Synthesis of archaeal glycolipid adjuvants-what is the optimum number of sugars?

Article abstract of DOI:10.1016/j.carres.2008.06.021

As part of a programme to optimize the use of archaeal-lipid liposomes (archaeosomes) as vaccine adjuvants, we present the synthesis and immunological testing of an oligomeric series of mannose glycolipids (Manp_1-5). To generate the parent arch

Full text of DOI:10.1016/j.carres.2008.06.021

Carbohydrate Research 343 (2008) 2349–2360
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of archaeal glycolipid adjuvants—what is the optimum
number of sugars?
*
Dennis M. Whitfield , Eva E. Eichler, G. Dennis Sprott
National Research Council, Institute for Biological Sciences, 100 Sussex Drive Ottawa, ON, Canada K1A 0R6
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 May 2008
Received in revised form 20 June 2008
Accepted 26 June 2008
Available online 4 July 2008
As part of a programme to optimize the use of archaeal-lipid liposomes (archaeosomes) as vaccine adju-
vants, we present the synthesis and immunological testing of an oligomeric series of mannose glycolipids
(Manp1–5). To generate the parent archaeol alcohol precursor, the polar lipids extracted from the archa-
eon Halobacterium salinarum were hydrolyzed to remove polar head groups, and the archaeol so gener-
ated partitioned into diethyl ether. This alcohol was then iteratively glycosylated with the donor 2-O-
acetyl-3,4,6-tri-O-benzyl-a/b-D-mannopyranosyl trichloroacetimidate to yield a-Manp-(1?2) oligomers.
Keywords:
Glycolipids
Synthesis
Archaea
Adjuvants
Liposomes
A starch-derived trimer was also synthesized as a control. To promote hydration and form stable archaeo-
somes, an archaeal anionic lipid archaetidylglycerol (AG) was included in a 4:1 molar ratio. Archaeo-
somes prepared from Manp1–2–AG were recovered at only 34–37%, whereas Manp3ꢀ4–AG recoveries
were 72–77%. Lipid recovery following hydration of Manp5–AG archaeosomes declined to 34%, indicating
an optimum of 3–4 Manp units for bilayer formation. The CD8+ T cell response in mice immunized with
Manp3–5 archaeosomes containing ovalbumin was highest for Manp4 and declined for Manp3 and Manp5,
revealing an optimum length of four unbranched units. The starch-derived trimer was more active than
the Manp oligomers, suggesting the involvement of either a general binding lectin on antigen-presenting
cells with highest affinity for triglucose or multiple lectin receptors.
Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
1. Introduction
chemistry, ether versus ester linkages from the glycerol backbone
to the alkyl chains, and having isoprenoid chains typically fully sat-
The control of disease states through the use of vaccination with
disease-associated antigens is a proven concept.1 Recent develop-
ments include the use of proteomics and genetic data mining to
determine candidate antigens from bacteria, viruses, parasites
and cancer cells as the commonest diseases to be studied by these
approaches.2 However, the complexity of the host response to the
introduction of such foreign antigens requires careful and detailed
studies to allow for the preparation of vaccine formulations that
lead to effective disease control. In most cases, antigen alone is
insufficient, and adjuvants that boost specific components of the
host’s immune response are necessary.3 This is an active area of
biomedical research. Our contribution to this area arose out of
the recognition that specialized liposomes named archaeosomes,4
composed of the total polar lipids from Archaea, could entrap anti-
gen and lead via both major histocompatibility complexes, MHC
class I and MHC class II pathways to strong immunological
responses in suitable animal models.5
urated.6 As well, some archaea biosynthesize C86 dimer species
where the methyl groups of the isoprenoid side chains are joined
such that the two glycerol head groups are at either end of the mol-
ecule, named caldarchaeol.7 In most archaeal species these head
groups are phosphodiester linked to small polar groups like serine,
glycerol, ethanolamine or glycosidically linked to short oligosac-
charides like the b-(1?6) glucose linked dimer gentiobiose. Recent
experiments from our group using synthetic glycolipids based on
the archaeol core lipid demonstrate that a major part of the CD8+
cytotoxic thymic lymphocyte (CTL) adjuvant properties are related
to the glyco-portion of such glycolipids.8
The combination of these newer studies and a larger number of
earlier studies with total polar lipids such as those from the species
Methanobrevibacter smithii have led to a model to describe the
interaction of archaeosomes with antigen-presenting cells (APCs).
APCs such as dendritic cells or macrophages can show adjuvant
effects by at least five mechanisms.8a,9 This can be via (1) an extra-
cellular depot effect where the highly stable archaeosomes slowly
dose out antigen, (2) a cell surface effect by targeting receptors on
APC which also leads to specific delivery of the entrapped antigen,
and cytostolic effects whereby antigens are sorted via endosomal
compartments into (3) MHC class II or (4) MHC class I presentation
Polar glycero-lipids of Archaea differ from those found in the
Bacteria and Eukarya domains of life by sn-2,3 versus sn-1,2 stereo-
* Corresponding author. Tel.: +1 613 993 5265; fax: +1 613 952 9092.
E-mail address: dennis.whitfield@nrc-cnrc.gc.ca (D. M. Whitfield).
0008-6215/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.06.021

2350
D. M. Whitfield et al. / Carbohydrate Research 343 (2008) 2349–2360
pathways and (5) a co-stimulatory effect, probably receptor medi-
ated, which leads to up-regulation of cytokines and other immuno-
logical regulators.
2. Results and discussion
2.1. Glycolipid synthesis
Previous studies had shown that APC targeting with M. smithii
total polar lipids probably does not employ lipid A-like pathways,
which are through toll receptors 2 or 4.10 Nor does it appear to go
Archaeol is readily available in gram quantities from the bio-
mass of Halobacterium salinarum grown in research scale reactors
by a simple process of lipid extraction, head group hydrolysis
and silica gel chromatography.8a The mannose donor (1) was cho-
sen based on previous synthetic work, and is easily made in multi-
gram quantities.20 Donor 1 was coupled with archaeol under
through CD1 receptors like a-Gal glycolipids that activate natural
killer (NK) cells.11 Targeting of total polar lipid archaeosomes may
proceed via archaetidyl serine (AS), the analogue of phosphatidyl-
serine (PS).12 PS is flipped from the inner leaflet of healthy cells to
the outer bilayer surface in apoptotic cells where it binds to recep-
tors on APCs as an integral part of the apoptosis process. Recent
evidence suggests that PS mediated APC targeting leads to down-
regulation instead of the required up-regulation of the immune
response.13 Therefore, we wondered if we could engineer targeting
to APCs14 via well-known receptors such as DC-sign15 and the man-
nose receptor16 by synthesizing glycolipids based on the archaeol
lipid and mannose-containing oligosaccharide head groups. These
mannose-containing lipids would be formulated as archaeosomes
without AS, and possibly achieve both targeting and cytokine up-
regulation as well as benefiting from the stability of archaeosomes.4
Other groups have prepared mannosylated liposomes and pre-
sented evidence for targeting APCs but not with archaeal lipids.17
standard glycosylation conditions to give
a-mannosylated lipid
2a in 47% yield as the only detectable anomer. The anomericity
was initially determined by the chemical shift of Manp H-5, which
was in the range 3.7–3.8 ppm as expected for a
-mannosides.21 The
acetyl group at O-2 is readily removed to give alcohol 2b in almost
quantitative yield. This can be used for chain extension or the
benzyl groups removed by hydrogenation. Our first attempt to re-
move the benzyl groups with a Pd–C catalyst, which had been used
successfully in the previous synthetic work with 1, led to a
complex mixture of products. By switching to Pearlman’s catalyst
(Pd(OH)2–C), which has been recommended for other mannose
oligomers,22 the benzyl groups could be easily removed and the
resulting tetraol 2c easily purified. This process was repeated
leading to Manp dimers 3a–c, trimers 4a–c, tetramers 5a–c and
pentamers 6a–c, see Scheme 1.
Monomer 2c, dimer 3c and trimer 4c readily dissolved in
CDCl3–CD3OD mixtures (typically 1:1, v/v) and gave NMR spectra
with sharp lines. Tetramer 5c and especially pentamer 6c on the
other hand in spite of numerous attempts and experiments with
different solvent mixtures gave broad lines, and definitive
assignments were not possible. Analysis of the 1D 1H or 13C spectra
was not particularly informative outside of identifying the
anomeric resonances. Except that the 13C pattern of the aliphatic
To this end we decided to synthesize
a-Manp-(1?2)-a-Manp
oligomers with 1–5 mannose residues attached glycosidically to
archaeol. There have been a number of successful syntheses of oli-
gosaccharides attached to archaeal lipids and related analogues but
none to our knowledge containing oligomannosides or using our
semi-synthetic approach.18 The
a-Manp-(1?2)-a-Manp disaccha-
ride is frequently found at the non-reducing termini of glycoconju-
gates known to be recognized by receptors on APCs.19 We present
the synthesis and characterization of these oligomers as well as
some biophysical and immunological data as adjuvants.
Scheme 1. Synthesis of Manp1 to Manp5 oligomers attached to archaeol.

D. M. Whitfield et al. / Carbohydrate Research 343 (2008) 2349–2360
2351
lipid portion was nearly identical in all lipid containing species.
The remaining 1H and 13C sugar resonances were assigned
by 2D 1H–1H COSY, and 1D or 2D 1H–1H TOCSY correlation
experiments. Subsequent 13C–1H correlation assigned the 13C
resonances. The glycerol resonances and the resonances of the
ether methylenes of the side chains were almost always over-
lapped with each other and with some of the sugar resonances.
Partial assignment could be achieved by 1H–1H COSY connecti-
vities to the rest of the phytanyl side chain or more effectively from
edited 13C–1H HSQC where methine connectivities can be
distinguished from methylene connectivities by the sign of the
crosspeaks. Finally inter-residue or sugar–lipid connectivities were
established by NOE measurements in order to distinguish
sugar residues from each other. The NOE also corroborated the
In what was designed as a negative control experiment we also
synthesized the -Glcp-(1?4)- -Glcp-(1?4)-b-Glcp-(1?O)-ar-
a
a
chaeol trimer derived from starch. A previous synthesis of O-2 gly-
cosubstituted 1,3-di-phytanyl-glycerol with the same trisaccharide
was used as a precedent for making the natural stereoisomer from
archaeol.23 Thus, the commercially available
a-Glcp-(1?4)-a-
Glcp-(1?4)-Glcp trimer was acetylated to give 7a. Subsequent
hemi-acetal formation with hydrazine and treatment with CCl3CN
and DBU led to trichloroacetimidate 7c via 7b. Glycosylation of
archaeol and deprotection formed 8a and 8b. These species were
also characterized as above, and the respective data are presented
in Tables 2–6.
a-anomericity of the linkages. A series of representative spectra
2.2. Archaeosome preparation and adjuvant properties
are shown in Figure 1a and b for Manp dimer 3c. Complete assign-
ments are presented in Tables 2–5 in Section 4. The composition of
all species was also confirmed by MALDI MS and compiled in Table
6. All lipid containing species gave characteristic [M+Na]+ ions
(Scheme 2).
Manp1ꢀ5-archaeols were dried, and hydration was attempted in
water at 65 °C. The Manp4 5c hydrated well, Manp3 4c and Manp5
6c hydrated moderately well with clumps occurring and the Manp1
2c and Manp2 3c hydrated with difficulty. Previously it was noted
Figure 1a. 1D 1H–1H TOCSY correlation experiments for 3c, Top, irradiate ManII H-1, middle, irradiate ManI H-1 and bottom, reference partial 1H NMR spectra, both spectra
with 135 ms mixing time.

2352
D. M. Whitfield et al. / Carbohydrate Research 343 (2008) 2349–2360
The archaeosomes formed from Manp3 4c, Manp4 5c and Glcp3
8b were stable for weeks in buffer at 4 °C.
Ovalbumin used as a test protein antigen in which the T cell epi-
tope is known26 was entrapped within the archaeosomes. Upon
subcutaneous injection at zero and 3 weeks, measuring antigen-
specific CD8+ CTL activity in splenic cells8b and titrating anti-OVA
antibody in mouse sera27 assessed adjuvant activity for both
MHC class I and class II pathways. Results are compared with
archaeosomes consisting of total polar lipid extracts from M.
smithii, as these are known to promote high CTL activity to an
entrapped antigen.28 These pure glycolipid adjuvants all showed
CTL activity that was only slightly less than the TPL from M. smithii,
that is, the adjuvant activity is predominantly from the glycolipids.
This is indicated by only low CTL activity for AG archaeosomes
(Fig. 2e).
The results of a CTL assay depicted in Figure 2 clearly show that
archaeosomes made from Manp3ꢀ4 4c and 5c are potent. However,
the supposed negative control Glcp3 8c was indeed more active
than 4c and 5c. Antibody responses for the same compounds dem-
onstrated that in addition to MHC class I, the Th2 arm of the MHC
class II pathway is also activated by these archaeosomes (data not
shown). While means of anti-OVA antibody titres were high for 4c,
5c and 8c compared to TPL from M. smithii, they were not signifi-
cantly higher (p >0.05). If these species function via the expected
pathways, then this result is surprising and contrasts sharply with
other results with mannose-containing liposomes where the non-
mannose-containing negative controls were indeed negative.29
A
separate communication also found that mannose residues on a
glycoprotein did not enhance processing and presentation of anti-
gens in dendritic cells. This failure was not attributed to failure of
internalization but rather to an inability to enter late endosomes
or lysosomal compartments.30 Our result suggests that up-
regulation of the CTL response by archaeosomes does not
proceed through mannose receptors alone, but includes
another mechanism.
3. Conclusion
We present a stepwise synthesis of
a-Manp-(1?2)-a-Manp
oligomers glycosidically attached to the lipid archaeol. Especially
the trimer and tetramer 4c and 5c show strong adjuvant activity
by both MHC class I and class II pathways. The observation
that the starch-derived trimer linked to archaeol 8c also shows
good adjuvant activity suggests the presence of a novel immuno-
stimulatory pathway for archaeal glycolipids. These novel lipids
can be prepared by relatively inexpensive chemical synthesis at
least in comparison to the more complex glycolipids like lipid A
Figure 1b. 1H–1H COSY (bottom, positive contours only) and 13C–1H HSQC (top,
both positive and negative contours plotted) spectra for 3c. For labels A = archaeol.
Table 1
Archaeosome characteristics following 0.45 lm sterilizing filtration
Lipids (mol %)
Size (nm SD)
% Lipids recovered
Manp2–AG (80:20)
Manp3–AG (80:20)
Manp4–AG (80:20)
Manp5–AG (80:20)
AG
78 36
101 57
85 52
105 50
91 53
34
77
72
34
88
analogues,31
QS-21A.33
a
-Gal ceramides32 and complex saponins like
4. Experimental
4.1. General methods
that vesicles composed of 1,3-di-O-phytanyl-2-O-(b-D-maltotrio-
Archaetidylglycerol was purified from Haloferax volcanii.34
4.2. Procedure A
syl)glycerol aggregated in the presence of salts, but with incorpo-
ration of the anionic glycolipid sulfonoquinovosyl diacylglycerol
vesicle stability was improved.24 By introducing an anionic
phospholipid, archaetidylglycerol at 20 mol %, the recovery of
archaeosomes was greatly improved, with the exception of Manp1
2c (Table 1). To achieve some yield of archaeosomes from Manp1, it
was necessary to prepare the vesicles by a detergent dialysis meth-
4.2.1. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-
tetramethylhexadecyloxy]propan-1-yl 2-O-acetyl-
3,4,6-tri-O-benzyl-a-D-mannopyranoside (2a)
od using the non-ionic detergent octyl b-D
-glucopyranoside.25 Be-
To a mixture of (2R)-2,3-bis[(3R,7R,11R)-3,7,11,15-tetramethylhex-
adecyloxy]propan-1-ol (archaeol) (720 mg, 1.1 mmol), 2-O-acetyl-
cause archaeosome yields were comparably high for Manp3–AG
(80:20 mol %) and Manp4–AG (80:20 mol %), and low for the others
only these compositions were used in subsequent animal trials.
3,4,6-tri-O-benzyl-a-D-mannopyranosyl trichloroacetimidate (1,
1.4 g, 2.2 mmol) and molecular sieves 4 Å (3 g) was added

Table 2
1H NMR data of mannose archaeol compounds—sugars and protecting groups
H-60 ðJ6;6
Þ
BnAr
BnCH2 (JH,H
)
AcCH3
OH (J2,OH)
0
0
Compound
residue
H-1 (J1,2
)
H-2 (J2,3
)
H-3 (J3,4
)
H-4 (J4,5
)
H-5 (J5,6
)
H-6 ðJ5;6
Þ
2a
2b
2c
4.82 br s
4.92 (1.2)
4.76 br s
5.36 (2.9)
4.06 br s
3.83 br s
3.93 (9.2)
3.87 m
3.87 (9.4)
3.87 m
3.79 br t
3.80 br t
3.51 m
3.78 br d
3.77 br d
3.76 br d
3.78 br d
7.3–7.1 m (15)
7.3–7.1 m (15)
—
4.82 (12.0) 4.66 (12.0) 4.65 br s (2)
4.48 (12.0) 4.45 (12.0)
2.10 s (3)
3.69 br d (11.2)
3.76 br d
4.82 (12.0) 4.68 abq (2) 4.66
(12.0) 4.52 d (2) (12.0)
2.4 br s
3.70 m
3.68 m
—
—
3a
ManI
4.86 br s
5.08 br s
3.99 br s
5.53 br s
3.89 m
3.96 m
3.85 m
3.85 m
3.90 m
3.79 m
3.7–3.8 m
3.7–3.8 m
3.7–3.8 m
3.7–3.8 m
7.3–7.1 m (30)
—
4.83 d 4.80 d 4.64 m (5) 4.54 (11.2)
4.46 (3) 4.35 (10.8)
—
2.10 s (3)
—
ManII
3b
ManI
4.92 br s
5.10 br s
3.99 br s
4.07 br s
3.87 m
3.81 m
3.79 m
3.79 m
3.88 m
3.72 m
3.7–3.8 m
3.7–3.8 m
3.7–3.8 m
3.7–3.8 m
7.3–7.1 m (30)
4.78 (10.4) 4.75 (11.2) 4.61 m
(4) 4.47 m (6)
—
ManII
3c
ManI
ManII
4.99 br s
4.91 br s
3.81 br s
3.92 br s
3.70 m
3.69 m
3.67 m
3.66 m
3.48 m
3.58 m
3.76 m
3.78 m
3.76 m
3.75 m
4a
ManI
ManII
ManIII
4.89 br s
5.18 br s
5.03 br s
3.98 br s
4.08 br s
5.52 br s
3.83 m
3.80 m
3.96 m
3.83 m
3.83 m
3.70 m
3.79 m
3.79 m
3.83 m
3.8–3.7 m
3.8–3.7 m
3.8–3.7 m
3.8–3.7 m
3.8–3.7 m
3.8–3.7 m
7.3–7.1 m (45)
7.3–7.1 m (45)
4.81 (2) 4.7–4.38 (15) 4.28 (12.0)
2.10 s (3)
4b
ManI
4.89 br s
3.97 m
3.75 m
3.67 m
3.74 m
3.7 m
3.64 m
4.78 m (3) 4.65 m (2) 4.59–4.42 m
(12) 4.30 (12.2)
2.35 br s
ManII
ManIII
5.19 br s
5.10 br s
4.09 m
4.09 m
3.81 m
3.81 m
3.82
3.82
3.8 m
3.8 m
3.7 m
3.7 m
3.64 m
3.64 m
4c
ManI
ManII
ManIII
4.94 br s
4.96 br s
5.22 br s
3.78 m
3.99 br s
3.93 br s
3.60 m
3.63 m
3.73 m
3.60 m
3.63 m
3.58 m
3.44 m
3.48 m
3.53 m
3.8–3.6 m
3.8–3.6 m
3.8–3.6 m
3.8–3.6 m
3.8–3.6 m
3.8–3.6 m
5a
ManI
ManII
ManIII
ManIV
4.86 br s
5.19 br s
5.22 br s
5.04 br s
4.00 m
4.10 m
4.10 m
5.55 br dd
3.82 m
3.77 m
3.77 m
3.75 m
3.66 m
3.75 m
3.75 m
3.75 m
3.67 m
3.76 m
3.76 m
3.76 m
3.7–3.4 m
3.7–3.4 m
3.7–3.4 m
3.7–3.4 m
3.7–3.4 m
3.7–3.4 m
3.7–3.4 m
3.7–3.4 m
7.3–7.1 m
4.83–4.30 m, 4.16 d (12.0)
2.12 s (3)
5b
ManI
ManII
ManIII
ManIV
4.92 br s
5.20 m
5.20 m
3.98 m
4.11 m
4.11 m
4.08 m
3.92–3.78 m
3.92–3.78 m
3.92–3.78 m
3.92–3.78 m
3.74 m
3.90 m
3.90 m
3.90 m
3.90–3.75 m
3.90–3.75 m
3.90–3.75 m
3.90–3.75 m
3.74–3.48 m
3.74–3.48 m
3.74–3.48 m
3.74–3.48 m
3.74–3.48 m
3.74–3.48 m
3.74–3.48 m
3.74–3.48 m
7.3–7.1 m (60)
4.82–4.26 m (23) 4.13 d (12.4)
2.3 br
5.11 br s
5c
ManI
ManII
ManIII
ManIV
4.97 br s
5.24 br s
5.27 br s
4.99 br s
3.85 m
3.99 m
3.99 m
3.95 m
3.80 m
3.80 m
3.80 m
3.70 m
3.48 m
3.62 m
3.65 m
3.70 m
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
(continued on next page)

Table 2 (continued)
H-60 ðJ6;6
Þ
BnAr
BnCH2 (JH,H
)
AcCH3
OH
(J2,OH)
0
0
Compound H-1 (J1,2
residue
)
H-2 (J2,3
)
H-3 (J3,4
)
H-4 (J4,5
)
H-5 (J5,6
)
H-6 ðJ5;6
Þ
6a
ManI
4.93 br s
3.97 m
n.d.
n.d.
n.d.
n.d.
n.d.
7.3–7.1 m
(75)
4.84 m (3) 4.77 (11.6) 4.66–4.30 m 2.03 s (3)
(23) 4.25 (11.0) 4.17 m (2)
ManII
ManIII
ManIV
ManV
5.17 br s
5.21 br s
5.22 br s
5.01 br s
4.07 m
4.07 m
4.07 m
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
5.51 br s
6b
ManI
4.93 br s
3.91 m
n.d.
n.d.
n.d.
n.d.
n.d.
7.3 7.0 m
(75)
4.83–4.28 m (28) 4.20 (11.2) 4.08 m
(1)
n.d.
ManII
ManIII
ManIV
ManV
5.18 br s
5.21 m
5.21 m
4.08 m
4.08 m
4.08 m
4.08 m
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
5.10 br s
6c
ManI
4.92 br s
5.20 br s
5.24 br s
5.24 br s
4.95 br s
3.80 m
3.94 m
3.94 m
3.94 m
3.92 m
3.74 m
3.76 m
3.76 m
3.76 m
3.65 m
3.46 m
3.58 m
3.62 m
3.49 m
3.52 m
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
ManII
ManIII
ManIV
ManV
8a
GlcI
4.57 d (8.0) 4.81 dd (9.4) 5.22 t (9.0) 3.93 m
3.68 ddd
(3.7)
3.93 m
4.43 br d
(4.1)
4.43 br d
(3.6)
4.29 dd (12.1)
4.16 dd (12.3)
2.15, 2.13, 2.07, 2.04, 2.01, 2.00, 1.98 (3),
1.96
GlcII
GlcIII
5.25 d (4.1) 4.72 dd
(10.3)
5.375 t
(9.0)
3.93 m
5.380 d
(4.1)
4.83 dd (9.9) 5.33 t (9.5) 5.04 t (9.0) 3.93 m (3.6) 4.23 dd
4.03 br d
(12.0)
8b
GlcI
4.27 d (7.8) 3.30 dd
(10.0)
5.09 d (3.7) 3.51–3.42 m 3.85 t (9.2) 3.51–3.42
m
3.61 t (9.0) 3.53 t (9.3) 3.32 ddd
(2.1)
3.84 dd (3.9) 3.77 dd (12.0)
3.81–3.75 m 3.7–3.58 m
3.81–3.75 m 3.81–3.75 m
GlcII
GlcIII
3.7–3.58 m
5.09 d (3.7) 3.51–3.42 m 3.73 m
3.26 t (9.8) 3.7–3.58 m

Table 3
13C NMR data of mannose archaeol compounds—sugars and protecting groups
Compound
residue
C1
C2
C3
C4
C5
C6
Ac C@O
Ac CH3
Bnip
Bn CH
Bn CH2
2a
2b
2c
97.9
68.7
68.3
67.7
78.2
80.2
72.1
74.2
74.2
72.1
71.3
71
68.8
68.8
62.2
170.5
—
21.1
—
138.5, 138.2, 137.9
138.4, 138.2, 137.9
128.4–127.5
128.5–127.5
75.1, 73.4, 71.8
75.0, 73.4, 72.0
99.4
101.1
73.5
3a
ManI
ManII
98.7
99.6
74.9
68.7
78.2
79.7
74.5
74.3
71.8
71.8
69.2
69
170.1
—
21.1
—
138.56, 138.52, 138.50, 138.4, 138.2, 138.0
138.6 (2), 138.4, 138.24, 123.2, 138.0
—
128.3–127.3
128.4–127.3
—
75.0 (2), 73.4, 73.2, 72.1, 71.9
75.0 (2), 73.3, 73.2, 72.3, 72.1
—
3b
ManI
ManII
98.8
101.1
74.6
68.5
79.7
80
74.3
74.3
71.5
71.8
69.2
69
3c
ManI
ManII
99.5
103.4
80.1
71.3
71.9
74
68.2
68.2
71.6
73.7
62.6
62.3
—
—
4a
ManI
ManII
ManIII
98.8
100.6
99.4
74.7
74.7
68.7
79.4
79.5
78.2
71.9
71.9
71.9
74.7
74.7
74.2
69.2
69.2
69.3
170.2
21.2
138.6 (3), 138.4 (3), 138.3, 138.2, 138.1
128.3–127.5
75.0, 73.3, 72.1
4b
ManI
ManII
ManIII
98.8
100.7
100.9
74.6
74.9
68.3
79.9
79.4
80.1
74.2
74.2
74.2
71.9
71.9
71.5
68.9
68.9
69.1
—
—
138.5, 138.3, 138.1, 138.0
128.4–127.4
75.0, 73.3, 73.2, 72.3, 72.0
4c
ManI
ManII
ManIII
99.6
101.6
103.1
79.6
79.4
71.2
71.6
71.7
71.9
68.3
68.5
67.9
74.13
74.05
73.6
62.6
62.7
62.2
—
—
—
—
—
5a
ManI
98.8
75.2
78.3
71.8
74.7
69.2
170.1
21.2
138.62 (2), 138.58, 138.55, 138.5 (2),
138.4 (3), 138.3, 138.2, 138.1
128.4–127.4
75.0, 73.3, 73.2, 71.92, 71.86, 71.8
ManII
ManIII
ManIV
100.7
101.1
99.4
75.4
75.5
68.8
79.3
79.3
79.3
71.8
71.7
72.3
74.7
74.6
74.3
69.35
69.35
69.41
5b
ManI
98.8
75.5
79.2
71.7
74.8
69.2
—
—
138.62 (3), 138.56 (2), 138.5(2), 138.4,
138.35, 138.31, 138.2, 138.1
128.4–127.4
74.9, 73.3, 73.2, 72.1, 71.8
ManII
ManIII
ManIV
100.9
100.9
101.1
75.1
75.1
68.5
79.3
79.4
80.1
71.6
71.6
72.4
74.8
74.7
74.3
68.8
68.8
69.4
5c
ManI
ManII
ManIII
ManIV
99.9
102
101.9
103.7
80.1
71.6
72
72
69
74.6
74.6
74.5
74.1
62.9
63.1
63
79.93
79.87
71.2
68.8
68.6
68.2
72.2
62.6
(continued on next page)

Table 3 (continued)
Compound
residue
C1
C2
C3
C4
C5
C6
Ac C@O
Ac CH3
21.2
Bnip
Bn CH
Bn CH2
6a
ManI
ManII
ManIII
ManIV
ManV
98.8
76.1
75.7
75.7
75.5
75.0
78.2
79
79.1
79.2
79.3
71.72
71.78
71.85
71.9
74.3
74.7
74.7
74.7
74.3
69.6
69.5
69.3
69.2
67.3
170.1
138.7, 138.5, 138.4, 138.3, 138.2, 138.1
128.4–127.2
75.2, 74.9, 73.3, 73.23, 73.19
101.2
101.3
101.3
99.4
71.9
6b
ManI
ManII
ManIII
ManIV
ManV
98.8
101.3
101.3
100.9
100.9
75.7
75.7
75.7
75.7
68.8
79.1
79.1
79.3
79.7
80.1
71.8
71.8
71.8
71.8
71.8
75.1
75.1
75
75
75
69.5
69.5
69.4
69.3
67.3
138.7, 138.6, 138.42, 138.36, 138.2, 138.1
128.4–127.2
75.1, 75.0, 74.3, 73.2, 72.4, 72.1
6c
ManI
ManII
ManIII
ManIV
ManV
99.6
101.6
101.6
101.6
103.3
79.5
79.6
79.6
79.6
72
71.4
71.5
71.5
71.5
71.6
68.4
68.4
68.4
68.4
68.4
74.3
74.3
74.3
74.3
74.3
62.8
62.8
62.8
62.8
62.8
8a
GlcI
100.5
72.2
75.4
72.4
71.7
63
170.59, 170.56, 170.51, 170.46,
170.3, 170.1, 169.8, 169.7,
169.5, 169.4
20.9, 20.8, 20.64, 20.61, 20.5
GlcII
GlcIII
95.7
95.6
70.5
70.4
71.9
70.1
73.8
67.8
68.4
68.8
62.3
61.3
8b
GlcI
GlcII
GlcIII
104.2
102.6
102.5
73.9
73.6
73.1
76.9
74.4
72.8
83.4
83.2
71
76
74.2
74.6
61.6
62.5
61.6

D. M. Whitfield et al. / Carbohydrate Research 343 (2008) 2349–2360
2357
Table 4
1H NMR data of lipid in archaeol compounds
Compound
aa0
b
cc0
dd0
ee0
ff0 + gg0
CH
CH2
CH3
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
8a
8b
3.67 m, 3.50 m
3.74 m, 3.53 m
3.68 m, 3.47 m
3.62 m, 3.37 m
3.65 m, 3.40 m
3.69 m, 3.43 m
3.63 m, 3.33 m
3.64 m, 3.35 m
3.65 m, 3.38 m
3.60 m, 3.40 m
3.60 m, 3.40 m
3.68 m, 3.42 m
3.59 m, 3.45 m
3.59 m, 3.38 m
3.48 br m
3.53 m
3.53 m
3.54 m
3.47 m
3.51 m
3.55 m
3.48 m
3.53 m
3.51 m
3.50 m
3.50 m
3.56 m
3.54 m
3.46 m
3.57 m
3.54 m
3.63 m
3.55 m
3.57 m
3.56 m
3.50 m
3.54 m
3.57 m
3.50 m
3.52 m
3.53 m
3.51 m
3.51 m
3.54 m
3.57 m, 3.50 m
3.71 m
3.58 m
3.54 m
3.62 m
3.55 m
3.55 m
3.45 m
3.40 m
3.44 m
3.46 m
3.40 m
3.43 m
3.41 m
3.40 m
3.40 m
3.46 m
3.50 m
3.38 m
3.47 m
3.42 m
3.53 m
3.42 m
3.45 m
3.44 m
3.38 m
3.40 m
3.46 m
3.36 m
3.39 m
3.40 m
3.38 m
3.38 m
3.45 m
3.49 m
3.34 m
3.43 m
3.40 m
3.48 m
1.52 m, 1.31 m
1.52 m, 1.35 m
1.50 m, 1.32 m
1.54 m, 1.29 m
1.57 m, 1.32 m
1.55 m, 1.33 m
1.56 m, 1.31 m
1.53 m, 1.30 m
1.51 m, 1.29 m
1.55 m, 1.30 m
1.55 m, 1.30 m
1.57 m, 1.34 m
1.60 m, 1.40 m
1.54 m, 1.39 m
1.54 m, 1.39 m
1.56 m, 1.32 m
1.59 m, 1.35 m
1.47 m, 1.30 m
1.54 m, 1.36 m
1.56 m, 1.33 m
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
1.4–1.0
0.8 m
0.85 m
0.8 m
1.48 m, 1.44 m, 1.31 m
1.52 m, 1.50 m, 1.26 m
1.49 m, 1.47 m, 1.33 m
1.52 m, 1.48 m, 1.36 m
1.51 m, 1.46 m, 1.33 m
1.44 m, 1.42 m, 1.30 m
1.50 m, 1.45 m, 1.33 m
1.50 m, 1.45 m, 1.33 m
1.50 m, 1.49 m, 1.35 m
1.50 m, 1.40 m
0.8 m
0.85 m
0.81 m
0.85 m
0.81 m
0.79 m
0.85 m
0.85 m
0.84 m
0.85 m
0.9 m
1.50 m, 1.40 m
1.53 m
1.48 m, 1.34 m
1.52 m, 1.36 m
0.9 m
0.8 m
0.85 m
3.86 m, 3.54 m
3.92 br dd (10.0), (2.7) 3.59 m
Table 5
13C NMR data of lipids in archaeol compounds
Compound
a
b
c
d
e
CH
CH2
CH3
2a
67.4 77.4 69
70.5 70.1 32.8, 29.9, 29.8, 28.0
39.4, 37.54, 37.47, 37.4, 37.3, 37.0, 36.7, 24.8, 24.5,
24.4
22.7, 22.6, 19.74, 19.70,
19.6
2b
67.1 77.5 68.9 70.6 70.1 32.8, 29.9, 29.8, 28.0
39.4, 37.53, 37.46, 37.4, 37.3, 37.0, 36.7, 24.8, 24.5,
24.4
22.7, 22.6, 19.74, 19.70,
19.6
2c
3a
67.5 78.5 69.6 71.3 70.7 33.5,30.6, 30.4, 30.3
67.2 77.5 68.9 70.8 70.1 32.8, 29.9, 29.8, 28.0
40.1, 38.1, 38.0, 37.7, 37.3, 25.5, 25.13, 25.08
39.4, 37.53, 37.46, 37.4, 37.3, 37.0, 36.7, 24.8, 24.5,
24.4
23.1, 23.0, 20.2
22.7, 22.6, 19.75, 19.71,
19.6
3b
3c
4a
4b
4c
5a
67.2 77.5 69
67.9 77.5 69.7 71.6 70.8 33.5, 30.6, 30.5, 28.7
67.2 77.4 69 70.9 70 32.8, 30.0, 29.8, 28.0
67.1 77.6 69.3 71.2 70.3 33.1, 30.2, 30.0, 28.2
67.9 77.7 68.5 71.2 69.7 33.5, 30.6, 30.4, 28.6
70.8 70.1 32.8, 29.9, 29.8, 28.0
39.3, 37.5, 37.4, 37.3, 37.0, 36.6, 24.8, 24.5, 24.4
40.1, 38.1, 38.0 37.7, 37.4, 25.5, 25.2, 25.1
39.4, 37.5, 37.3, 37.1, 36.7, 24.8, 24.5, 24.4
39.6, 37.7, 37.5,37.3, 36.9, 25.1, 24.7, 24.6
40.1, 38.1, 37.9, 37.6, 37.3, 25.5, 25.12, 25.07
39.4, 375, 37.3, 37.1, 36.7, 24.8, 24.5, 24.4
22.7, 22.6, 19.7, 19.6
23.14, 23.06, 20.2
22.7, 22.6, 19.8
23.0, 22.9, 20.0
23.1, 23.0, 20.2, 20.1
22.7, 22.6, 19.74, 19.68,
19.6
67.3 78.3 69.2 71
70.1 32.8, 30.0, 29.8, 28.0
5b
5c
6a
6b
67.3 77.6 69
71.6 71
32.8, 30.0, 29.8, 28.0
39.4, 37.5, 37.3, 37.1, 36.7, 24.8, 24.5, 24.4
40.3, 38.43, 38.36, 38.3, 38.2, 38.0, 37.7, 25.7, 25.3
39.4, 37.6, 37.5, 37.3, 27.1, 36.7, 24.8, 24.5, 24.4
39.3, 37.5, 37.3, 37.1, 36.7, 24.8, 24.5, 24.4
22.7, 22.6, 19.7, 19.6
23.2, 23.1, 20.4, 20.3
22.7, 22.6, 19.8, 19.7
22.7, 22.6, 19.8, 19.7, 19.6
68.4 78.9 69.8 71.7 70.9 33.8, 30.8, 30.7, 28.9
68.7 77.6 70.1 71.9 71 32.8, 30.0, 29.8, 29.7
68.5 77.6 69 71
70.1 32.83, 32.81, 31.9, 30.0, 29.8, 29.7,
29.4
6c
68
78.7 69.7 71.2 70.8 33.5, 30.6, 30.5, 30.3, 28.7
40.1, 39.6, 38.13, 38.09, 38.0, 37.7, 37.4, 25.5, 25.15,
25.10
39.3, 37.41, 37.37, 37.2, 37.1, 36.5, 24.8, 24.4, 24.3,
23.1, 23.0, 20.23, 20.15
22.7, 22.6, 19.71, 19.67
8a
8b
70.4 77.7 69.3 70.4 70.1 32.8, 29.9, 29.8, 27.9
69.8 78.4 69.4 71.2 70.8 33.5, 30.6, 30.5, 28.7
40.01, 38.13, 38.09, 38.0, 37.7, 37.3, 25.5, 25.15, 25.09 23.1, 23.0, 20.3 20.2
CH2Cl2 (15 mL). After stirring at rt under an argon atmosphere for
1 h, triethylsilyltrifluoromethanesulfonate (25 L, 0.11 mmol) was
added and the stirring continued for 40 min. The reaction was
quenched with diisopropylethylamine (100 L). The whole reac-
4.3.2. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-
tetramethylhexadecyloxy]propan-1-yl 2-O-(2-O-acetyl-3,4,6-
l
tri-O-benzyl-a-D-mannopyranosyl)-3,4,6-tri-O-benzyl-a-D-
l
mannopyranoside (3a)
tion was adsorbed on silica gel and then purified by silica gel chro-
matography eluting with 9:1 hexanes–EtOAc to yield pure product
as a viscous oil (0.58 g, 47%) plus some mixed fractions.
Compound 3a (1.04 g, 63%) was prepared from 2b (1.15 g, 0.206
mmol) using procedure A and purified by silica gel eluting with 9:1
hexanes–EtOAc followed by 85:15 hexanes–EtOAc.
4.3.3. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexa-
4.3. Procedure B
decyloxy]propan-1-yl 2-O-(3,4,6-tri-O-benzyl-a-D-manno-
pyranosyl)-3,4,6-tri-O-benzyl- -mannopyranoside (3b)
a
-D
4.3.1. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexa-
decyloxy]propan-1-yl 3,4,6-tri-O-benzyl-a-D-mannopyranoside
(2b)
Compound 3b (0.98 g, 88%) was prepared from 3a (1.14 g, 0.73
mmol) using procedure B and purified by silica gel eluting with
85:15 hexanes–EtOAc.
Compound 2a (0.58 g, 0.51 mmol) was dissolved in a mixture of
dry CH3OH (10 mL) and CH2Cl2 (2 mL). Then, 1 M NaOCH3 (0.5 mL)
was added and the stirring continued for 4 h. The reaction mixture
was diluted with CH2Cl2 (150 mL) and washed 2ꢁ with NH4Claq
followed by saturated NaClaq. After drying with Na2SO4, filtration
and evaporation the residue was purified by column chromatogra-
phy on silica gel eluting with 5:1 hexanes–EtOAc to yield pure
compound as a viscous oil (520 mg, 93%).
4.3.4. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-
tetramethylhexadecyloxy]propan-1-yl 2-O-(2-O-acetyl-3,4,6-
tri-O-benzyl-
a-
D-mannopyranosyl)-2-O-(3,4,6-tri-O-benzyl-
a-D-
mannopyranosyl)-3,4,6-tri-O-benzyl
a-D-mannopyranoside (4a)
Compound 4a (440 mg, 38%) was prepared from 3b (880 mg,
0.58 mmol) using procedure A and purified by silica gel eluting
with 85:15 hexanes–EtOAc.

2358
D. M. Whitfield et al. / Carbohydrate Research 343 (2008) 2349–2360
Table 6
4.3.5. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexadecyl-
oxy]propan-1-yl 2-O-(3,4,6-tri-O-benzyl- -mannopyranosyl)-
2-O-(3,4,6-tri-O-benzyl- -mannopyranosyl)-3,4,6-tri-O-
benzyl- -mannopyranoside (4b)
Compound 4b (370 mg, 90%) was prepared from 4a (420 mg,
0.21 mmol) using procedure B and purified by silica gel eluting
with 85:15 hexanes–EtOAc.
MS data for archaeol compounds
a-D
a-D
Compound Formula
MW
calcd
MS MALDI
[
a
]
c (solvent)
D
a-D
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
C72H118O9
C70H116O8
C49H98O8
1127.23 1149.69 [M+Na]+
1165.65 [M+K]+
1085.7
22
21
0.3 CHCl3
1.1 CHCl3
1107.78 [M+Na]+
1123.71 [M+K]+
814.73 837.52 [M+Na]+
853.38 [M+K]+
19.5 0.8 CHCl3
18.7 0.4 CHCl3
23.3 0.3 CHCl3
28.3 0.6 CHCl3
33.5 0.2 CHCl3
29.8 0.7 CHCl3
42.3 0.6 CHCl3
19.1 0.9 CHCl3
20.5 1.4 CHCl3
32.5 1.2 CHCl3
20.5 1.2 CHCl3
39.3 2.2 CHCl3
4.3.6. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-
tetramethylhexadecyloxy]propan-1-yl 2-O-(2-O-acetyl-3,4,6-
C99H146O14
C97H144O13
C55H108O13
1559.07 1583.17 [M+Na]+
1599.13 [M+K]+
tri-O-benzyl-
-mannopyranosyl)-2-O-(3,4,6-tri-O-benzyl-
mannopyranosyl)-3,4,6-tri-O-benzyl -mannopyranoside (5a)
a-D-mannopyranosyl)-2-O-(3,4,6-tri-O-benzyl-
1518.21 1540.75 [M+Na]+
1556.71 [M+K]+
a-D
a-D-
a-D
976.78 999.83 [M+Na]+
1015.79 [M+K]+
Compound 5a (178 mg, 73%) was prepared from 4b (190 mg,
0.11 mmol) using procedure A and purified by silica gel eluting
with 85:15 hexanes–EtOAc.
C126H174O19 1991.27 2015.16 [M+Na]+
2031.12 [M+K]+
C124H172O18 1949.25 1972.89 [M+Na]+
1988.84 (M+K)+
4.3.7. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethyl-
C61H118O18
1139.61 1161.90 [M+Na]+
1173.83 [M+K]+
hexadecyloxy]propan-1-yl-2-O-3,4,6-tri-O-benzyl-
pyranosyl-2-O-(3,4,6-tri-O-benzyl- -mannopyranosyl)-2-O-
(3,4,6-tri-O-benzyl- -mannopyranosyl)-3,4,6-tri-O-benzyl-
-mannopyranoside (5b)
Compound 5b (93 mg, 55%) was prepared from 5a (172 mg, 0.61
a-D-manno-
C153H202O24 2423.46 2447.60 [M+Na]+
2463.59 (M+K)+
a-D
a-D
a-
C151H200O23 2383.26 2405.74 [M+Na]+
2421.71 [M+K]+
D
C67H128O23
1301.75 1324.11 [M+Na]+
1327.13 [M+K]+
mmol) using procedure B and purified by silica gel eluting with
85:15 hexanes–EtOAc.
C180H230O29 2855.65 2880.77 [M+Na]+
2896.70 [M+K]+
C178H228O28 2813.64 2838.57 [M+Na]+
2854.55 [M+K]+
4.3.8. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-
tetramethylhexadecyloxy]propan-1-yl 2-O-(2-O-acetyl-3,4,6-
C73H138O28
1462.94 1485.99 [M+Na]+
20.8 0.4 CH2Cl2–
CH3OH 1:1 v:v
tri-O-benzyl-
-mannopyranosyl)-2-O-(3,4,6-tri-O-benzyl-
mannopyranosyl)-2-O-(3,4,6-tri-O-benzyl-
mannopyranosyl)-3,4,6-tri-O-benzyl -mannopyranoside (6a)
a-D-mannopyranosyl)-2-O-(3,4,6-tri-O-benzyl-
1501.86 [M+K]+
1558.94 1582.0 [M+Na]+
1139.61 1161.7 [M+Na]+
1177.6 [M+K]+
a-D
a-D-
a-D-
8a
8b
C81H138O28
C61H118O18
a-D
Compound 6a (163 mg, 68%) was prepared from 5b (200 mg,
0.084 mmol) using procedure A and purified by silica gel eluting
with 85:15 hexanes–EtOAc.
Scheme 2. Synthesis of starch-derived trimer attached to archaeol.

D. M. Whitfield et al. / Carbohydrate Research 343 (2008) 2349–2360
2359
alyst) (150 mg) was added and the mixture hydrogenated using a
Parr apparatus at 50 psi of H2 with shaking for 64 h. The catalyst
was removed by filtration through a bed of Celite, and was well
washed with EtOAc and CH3OH. The combined filtrates were evap-
orated and then purified by silica gel chromatography eluting with
7:1:1 EtOAc–CH3OH–H2O to yield a waxy solid (62 mg, 83%).
4.4.2. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-
tetramethylhexadecyloxy]propan-1-yl 2-O-
(a-D
-mannopyranosyl)
a-D-mannopyranoside (3c)
Compound 3c (53 mg, 82%) was prepared from 3b (100 mg,
0.066 mmol) using procedure C.
4.4.3. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-
tetramethylhexadecyloxy]propan-1-yl 2-O-
(a-D
-mannopyranosyl)-2-O-(
a-D-mannopyranosyl)-a-D-
mannopyranoside (4c)
Compound 4c (41 mg, 70%) was prepared from 4b (100 mg,
0.051 mmol) using procedure C.
4.4.4. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-
tetramethylhexadecyloxy]propan-1-yl 2-O-(
mannopyranosyl)-2-O-( -mannopyranosyl)-2-O-
-mannopyranosyl)- -mannopyranoside (5c)
Compound 5c (43 mg, 66%) was prepared from 5b (120 mg,
a-D-
a-D
(a-D
a-D
0.050 mmol) using procedure C.
4.4.5. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-
tetramethylhexadecyloxy]propan-1-yl 2-O-(
a-D-
mannopyranosyl)-2-O-(
-mannopyranosyl)-
Compound 6c (28 mg, 65%) was prepared from 6b (83 mg, 0.029
a
-
D
-mannopyranosyl)-2-O-
(a-D
a-D-mannopyranoside (6c)
mmol) using procedure C.
4.4.6. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-
tetramethylhexadecyloxy]propan-1-yl 4-O-(2,3,4,6-tetra-O-a-D-
glucopyranosyl)-4-O-(2,3,6-tri-O-acetyl-
2,3,6-tri-O-acetyl-b- -glucopyranoside (8a)
To known23 4-O-(2,3,4,6-tetra-O-
-glucopyranosyl)-4-O-
(2,3,6-tri-O-acetyl- -glucopyranosyl)-2,3,6-tri-O-acetyl-b- -glu-
a-D-glucopyranosyl)-
D
a-D
a
-
D
D
Figure 2. CTL MHC class I immune responses in mice immunized with various
synthetic glycosylarchaeols. (A) CTL activity is presented as % lysis of antigen-
specific EG.7 target cells. (B) Antigen specificity was confirmed by measuring CTL
activity for non-specific EL-4 target cells. Vaccines used to immunize mice were
OVA entrapped in (a), total polar lipid archaeosomes from M. smithii or in
archaeosomes containing 20 mol % AG with 80 mol % of either (b), starch-derived
trimer 8b; (c) Manp tetramer 5c; or (d) Manp trimer 4c. (e) OVA entrapped in
glycolipid-free archaeosomes consisting of AG–cholesterol (80:20 mol %); (f) naive.
copyranosyl trichloroacetimidate (280 mg, 0.29 mmol), 4 molecu-
lar sieves (300 mg) and archaeol (78 mg, 0.12 mmol) was added
CH2Cl2 (3 mL), and the mixture stirred for 1 h at rt under an atmo-
sphere of argon. To this was added triethylsilyltrifluoromethane-
sulfonate (3 lL, 0.013 mmol) and the mixture stirred for 40 min
when TLC in 1:1 hexanes–EtOAc (Rf = 0.5) indicated the reaction
was complete. The reaction was quenched with diisopropylethyl-
amine (10 lL), filtered with rinsing with CH2Cl2. The combined fil-
trates were concentrated and the residue purified by silica gel
chromatography eluting with 2:1 hexanes–EtOAc to yield pure
8a as a waxy solid (99 mg, 53%).
4.3.9. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexa-
decyloxy]propan-1-yl-2-O-(3,4,6-tri-O-benzyl-
pyranosyl)-2-O-(3,4,6-tri-O-benzyl- -manno-pyranosyl)-2-O-
(3,4,6-tri-O-benzyl- -mannopyranosyl)-2-O-(3,4,6-tri-O-benzyl-
-mannopyranosyl)-3,4,6-tri-O-benzyl- -mannopyranoside
(6b)
a-D-manno-
a-D
4.5. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-
a-D
tetramethylhexadecyloxy]propan-1-yl 4-O-(
a-D-glucopyr-
a-
D
a-D
anosyl)-4-O-( -glucopyranosyl)-b- -glucopyranoside (8b)
a-
D
D
Compound 6b (88 mg, 90%) was prepared from 6a (134 mg,
0.086 mmol) using procedure B and purified by silica gel eluting
with 75:25 hexanes–EtOAc.
Compound 8a (134 mg, 0.086 mmol) was deacetylated follow-
ing procedure B and purified by silica gel chromatography eluting
with CHCl3–CH3OH–H2O 10:3:0.3 to yield pure 8b (88 mg, 90%).
4.4. Procedure C
4.6. NMR and MS tables
4.4.1. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethyl-
hexadecyloxy]propan-1-yl a-D-mannopyranoside (2c)
Compound 2b (100 mg, 0.051 mmol) was dissolved in EtOAc
(10 mL) and, after purging with argon, Pd(OH)2–C (Pearlman’s cat-
1H and 13C NMR of 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b and 8a
were obtained in CDCl3 solution (referenced to residual CHCl3 at
7.26 ppm 1H and 77.0 ppm central resonance 13C), whereas those

2360
D. M. Whitfield et al. / Carbohydrate Research 343 (2008) 2349–2360
of 2c, 3c, 4c, 5c, 6c and 8b were obtained in 1:1 (v:v) solutions of
CD3OD–CDCl3 (referenced to residual CHD2OD at 3.31 ppm 1H and
49.15 ppm central resonance 13C). Chemical shifts are in ppm and
coupling constants in hertz. Subscript Roman Numerals refer to the
residue number with I the reducing termini. NMR was performed
on either a Varian 400 MHz or 200 MHz spectrometers.
References
1. (a) Aguilar, J. C.; Rodriguez, E. G. Vaccine 2007, 25, 3752–3762; (b) Medzhitov,
R. Nature 2007, 449, 819–826.
2. Rappuoli, R. Nat. Biotechnol. 2007, 25, 1361–1366.
3. Kim, S. K.; Ragupathi, G.; Cappello, S.; Kagan, E.; Livingstone, P. O. Vaccine 2001,
19, 530–537.
4. Sprott, G. D.; Dicaire, C. J.; Fleming, L. P.; Patel, G. B. Cells Mater. 1996, 6, 143–
155.
5. (a) Tolson, D. L.; Latta, R. K.; Patel, G. B.; Sprott, G. D. J. Liposome Res. 1996, 6,
755–776; (b) Krishnan, L.; Sad, S.; Patel, G. B.; Sprott, G. D. J. Immunol. 2001,
166, 1885–1893.
4.7. Archaeosome preparation and characterization
Archaeosomes were prepared by drying lipids from CHCl3–
CH3OH (2:1) and hydrating 20–30 mg lipids at 40 °C in 2 mL of
pyrogen-free water containing the test antigen OVA dissolved at
10 mg/mL. In all cases AG was mixed in CHCl3–CH3OH with the
synthetic glyco-archaeols. An AG–cholesterol (80:20 mol %)
archaeosome with OVA entrapped was prepared as above. The size
of the vesicles in the preparations was decreased by sonication in a
sonic water bath at 40 °C. Antigen not entrapped was removed by
centrifuging (200,000g, Rmax for 30 min) and washing three times
from 8 mL volumes of PBS. Archaeosome preparations were filter
6. Kates, M. Biochem. Soc. Symp. 1992, 58, 51–72.
7. Nishihara, M.; Morii, H.; Koga, Y. J. Biochem. (Tokyo) 1987, 101, 1007–1015.
8. (a) Sprott, G. D.; Dicaire, C. J.; Côtè, J.-P.; Whitfield, D. M. Glycobiology 2008, 18,
559–565; (b) Krishnan, L.; Sad, S.; Patel, G. B.; Sprott, G. D. J. Immunol. 2000,
165, 5177–5185.
9. Krishnan, L.; Sprott, G. D. Vaccine 2008, 26, 2043–2055.
10. Krishnan, L.; Gurnani, K.; Dicaire, C. J.; van Faassen, H.; Zafer, A.; Kirschning, C.
J.; Sad, S.; Sprott, G. D. J. Immunol. 2007, 178, 2396–2406.
11. (a) de la Salle, H.; Mariotti, S.; Angenieux, C.; Gilleron, M.; Garcia-Alles, L.-F.;
´
Malm, D.; Berg, T.; Paoletti, S.; MaZtre, B.; Mourey, L.; Salamero, J.; Cazenave, J.
P.; Hanau, D.; Mori, L.; Puzo, G.; De Libero, G. Science 2005, 310, 1321–1324; (b)
Fujio, M.; Wu, D.; Garcia-Navarro, R.; Ho, D. D.; Tsuji, M.; Wong, C.-H. J. Am.
Chem. Soc. 2006, 128, 9022–9023; (c) Franck, R. W.; Tsuji, M. Acc. Chem. Res.
2006, 39, 692–701; (d) McGreal, E. P.; Rosas, M.; Brown, G. D.; Zamze, S.; Wong,
S. Y. C.; Gordon, S.; Martinez-Pomares, L.; Taylor, P. R. Glycobiology 2006, 16,
422–430.
sterilized using 0.45
immunizations, dilutions were made to achieve 15
l
m filters and stored at 4 °C. Just prior to
OVA/
lg
0.1 mL of PBS buffer (10 mM sodium phosphate, 160 mM NaCl,
pH 7.1). Quantification of antigen loading by SDS polyacrylamide
gel electrophoresis was as described, where antigen loading was
based on salt corrected dry weights.35 Average diameters were
determined by particle size analysis using a 5 mW He–Ne laser (Ni-
comp 370). All glassware was rendered pyrogen-free by heating for
6 h at 180 °C.
12. Gurnani, K.; Kennedy, J.; Sad, S.; Sprott, G. D.; Krishnan, L. J. Immunol. 2004, 173,
566–578.
13. Hoffmann, P. R.; Kench, J. A.; Vondracek, A.; Kruk, E.; Daleke, D. L.; Jordan, M.;
Marrack, P.; Henson, P. M.; Fadok, V. A. J. Immunol. 2005, 174, 1393–1404.
14. (a) Grandjean, C.; Angyalosi, G.; Loing, E.; Adriaenssens, E.; Melnyk, O.; Pancru˜,
V.; Auriault, C.; Gras-Masse, H. ChemBioChem 2001, 2, 747–757; (b) Düffels, A.;
Green, L. G.; Ley, S. V.; Miller, A. D. Chem. Eur. J. 2000, 6, 1416–1430.
15. Powlesland, A. S.; Ward, E. M.; Sadhu, S. K.; Guo, Y.; Taylor, M. E.; Drickamer, K.
J. Biol. Chem. 2006, 281, 20440–20449.
16. East, L.; Isacke, C. M. Biochim. Biophys. Acta 2002, 1572, 364–386.
17. (a) Sugimoto, M.; Ohishi, K.; Fukasawa, M.; Shikata, K.; Kawai, H.; Itakura, H.;
Hatanaka, M.; Sakabara, R.; Ishiguro, M.; Nakata, M.; Mizuochi, T. FEBS Lett.
1995, 363, 53–56; (b) Liu, X.; Stocker, B. L.; Seeberger, P. H. J. Am. Chem. Soc.
2006, 128, 3638–3648; (c) Jain, S.; Vyas, S. P. J. Liposome Res. 2006, 16, 331–345.
18. (a) van Boeckel, C. A. A.; Westerduin, P.; van Boom, J. H. Carbohydr. Res. 1984,
133, 219–234; (b) Goodby, J. W.; Görtz, V.; Cowling, S. J.; Mackenzie, G.; Martin,
P.; Plusquellec, D.; Benvegnu, T.; Boullanger, P.; Lafont, D.; Queneau, Y.;
Chambert, S.; Fitremann, J. Chem. Soc. Rev. 2007, 36, 1971–2032.
19. Feinberg, H.; Castelli, R.; Drickamer, K.; Seeberger, P. H.; Weis, W. I. J. Biol.
Chem. 2007, 282, 4202–4209.
20. Douglas, S. P.; Whitfield, D. M.; Krepinsky, J. J. J. Am. Chem. Soc. 1995, 117,
2116–2117.
21. Karelin, A. A.; Tsvetkov, Yu. E.; Kogan, G.; Bystricky, S.; Nifantiev, N. E. Russ. J.
Bioinorg. Chem. 2007, 33, 119–130.
22. Mathew, F.; Mach, M.; Hazen, K. C.; Fraser-Reid, B. Synlett 2003, 1319–1322.
23. Minamikawa, H.; Hato, M. Langmuir 1997, 13, 2571–2654.
24. Baba, T.; Minamikawa, H.; Hato, M.; Handa, T. Biophys. J. 2001, 81, 3377–3386.
25. Schwendener, R. A.; Asanger, M.; Weder, H. G. Biochem. Biophys. Res. Commun.
1981, 100, 1055–1062.
4.8. Animal usage
To determine adjuvant activity, OVA entrapped in archaeo-
somes (OVA-archaeosomes) was used to immunize female
C57BL/6 mice on days 0 and 21 (8 weeks old on first injection).
Injections were subcutaneous at the tail base with 0.1 mL PBS con-
taining 15 lg OVA entrapped in 0.2–0.63 mg lipids. Blood samples
were collected on week 7 from the tail vein for anti-OVA antibody
titration done by Elisa.27 Spleens were collected on week 7 from
duplicate mice to determine CTL activity using the Cr51-assay with
specific and non-specific targets EG.7 and EL-4, respectively.8b All
protocols were approved by the Institutional Animal Care Commit-
tee, and were in compliance with guidelines set by the Canadian
Council on Animal Care.
26. Rotzschke, O.; Falk, K.; Stevanovic, S.; Jung, G.; Walden, P.; Rammensee, H. G.
Eur. J. Immunol. 1991, 21, 2891–2894.
4.9. Statistical analysis
27. Krishnan, L.; Dicaire, C. J.; Patel, G. B.; Sprott, G. D. Infect. Immun. 2000, 68, 54–
63.
28. Krishnan, L.; Gurnani, K.; Dicaire, C. J.; van Fassen, H.; Zafer, A.; Kirschning, C. J.;
Sad, S.; Sprott, G. D. J. Immunol. 2007, 178, 2396–2406.
Means are reported as means S.D., and significance between
means (P <0.05) compared by two-tailed t-test.
29. Murthy, B. N.; Voelcker, N. H.; Jayaraman, N. Glycobiology 2006, 16, 822–832.
30. Napper, C. E.; Taylor, M. E. Glycobiology 2004, 14, 7C–12C.
31. Bazin, H. G.; Bess, L. S.; Livesay, M. T.; Ryter, K. T.; Johnson, C. L.; Arnold, J. S.;
Johnson, D. A. Tetrahedron Lett. 2006, 47, 2087–2092.
32. Fujio, M.; Wu, D.; Garcia-Navarro, R.; Ho, D. D.; Tsuji, M.; Wong, C.-H. J. Am.
Chem. Soc. 2006, 128, 9022–9023.
33. Kim, Y. J.; Wang, P.; Navarro-Villalobos, M.; Rohde, B. D.; Derryberry, J. M.; Gin,
D. Y. J. Am. Chem. Soc. 2006, 128, 11906–11915.
34. Sprott, G. D.; Larocque, S.; Cadotte, N.; Dicaire, C. J.; McGee, M.; Brisson, J. R.
Biochim. Biophys. Acta 2003, 1633, 179–188.
Acknowledgements
Mr. John Shelvey grew Halobacterium salinarum biomass in a
250 L fermenter in our cell culture facility. Dr. Henryk Borowry-
Borokowski from the laboratory of D.M.W. synthesized the Glcp3
derivatives. We thank Chantal Dicaire for performing the CTL and
Elisa assays in the laboratory of G.D.S. This article is publication
#42525 of the National Research Council of Canada.
35. Sprott, G. D.; Patel, G. B.; Krishnan, L. Methods Enzymol. 2003, 373, 155–172.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2008.06.021.