, 1H, J5,6a 1.5, J6a,6b 9.0Hz, H6a),
4.44 (d, 1H, J5,6b 0.0Hz, H6b), 4.52–4.56 (m, 1H, H5),
5.31 (d, 1H, J1,2 1.8Hz, H1), 5.62 (dd, 1H, J2,3 3.3Hz,
H2), 5.83 (dd, 1H, J3,4ꢀ4,5 10.0Hz, H4), 5.92 (dd, 1H,
H3), 7.06–7.55, 7.71–8.04 (2m, 25H, Ph). (B) CAN
(58mg, 105lmol) was added to the mannoside 28
(25mg, 30lmol) as described in (A), except for perform-
ing the reaction at 30ꢁC, to yield the hemiacetal 29 as a
colourless oil (14mg, 64%). This sample was spectro-
scopically identical to that produced in (A).
1
as a colourless oil (710mg, 83%, two steps). H NMR
(400MHz, CDCl3) d 3.72 (dd, 1H, J5,6a 3.4, J6a,6b
13.0Hz, H6a), 3.76 (s, 3H, OMe), 3.79 (dd, 1H, J5,6b
2.2Hz, H6b), 4.16–4.20 (m, 1H, H5), 5.69 (d, 1H, J1,2
1.8Hz, H1), 5.84 (dd, 1H, J2,3 3.4Hz, H2), 5.91 (dd,
1H, J3,4ꢀ4,5 10.1Hz, H4), 6.16 (dd, 1H, H3), 6.82–8.11
(m, 19H, ArH); 13C NMR (100MHz, CDCl3) d 55.9,
61.3, 67.3, 69.7, 70.7, 71.7, 97.0, 115.0, 118.0, 128.6,
128.7, 128.9, 129.3, 129.4, 129.9, 130.1, 130.2, 133.5,
133.8(6), 133.9(4), 150.1, 155.6, 165.7, 166.8. (C) Et3N
(767lL, 5.50mmol) was added to a solution of the alco-
hol 51 (658mg, 1.10mmol) and diphenyl phosphoro-
chloridate (570lL, 2.75mmol) in 1,2-DCE (10mL) and
the combined mixture stirred (0ꢁC!rt, o/n). The mix-
ture was subjected to workup (CHCl3) and FC
(10–40% EtOAc/hexane) to yield the diphenyl phos-
3.11. 2,3,4-Tri-O-benzoyl-6-O-diphenylphosphoryl-a-D-
mannopyranosyl trichloroacetimidate (31)
K2CO3 (374mg, 2.70mmol) was added to a stirred solu-
tion of the hemiacetal 29 (353mg, 0.490mmol) and tri-
chloroacetonitrile (1.00mL, 10.0mmol) in 1,2-DCE
(5mL) and the combined mixture stirred (0ꢁC,
10min). After this time, the mixture was concentrated
and the residue subjected to FC (10–20% EtOAc/hex-
ane) to yield the imidate 31 as a pale yellow oil
(382mg, 90%). 1H NMR (400MHz, CDCl3) d 4.41–
4.55 (m, 3H, H5,6a,6b), 5.86 (dd, 1H, J1,2 2.0, J2,3
3.3Hz, H2), 5.91 (dd, 1H, J3,4 10.1Hz, H3), 6.02 (dd,
1H, J4,5 10.1Hz, H4), 6.53 (d, 1H, H1), 7.08–8.06 (m,
1
phate 28 as a colourless oil (851mg, 93%). H NMR
(400MHz, CDCl3) d 3.73 (s, 3H, OMe), 4.42–4.48 (m,

J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
6071
25H, Ph), 8.81 (br s, 1H, NH); 31P NMR (162MHz,
JH,H 9.6Hz, cHexCH2), 3.35 (dd, 1H, JH,cHex(CH)
7.6Hz, cHexCH2), 3.46 (ddd, 1H, J4,5 9.7, J5,6a
2.0, J5,6b 5.7Hz, H5I), 3.51 (dd, 1H, J3,4 9.8Hz,
H4I), 3.58 (dd, 1H, J6a,6b 12.4Hz, H6bI), 3.61–3.66 (m,
1H, H5II), 3.68–3.72 (m, 3H, H6aI,3II,4II), 3.74
(dd, 1H, J2,3 3.3Hz, H3I), 3.78–3.83, 3.89–3.96 (2m,
4H, H2I,2II,6aII,6bII), 4.84 (d, 1H, J1,2 1.4Hz, H1I),
4.88 (d, 1H, J1,2 1.6Hz, H1II); 31P NMR (162MHz,
D2O) d 2.7 (PO(ONa)2);HRMS calcd for C 19H35O14P
[M+H]+ 519.1837, found 519.1789;CE: tm = 21.28min.
CDCl3)
d
ꢁ11.5 (PO(OPh)2). HRMS calcd for
C39H32O11P [M+HꢁCCl3CONH2]+ 707.1682, found
707.1348.
3.12. Benzyl 2-O-(2,3,4-tri-O-benzoyl-6-O-diphenylphos-
phoryl-a-D-mannopyranosyl)-3,4,6-tri-O-benzyl-a-D-
mannopyranoside (32)
TMSOTf (24lL, 0.13mmol) was added to the imidate
31 (116mg, 0.13mmol) and alcohol 10 (60mg,
0.11mmol) as described for the preparation of 25/26 to
yield the mannobioside 32 as a colourless oil (110mg,
79%). 1H NMR (400MHz, CDCl3) d 3.74 (dd,
1H, J5,6a 2.0, J6a,6b 10.6Hz, H6aI), 3.79 (dd, 1H, J5,6b
5.1Hz, H6bI), 3.86 (ddd, 1H, J4,5 9.2Hz,
H5I), 3.97 (dd, 1H, J2,3 2.8, J3,4 9.1Hz, H3I), 4.02
(dd, 1H, H4I), 4.05–4.06 (m, 1H, H2I), 4.37–4.50 (m,
7H, H5I, 5II, PhCH2a-c), 4.56, 4.86 (AB quartet, JA,B
11.6Hz, PhCH2d), 4.61 (dd, 1H, J5,6a 2.9, J6a,6b
11.8Hz, H6aII), 4.69 (dd, 1H, J5,6b 5.4Hz,
H6bII), 4.71 (B of AB quartet, JA,B 10.9Hz, PhCH2c),
5.13 (d, 1H, J1,2 1.8Hz, H1I), 5.20 (d, 1H, J1,2 1.6Hz,
H1II), 5.86–5.94 (m, 3H, H2II, 3II, 4II), 7.14–8.05
(m, 45H, Ph); 13C NMR (100MHz, CDCl3) d 66.8,
67.6 (d, JC,P 4.6Hz), 69.3, 69.6, 70.0 (d, JC,P 7.3Hz),
70.2, 70.4, 72.2, 72.8, 73.5, 75.1, 75.5, 76.6, 79.9, 98.4,
99.6, 120.2, 120.2(8), 120.3(2), 125.4(6), 125.5(0),
127.7, 127.7(7), 127.8(3), 127.9, 128.0, 128.1, 128.4,
128.4(7), 128.5(4), 128.5(8), 128.6(2), 128.7, 128.8,
129.0, 129.4, 129.6, 129.8(7), 129.9(2), 130.0(9),
130.1(3), 133.3, 133.5(9), 133.6(3), 137.4, 138.4, 138.5,
138.6, 150.5(9), 150.6(2), 150.6(6), 150.6(9), 165.3, 165.4,
165.6; 31P NMR (162MHz, CDCl3) d ꢁ11.3 (PO(OPh)2);
HRMS calcd for C73H67O17P [M+H]+ 1247.4189, found
1247.4105.
3.14. Benzyl 3,4,6-tri-O-benzoyl-a-D-mannopyranoside
(34)
(A) A small piece of sodium metal (50mg) was added to
a suspension of 3,4,6-tri-O-acetyl-1,2-O-(benzyloxyethyl-
idene)-b-D-mannopyranose20 (5.21g, 11.8mmol) and the
combined mixture was stirred (1h). The solid slowly dis-
solved and after 1h, the mixture was neutralized by the
addition of AGꢂ 50W-X8 resin (H+ form) and filtered.
The solvent was evaporated and co-evaporated
(2 · 100mL MeCN) and the residual oil used in the fol-
lowing reaction without further purification. (B) Ben-
zoyl chloride (4.83mL, 41.6mmol) was added dropwise
to a cooled (0ꢁC) solution of the crude product from
(A) in pyridine (30mL) and the combined mixture was
stirred (0ꢁC!rt, o/n). After this time, the mixture was
cooled (0ꢁC) and MeOH (5mL) was introduced with
continued stirring (5min). The solvent was evaporated
and the residue was subjected to workup (EtOAc), FC
(10–40% EtOAc/hexanes) and recrystallization to yield
3,4,6-tri-O-benzoyl-1,2-O-(benzyloxyethylidene)-b-D-
mannopyranose (52) as colourless needles (3.26g, 57%,
1
two steps), mp 140–141ꢁC (EtOAc/hexanes); H NMR
(400MHz, CDCl3) d 1.84 (s, 3H, ortho-CH3), 4.07
(ddd, 1H, J4,5 9.4, J5,6a 3.3, J5,6b 4.7Hz, H5), 4.46 (dd,
1H, J6a,6b 12.1Hz, H6b), 4.53, 4.57 (AB quartet, JA,B
11.3Hz, PhCH2), 4.62 (dd, 1H, H6a), 4.85 (dd, 1H,
J1,2 2.7, J2,3 3.8Hz, H2), 5.56 (dd, 1H, J3,4 9.8Hz,
H3), 5.67 (d, 1H, H1), 5.91 (dd, 1H, J4,5 9.7Hz, H4),
7.16–8.02 (m, 20H, ArH); 13C NMR (100MHz, CDCl3)
d 25.0, 63.5, 65.1, 66.6, 71.6, 72.0, 76.8, 97.9, 124.6,
127.8, 128.0, 128.5, 128.6, 129.1, 129.2, 129.9, 130.2,
133.3, 133.6, 133.7, 137.6, 165.4, 166.2, 166.4. (C)
TMSOTf (424lL, 2.34mmol) was added to the ortho-
ester 52 (972mg, 1.56mmol) and benzyl alcohol
(180lL, 1.71mmol) as described for the preparation of
25 to yield a colourless oil. This residue was co-evapo-
rated (2 · 10mL MeCN) and used in the next reaction
without further purification or characterization. A small
sample was purified by FC (10–40% EtOAc/hexanes),
yielding benzyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-man-
nopyranoside (53) as a colourless oil. 1H NMR
(400MHz, CDCl3) d 2.10 (s, 3H, Ac), 4.39 (ddd, 1H,
J4,5 9.7, J5,6a 2.8, J5,6b 5.3Hz, H5), 4.47 (dd, 1H, J6a,6b
12.0Hz, H6b), 4.57 (dd, 1H, H6a), 4.63, 4.80 (AB quar-
tet, JA,B 11.8Hz, PhCH2), 5.00 (d, 1H, J1,2 1.8Hz, H1),
5.52 (dd, 1H, J2,3 3.3Hz, H2), 5.82 (dd, 1H, J3,4 10.0Hz,
H3), 5.91 (dd, 1H, H4), 7.30–7.54, 7.86–8.06 (2m, 20H,
Ph); 13C NMR (100MHz, CDCl3) d 21.0, 63.5, 65.5,
67.4, 69.3, 69.9, 70.1, 96.8, 127.2, 127.8, 128.3, 128.4,
128.5(9), 128.6(3), 128.7, 128.8, 129.1, 129.4, 129.8(6),
129.8(9), 130.0(1), 130.0(3), 133.3, 133.5, 133.6, 136.5,
3.13. Attempted deprotection of the tetrabenzyl ether (32):
Methylcyclohexyl 2-O-(6-O-phospho-a-D-mannopyranos-
yl)-a-D-mannopyranoside, disodium salt (33)
Pd (20mg of 10% on C) was added to a solution of the
tetrabenzyl ether 32 (100mg, 83.5mmol) in MeOH
(2mL) and EtOAc (2mL) containing AcOH (50lL)
and the combined mixture was vigorously stirred under
hydrogen (100psi, 48h). The mixture was filtered and
Pd(OH)2 (20mg of 10% on C) was introduced and stir-
ring continued under hydrogen (100psi, 48h). The mix-
ture was filtered and PtO2 (10mg) was introduced and
stirring continued under hydrogen (100psi, 48h). The
mixture was filtered and the solvent evaporated. The res-
idue was dissolved in MeOH (2mL) and NaOH (1mL of
1M, 1.0mmol) and the combined mixture was stirred (rt,
o/n). The mixture was acidified to pH2.0 by the addition
of AGꢂ 50W-X8 (H+ form) and filtered. The filtrate was
neutralized with Na2CO3 (1M) and the solvent evapo-
rated. The residue was subjected to gel filtration chroma-
tography (Bio-Gel P-2;0.1M NH HCO3;170mL/h) to
4
yield, after lyophilization, the methylcyclohexyl manno-
bioside 33 as a colourless powder (9.6mg, 23%). 1H
NMR (400MHz, D2O) d 0.75–0.85, 0.99–1.11, 1.45–
1.62 (3m, 11H, cHex), 3.23 (dd, 1H, JH,cHex(CH) 5.8,

6072
J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
165.6, 165.8, 166.3, 170.0. (D) AcCl (0.5mL) was added
to a solution of the crude product from (C) in 1,2-DCE
(10mL) and MeOH (20mL) and the combined mixture
was stirred (rt, o/n). The mixture was cooled (0ꢁC)
and Et3N (1mL) was introduced with continued stirring
(5min) prior to evaporation of the solvent. The residue
was subjected to workup (EtOAc) and FC (10–40%
EtOAc/hexanes) to yield the alcohol 34 as a colourless
oil (418mg, 40%, two steps). 1H NMR (400MHz,
CDCl3) d 4.33 (dd, 1H, J1,2 1.8, J2,3 3.0Hz, H2), 4.38
(ddd, 1H, J4,5 9.9, J5,6a 3.0, J5,6b 5.4Hz, H5), 4.47 (dd,
1H, J6a,6b 12.0Hz, H6b), 4.55 (dd, 1H, H6a), 4.62,
4.81 (AB quartet, JA,B 11.9Hz, PhCH2), 5.03 (d, 1H,
H1), 5.70 (dd, 1H, J3,4 9.9Hz, H3), 5.94 (dd, 1H, H4),
7.29–7.52, 7.90–8.03 (2m, 20H, ArH); 13C NMR
(100MHz, CDCl3) d 63.7, 67.3, 69.2, 69.6(7), 69.7(0),
72.9, 98.8, 128.4, 128.6, 128.8, 129.3, 129.40, 129.9,
129.9(7), 129.9(9), 133.3, 133.5(2), 133.5(4), 136.8,
165.7(9), 165.8(3), 166.4;HRMS calcd for C 34H30O9
[M+H]+ 583.1963, found 583.1939.
KOH (1mL of 2.0M) was added to the product from
(B) in THF (1.2mL) and the combined mixture was stir-
red (rt, o/n). After this time the mixture was acidified
with AGꢂ 50W-X8 resin (H+ form) and filtered. The fil-
trate was neutralized with Na2CO3 (1M) and the solvent
evaporated. The residue was subjected to gel filtration
chromatography (Biogel P2;0.1M NH HCO3;170mL/
4
h) to yield, after lyophilization, an anomeric mixture
(17:3) of the a-1,2-mannobioside (5) as a colourless
powder (1.0mg, 30%, three steps). 1H NMR
(400MHz, D2O) d 3.45–3.80 (m, 11H, H3I,4I,5I,6aI,
6bI,2II,3II,4II,5II,6aII,6bII), 3.92 (d, 1H, J1,2 1.8, J2,3
3.4Hz, H2I-major), 3.96 (d, 1H, J1,2 1.8, J2,3 3.4Hz,
H2I-minor), 4.76 (d, 1H, H1I-minor), 4.89 (d, 1H, H1I-
major), 4.99 (d, 1H, H1II-minor), 5.23 (d, 1H, H1II-
major); 13C NMR (major anomer;400MHz, D 2O) d
61.1, 61.2, 67.0, 67.2, 70.1, 70.5, 72.6, 73.0, 73.4, 79.3,
92.6, 102.3; 31P NMR (162MHz, D2O) d ꢁ1.5(7)
(PO(ONa)2-minor), ꢁ1.6(4) (PO(ONa)2-major);HRMS
calcd for C12H22O14P [M+H]+ 422.0820, found
422.0812;CE: tm = 22.13min.
3.15. Benzyl 2-O-(2,3,4-tri-O-benzoyl-6-O-diphenylpho-
rosphoryl-a-D-mannopyranosyl)-3,4,6-tri-O-benzoyl-a-D-
mannopyranoside (35)
Method B: The hexabenzoate 35 (15mg, 11.6lmol) in
THF was first treated with KOH with the inclusion of
18-crown-6 (13.1mg, 49.6lmol), and then Pd(OH)2/C
and PtO2 as described in the previous method [steps
(C), (A) and then (B), except that the reaction was per-
formed over a short period (1h)] to yield, after lyophiliza-
tion, the phosphomannobioside 5 as a colourless powder
(2.4mg, 49%, three steps). This sample was identical in all
respects to that produced in the previous method.
TMSOTf (22.5lL, 123lmol) was added to the imidate
31 (107mg, 123lmol) and alcohol 34 (60mg, 103lmol)
as described for the preparation of 25/26 to yield the
1
mannobioside 35 as a colourless oil (112mg, 84%). H
NMR (400MHz, CDCl3)
d
4.29–4.66 (m, 7H,
H2I,5I,6aI,6bI,5II,6aII,6bII), 4.61, 4.79 (AB quartet, JA,B
11.7Hz, PhCH2), 5.13 (d, 1H, J1,2 1.6Hz, H1II), 5.26
(d, 1H, J1,2 1.7Hz, H1I), 5.86–5.91, 5.95–6.02 (2m, 5H,
H3I,4I,2II,3II,4II), 7.00–7.54, 7.81–8.09 (2m, 45H, Ph);
13C NMR (100MHz, CDCl3) d 63.8, 66.7, 67.5 (d,
JC,P 5.3Hz), 67.8, 69.3, 69.8, 70.2, 70.4 (d, JC,P
7.7Hz), 71.1, 98.3, 99.9, 120.2, 125.5, 125.5(6),
125.5(7), 128.3(0), 128.3(4), 128.5, 128.6(7), 128.7(0),
128.7(3), 128.8, 128.9, 129.1, 129.2(9), 129.3(3),
129.8(7), 129.9(1), 129.9(3), 129.9(6), 129.9(8), 130.0(4),
130.0(8), 130.1(2), 130.2, 133.3(2), 133.3(4), 133.4,
133.5, 133.6, 133.7, 136.8, 165.1, 165.2, 165.4, 165.6,
165.8, 166.6; 31P NMR (162MHz, CDCl3) d ꢁ11.5
(PO(OPh)2);HRMS calcd for C 73H62O20P [M+H]+
1289.3572, found 1289.3599.
3.17. Methyl 3-O-(2,3,4-tri-O-benzyl-6-O-diphenylphos-
phoryl-D-mannopyranosyl)-2,4,6-tri-O-benzoyl-a-D-man-
nopyranoside (37/38)
TMSOTf (56lL, 310lmol) was added to the imidate 24
(256mg, 310lmol) and alcohol 13 (150mg, 266lmol) as
described for the preparation of 25/26 to yield two frac-
tions. Firstly, the amide 27 was produced as a pale yellow
coloured oil (53mg, 21% on 24). This sample was spec-
troscopically identical to that produced during the synthe-
sis of 25/26. Next, an anomeric mixture (2:1) of the
disaccharides 37/38 was produced as a colourless oil
1
(226mg, 72%). Partial H NMR (400MHz, CDCl3) d
3.40 (s, 3H, OMe-major), 3.44 (s, 3H, OMe-minor), 4.85
(d, 1H, J1,2 1.7Hz, H1I-major), 4.92 (d, 1H, J1,2 1.6Hz,
H1I-minor), 5.01 (d, 1H, J1,2 1.7Hz, H1II-major); 31P
NMR (162MHz, CDCl3) d ꢁ11.5 (PO(OPh)2-minor),
ꢁ11.4 (PO(OPh)2-major). ESMS: m/z 1171.4 [M+H]+.
3.16. 2-O-(6-O-Phospho-a-D-mannopyranosyl)-D-manno-
pyranose, disodium salt (5)
Method A: (A) Pd(OH)2 (2mg of 10% on C) was added
to a solution of the benzyl ether 35 (10mg, 7.8lmol) in
THF (1mL) and AcOH (2.5lL) and the combined mix-
ture was vigorously stirred under hydrogen (100psi,
72h). The mixture was filtered and the solvent evapo-
rated and co-evaporated (2 · 1mL H2O) to yield a col-
3.18. Methyl 3-O-(2,3,4-tri-O-benzoyl-6-O-diphenylphos-
phoryl-a-D-mannopyranosyl)-2,4,6-tri-O-benzoyl-a-D-
mannopyranoside (39)
1
ourless oil. Partial H NMR (400MHz, D2O) d 5.13
TMSOTf (15lL, 82lmol) was added to the imidate 31
(42mg, 48lmol) and alcohol 13 (25mg, 44lmol) as de-
scribed for the preparation of 25/26 to yield the hexa-
(d, 1H, J1,2 1.7Hz, H1II), 5.58 (d, 1H, J1,2 1.8Hz,
H1I). (B) The product from (A) was dissolved in THF
(1mL) and treated with PtO2 (5mg) and the combined
mixture was vigorously stirred under hydrogen
(100psi, o/n). After this time, the filtrate was neutralized
with Na2CO3 (1M) and the solvent evaporated. (C)
1
benzoate 39 as a colourless oil (44mg, 78%). H NMR
(400MHz, CDCl3) d 3.42 (s, 3H, OMe), 4.24–4.30 (m,
2H, H5I,6aII), 4.41–4.45 (m, 3H, H6bI,5II,6bII), 4.58
(dd, 1H, J2,3 3.4, J3,4 9.9Hz, H3I), 4.69 (dd, 1H,

J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
6073
J5,6 2.7, J6,6 12.2Hz, H6aI), 4.92 (d, 1H, J1,2
1.6Hz, H1I), 5.16 (d, 1H, J1,2 1.8Hz, H1II), 5.26 (dd,
1H, J2,3 3.3Hz, H2II), 5.60–5.64 (m, 2H, H2I,3II),
5.86 (dd, 1H, J3,4 10.0, J4,5 10.0Hz, H4II), 5.96 (dd,
1H, J4,5 10.1Hz, H4I), 7.05–8.18 (m, 40H, ArH); 13C
NMR (100MHz, CDCl3) d 55.6, 63.2, 66.2, 67.0 (d,
JC,P 5.4Hz), 68.8, 69.0, 69.5, 70.2 (d, JC5,P 8.7Hz),
70.3, 71.8, 75.8, 98.8, 99.5, 120.2(8), 120.3(3), 120.3(6),
120.4(0), 125.3(0), 125.3(2), 125.3(7), 125.3(9), 128.3,
128.5, 128.5(7), 128.6(0), 128.6(4), 128.9, 129.1, 129.2,
129.2(8), 129.3(4), 129.5, 129.7(8), 129.8(2), 129.8(3),
129.9, 129.9(6), 130.0(2), 130.1, 130.2, 130.4, 133.1,
133.2, 133.4(7), 133.4(9), 133.8, 164.9, 165.2, 165.5,
166.1, 166.4; 31P NMR (162MHz, CDCl3) d ꢁ11.4
(PO(OPh)2);HRMS calcd for C 67H57O20P [M+H]+
1213.3254, found 1213.3208.
(500mg of 3A powder) and the combined mixture was
stirred (rt, o/n). The mixture was filtered and evaporated
and the residue subjected to FC (15% EtOAc/hexanes)
to yield the imidate 41 as a colourless oil (647mg,
˚
1
48%, two steps). H NMR (200MHz, CDCl3) d 1.06
(s, 9H), 1.92, 2.01, 2.17 (3s, 3 · 3H), 3.70–3.80 (ABX,
2H), 4.05 (dt, 1H, J 3.3, 9.9Hz), 5.41 (dd, 1H, J 3.3,
9.9Hz), 5.45 (dd, 1H, J 1.5, 3.3Hz), 5.57 (t, 1H, J
9.9Hz), 6.33 (d, 1H, J 1.8Hz), 7.30–7.80 (m, 10H),
8.75 (s, 1H).
3.21. Benzyl 2-O-(2,3,4-tri-O-acetyl-6-O-tert-butyldiphen-
ylsilyl-a-D-mannopyranosyl)-3,4,6-tri-O-benzyl-a-D-man-
nopyranoside (44)
TMSOTf (15lL, 82lmol) was added to the imidate 41
(100mg, 145lmol) and alcohol 10 (60mg, 111lmol) as
described for the preparation of 25/26 to yield the man-
nobioside 44 as a colourless oil (117mg, 99%). 1H NMR
(200MHz, CDCl3) d 1.09 (s, 9H), 1.91, 2.03, 2.12 (3s,
3 · 3H), 3.60–4.05 (m, 8H), 4.12 (t, 1H, J 2.0Hz),
4.36–4.88 (m, 8H), 4.99 (d, 1H, J 1.8Hz), 5.14 (d, 1H,
J 1.5Hz), 5.40–5.50 (m, 3H), 7.10–7.80 (m, 35H).
3.19. Methyl 3-O-(6-O-phospho-a-D-mannopyranosyl)-a-
D-mannopyranose (8)
Method A: The hexabenzoate 39 (82mg, 64lmol) was
treated first with PtO2 and then KOH in THF as
described for the preparation of 5 [steps (B) and (C)]
to yield, after lyophilization, the phosphomannobioside
8 as a colourless powder (14mg, 48%, two steps).
1H NMR (400MHz, D2O) d 3.25 (s, 3H, OMe),
3.49 (ddd, 1H, J4,5 10.0, J5,6a 2.1, J5,6b 5.8Hz,
H5I), 3.55–3.63 (m, 3H, H4I,6I,4II), 3.68 (dd,
3.22. Benzyl 2-O-(2,3,6-tri-O-acetyl-a-D-mannopyranos-
yl)-3,4,6-tri-O-benzyl-a-D-mannopyranoside (45)
A combined mixture of TBAF (170lL of 1M in THF,
170lmol), acetic acid (20lL) and the silyl ether 44
(37mg, 35lmol) in THF (1mL) was stirred (rt, 2d).
EtOAc (10mL) was added and the solution was succes-
sively washed (satd aq NaCl), dried (Na2SO4), filtered
and evaporated. The residue was subjected to FC (25–
50% EtOAc/hexanes) to yield the alcohol 45 as a colour-
1H,
J2,3
3.4,
J3,4
9.5Hz, H3I),
3.72–3.76
(m, 3H, H6I,3II,5II), 3.85–3.87 (m, 3H, H2II,6aII,6bII),
3.98 (dd, 1H, J1,2 1.8Hz, H2I), 4.58 (d, 1H, H1I), 4.92
(d, 1H, J1,2 1.6Hz, H1II); 13C NMR (100MHz, D2O) d
54.9, 61.0, 63.8 (d, JC,P 5.3Hz), 66.1, 66.5, 69.5, 70.2,
70.3, 72.6 (d, J5,P 6.9Hz), 72.7, 79.3, 101.1, 102.8; 31P
NMR (162MHz, D2O) d 2.7 (PO(ONa)2);HRMS calcd
for C13H25O14P [M+H]+ 437.1055, found 437.1040;CE:
tm = 21.46min.
1
less oil (18mg, 63%). H NMR (200MHz, CDCl3) d
2.03–2.05 (m, 9H), 3.60–4.20 (m, 11H), 4.37 (dd, 1H, J
4.4, 12.4Hz), 4.39–4.65 (m, 8H), 4.76 (t, 1H, J 9.9Hz),
4.92 (br s, 2H), 5.20 (dd, 1H, J 3.3, 9.9Hz), 5.38 (dd,
1H, J 1.5, 3.3Hz), 7.10–7.40 (m, 20H).
Method B: The hexabenzoate 39 (10mg, 7.8lmol) was
treated first with KOH in THF and then PtO2 as de-
scribed for the preparation of 5 [steps (C) and (B)] to
yield the phosphomannobioside 8 as a colourless pow-
der (2.1mg, 49%, two steps). This sample was identical
in all respects to that produced in the previous method.
3.23. Benzyl 2-O-(2,3,4-tri-O-benzoyl-a-D-mannopyranos-
yl)-3,4,6-tri-O-benzyl-a-D-mannopyranoside (48)
(A) TMSOTf (123lL, 0.68mmol) was added to the imi-
date 4332 (464mg, 0.68mmol)32 and alcohol 10 (308mg,
0.57mmol) as described for the preparation of 25/26 to
yield a colourless oil. This residue was co-evaporated
(2 · 10mL MeCN) and used in the next reaction without
further purification or characterization. (B) Acetyl chlo-
ride (1.0mL) was added to a solution of the product
from (A) and MeOH (10mL) in 1,2-DCE (5mL) and
the combined mixture was stirred (rt, o/n). The mixture
was cooled and neutralized (Et3N) and the solvents were
evaporated. The residue was subjected to workup
(EtOAc) and FC (10–40% EtOAc/hexane) to yield the
alcohol 48 as a colourless oil (422mg, 73%, two steps).
1H NMR (400MHz, CDCl3) d 3.60–3.61 (m, 2H,
H6aII,6bII), 3.72 (dd, 1H, J5,6a 2.0, J6a,6b 10.4Hz,
H6aI), 3.77 (dd, 1H, J5,6b 5.2Hz, H6bI), 3.82–3.88 (m,
1H, H5II), 3.99–4.02 (m, 3H, H2I,3I,4I), 4.11–4.16 (m,
1H, H5II), 4.46–4.74 (m, 7H, PhCH2), 4.85 (B of
AB quartet, JA,B 11.0Hz, PhCH2), 5.03 (d, 1H, J1,2
1.6Hz, H1I), 5.21 (d, 1H, J1,2 1.2Hz, H1II), 5.73 (dd, 1H,
3.20. 2,3,4-Tri-O-acetyl-6-O-tert-butyldiphenylsilyl-a-D-
mannopyranosyl trichloroacetimidate (41)
(A) Imidazole (167mg, 2.45mmol) and tert-butyldiphen-
ylsilyl chloride (640lL, 5.61mmol) were added to a
solution of the alcohol 17 (785mg, 2.25mmol)25 in 1,2-
DCE (10mL) and the combined mixture was stirred
(1h). The mixture was filtered and the filtrate subjected
to FC (25% EtOAc/hexanes) to yield, presumably the
silyl ether 40, as a colourless oil (1.19g). (B) Benzylam-
ine (856lL, 7.83mmol) was added to a solution of the
tetraacetate 40 (1.15g, 1.96mmol, max.) and in Et2O
(20mL) and the combined mixture was stirred (rt,
o/n). The mixture was subjected to workup (Et2O).
The solution was used in the following reaction without
further purification or characterization. (C) Cesium car-
bonate (1.3g, 3.7mmol) was added to the product from
(B), trichloroacetonitrile (1mL, 10mmol) and mol sieves

6074
J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
J3,4ꢀ4,5 10.4Hz, H4II), 5.85 (dd, 1H, J2,3 3.2Hz, H2II),
5.99 (dd, 1H, J3,4 10.0Hz, H3II), 7.10–7.60, 7.78–8.06
(2m, 35H, Ph);ESMS: m/z 1015.5 [M+H]+.
idate (80lL, 0.390mmol) as described for the prepara-
tion of 32 to yield the diphenyl phosphate 39 as a
colourless oil (94mg, 90%). This sample was identical
in all respects to that prepared by the previous
method.
3.24. Alternative synthesis of benzyl 2-O-(2,3,4-tri-O-
benzoyl-6-O-diphenylphosphoryl-a-D-mannopyranosyl)-
3,4,6-tri-O-benzyl-a-D-mannopyranoside (32)
Acknowledgements
Et3N (96lL, 0.69mmol) was added to a solution of the
alcohol 48 (140mg, 0.14mmol) and diphenyl phosphoro-
chloridate (86lL, 0.42mmol) in 1,2-DCE (5mL) and the
combined mixture was heated (80ꢁC, 3h). The mixture
was diluted (CHCl3) and subjected to workup and FC
(10–40% EtOAc/hexane) to yield the diphenyl phos-
phate 32 as a colourless oil (157mg, 91%). This sample
was identical in all respects to that prepared by the pre-
vious method.
Drs. Ligong Liu and Ian Bytheway (Progen Industries
Ltd.) are thanked for useful discussions and critical
reading of this manuscript. Dr. Sue Boyd (Centre for
Magnetic Resonance, Griffith University, Nathan Cam-
pus, Qld) is also thanked for useful discussions. This
work was funded in part by an AusIndustry START
grant to Progen Industries Ltd.
3.25. Methyl 3-O-(6-O-acetyl-2,3,4-tri-O-benzoyl-a-D-
mannopyranosyl)-2,4,6-tri-O-benzoyl-a-D-mannopyrano-
side (49)
References and notes
1. Parish, C. R.;Freeman, C.;Brown, K. J.;Francis, D. J.;
Cowden, W. B. Cancer Res. 1999, 59, 3433.
TMSOTf (38.6lL, 0.213mmol) was added to the imi-
date 43 (145mg, 0.213mmol) and alcohol 13 (94mg,
0.164mmol) as described for the preparation of 25/26
to yield the acetate 49 as a colourless oil (142mg,
2. Ferro, V.;Don, R. Australas. Biotechnol. 2003, 13, 38.
3. Cochran, S.;Li, C.;Fairweather, J. K.;Kett, W. C.;
Coombe, D. R.;Ferro, V. J. Med. Chem. 2003, 46, 4601.
4. Vlodavsky, I.;Friedmann, Y. J. Clin. Invest. 2001, 108,
341.
1
79%). H NMR (400MHz, CDCl3) d 2.06 (s, 3H, Ac),
3.45 (s, 3H, OMe), 4.13 (dd, 1H, J5,6a 2.4, J6a,6b
12.1Hz, H6aII), 4.18 (dd, 1H, J5,6b 5.2Hz, H6bII), 4.25
(ddd, 1H, J4,5 10.1, J5,6a 2.7, J5,6b 4.5Hz, H5I), 4.36
(ddd, 1H, J4,5 9.8Hz, H5II), 4.43 (dd, 1H, J6a,6b
12.1Hz, H6bI), 4.59 (dd, 1H, J2,3 3.4, J3,4 9.8Hz,
H3I), 4.68 (dd, 1H, H6aI), 4.98 (d, 1H, J1,2
1.5Hz, H1I), 5.25–5.28 (m, 2H, H1II,2II), 5.59 (dd, 1H,
H2I), 5.62 (dd, 1H, J2,3 3.2, J3,4 9.8Hz, H3II),
5.74 (dd, 1H, H4II), 5.97 (dd, 1H, H4I), 7.14–7.82,
8.02–8.18 (2m, 30H, Ph);HRMS calcd for C 57H51O18
[M+H]+ 1023.3075, found 1023.3091.
5. Parish, C. R.;Freeman, C.;Hulett, M. D.
Biophys. Acta 2001, 1471, M99.
6. Wall, D.;Douglas, S.;Ferro, V.;Cowden, W.;Parish, C.
Thromb. Res. 2001, 103, 325.
Biochim.
7. Demir, M.;Iqbal, O.;Hoppensteadt, D. A.;Piccolo, P.;
Ahmad, S.;Schultz, C. L.;Linhardt, R. J.;Fareed, J. Clin.
Appl. Thromb. Hemost. 2001, 7, 131.
8. Piccolo, P.;Iqbal, O.;Demir, M.;Ma, Q.;Gerbutavicius,
R.;Fareed, J. Clin. Appl. Thromb. Hemost. 2001, 7, 149.
9. Francis, D. J.;Parish, C. R.;McGarry, M.;Santiago, F.
S.;Lowe, H. C.;Brown, K. J.;Bingley, J. A.;Hayward, I.
P.;Cowden, W. B.;Campbell, J. H.;Campbell, G. R.;
Chesterman, C. N.;Khachigian, L. M. Circ. Res. 2003, 92,
e70.
10. Nyberg, K.;Ekblad, M.;Bergstro ¨m, T.;Freeman, C.;
Parish, C. R.;Ferro, V.;Trybala, E. Antiviral Res. 2004,
63, 15.
11. Yu, G.;Gunay, N. S.;Linhardt, R. J.;Toida, T.;Fareed,
J.;Hoppensteadt, D. A.;Shadid, H.;Ferro, V.;Li, C.;
Fewings, K.;Palermo, M. C.;Podger, D. Eur. J. Med.
Chem. 2002, 37, 783.
3.26. Methyl 3-O-(2,3,4-tri-O-benzoyl-a-D-mannopyranos-
yl)-2,4,6-tri-O-benzoyl-a-D-mannopyranoside (50)
Acetyl chloride (1.0mL) was added to the acetate 49
(120mg, 0.110mmol) as described for the preparation
of 48 to yield the alcohol 50 as a colourless glass
1
(95mg, 82%). H NMR (400MHz, CDCl3) d 3.46 (s,
3H, OMe), 3.53 (dd, 1H, J5,6a 3.5, J6a,6b 13.0Hz,
H6aII), 3.72 (dd, 1H, J5,6b 2.1Hz, H6bII), 4.02–4.06
(m, 1H, H5II), 4.26 (ddd, 1H, J4,5 10.0, J5,6a 2.6, J5,6b
4.5Hz, H5I), 4.44 (dd, 1H, J6a,6b 12.2Hz, H6bI), 4.57
(dd, 1H, J2,3 3.4, J3,4 9.4Hz, H3I), 4.68 (dd, 1H,
H6aI), 4.93 (d, 1H, J1,2 1.7Hz, H1I), 5.27–5.29 (m, 2H,
H1II,2II), 5.57 (dd, 1H, H2I), 5.67 (dd, 1H, J3,4–4,5
9.9Hz, H4II), 5.71 (dd, 1H, J2,3 3.2Hz, H3II), 5.98 (dd,
1H, H4I), 7.13–7.82, 8.12–8.15 (2m, 30H, Ph);ESMS:
m/z 981.4 [M+H]+.
12. Ferro, V.;Fewings, K.;Palermo, M. C.;Li, C. Carbohydr.
Res. 2001, 332, 183.
13. Parolis, L. A.;Parolis, H.;Kenne, L.;Meldal, M.;Bock,
K. Carbohydr. Res. 1998, 309, 77.
14. Ferro, V.;Li, C.;Fewings, K.;Palermo, M. C.;Linhardt,
R.;Toida, T. Carbohydr. Res. 2002, 337, 139.
15. Matta, K. L.;Chowdhary, M. S.;Jain, R. K.;Abbas, S. A.
Carbohydr. Res. 1986, 150, C1.
16. Srivastava, O. P.;Hindsgaul, O. J. Org. Chem. 1987, 52,
2869.
17. Christensen, M. K.;Meldal, M.;Bock, K. J. Chem. Soc.,
Perkin Trans. 1 1993, 1453.
18. Srivastava, O. P.;Hindsgaul, O. Carbohydr. Res. 1987,
161, 195.
19. Srivastava, O. P.;Hindsgaul, O. Carbohydr. Res. 1987,
161, 324.
3.27. Alternative synthesis of methyl 3-O-(2,3,4-tri-O-
benzoyl-6-O-diphenylphosphoryl-a-D-mannopyranosyl)-
2,4,6-tri-O-benzoyl-a-D-mannopyranoside (39)
The alcohol 50 (76mg, 0.73mmol) was treated with
Et3N (75lL, 0.73mmol) and diphenyl phosphorochlor-
20. Ogawa, T.;Yamamoto, H. Carbohydr. Res. 1982, 104,
271.

J. K. Fairweather et al. / Bioorg. Med. Chem. 12 (2004) 6063–6075
6075
21. Wang, W.;Kong, F. J. Org. Chem. 1998, 63, 5744.
22. Chen, L.;Kong, F. J. Carbohydr. Chem. 2002, 21, 341.
23. Posternak, T.;Rosselet, J. P. Helv. Chim. Acta 1953, 36,
1614.
28. Mulard, L. A.;Costachel, C.;Sansonetti, P. J. J. Carbo-
hydr. Chem. 2000, 19, 849.
29. Dondoni, A.;Marra, A.;Massi, A. J. Org. Chem. 1999,
64, 933.
24. Reynolds, D. D.;Evans, W. L. J. Am. Chem. Soc. 1940,
62, 66.
30. Andrews, J. S.;Pinto, B. M. Carbohydr. Res. 1995, 270,
51.
25. Mori, M.;Ito, Y.;Ogawa, T. Carbohydr. Res. 1989, 192,
131.
26. Fukuyama, T.;Laird, A. A.;Hotchkiss, L. M. Tetrahe-
dron Lett. 1985, 26, 6291.
27. Hoffmann, M. G.;Schmidt, R. R. Liebigs Ann. Chem.
1985, 2403.
31. Sabesan, S.;Neira, S. Carbohydr. Res. 1992, 223, 169.
32. Heng, L.;Ning, J.;Kong, F. J. Carbohydr. Chem. 2001,
20, 285.
33. Iselin, B. M. J. Am. Chem. Soc. 1949, 71, 3822.
34. Amer, H.;Hofinger, A.;Kosma, P. Carbohydr. Res. 2003,
338, 35.