Development of new glycosylation methodologies for the synthesis of archaeal-derived glycolipid adjuvants

Carbohydrate Research 345 (2010) 214–229

Contents lists available at ScienceDirect

Carbohydrate Research

journal homepage: w ww.elsevier.com/locate/carres

Development of new glycosylation methodologies for the synthesis of

archaeal-derived glycolipid adjuvants

*

Dennis M. Whitﬁeld , Siu H. Yu, Chantale J. Dicaire, G. Dennis Sprott

National Research Council, Institute for Biological Science, 100 Sussex Drive, Ottawa, Canada ON K1A 0R6

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 4 July 2009

Received in revised form 9 October 2009

Accepted 16 October 2009

Available online 6 November 2009

To commercialize the production of glycolipid adjuvants, their synthesis needs to be both robust and

inexpensive. Herein we describe a semi-synthetic approach where the lipid acceptor is derived from

the biomass of the archaeon Halobacterium salinarum, and the glycosyl donors are chemically synthe-

sized. This work presents some preliminary results using the promoter system N-iodosuccinimide

(NIS) and a stable 0.25 M solution of boron triﬂuoride–triﬂuoroethanol (BF₃ꢀTFE₂) in dichloromethane.

This promoter system allows for the use of peracetyl alkyl(aryl)thioglycosides that are available in high

yield from inexpensive disaccharide starting materials by promoting clean glycosylation reactions from

which pure product can be easily isolated. Conventional glycosylation using NIS–silver triﬂuoromethane-

sulfonate (AgOTf) leads to extensive acetyl transfer to the archaeol acceptor and numerous byproducts

that make puriﬁcation complicated. As part of preliminary structure–adjuvant activity studies, we

describe the one-pot synthesis of a gentiobiose b-Glcp-(1?6)-Glcp-SEt donor with an O-2 benzoyl group,

which can be used to prepare a disaccharide attached to archaeol in 85% overall yield, and the related

glycolipid trisaccharide b-Glcp-(1?6)-b-Glcp-(1?6)-b-Glcp-(1?O)-archaeol. The synthesis of the iso-

Keywords:

Glycosylation

Glycolipids

Adjuvants

MHC Type 1

CTL

meric b-Glcp-(1?6)-a-Glcp-(1?O)-archaeol featuring a >10:1 a/b a-selective glycosylation using the

promoter system N-phenylselenylphthalimide–triﬂuoromethanesulfonic acid (TfOH) is also presented,

along with the adjuvant properties of the corresponding archaeosomes (liposomes comprised entirely

of combinations of isoprenoid archaeal-like lipids). These new vaccine formulations extend previous

observations that glycolipids are integral to the activation of MHC type I pathways via CD8⁺antigen-spe-

ciﬁc T-cells. The b-Glcp-(1?6)-b-Glcp-(1?6)-b-Glcp-(1?O)-archaeol trisaccharide is shown to be more

active than the Glcp-(1?6)-b-Glcp-(1?O)-archaeol disaccharide.

1. Introduction

parent C₄₃compound is known as archaeol.⁴Most species of meth-

anogen and thermoacidophilic archaea also contain C₈₆diols

Recent studies from our group have shown that synthetic glyco-

lipids based on oligosaccharides glycosidically linked to archaeol

can activate the immune system by both MHC type I and MHC type

II pathways.¹Furthermore, trisaccharides and tetrasaccharides

have been shown to form the most stable liposomes, named

archaeosomes.²Disaccharides formed stable archaeosomes if they

were mixed with ester-based di-palmitoyl-phosphorylglycerol

(DPPG) and cholesterol in proportions 25:55:20.

The name archaeosomes is derived from the domain Archaea

and the principal components are lipids derived from bacterial spe-

cies from this domain.³These lipids contain glycerol of the oppo-

site 2,3-sn-glycerol substitution stereochemistry to the 1,2-ester

substitution in most mammalian lipids. The lipids are further char-

acterized by C₂₀phytanyl side chains with the 3R,7R,11R stereo-

chemistry, which are ether-linked to the glycerol moiety. The

known as caldarchaeol, which are dimerically linked through the

terminal methyl groups of the side chains.⁵In some species the al-

kyl side chains are further derivatized to contain variable numbers

of ﬁve-membered rings. Because cholesterol is prone to oxidation,

it is not a desirable component for mixtures intended to be used in

humans and so we are attempting to ﬁnd lipid compositions that

do not require cholesterol for stability. As mentioned above, one

approach is to use tri- or tetrasaccharide head groups as one of

the components of the archaeosomes. Secondly, we wished to

examine a range of linkages including both anomers at the archae-

ol. Lastly, the high activity of these glycolipids suggests that they

are suitable for commercial use as adjuvants. For these reasons,

we wished to develop highly efﬁcient synthetic methods. As previ-

ously reported, the Glcp-(1?6)-b-Glcp-(1?O)-archaeol glycolipid

was among the most immunologically active. In this report we de-

scribe our efforts in three main areas, namely, (1) a new glycosyl-

ation method that leads to high yields of the requisite glycolipids

from easily available peracetylated alkyl(aryl)thio-glycosides,

* Corresponding author. Tel.: +1 613 993 5265; fax: +1 613 952 9092.

E-mail address: Dennis.Whitﬁeld@nrc-cnrc.gc.ca (D.M. Whitﬁeld).

doi:10.1016/j.carres.2009.10.011

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

215

which could, in turn, be accessed from readily accessible disaccha-

ride, (2) the development of highly -selective glycosylation

conditions en route to the anomer b-Glcp-(1?6)- -Glcp-(1?O)-

archaeol, and (3) the synthesis of the b-Glcp-(1?6)-b-Glcp-

(1?6)-b-Glcp-(1?O)-archaeol trimeric homologue.

ﬂash chromatography using 85:15 chloroform–methanol as the

eluant.

a

The archaeol lipid is isolated from Halobacterium salinarum by

lipid extraction, acid hydrolysis, and silica gel ﬂash chromatogra-

phy initially using 9:1 hexanes–ethyl acetate.¹Early studies indi-

cated that traces of unreacted, non-archaeol lipid were found

after glycosylations. Upon further experimentation, these trace

impurities were largely eliminated by using a slightly acidic chro-

matography solvent system of hexanes–t-butylmethylether–acetic

acid (80:20:0.5, v/v/v), such that conventional NIS–AgOTf

glycosylations could be run until no more archaeol was present.

However, these glycosylation reactions produced more spots on

TLC than what can be accounted for by the products and starting

materials.

2. Results and discussion

2.1. Glycolipid synthesis

Our strategy was to synthesize the requisite glycolipids in the

simplest possible manner such that the syntheses could easily be

scaled to industrial quantities. Preliminary results had shown that

at least a disaccharide was necessary to form stable archaeo-

somes.¹Thus, we obtained the following readily available disac-

charides: lactose, maltose, cellobiose, melibiose, and isomaltose

and acetylated them using the acetic anhydride–sodium acetate

method.⁶This method is inexpensive and in all cases tested, the

desired b-acetates could be puriﬁed by crystallization. Subse-

quently, the anomeric acetates were converted to ethylthio or

phenylthio glycosides under standard BF₃ꢀOEt₂–EtSH or PhSH con-

ditions⁷to yield 1a–5a.⁸After ﬂash chromatography puriﬁcation,

disaccharides 1a–5a were attached to the lipid by glycosylation

under standard N-iodosuccinimide–silver triﬂuoromethanesulfo-

nate (NIS–AgOTf)⁹conditions to yield 1b–5b. In most cases, mod-

erate isolated yields in the 15–50% range were obtained after

simple ﬂash chromatography (Scheme 1). The acetyl groups were

easily removed under Zemplen (NaOMe–MeOH) conditions¹⁰and

the resulting disaccharide glycolipids (1c–5c) were puriﬁed by

When we tried the NIS–AgOTf-promoted glycosylation with the

peracetyl phenylthio glycoside of melibiose [a-Galp-(1?6)-Glcp],

4a, the TLC of the reaction mixture revealed a multitude of spots

and the desired peracetylated glycolipid (4b) could only be isolated

as an impure mixture in an estimated 14% yield (Fig. 1). By running

the reaction at higher temperature, the yield could be slightly im-

proved. None of the extraneous spots could be isolated in pure

form but all contained sugar and only traces of lipid. One lipid-con-

taining side product could be isolated and identiﬁed as the mono-

acetate of archaeol by NMR and MS. Thus, the problem in all cases

is likely the acyl transfer side reaction, for which a mechanism is

postulated based on proton transfer to O-2 from an ion–dipole

complex between the nucleophile and the oxacarbenium ion.¹¹

This side reaction can be minimized by, for example, switching

to benzoyl protection¹²(see below) but at the expense of extra

OAc

AcO

4'

O

OAc

1'

4

lactose (4' ax, 1' eq)

O

AcO

or

SY

AcO

maltose (4' eq, 1' ax)

or

cellobiose (4' eq, 1' eq)

OAc

1a

2a

lactose (Y = Et)

maltose (Y = Ph)

1) Ac₂O

NaOAc

1) Archaeol

NIS/AgOTf or BF₃·TFE₂

3a

cellobiose (Y = Ph)

or

2) BF₃·OEt₂

2) NaOMe

MeOH/CH₂Cl₂

EtSH or PhSH

OAc

AcO 4'

AcO

O

1'

6

melibiose (4' ax, 1' ax)

or

isomaltose (4' eq, 1' ax)

O

Ac

O

AcO

SY

OAc

4a

melibiose (Y = Ph)

5a isomaltose (Y = Ph)

OR

RO

4'

O

OR

O

1'

4

O

R

O

RO

OR

1-3b

1-3c

R = H

R = Ac;

or

OR

RO 4'

RO

O

1'

6

O

RO

O

R

O

OR

4-5b

4-5c

R = H

R = Ac;

Scheme 1. Synthesis of disaccharides attached to archaeol.

216

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

Figure 1. A thin layer chromatogram (TLC) of the NIS–Lewis acid promoted glycosylation reactions of 4a and archaeol developed in hexanes–ethyl acetate–dichloromethane,

6:3:1. Spots were visualized by spraying with 5% sulfuric acid and heating. Identiﬁed spots are indicated with arrows. All reactions were run under identical conditions

*

optimized for NIS–AgOTf. Yield obtained from glycosylation run at room temperature.

synthetic steps. A selection of other glycosylation conditions was

also tested but all led to similar low yields accompanied by exten-

sive acyl transfer. These conditions include, with yields of 4b in

brackets: PhSeNPhth–TfOH (11%), preactivation conditions, TolS-

Cl–AgOTf (21%), and dimethyl disulﬁde–triﬂic anhydride¹³(14%).

Independent to the above considerations, we were intrigued by

the report from the Olah group that a 1:2 BF₃–triﬂuoroethanol

(TFE) complex could act as an efﬁcient Lewis acid.¹⁴In fact, we

wondered if this would be applicable to glycosylation chemistry

since the anion formed after proton transfer from this complex

should be highly delocalized and hence a very weak nucleophile.

Because proton transfer is mechanistically linked to acyl transfer,

it is hypothesized that changing the acidity of the promoter could

improve the glycosylation yields. Boron triﬂuoride diethyletherate

in conjunction with NIS has been reported to catalyze glycosyla-

tions with thioglycosides.¹⁵We postulated that the above complex

may be even better. As a convenient method of preparation, we

simply mixed BF₃ꢀOEt₂and TFE in a 1:2 ratio in dichloromethane

under argon at ꢁ40 °C, followed by a slight concentration under

vacuum (about 5 torr) at ꢁ40 °C for 20 min to obtain an overall

concentration of 0.25 M in boron. As a test case, we examined

the glycosylation at 0 °C between the donor ethyl 2,3,4,6-tetra-O-

ported in Table 1. All relevant chemical reporters show shifts

d) in the mixture consistent with complex formation. This spe-

(D

cies is, however, clearly different from that reported by Olah and

co-workers because diethylether is still present in our solution.

This 0.25 M CH₂Cl₂solution could be stored for months at ꢁ20 °C

without discoloration or loss of activity as assessed using the above

test reaction. Further testing with the same reaction found that the

reaction even proceeded at ꢁ40 °C. As well, the promoter mixture

could be added before the acceptor and the reaction still proceeded

efﬁciently. Toluene and acetonitrile were found to be compatible

as co-solvents. In our hands, this promoter solution is easier to

handle by standard inert atmosphere syringe techniques than

either of the reagents NIS–TfOH or NIS–AgOTf. As well, the TFE

could be replaced by other ﬂuorinated alcohols such as hexaﬂuoro-

isopropanol, HFIP, with comparable or better results (Fig. 1). This

ﬂexibility allows for ﬁne tuning of the acidity of the promoter. Fur-

ther results with these reagents will be reported in due course.

Returning to our glycolipid synthesis, we ﬁrst tested these new

conditions with the cellobiose donor 3a and archaeol, where the

disaccharide glycolipid 3b could be isolated in 65% yield with no

detectable side reactions. As for the problematic melibiose glyco-

sylation to generate 4b, the new promoter system not only more

than doubled the yield (38%) compared to the NIS–AgOTf method

(14%), the product purity was also improved. The side reactions

seem to be similar as judged by TLC but diminished. These results

suggest that the proton transfer to O-2 is minimized with NIS–

BF₃ꢀOEt₂:2TFE as compared with NIS–AgOTf. In the latter case the

intermediacy of glycosyl triﬂates has been postulated but not pro-

ven.¹⁸Last but not least, a scaled-up NIS–BF₃ꢀOEt₂:2TFE-promoted

glycosylation between archaeol (400 mg, 0.612 mmol) and lactose

disaccharide 1a gave an isolated yield of 88%, from which close to

700 mg of glycolipid 1b was obtained.

benzoyl-1-thio-b-

D

-galactopyranoside (6)¹⁶and the acceptor

-mannopyranoside (7)¹⁷using

methyl 2,3,4-tri-O-benzyl-

a

-D

2.5 equiv of NIS and 1.0 equiv of the BF₃ꢀOEt₂:2TFE solution

(Scheme 2). Indeed, the expected b-Galp-(1?6)-Manp disaccharide

(8) was obtained in good yield with signiﬁcantly fewer side

reactions.

To characterize the reagent ¹H, ¹³C, and ¹⁹F NMR spectra were

recorded in CD₂Cl₂solutions prepared as mentioned above in

CH₂Cl₂. The results are compared with the separate reagents mea-

sured in the same solvent at the same concentration and are re-

BzO

OBz

BzO

OBz

O

NIS/AgOTf or BF₃·TFE₂

SEt

BzO

O

or

O

BzO

OBn

O

6

+

HO

BzO

PhSeNPhth/TfOH

BnO

OBn

O

BnO

8

OMe

BnO

OMe

Scheme 2. Synthesis of b-Galp-(1?6)-Manp test disaccharide.

7

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

217

Table 1

¹H (399.89 MHz), ¹³C (100.55 MHz), and ¹⁹F (188.3 MHz) NMR characterization of the BF₃ꢀOEt₂–TFE 1:2 reagent at 0.25 M in CD₂Cl₂

Nucleus^a

d

D

d

D

d

D

d

Dd

BF₃ꢀOEt₂OCH₂(BF₃)

4.15 q

BF₃ꢀOEt₂OCH₂CH₃

1.39 t

TFE CH₂

TFE OH (CF₃)

OCH₂(BF₃)

OCH₂CH₃

CH₂

OH (CF₃)

¹H

3.95 dq

2.38 t

0.167

0.036

0.044

2.92 br s

J = 7.1

J = 9.2

J = 7.2

¹³C

70.2 s

13.7 s

61.6 q

J = 35

123 q

J = 280

0.581

0.166

ꢁ0.124

0.175

0.105

¹⁹F

ꢁ153

ꢁ77.0 t

J = 9.2

ꢁ0.543

Downﬁeld chemical shifts are positive in ppm.

a

¹H referenced to CDHCl₂, ¹³C referenced to CD₂Cl₂, and ¹⁹F referenced externally.

Next we planned to make b-Glcp-(1?6)-

a

-Glcp-(1?O)-archae-

formation. The choice of this combination was based on previous

observations that in situ-generated phenylselenyltriﬂate is a good

promoter system for thioglycosides in some cases.²⁵The use of

ol because it has been reported that the anomericity of a glycosidic

linkage to glycerol-based lipids changes the orientation of the su-

gar head group with respect to the lipid bilayer.¹⁹If binding to a

receptor on Antigen Presenting Cells (APCs) is involved in the ac-

tion of archaeol-derived glycolipids, then this change in orientation

should affect activity. In all the above examples the linkage of b-

Gal or b-Glc to the archaeol was insured by neighboring group par-

ticipation.²⁰Thus, a donor with a non-participating group at O-2

was needed. It is also well known that bulky groups at O-6²¹of

N-phenylselenylphthalimide in a-selective glycosylations has been

reported using Mg(ClO₄)₂as activator and some other closely related

conditions.²⁶Other groups have also explored analogous combina-

tions of N-phenylthio derivatives to promote glycosylations with

thioglycosides.²⁷Indeed, this new combination promotes the test

reaction between 6 and 7 (Scheme 2) in about 70% yield. Application

of these conditions to the reaction between 10 and archaeol resulted

D

-glucopyranosyl donors favor

These considerations led to the use of ethyl 2,3,4-tri-O-benzyl-6-

O-t-butyldiphenylsilyl-1-thio-b- -glucopyranoside as the donor

a

-glycosylation by steric effects.²²

in a highly

a

-selective glycosylation from which the product 11a

/b ratio as estimated

could be isolated in 60% yield (Scheme 3). The

a

D

fromthe¹HNMRspectrumofthecrudereactionmixturewasgreater

than 10:1. Interestingly, application of the NIS–(BF₃:2TFE)

conditions described above led to almost the reverse selectivity with

b-isomer 12b being isolated in 55% yield after glycosylation and

desilylation. This total change in stereospeciﬁcity corroborates the

hypothesis that the counterion of the glycopyranosyl oxacarbenium

ion is an important factor for facial selectivity.²⁸

(10) which in turn was synthesized from commercially available

diacetate 9 by standard chemistry.²³Glycosylation under standard

NIS–TfOH conditions²⁴led to an

a

/b ratio of only 70:30 and the

two anomers (11a and 11b) were close in R_fwith the desired

anomer slightly less polar of the two. As well, a third impurity, ten-

tatively identiﬁed as the hydrolyzed donor, also eluted just before

a

-

the

a

-anomer. In fact, pure compounds could only be obtained in

Subsequently, 2,3,4,6-tetra-O-benzoyl-b-D-glucopyranosyl tri-

low yield after repetitive puriﬁcation by preparative TLC. Prepara-

tively, it was easiest to obtain pure 12a after desilylation rather

than rigorously purifying 11a.

Through experimentation, N-phenylselenylphthalimide–TfOH

was found to be a better glycosylation promoter for the above trans-

chloroacetimidate (13)²⁹was reacted with alcohol 12a to yield

disaccharide 14a using BF₃ꢀOEt₂as the promoter. The protecting

groups were removed by ﬁrst treating under Zemplen conditions

to give 14b and then using hydrogenolysis with Pd(OH)₂on C

(Pearlman’s catalyst) to give 14c.

1)Archaeol

OTBDPS

OAc

1) BF₃·OEt₂/EtSH

2) NaOMe/MeOH

PhSeNPhth/TfOH

O

BnO

2) TBAF/THF

3) TBDPSiCl

imidazole/CH₂Cl₂

BnO

SEt

OAc

9

10

1) BF₃·OEt₂

OBz

OR

O

BnO

O

BzO

TCI

BzO

Bn

O

BzO

13

O

2) NaOMe, MeOH/CH₂Cl₂

3) H₂, Pd(OH)₂/C

12a

12b

R = H α

R = H β

11a

11b

R = TBDPSi α

R = TBDPSi β

O²

R

²RO

O

²RO

¹RO

O

14a ¹

14b ¹

14c ¹

R = Bn, ²R = Bz

R = Bn, ²R = H

R = H, ²R = H

¹RO

O

Scheme 3. Synthesis of b-Glcp-(1?6)-a-Glcp-(1?O)-archaeol disaccharide.

218

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

OH

IRA^-743

resin

OH

PhB

1) Phenylboronic

acid

O

HO

BzO

O

HO

O

SEt

BzO

2) BzCl/Py

OBz

OH

OBz

17

15

16

OAc

1) Archaeol

TolSCl/AgOTf

or

O

1) TolSCl/AgOTf

-78°C then -40°C

AcO

BzO

O

NIS/BF₃

·

TFE₂

Bz

O

AcO

BzO

2) Ac₂O/Py

SEt

2) NaOMe/MeOH

18

OBz

²R

O

²R

O

¹R

O

¹R

²R

O

¹R

O

19a ¹

R = Bz, ²R = Ac

¹R

O

19b ¹R = H, ²R = H

Scheme 4. Synthesis of b-Glcp-(1?6)-Glcp-(1?O)-archaeol disaccharide.

As mentioned above, the gentiobiose-derived b-Glcp-(1?6)-b-

(BF₃:2TFE) conditions led to a yield of greater than 85% and 19a

was isolated highly pure after a simple chromatographic step.

The higher yield in this case compared to that of the peracetates

1a–5a is almost undoubtedly due to the benzoyl group at O-2

which minimizes acyl transfer. Treatment under Zemplen condi-

tions led to the target 19b which in some cases was sufﬁciently

pure to use without further puriﬁcation. This disaccharide is iden-

tical to the one found in Methanobrevibacter smithii where it ac-

counts, in both archaeol and caldarchaeol forms, for about

42 wt % of the total polar lipids (TPL).³

The trisaccharide homologue could then be constructed from

the same compounds by a slight variation in the order of reactions

(Scheme 5). Thus, donor 16 was reacted with archaeol and, after

cleavage of the phenylboronate diol, 20b was puriﬁed in 60% over-

all yield. Without further protecting group manipulations, diol 20b

was reacted with donor 18 to yield protected trisaccharide 21a.

Subsequent treatment under Zemplen conditions led to the target

21b which after puriﬁcation was ready for use. All compounds

were characterized by ¹H and ¹³C NMR spectroscopies and MS

spectrometry. The corresponding data for all lipid-containing com-

pounds are compiled in Tables 3–7 in Section 4. Due to spectral

overlap, particularly of the glycerol and phytanyl methylenes at-

tached to glycerol, gHSQC ¹³C–¹H and gCOSY ¹H–¹H and TOCSY

¹H–¹H 2D correlation experiments were necessary to assign the

resonances. For all the compounds reported in this work, the

Glcp-(1?O)-archaeol was among the best adjuvants.^1aCombined

with our observations that both mannose-derived trimers and tet-

ramers formed stable archaeosomes with high adjuvant activity,²it

seemed highly plausible that the trimer b-Glcp-(1?6)-b-Glcp-

(1?6)-b-Glcp-(1?O)-archaeol might also be a good adjuvant.

Our synthetic strategy of using disaccharide donors suggested that

a b-Glcp-(1?6)-b-Glcp disaccharide donor should also be useful.

Unfortunately, free gentiobiose is currently considerably more

expensive than any of the disaccharides used in Scheme 1. There-

fore, we decided to make use of the well-established synthetic

strategy starting from ethyl 1-thio-b-D-glucopyranoside (15) and

2,3-dibenzoyl-4,6-phenylboranyldiester intermediate (16)³⁰to

prepare diol 17,³¹as illustrated in Scheme 4. The phenylboronate

16 is a solid that is conveniently puriﬁed by precipitation and

therefore only diol 17 requires chromatography. Donor 17 was

subsequently pre-activated at low temperature with in situ-gener-

ated toluylsulfenyltriﬂate and then allowed to react with another

molecule of itself. The crude product was partially puriﬁed, acety-

lated, and then puriﬁed to give disaccharide donor 18 in 37% over-

all yield. It is worth noting that the absolute yield is, in fact, 50%

due to the reaction stoichiometry and therefore this synthetic

route is quite efﬁcient. Donor 18 could be reacted with archaeol

using the standard toluylsulfenyltriﬂate preactivation conditions

to give glycoside 19a in 64% yield. Switching to the new NIS–

²R

O

1) Archaeol

TolSCl/AgOTf

-78°C then -40°C

²R

O

O³

¹R

O

R

16

¹R

R = Bz, ²R = PhB

O

³R =

2) IRA^-743 resin

20a ¹

O

20b ¹R = Bz, ²R = H

O²

R

4

21a ¹

R = Bz, ²R = Ac, R = H

²R

O

¹R

O

18

21b ¹

R, ²R, ⁴R = H

¹R

1) TolSCl/AgOTf

-78°C then -40°C

²R

¹R

O

¹R

O

⁴R

O

2) NaOMe/MeOH

O

O³

R

¹R

O

¹R

O

Scheme 5. Synthesis of b-Glcp-(1?6)-b-Glcp-(1?6)-b-Glcp-(1?O)-archaeol trisaccharide.

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

219

Table 2

Data for OVA-archaeosomes composed of semi-synthetic isoprenoid lipids

Entry

Archaeosome

(mol % composition)

Average diameter

(nm)

OVA loading

g/mg)

(l

1

2

3

4

5

6

7

8

9

10

11

12

13

PGP-Me(22)

92 54

90 52

141 57

76 41

97 46

130 53

Unstable

134 64

176 96

169 86

94 55

98 53

107 39

14.2

21.1

26.7

16.9

14.7

29.6

—

41.0

87.0

92.0

30.0

45.8

39.2

Gentiotriosyl-A(21b)/PGP-Me(22) (45/55)

Gentiobiosyl-A(19b)/PGP-Me(22) (45/55)

Mannotriosyl-A(23a)/PGP-Me(22) (45/55)

Maltotriosyl-A(24)/PGP-Me(22) (45/55)

Lactosyl-A(1c)/PGP-Me(22) (45/55)

Gentiobiosyl-A(19b)/DPPG (35/65)

Gentiobiosyl-A(19b)/DPPG/chol (35/45/20)

Gentiobiosyl-A(19b)/DPPG/chol (25/55/20)

Gentiobiosyl-aA(14c)/DPPG/chol (25/55/20)

Mannosyl₄-A(23b)/DPPG/chol (45/35/20)

Gentiotriosyl-A(21b)/DPPG/chol (35/45/20)

Gentiotriosyl-A(21b)/PGP-Me(22)/chol (35/35/30)

expected connectivities and anomericities could be unambiguously

determined especially in the acylated derivatives. As well, for lipid-

derivative 19b showed the highest activities among the disaccha-

rides. The MHC type I pathway has been implicated in providing

protection against intracellular pathogens and for anti-cancer vac-

cines. In all cases, the observed adjuvant activity was considerably

higher than what can be attributed to enhanced uptake by APCs of

a particulate entrapped antigen. The details of this enhancement in

adjuvant activity are under active investigation in our institute and

preliminary results suggest a role in phagocytosis and intracellular

trafﬁcking, as well as the possibility that the glyco head group may

lead to the up-regulation of genes through cell-signaling events.¹

containing species, the MH⁺or MNH₄molecular ions are diagnos-

þ

tic (Table 7).

2.2. Adjuvant activity

2.2.1. Background

It is well recognized that to produce anti-cancer vaccines, pow-

erful adjuvants must be used. This is because the tumor associated

antigens are often self antigens or closely resemble self antigens

whose antigenicity is strongly down-regulated by the immune sys-

tem to prevent autoimmunity.³²In a previous communication, we

have shown that the disaccharides 1c–5c, 14c, and 19b incorpo-

rated in archaeosomes function as adjuvants in a model system

based on the protein antigen ovalbumin (OVA) by both MHC type

I and MHC type II pathways. Lactose derivative 1c and gentiobiose

Our previous studies also compared a-linked 14c with b-linked

19b. In this regard, the adjuvant activity, especially the CTL activity

associated with the MHC class I pathway, was consistently better

for 19b. These results corroborate the assumption that the

adjuvant activity of these glycolipids is at least in part receptor

mediated. If the adjuvant effects were solely the result of favorable

physico-chemical properties (compare entries 9 and 10 in Table 2)

Figure 2. Adjuvant activity in mice immunized with OVA-archaeosomes composed of PGP-Me and various semi-synthetic glyco isoprenoid archaeols. C57BL/6 mice were

immunized subcutaneously on days 0 and 21 with 0.1 mL PBS containing 15 g OVA, or 15 g OVA entrapped in archaeosomes of various lipid compositions (Table 2).

l

Antigen-speciﬁc T cell frequencies are shown as Elispot assays in two separate animal trials (panels A and B). Antigen non-speciﬁc control responses are shown as ꢁ (no

SIINFEKL peptide), and antigen-speciﬁc responses as + (with SIINFEKL peptide). Anti-OVA antibody titers in the sera of mice (n = 4) are shown in panel C. Signiﬁcant

differences in panels A and B (P <0.05) were found between a and b (P 0.0053), a and c (P 0.0037), a and e (P 0.0032), g and h (P 0.0001), g and i (P 0.0093), c and e (P 0.0204), d

and e (P 0.0135), h and i (P 0.0249), and, a and f (P 0.041). The differences were not signiﬁcant between c and d (P 0.8898). In panel C, signiﬁcant differences (P <0.05) occurred

between b and c (P 0.0415), b and f (P 0.0125), e and f (P 0.0469), and ‘a’ compared to each other immunogens b–f (P values 0.0132, 0.0053, 0.0278, 0.0057, and 0.0041,

respectively).

220

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

of the archaeosomes, such isomeric difference as in 14c and 19b

should have no effect on immune responses. To further test the fac-

tors favorable for adjuvant activity, we performed some similar

studies with trisaccharide 21b. As shown in Figure 2, in the same

OVA-based test system, archaeosomes prepared from 21b are at

least as active as, and generally signiﬁcantly better, than those

from 19b.

both these formulations were signiﬁcantly superior to 24. Scheme 6

shows the structures of 23a,b and 24 which was previously pre-

pared as a control for 23a.²Further shown in Figure 2B, gentiotri-

osylarchaeol 21b produced signiﬁcantly higher T cell frequencies

than either gentiobiosylarchaeol 19b or lactosylarchaeol 1c. This

two component formulation of 21b and 22 almost matches the

activity of the complete mixture of TPL from M. smithii, the positive

control.⁴⁰CTL assays (not shown) conﬁrmed these results with the

exception that in CTL assays there was no signiﬁcant difference be-

tween the gentiotriosylarchaeol and lactosylarchaeol formulations,

which were both notably greater than gentiobiosylarchaeol/PGP-

Me. Class II presentation of the OVA antigen was also measured.

Anti-OVA antibody titers in the sera of mice were similar to the ti-

ters found with OVA-archaeosomes prepared from the TPL of M.

smithii.³³However, titers were signiﬁcantly higher in pure OVA–

PGP-Me archaeosomes than for all other archaeosome composi-

tions shown (Fig. 2C).

To summarize, the semi-synthetic isoprenoid lipids synthesized

in this study can be formulated as stable archaeosomes that entrap

protein antigen, and have varying degrees of adjuvant activity. Sta-

bility of the lipid membrane is an important feature, because the

antigen must remain associated with the lipid carrier to deliver

antigen effectively.³⁴The low activity of maltotriosylarchaeol com-

pared with gentiotriosylarchaeol and mannotriosylarchaeol is in

agreement with our previous conclusion that subtleties in the gly-

co isoprenoid lipid head group alter the MHC class I response to an

entrapped antigen to various degrees.^1aStraight chain mannosylar-

chaeols varying from one to ﬁve mannose residues were synthe-

sized and used to form stable vesicles that activated the MHC

class I immune response optimally with three or four mannose

units.²This ﬁnding is further supported herein, by demonstrating

that enhanced MHC class I adjuvant activity occurs for 21b versus

19b.

In PGP-Me adjuvant formulations with OVA, a trend of inverse

relation between MHC class I and MHC class II responses appears

to be emerging. In pure PGP-Me adjuvants, the antibody response

(Fig. 2C) is high relative to glycolipid/PGP-Me formulations. Fur-

thermore, it appears clearly that glyco isoprenoid lipids enhance

the MHC class I response (Fig. 2A and B). It may prove exciting to

explore this inverse relationship further by expanding these data

to include new isoprenoid phospholipid structures. Recently, gly-

coarchaeal lipids have been implicated in dramatic fusion of TPL

(glycolipid-containing) archaeosomes that occurs under the spe-

ciﬁc conditions found in the acidic phagolysosome. It was pro-

posed that this fusogenic activity may explain antigen delivery

for MHC class I presentation by fusion and exocytosis of en-

trapped antigen from glycolipid archaeosomes found within the

phagolysosome to the cytosol of antigen-presenting cells.³⁵

Hence, the inverse relationship observed between antibody re-

sponses and CD8⁺T cell responses may reﬂect competition for

limited amounts of antigen between these two presentation

pathways.

2.2.2. Archaeosomes from semi-synthetic isoprenoid lipids

The polar isoprenoid lipids synthesized in this study could be

formulated as stable archaeosomes that entrapped water-soluble

antigen OVA (Table 2). Disaccharides in the same formulation

had similar sizes and entrapped similar amounts of OVA (entries

3 and 6). Similarly, trisaccharides exhibited analogous physical

behavior (entries 2, 4, and 5). Charge repulsion in neutral glycolipid

archaeosomes could be provided by dipalmitoyl lipids such as

DPPG provided that cholesterol was included to stabilize the

vesicles. Optimization led to the routine use of the following glyco-

lipid–DPPG–cholesterol ratio of 35%:45%:20%. Further experimen-

tation showed that the anionic archaeal lipid PGP-Me (22,

Scheme 6) mixed with glycolipids formed stable archaeosomes in

the absence of cholesterol. Stable archaeosomes composed entirely

of PGP-Me could be formulated albeit with lesser amounts of OVA

entrapped than for many of the other formulations (Table 2,

entry 1).

2.2.3. Adjuvant activity

Mice immunized subcutaneously at zero and three weeks with

OVA (15

lg/injection) entrapped in archaeosomes developed im-

mune responses much greater than with the non-adjuvanated

antigen (Fig. 2). Antigen-speciﬁc CD8⁺T cell frequencies are shown

for two separate animal trials in panels A and B. As shown in Fig-

ure 2A, the PGP-Me adjuvant produced a signiﬁcantly decreased

activity compared with inclusion into the PGP-Me formulation of

gentiotriosylarchaeol 21b, mannotriosylarchaeol 23a, or maltotrio-

sylarchaeol 24. Inclusion of gentiotriosylarchaeol 21b was insignif-

icantly different in comparison to mannotriosylarchaeol 23a, but

OH

O

P

O^-

HO

O

P

O^-

O

OR

n = 2 or 3

MeO

O

HO

22

PGP-Me

OR

23a

23b

Mannotriosyl-A

Mannosyl₄-A

OH

O

HO

OH

H

O

HO

OH

HO

O

3. Conclusions

HO

OR

24

Maltotriosyl-A

H

O

This work presented two modiﬁed methods for glycosylation

reactions with the biomass-derived lipid archaeol. One method

based on N-phenylselenylphthalimide–triﬂic acid leads to a highly

a

-selective glycosylation. The second method is based on N-iodo-

O

succinimide activation of acylated thiosugar donors by a solution

of boron triﬂuoride etherate and triﬂuoroethanol, which leads to

a clean glycosylation reaction at least in comparison with NIS–

AgOTf-based methods. Further developments of preactivation-

based synthesis of thioglycosides leads to an efﬁcient synthesis

of b-Glcp-(1?6)-b-Glcp-(1?O)-archaeol disaccharide and its

R =

Scheme 6. Structures of anionic archaeal phospholipid PGP-Me (22) and previously

synthesized²glycolipids 23a,b and 24. At physiologic pH, PGP-Me has two negative

charges.

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

221

trimeric homologue b-Glcp- (1?6)-b-Glcp-(1?6)-b-Glcp-(1?O)-

4.3. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhex-

adecyloxy]propan-1-yl 4-O-[2,3,4,6-tetra-O-acetyl-b-

galactopyranosyl]-2,3,4-tri-O-acetyl-b- -glucopyranoside (1b)

archaeol. These methods should be amenable to industrial scale

synthesis of these glycolipids. It is further shown that lipid formu-

lations that contain 100% archaeal-derived lipids can be formu-

lated that activate both type I and type II MHC pathways.

Preliminary evidence for a bias of the anionic lipid PGP-Me toward

type II activation versus a bias for type I activation for glycolipids is

presented. The possible receptors on APCs and the mechanisms of

this activation are currently under investigation.

D-

D

Compound 1b was prepared from 1a using the procedure A-1.

See NMR and MS tables in Section 4.28 for characterization data.

4.4. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexadecyl-

oxy]propan-1-yl 4-O-b-D-galactopyranosyl-b-D-glucopyranoside

(1c)

4. Experimental

4.4.1. Procedure B

Compound 1b (84 mg, 0.066 mmol) was dissolved in dry

CH₃OH (3.9 mL) and CH₂Cl₂(3.9 mL). To this solution was added

1 M methanolic NaOCH₃(0.40 mL) and stirring was continued for

16 h at room temperature. The mixture was cooled in an ice bath

and neutralized with Rexyn 101(H) resin that had been washed

with water and CH₃OH. The solids were removed by ﬁltration with

CH₃OH rinse. The combined ﬁltrates were concentrated and the

residue was puriﬁed by silica gel chromatography eluting with

CHCl₃–CH₃OH 85:15 to yield pure 1c (46 mg, 71%). All yields using

this method were in the 70–95% range. See NMR and MS tables in

Section 4.28 for characterization data.

4.1. General methods

The ¹H NMR spectra were obtained on a Varian-400 (400 MHz)

spectrometer with tetramethylsilane or the residue signal of the

solvent as the internal standard. The ¹³C NMR spectra were

recorded on a Varian-400 (100.55 MHz). Optical rotations were

measured at 20 °C in a 1 dm cell on a PerkinElmer 343 polarimeter.

Thin layer chromatography was performed on pre-coated plates of

Silica Gel (60-F₂₅₄, E. Merck, Darmstadt) and visualized with

H₂SO₄–H₂O (1:20 v/v) followed by heating. Unless otherwise sta-

ted, column chromatography was performed on Silica Gel 60

(230–400 mesh, Merck). All solvents and reagents were puriﬁed

and dried according to standard procedures.

4.5. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexade-

cyloxy]propan-1-yl 4-O-[2,3,4,6-tetra-O-acetyl-

a-D-gluco-

pyranosyl]-2,3,4-tri-O-acetyl-b- -glucopyranoside (2b)

D

4.2. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-

tetramethylhexadecyloxy]propan-1-yl 6-O-[2,3,4,6-tetra-O-

acetyl-a-D-glucopyranosyl]-2,3,4-tri-O-acetyl-b-D-

glucopyranoside (5b)

4.2.1. Procedure A-1

Compound 2b was prepared from 2a using either procedure A-1

or procedure A-2. See NMR and MS tables in Section 4.28 for char-

acterization data.

4.6. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexadecyloxy]-

propan-1-yl 4-O-a-D-glucopyranosyl-b-D-glucopyranoside (2c)

Archaeol (85 mg; 0.13 mmol) and phenyl 6-O-[2,3,4,6-tetra-O-

acetyl- -glucopyranosyl]-2,3,4-tri-O-acetyl-1-thio-b- -glucopy-

a-

D

Compound 2c was prepared from 2b using procedure B. See

ranoside (5a 153 mg, 1.6 equiv) were dissolved in CH₂Cl₂(3.0 mL)

and cooled in an ice bath under an argon atmosphere along with

powdered 4 Å molecular sieves (200 mg). To this mixture was

added NIS (74 mg, 2.5 equiv) followed by AgOTf (33 mg, 1.0 equiv)

and the stirring was continued for 0.5 h. The reaction was

quenched by the addition of diisopropylethylamine (excess) and

the mixture ﬁltered and rinsed with CH₂Cl₂. The organic layers

were concentrated and the residue was puriﬁed by silica gel chro-

matography eluting with hexanes–EtOAc–CH₂Cl₂6:3:1 to yield the

peracetylated disaccharide b-linked to archaeol, 5b, (19 mg, 15%).

All yields using this method were in the 14–50% range. See NMR

and MS tables in Section 4.28 for characterization data.

NMR and MS tables in Section 4.28 for characterization data.

4.7. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhex-

adecyloxy]propan-1-yl 4-O-[2,3,4,6-tetra-O-acetyl-b-

D-

glucopyranosyl]-2,3,4-tri-O-acetyl-b- -glucopyranoside (3b)

D

Compound 3b was prepared from 3a using procedure A-1. See

NMR and MS tables in Section 4.28 for characterization data.

4.8. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexadecyl-

oxy]propan-1-yl 4-O-[2,3,4,6-tetra-O-acetyl-b-D-glucopyran-

osyl-b- -glucopyranoside (3c)

D

4.2.2. Procedure A-2

Compound 3c was prepared from 3b using procedure B. See

NMR and MS tables in Section 4.28 for characterization data.

Archaeol (51 mg; 0.078 mmol) and SPh donor (5a, 79 mg,

1.4 equiv) were dissolved in CH₂Cl₂(1.5 mL) and cooled in an

ice bath under an argon atmosphere. To this solution was added

NIS (44 mg, 2.5 equiv) followed by a 0.25 M solution of BF₃ꢀOEt₂–

4.9. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexade-

cyloxy]propan-1-yl 6-O-[2,3,4,6-tetra-O-acetyl-a-D-galactopyr-

triﬂuoroethanol [156

ﬂuoroethanol (360

tonitrile bath) to which was added BF₃ꢀOEt₂(314

lL, 0.5 equiv; made from a solution of tri-

anosyl]-2,3,4-tri-O-acetyl-b- -glucopyranoside (4b)

D

l

L) in CH₂Cl₂(10.0 mL) at –45 °C (dry ice ace-

lL) followed by

Compound 4b was prepared from 4a using either procedure A-1

or procedure A-2. See NMR and MS tables in Section 4.28 for char-

acterization data.

treatment under vacuum, about 5 torr, for 20 min] Stirring was

continued for 1 h. The reaction mixture was then diluted with

CH₂Cl₂, and the reaction was quenched by the addition of satu-

rated aqueous NaHCO₃followed by 10% aqueous Na₂S₂O₃. After

drying with anhydrous Na₂SO₄, the organic phase was ﬁltered

and concentrated. The residue was puriﬁed by silica gel chroma-

tography eluting with hexanes–EtOAc–CH₂Cl₂6.5:2.5:1 to yield

5b (42 mg, 42%). See NMR and MS tables in Section 4.28 for char-

acterization data.

4.10. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexade-

cyloxy]propan-1-yl 6-O-a-D-galactopyranosyl-b-D-glucopyran-

oside (4c)

Compound 4c was prepared from 4b using procedure B. See

NMR and MS tables in Section 4.28 for characterization data.

222

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

a

4.11. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexadecyl-

1 ), 81.9 (C-2^b), 80.0 (C-2 ), 79.9 (C-5^b), 77.72 (C-4^b), 77.69 (C-

a

oxy]propan-1-yl 6-O-[-

a-

D-glucopyranosyl]-b-

D-glucopyran-

4 ), 75.91 (BnCH₂), 75.86 (BnCH₂), 75.5 (BnCH₂), 75.15 (BnCH₂),

a

oside (5c)

75.1 (BnCH₂), 72.3 (BnCH₂), 71.9 (C-5 ), 63.0 (C-6 ), 62.8 (C-6^b),

34.7, 31.6 (CCH₃t-butyl), 26.8 (CH₃t-butyl), 24.3 (CH₂S^b), 23.2

a

Compound 5c was prepared from 5b using procedure B. See

NMR and MS tables in Section 4.28 for characterization data.

(CH₂S ), 15.1 (CH₂CH₃S^b), 14.6 (CH₂CH₃S ); HRMS calcd for

C₄₅H₅₆N₁O₅Si₁S₁(M+NH₄)⁺: 750.3648. Found: 750.3647.

4.12. Methyl 6-O-[2,3,4,6-tetra-O-benzoyl-b-

D

-

4.14. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-

galactopyranosyl]-2,3,4-tri-O-benzyl- -mannopyranoside (8)

a

-

D

tetramethylhexadecyloxy]propan-1-yl 2,3,4-tri-O-benzyl-6-O-(t

-butyldiphenylsilyl)-

bis[(3R,7R,11R)-3,7,11,15-tetramethylhexadecyloxy]propan-1-

yl 2,3,4-tri-O-benzyl- -glucopyranoside (12a)

a-D-glucopyranoside (11a) and (2R)-2,3-

Donor (6, 100 mg, 0.15 mmol, 1.5 equiv), acceptor (7, 48 mg,

0.10 mmol), and powdered 4 Å molecular sieves (ꢂ200 mg) were

dispersed in CH₂Cl₂(2 mL) under an atmosphere of argon and

cooled in an ice bath. Then NIS (56 mg, 2.5 equiv) was added fol-

a-D

Archaeol (140 mg; 0.21 mmol) and thioglycoside donor 10

(236 mg, 1.5 equiv) were dissolved in CH₂Cl₂(2.6 mL) and cooled

in an ice bath under an argon atmosphere along with powdered

4 Å molecular sieves (300 mg). To this mixture was added N-sele-

nophenylphthalimide (116 mg, 1.8 equiv) followed by triﬂuoro-

lowed by addition of 0.25 M BF₃ꢀOEt₂ꢀTFE₂(206

lL, 0.5 equiv; as

prepared in procedure A-2) and stirring was continued for

10 min. The reaction was quenched by the addition of aqueous

NaHCO₃, the reaction mixture was diluted with CH₂Cl₂, and

washed with 10% aqueous Na₂S₂O₃until the red color disappeared.

The organic phase was separated using a separatory funnel, dried

over Na₂SO₄, ﬁltered, and concentrated. The residue was puriﬁed

by ﬂash chromatography eluting with hexanes–EtOAc 1:1 to yield

methanesulfonic acid (32 lL, 1.7 equiv). Stirring was continued

for 3 h and the reaction was quenched by the addition of diisopro-

pylethylamine (excess). The mixture was ﬁltered and rinsed with

CH₂Cl₂. The organic layers were concentrated and the residue

was puriﬁed by silica gel chromatography eluting with CH₂Cl₂–

cyclohexane–t-butylmethylether 49:49:2 to yield a mixture of

11a and 11b. This mixture was dissolved in anhydrous tetrahydro-

furan (4 mL) and a 1 M solution of tetrabutylammonium ﬂuoride in

8 as a white foam (85 mg, 83%): [a]

_D67.0 (c, 0.063, CHCl₃), ¹H NMR

CDCl₃8.07 br d (2H, J_H,H= 7.8 Hz, Bz_o), 7.99 br d (2H, J_H,H= 8.1 Hz,

Bz_o), 7.87 br d (2H, J_H,H= 8.1 Hz, Bz_o), 7.75 br d (2H, J_H,H= 8.2 Hz,

Bz_o), 7.81 br d (2H, J_H,H= 7.2 Hz, Bz_o), 7.59 m (1H, Bz_p), 7.52–

7.16 m (26H, ArH), 5.96 br d (1H, J_4,5<1 Hz, H-4^II), 5.82 dd (1H,

J_2,3= 10.7 Hz, H-2^II), 5.58 dd (1H, J_3,4= 3.2 Hz, H-3^II), 4.90 d (1H,

J_1,2= 7.8 Hz, H-1^II), 4.74 d (1H, J_H,H= 11.2 Hz, BnCHH), 4.67 dd

THF (400 lL) was added. The resulting mixture was heated at 50 °C

for 16 h under an atmosphere of argon. The reaction mixture was

then concentrated and the residue was puriﬁed by silica gel chro-

matography eluting with CH₂Cl₂–cyclohexane–t-butylmethylether

48:48:4 to yield 12a (108 mg; 48%). A small amount of 11a was

repuriﬁed by preparative TLC with CH₂Cl₂–cyclohexane–t-butyl-

methylether 49:49:2 (developed 2ꢃ) for an analytical sample:

(1H, J_6,6= 11.0 Hz, J_5,6= 6.4 Hz, H-6^II), 4.63 s (2H, BnCH₂), 4.50 s

0

(2H, BnCH₂), 4.47 d (1H, J_1,2<1 Hz, H-1^I), 4.44 d (1H, J_H,H= 11.2 Hz,

II

BnCHH), 4.38 dd (1H, J_5,6= 7.3 Hz, J_6,6= 11.0 Hz, H-6⁰), 4.28 m

0

(2H, H-5^II, H-6^I), 3.76 m (3H, H-3^I, H-5^I, H6⁰), 3.66 m (2H, H-4^I,

I

H-2^I), 2.96 s (3H, OCH₃); ¹³C NMR CDCl₃166.1 (C@O Bz), 165.6

(2C, C@O Bz), 165.2 (C@O Bz), 138.40 (Bn_ip), 138.36 (Bn_ip), 138.3

(Bn_ip), 133.5 (Bz_p), 133.2 (2C, Bz_p), 133.0 (Bz_p), 130.0 and 129.8

(4C, Bz_m), 129.6–127.5 (ArC), 102.2 (C-1^II), 98.6 (C-1^I), 80.2 (C-3^I),

75.0 (C-4^I), 74.9 (BnCH₂), 74.3 (C-2^I), 72.6 (BnCH₂), 72.0 (BnCH₂),

71.7 (C-3^II), 71.3 (C-5^I), 71.2 (C-5^II), 69.9 (C-6^I), 69.8 (C-2^II), 68.1

(C-4^II), 61.9 (C-6^II), 54.3 (OCH₃); HRMS calcd for C₆₂H₆₂N₁O₁₅

(M+NH₄)⁺1060.4119. Found: 1060.4214.

[a]

24.8 (c, 0.067, CHCl₃), ¹H NMR CDCl₃7.69 br d (2H, Ph_o),

D

7.62 br d (2H, Ph_o), 7.41–7.24 m (16H, ArH), 7.16 m (4H, Ph_m),

4.97–4.62 m (6H, BnCH₂), 4.88 d (1H, J_1,2= 4.8 Hz, H-1), 4.00 br t

(1H, H-3), 3.88 m (2H, H-6, H-6⁰), 3.76 m (1H, H-5), 3.67–3.49 m

(9H, H-2, H-4, CH₂O, CH₂O, CHO, OCH₂), 3.43 br t (2H, OCH₂)

1.62–1.48 m (6H, CH, CH₂), 1.38–1.22 m (42H, CH, CH₂), 0.88–

0.78 m (30H, CH₃); HRMS calcd for C₈₆H₁₃₈N₁O₈Si₁(M+NH₄)⁺:

1341.0190. Found: 1341.0305. Compound 12a:

[

a

]

10.3 (c,

D

0.132, CHCl₃), ¹H NMR CDCl₃7.40–7.24 m (15H, ArH), 4.97 d (1H,

J_H,H= 11.8 Hz, BnCH₂), 4.89 d (1H, J_H,H= 11.1 Hz, BnCH₂), 4.81 d

(1H, J_H,H= 11.8 Hz, BnCH₂), 4.80 d (1H, J_1,2= 3.8 Hz, H-1), 4.71 q

(2H, BnCH₂), 4.65 d (1H, J_H,H= 11.1 Hz, BnCH₂), 4.00 br t (1H,

4.13. Ethyl 2,3,4-tri-O-benzyl-6-O-(t-butyldiphenylsilyl)-1-thio-

D-glucopyranoside (10)

Ethyl 2,3,4-tri-O-benzyl-1-thio-

D

-glucopyranoside³⁶(1.13 g,

J_3,4= 9.1 Hz, H-3), 3.81 dd (1H, J_5,6= 2.3 Hz, J_6,6= 12.3 Hz, H-6),

0

2.28 mmol) was dissolved in CH₂Cl₂(15 mL) and cooled in an ice

bath under an argon atmosphere. To this solution was added imid-

azole (689 mg, 4.5 equiv) followed by t-butyldiphenylchlorosilane

(0.894 mL, 1.5 equiv) and the mixture was allowed to stir and

warm to room temperature over 16 h. The reaction mixture was

concentrated and the residue was puriﬁed by silica gel chromatog-

raphy eluting with CH₂Cl₂followed by 1% t-butylmethylether in

3.76 m (1H, H-5), 3.69 m (1H, H-6⁰), 3.67–3.45 m (9H, CH₂O,

CH₂O, CHO, OCH₂, H-4, H-2), 1.62–1.48 m (6H, CH, CH₂), 1.38–

1.22 m (42H, CH, CH₂), 0.88–0.78 m (30H, CH₃); ¹³C NMR CDCl₃

138.8 (Bn_ip), 138.34 (Bn_ip), 138.31 (Bn_ip), 128.4–127.6 (ArC), 97.2

(C-1), 81.9 (C-3), 80.2 (C-2), 77.8 (CHO), 77.2 (C-4), 76.7 (BnCH₂),

75.6 (BnCH₂), 72.9 (BnCH₂), 70.8 (C-5,JCH₂), 70.6 (JCH₂), 69.0

(CH₂O), 67.9 (CH₂O), 61.9 (C-6), 39.4 (CH₂), 37.6 (CH₂), 37.52

(CH₂), 37.47 (CH₂), 37.44 (CH₂), 37.29 (CH₂), 37.2 (CH₂), 36.7

(CH₂), 32.8 (CH), 30.0 (CH), 29.8 (CH), 28.0 (CH), 24.8 (CH₂), 24.5

(CH₂), 24.4 (CH₂), 19.75 (CH₃), 19.68 (CH₃); HRMS calcd for

C₇₀H₁₂₀N₁O₈(M+NH₄)⁺: 1102.9013. Found: 1102.9153.

CH₂Cl₂to yield 10 as a

a

/b (1:0.46) mixture: ¹H NMR CDCl₃(num-

/b mixture) 7.76–7.65 m (ArH),

ber of protons not assigned due to

a

7.41–7.24 m (ArH), 7.15 m (ArH), 5.45 d (J_1,2= 5.3 Hz, H-1 ), 4.94–

4.58 m (BnCH₂), 4.48 d (J_1,2= 9.7 Hz, H-1^b), 4.13 m (H-5 ), 3.92–

a

b

3.80 m (H-2 , H-3 , H-6 , H-6⁰a,H-6^b, H-6⁰), 3.75 t (J_4,5= 9.1 Hz,

H-4^b), 3.67 t (J_3,4= 8.7 Hz, H-3^b), 3.57 t (J_3,4= 9.8 Hz, J_4,5= 9.9 Hz,

4.15. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexade-

cyloxy]propan-1-yl 2,3,4-tri-O-benzyl-6-O-(t-butyldiphenylsi-

H-4 ), 3.47 dd (J_2,3= 9.4 Hz, H-2^b), 3.37 m (H-5^b), 2.74 m (CH₂S ),

a

2.52 m (CH₂S^b), 1.32 t (J = 7.6 Hz, CH₃CH₂S ), 1.25 t (J = 7.6 Hz,

lyl)-

3,7,11,15-tetramethylhexadecyloxy]propan-1-yl 2,3,4-tri-O-

benzyl- -glucopyranoside (12b)

a-D-glucopyranoside (11b) and (2R)-2,3-bis[(3R,7R,11R)-

a

CH₃CH₂S^b), 1.04 s (CH₃t-butyl), 1.02 s (CH₃t-butyl); ¹³C NMR CDCl₃

138.7 (Bn_ip), 138.3 (Bn_ip), 138.0 (Bn_ip), 138.4 (Bn_ip^b), 138.14

(Bn_ip^b), 138.10 (Bn_ip^b), 135.9 (Ph_p), 135.8 (Ph_p), 135.6 (Ph_p), 133.6

a-D

a

(Ph_ip), 133.2 (Ph_ip), 134.8 (Ph_ip^b), 133.1 (Ph_ip^b), 129.5 m (ArC),

Archaeol (50 mg; 0.076 mmol) and thioglycoside donor 10

(84 mg, 1.5 equiv) were dissolved in CH₂Cl₂(1.5 mL) and cooled

a

128.5–127.5 (ArC), 86.7 (C-3^b), 84.4 (C-1^b), 82.7 (C-3 ), 82.2 (C-

a

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

223

to ꢁ60 °C under an argon atmosphere. To this solution was added

ular sieves (100 mg) and the mixture was cooled in an ice bath un-

L,

.

NIS (43 mg, 2.5 equiv) followed by 0.25 M BF₃ꢀOEt₂TFE₂(153

lL,

der an atmosphere of argon. To this mixture BF₃ꢀOEt₂(8

l

0.5 equiv; as prepared in procedure A-2) and the stirring was con-

tinued for 16 h. The reaction mixture was diluted with CH₂Cl₂,

washed with 10% aqueous Na₂S₂O₃followed by saturated aqueous

NaHCO₃in a separatory funnel. The combined aqueous phase was

re-extracted with CH₂Cl₂. The combined organic phase was then

dried with Na₂SO₄, ﬁltered, and concentrated. The residue was

puriﬁed by silica gel chromatography eluting with CH₂Cl₂–cyclo-

hexane–t-butyl methyl ether 49:49:2 to yield a mixture of 11a

and 11b. This mixture was dissolved in anhydrous tetrahydrofuran

(2 mL) and a 1 M solution of tetrabutylammonium ﬂuoride in THF

1 equiv) was added and the stirring was continued for 2 h. The

reaction was quenched by the addition of excess diisopropylethyl-

amine, ﬁltered, and concentrated. The residue was puriﬁed by sil-

ica gel chromatography eluting with 2.5% acetone in toluene to

yield 14a (63 mg; 85%). [a]

16.1 (c, 0.219, CHCl₃), ¹H NMR CDCl₃

D

7.92 br d (2H, J_H,H= 7.2 Hz, Bz_o), 7.89 m (4H, Bz_o), 7.81 br d (2H,

J_H,H= 7.2 Hz, Bz_o), 7.53–7.16 m (25H, ArH), 7.00 m (2H, ArH), 5.87

br t (1H, J_3,4= 9.7 Hz, H-3^II), 5.66 br t (1H, J_4,5= 10.2 Hz, H-4^II),

5.60 br t (1H, J_2,3= 9.7 Hz, H-2^II), 4.86 d (1H, J_H,H= 10.8 Hz, BnCH₂),

4.78 d (1H, J_1,2= 3.5 Hz, H-1^I), 4.76 d (1H, J_1,2= 7.9 Hz, H-1^II),

4.63 m (4H, BnCH₂, H-6^II), 4.51 dd (1H, J_5,6= 5.0 Hz, J_6,6= 12.0 Hz,

0

(200 lL) was added. The resulting mixture was heated at 50 °C for

II

16 h under an atmosphere of argon. The reaction mixture was then

concentrated and the residue was puriﬁed by silica gel chromatog-

raphy eluting with CH₂Cl₂–cyclohexane–t-butylmethylether

48:48:4 to yield 12b (45.8 mg; 55%). A small amount of 11b was

repuriﬁed by preparative TLC with CH₂Cl₂–cyclohexane–t-butyl-

methylether 49:49:2 (developed 2ꢃ) for an analytical sample:

H-6⁰), 4.42 d (1H, J_H,H= 11.3 Hz, BnCH₂), 4.25 d (1H, J_H,H= 11.3 Hz,

BnCH₂), 4.17 br d (1H, H-6^I), 4.06 m (1H, H-5^II), 3.88 br t (1H,

I

J_2,3= 9.4 Hz, J_3,4= 9.0 Hz, H-3^I), 3.79 m (2H, H-5^I, H6⁰), 3.57 m

(5H, CH₂O, CHO, OCH₂), 3.44 m (6H, CH₂O, OCH₂, H-4^I, H-2^I),

1.61–1.48 m (6H, CH, CH₂), 1.38–1.01 m (42H, CH, CH₂), 0.88–

0.81 m (30, CH₃); ¹³C NMR CDCl₃166.1 (C@O Bz), 165.8 (C@O

Bz), 165.1 (C@O Bz), 164.9 (C@O Bz), 138.9 (Bn_ip), 138.5 (Bn_ip),

138.4 (Bn_ip), 133.4 (Bz_p), 133.2 (Bz_p), 133.1 (Bz_p), 133.0 (Bz_p),

129.8–127.2 (ArC), 101.3 (C-1^II), 97.1 (C-1^I), 81.7 (C-3^I), 79.9 (C-

2^I), 77.8 (CHO), 77.2 (C-4^I), 75.3 (BnCH₂), 74.4 (BnCH₂), 72.9 (C-

3^II), 72.7 (BnCH₂), 72.2 (C-5^II), 71.8 (C-2^II), 70.7 (CH₂O), 70.0

(JCH₂), 69.8 (C-4^II), 69.3 (C-5^I), 69.0 (JCH₂), 68.1 (C-6^I), 67.7

(CH₂O), 63.3 (C-6^II), 39.4 (CH₂), 37.6 (CH₂), 37.45 (CH₂), 37.42

(CH₂), 37.3 (CH₂), 37.2 (CH₂), 36.7 (CH₂), 32.8 (CH), 30.0 (CH),

29.8 (CH), 28.0 (CH), 24.8 (CH₂), 24.5 (CH₂), 24.4 (CH₂), 22.7

(CH₃), 22.6 (CH₃), 19.73 (CH₃), 19.71 (CH₃), 19.67 (CH₃); HRMS

calcd for C₁₀₄H₁₄₆N₁O₁₇(M+NH₄)⁺: 1681.0590. Found: 1681.0718.

[a]

_D1.4 (c, 0.051, CHCl₃), ¹H NMR CDCl₃7.75 br d (2H, J_H,H= 6.3 Hz,

Ph_o), 7.69 br d (2H, J_H,H= 6.6 Hz, Ph_o), 7.44–7.24 m (16H, ArH), 7.17

m (2H, Ph), 5.02 d (1H, J_H,H= 10.9 Hz, BnCHH), 4.92 d (1H,

J_H,H= 10.7 Hz, BnCHH), 4.89 d (1H, J_H,H= 11.1 Hz, BnCHH), 4.80 d

(1H, J_H,H= 10.7 Hz, BnCHH), 4.74 d (1H, J_H,H= 10.7 Hz, BnCHH),

4.68 d (1H, J_H,H= 11.1 Hz, BnCHH), 4.46 d (1H, J_1,2= 7.8 Hz, H-1),

4.03 dd (1H, J_H,H= 11.0 Hz, J_H,H= 3.8 Hz, OCHH), 3.93 br s (2H, H-

6, H-6⁰), 3.75 br t (1H, J_3,4= 9.3 Hz, H-3), 3.71–3.63 m (5H, H-2,

OCHH, CHO, CH₂O), 3.57–3.42 m (5H, H-3, CH₂O, CH₂O, OCH₂),

3.43 br d (1H, H-5) 1.58–1.51 m (6H, CH, CH₂), 1.38–1.22 m

(42H, CH, CH₂), 0.87–0.81 m (30H, CH₃); ¹³C NMR CDCl₃138.7

(Bn_ip), 138.6 (Bn_ip), 138.3 (Bn_ip), 135.9 (Ph_o), 135.6 (Ph_o), 133.7

(Ph_ip), 133.1 (Ph_ip), 129.6–127.5 (ArC), 103.8 (C-1), 84.8 (C-3),

82.5 (C-4), 78.0 (CHO), 77.6 (C-2), 75.9 (C-5), 75.7 (BnCH₂), 75.1

(BnCH₂), 74.7 (BnCH₂), 71.1 (JCH₂), 70.1 (JCH₂), 69.0 (CH₂O),

69.6 (CH₂O), 62.7 (C-6), 39.4 (CH₂), 37.6 (CH₂), 37.5 (CH₂), 37.4

(CH₂), 37.3 (CH₂), 30.0 (CH), 29.9 (CH), 29.7 (CH), 28.0 (CH), 24.8

(CH₂), 24.5 (CH₂), 24.4 (CH₂), 22.7 (CH₂), 22.6 (CH₂), 19.75 (CH₃),

19.67 (CH₃), 19.3 (CH₃), 32.8 (CCH₃t-butyl), 26.8 (CH₃t-butyl);

HRMS calcd for C₈₆H₁₃₈N₁O₈Si₁(M+NH₄)⁺: 1341.0190. Found:

4.17. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexadecyl-

oxy]propan-1-yl 6-O-b-D-glucopyranosyl-a-D-glucopyranoside

(14c)

Disaccharide (14a, 17 mg; 0.010 mmol) was ﬁrst treated using

procedure B to yield (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetrameth-

ylhexadecyloxy]propan-1-yl 6-O-b-

D-glucopyranosyl-2,3,4-tri-O-

benzyl-

a

-D

-glucopyranoside (14b) [partial ¹H NMR CDCl₃4.70 d

1341.0299. Compound 12b: [

a

]

D

1.11 (c, 0.006, CH₂Cl₂), ¹H NMR

(1H, J_1,2= 3.5 Hz, H-1^I) and 4.28 d (1H, J_1,2= 7.6 Hz, H-1^II)], which

was then dissolved in EtOAc (10 mL) and after purging with argon,

Pd(OH)₂–C (Pearlman’s catalyst) (150 mg) was added and the mix-

ture was hydrogenated using a Parr apparatus at 50 psi of H₂with

shaking for 64 h. The catalyst was removed by ﬁltration through a

bed of Celite and was washed well with EtOAc and CH₃OH. The

combined ﬁltrates were evaporated and then puriﬁed by silica

gel chromatography eluting with CHCl₃–CH₃OH l 85:15 to yield

14c (8 mg; 82%). See NMR and MS tables in Section 4.28 for char-

acterization data.

CDCl₃7.40–7.24 m (15H, ArH), 4.97 d (1H, J_H,H= 11.6 Hz, BnCH₂),

4.93 d (1H, J_H,H= 11.2 Hz, BnCH₂), 4.86 d (1H, J_H,H= 10.8 Hz,

BnCH₂), 4.79

d

(1H, J_H,H= 11.6 Hz, BnCH₂), 4.71

d

(1H,

J_H,H= 11.2 Hz, BnCH₂), 4.63 d (1H, J_H,H= 10.8 Hz, BnCH₂), 4.49 d

(1H, J_1,2= 7.6 Hz, H-1), 3.97 dd (1H, J_H,H= 10.2 Hz, J_H,H= 3.6 Hz,

0

OCHH), 3.85 dd (1H, J_5,6= 2.0 Hz, J_6,6= 12.0 Hz, H-6), 3.72–3.40 m

(12H, CH₂O, CH₂O, CHO, OCH₂, OCHH, H-6⁰, H-3, H-4, H-2), 3.75

br m (1H, H-5), 1.62–1.48 m (6H, CH, CH₂), 1.38–1.22 m (42H,

CH, CH₂), 0.88–0.78 m (30H, CH₃); ¹³C NMR CDCl₃138.5 (Bn_ip),

138.4 (Bn_ip), 138.0 (Bn_ip), 128.5–127.6 (ArC), 104.0 (C-1), 84.5 (C-

3), 82.2 (C-2), 78.1 (CHO), 77.6 (C-4), 75.7 (BnCH₂), 75.0 (BnCH₂),

75.0 (C-5), 74.7 (BnCH₂), 70.3 (JCH₂), 70.1 (2C, CH₂O), 68.9

(CH₂O), 62.1 (C-6), 39.4 (CH₂), 37.54 (CH₂), 37.50 (CH₂), 37.46

(CH₂), 37.41 (CH₂), 37.1 (CH₂), 36.6 (CH₂), 32.8 (CH), 29.9 (CH),

29.8 (CH), 29.7 (CH), 28.0 (CH), 24.8 (CH₂), 24.5 (CH₂), 24.37

(CH₂), 24.35 (CH₂), 22.7 (CH₃), 22.6 (CH₃), 19.74 (CH₃), 19.69

(CH₃), 19.66 (CH₃); HRMS calcd for C₇₀H₁₁₇O₈(M+H)⁺:

1085.8748. Found: 1085.8788.

4.18. Ethyl 6-O-(4,6-di-O-acetyl-2,3-di-O-benzoyl-b-D-

glucopyranosyl)-4-O-acetyl-2,3-di-O-benzoyl-1-thio-b-D-

glucopyranoside (18)

To ethyl 2,3-di-O-benzoyl-1-thio-b-D-glucopyranoside (17,

3.0 g; 6.93 mmol) [prepared by deboronylation with IRA743 resin

of 16: ¹H NMR CDCl₃7.93 d (4H, J_H,H= 6.8 Hz, Bz_o) 7.49 m (2H,

Bz_p), 7.35 m (4H, Bz_m) 5.42 m (2H, H-2, H-3), 4.73

d (1H,

0

J_1,2= 9.8 Hz, H-1), 4.00 dd (1H, J_5,6= 3.4 Hz, J₆₆= 12.1 Hz, H-6),

0

4.16. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhex-

3.96 br t (1H, J_3,4, J_4,5= 9.4 Hz, H-4), 3.87 dd (1H, J_5,6= 4.9 Hz, H-

adecyloxy]propan-1-yl 6-O-[2,3,4,6-tetra-O-benzoyl-b-

D

-

6⁰), 3.60 ddd (1H, H-5), 3.17 br s (2H, OH), 2.73 m (2H, SCH₂),

1.25 t (3H, J_HH= 7.2 Hz, SCH₂CH₃)] and 4 Å molecular sieves (3 g)

was added CH₂Cl₂(30 mL) and the mixture was cooled in a dry

ice acetone bath (about ꢁ78 °C) under an atmosphere of argon.

glucopyranosyl]-2,3,4-tri-O-benzyl- -glucopyranoside (14a)

a-D

Alcohol (12a, 64 mg; 0.059 mmol) and 2,3,4,6-tetra-O-benzoyl-

-glucopyranosyl trichloroacetimidate (13, 66 mg; 1.5 equiv)

were dissolved in CH₂Cl₂(2.5 mL) along with powdered 4 Å molec-

a-

D

To this mixture were added p-toluenesulfenylchloride (667 lL;

4.6 mmol)) and silver triﬂuoromethanesulfonate (1.188 g;

224

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

4.6 mmol) and the mixture was stirred at this temperature for 1 h.

The mixture was then transferred to a dry ice acetonitrile bath

(about ꢁ45 °C) and the mixture was stirred for 1.75 h. The reaction

was quenched by the addition of diisopropylethylamine (1 mL),

and the reaction mixture was ﬁltered, followed by rinsing with

CH₂Cl₂and CH₂Cl₂–EtOAc 50:50. After concentration of the com-

bined ﬁltrates, the residue was puriﬁed by chromatography on sil-

ica gel eluting with hexanes–EtOAc–CH₂Cl₂6:3:1 to yield a viscous

oil (1.55 g). The product has a lower R_famong the two most preva-

lent products. This oil was dissolved in pyridine (15 mL), cooled in

an ice bath under an atmosphere of argon, and acetic anhydride

(7.5 mL) was added. The mixture was left to stir and warm to room

temperature overnight. After evaporation, the residue was puriﬁed

by medium pressure liquid chromatography on silica gel eluting

with hexanes–EtOAc–CH₂Cl₂6:3:1 to yield an amorphous white

4.21. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexade-

cyloxy]propan-1-yl 2,3-di-O-benzoyl-b- -glucopyranoside (20b)

D

Ethyl 2,3-di-O-benzoyl-4,6-phenylboranyl-b-D-1-thio-glucopy-

ranoside²⁸(16, 275 mg; 0.53 mmol - 2 equiv with respect to ar-

chaeol) was activated and reacted with archaeol as described

above for 18 except no molecular sieves were added. The crude

product was treated by shaking overnight in acetonitrile (about

25 mL) with IRA-743 resin (about 3 g) which had been soaked

and rinsed extensively with acetonitrile. The resin was removed

by ﬁltration, rinsed with CH₂Cl₂and acetonitrile, and the combined

ﬁltrates were evaporated to dryness. The residue was puriﬁed by

silica gel chromatography eluting with hexanes–EtOAc–CH₂Cl₂

7:2:1 to yield pure 20b as a viscous oil (151 mg, 56%). See NMR

and MS tables in Section 4.28 for characterization data.

solid (1.2 g; 37%). [

a]

68.3 (c, 0.030, CHCl₃), ¹H NMR CDCl₃

D

7.93 m (8H, Bz_o), 7.49 m (4H, Bz_p), 7.35 m (8H, Bz_m), 5.65 t (1H,

J_3,4= 9.4 Hz, H-3^I), 5.59 t (1H, J_3,4= 9.5 Hz, H-3^II), 5.44 dd (1H,

J_2,3= 10.0 Hz, H-2^II), 5.37 t (1H, J_4,5= 9.7 Hz, H-4^II), 5.33 dd (1H,

J_2,3= 10.0 Hz, H-2^I), 5.08 t (1H, J_4,5= 9.7 Hz, H-4^I), 4.89 d (1H,

4.22. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexadecyl-

oxy]propan-1-yl 6-O-(4,6-di-O-acetyl-2,3-di-O-benzoyl-b-

copyranosyl)-6-O-(4-O-acetyl-2,3-di-O-benzoyl-b- -glucopyran-

osyl)-2,3-di-O-benzoyl-b- -glucopyranoside (21a)

D-glu-

D

J_1,2= 7.9 Hz, H-1^II), 4.57 d (1H, J_1,2= 10.0 Hz, H-1^I), 4.37 dd (1H,

II

J_5,6= 5.0 Hz, J₆₆= 12.3 Hz, H-6^II), 4.21 dd (1H, J_5,6= 2.1 Hz, H-6⁰),

Donor (18, 92 mg, 0.099 mmol) was activated as mentioned

above in procedure for 19a and reacted with diol acceptor (20b,

67 mg; 0.066 mmol) for 2 h in a dry ice acetonitrile bath (about

ꢁ45 °C). The residue was puriﬁed by silica gel chromatography

eluting with hexanes–EtOAc–CH₂Cl₂7:2:1 to yield pure 21a as a

waxy solid (90 mg; 72%). See NMR and MS tables in Section 4.28

for characterization data.

0

3.95 br d (1H, H-6^I), 3.88 ddd (1H, H-5^II), 3.82 m (1H, H-5^I), 3.75

I

dd (1H, J_5,6= 7.3 Hz, J_6,6= 11.1 Hz, H-6⁰), 2.51 m (2H, SCH₂), 2.14

s (3H, AcCH₃), 1.94 s (3H, AcCH₃), 1.92 s (3H, AcCH₃), 1.05 t (3H,

J_HH= 7.2 Hz, SCH₂CH₃); ¹³C NMR CDCl₃170.7 (C@O Ac), 169.7

(C@O Ac), 169.4 (C@O Ac), 165.8 (C@O Bz), 165.6 (C@O Bz),

165.1 (2C, C@O Bz), 133.4 (Bz_p), 133.35 (Bz_p), 133.28 (Bz_p), 133.2

(Bz_p), 129.8 (Bz_m), 129.2 (Bz_ip), 129.1 (Bz_ip), 128.80 (Bz_ip), 128.76

(Bz_ip), 128.4 (Bz_o), 101.0 (C-1^II), 83.3 (C-1^I), 77.7 (C-5^I), 74.1 (C-

3^I), 73.0 (C-3^II), 72.2 (C-5^II), 71.6 (C-2^II), 70.4 (C-4^I), 69.1 (C-4^II),

68.41 (C-6^I), 68.36 (C-4^I), 61.9 (C-6^II), 24.0 (SCH₂), 20.8 (CH₃Ac),

20.54 (CH₃Ac), 20.52 (CH₃Ac), 14.7 (SCH₂CH₃); HRMS calcd for

C₄₈H₅₂N₁O₁₇S₁(M+NH₄)⁺946.2955. Found: 946.3036.

0

4.23. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhex-

adecyloxy]propan-1-yl 6-O-(b-D-glucopyranosyl)-6O-(b-D-

glucopyranosyl)-b- -glucopyranoside (21b)

D

The acyl groups were removed from 21a (80 mg; 0.042 mmol)

using procedure B. TLC analysis of the product indicated an uniden-

tiﬁed impurity and so the product was puriﬁed by preparative TLC

eluting with CHCl₃–MeOH–CH₃COOH–H₂O 85:22.5:10:4 to yield

pure 21b as a waxy solid (40 mg; 83%). See NMR and MS tables

in Section 4.28 for characterization data.

4.19. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexadecyl-

oxy]propan-1-yl 6-O-(4,6-di-O-acetyl-2,3-di-O-benzoyl-b-D-

glucopyranosyl)-4-O-acetyl-2,3-di-O-benzoyl-b-D-glucopyran-

oside (19a)

To donor (18, 236 mg, 0.255 mmol) and 4 Å molecular sieves was

added CH₂Cl₂(2 mL) and the mixture was cooled in a dry ice CH₃OH

bath (about ꢁ60 °C) under an atmosphere of argon. To this mixture

4.24. PGP-Me (archaetidylglycerol methylphosphate)

PGP-Me (Scheme 6) was puriﬁed by silica gel ﬂash chromatog-

raphy from Haloferax volcanii lipids. This source for PGP-Me had

the advantages of high abundance, 44% of the polar lipids, and gave

reasonably good separation from the other polar lipids present.³⁵

H. volcanii (ATCC 29605) was grown aerobically at 30 °C in medium

ATCC 974 containing 12.5% NaCl. Lipids were extracted from the

biomass and total polar lipids (TPLs) were collected by acetone

precipitation.³⁷TPLs (about 1 g) were pre-adsorbed on silica gel

(24 g) with 2:1 CHCl₃–MeOH. After solvent evaporation, the result-

ing silica gel/TPL mixture was loaded on a dry silica gel (140 g) col-

umn followed by elution using CHCl₃–MeOH–CH₃COOH–H₂O

(85:22.5:10:4, v/v) by gravity until the whole column was moist-

ened. The elution was then performed under pressure while frac-

tions were collected. Product visualization on TLC was done

using iodine and the desired PGP-Me (136.2 mg) was obtained

after combining the fractions and concentration.

were added p-toluenesulfenylchloride (37 lL; 0.255 mmol) and

silvertriﬂuoromethanesulfonate(65 mg; 0.255 mmol), andthemix-

ture was stirred at this temperature for 1 h. Archaeol (111 mg;

0.17 mol), which was dissolved in CH₂Cl₂(1.5 mL), was then added.

After10 min, thecoolingbathwasremovedandthereactionmixture

was allowed to warm to room temperature. After 40 min, the reac-

tion was quenched by the addition of diisopropylethylamine

(0.1 mL), and the reaction mixture was ﬁltered, followed by rinsing

with CH₂Cl₂and CH₂Cl₂–EtOAc 50:50. The combined ﬁltrates were

then concentrated and the residue was puriﬁed by chromatography

on silica gel eluting with hexanes–EtOAc–CH₂Cl₂7:2:1 to yield pure

19a as a viscous oil (161 mg; 64%). See NMR and MS tables in Section

4.28 for characterization data.

Compound 19a was also synthesized from 18 via procedure A-2

in a yield of 85%.

4.20. (2R)-2,3-Bis[(3R,7R,11R)-3,7,11,15-tetramethylhexadecyl-

4.25. Archaeosome preparation and characterization

oxy]propan-1-yl 6-O-(b-D-glucopyranosyl)-b-D-glucopyranoside

(19b)

Polar lipids dissolved in CHCl₃–MeOH (2:1) were combined in

the desired mol% and dried ﬁrst with a ﬂow of N₂, then under

vacuum for 1 h. Archaeosomes (liposomes partially or wholly

composed of archaeal-like lipids) were prepared by hydrating

20–30 mg lipids at 40 °C in 2 mL of PBS buffer (10 mM sodium

Disaccharide (19a 150 mg; 0.099 mmol) was subjected to

Procedure B to yield 19b (95 mg; 98%). See NMR and MS tables

in Section 4.28 for characterization data.

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

225

phosphate, 160 mM NaCl, pH 7.1) containing the test antigen OVA

dissolved at 10 mg/mL. To provide the charge required to prepare

stable archaeosomes from synthetic neutral glycolipids,¹the

diphospho-lipid PGP-Me was often included. In some cases choles-

terol (Sigma), DPPG (Sigma), or other synthetic archaeal phospho-

lipids were mixed in CHCl₃–CH₃OH with the synthetic

glycosylarchaeols. The size of the vesicles in the preparations

was decreased by sonication in a sonic water bath (Fisher Scien-

tiﬁc) at 40 °C. Antigen not entrapped was removed by centrifuga-

tion at 200,000g (R_max) and washing pellets twice with 12-mL

volumes of PBS. Antigen loading was quantiﬁed by SDS–polyacryl-

amide gel electrophoresis and was based on salt-corrected dry

weights.³⁸Average diameters of archaeosomes were determined

by particle size analysis using a 5 mW He/Ne laser (Nicomp 370).

sayed by Elispot performed in triplicate using splenic cells, as pre-

viously described.³⁹The samples of the same splenic cell

populations were used for CTL assays by the standard Cr⁵¹-assay

performed in triplicate with speciﬁc and non-speciﬁc targets

EG.7 and EL-4, respectively.⁴⁰All protocols were approved by the

Institutional Animal Care Committee, and murine testing was

according to the guidelines set by the Canadian Council on Animal

Care.

4.27. Statistical analysis

Elispot data are reported as means S.E.M. and signiﬁcance be-

tween means (P <0.05) was compared by two-tailed t test. Two-

tailed P values are reported.

4.26. Animal usage

4.28. NMR and MS tables

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-

¹H and ¹³C NMR of 1b–5b, 19a, 20b, and 21a were obtained in

CDCl₃solution (referenced to residual CHCl₃at 7.26 ppm ¹H and

77.0 ppm central resonance ¹³C) whereas those of 1c–5c, 14c,

19b, and 21b were obtained in 1:1 (v/v) solutions of CD₃OD–CDCl₃

(referenced to residual CHD₂OD at 3.31 ppm for ¹H and 49.15 ppm

for ¹³C). Chemical shifts are in ppm and coupling constants in Hz.

NMR was performed on either a Varian 400 MHz or a Varian 200

MHz spectrometers. The NMR data are compiled in Tables 3–6.

Table 7 contains the HRMS and optical rotation data (Scheme 7).

taining 15 lg OVA entrapped in archaeosomes. Blood samples

were collected from the tail vein 18 days after the second injection

for anti-OVA antibody titration done by ELISA.³¹Spleens were col-

lected from duplicate mice to determine antigen-speciﬁc CD8⁺

T

cell responses. Antigen-speciﬁc CD8⁺T cell frequencies were as-

Table 3

¹H NMR data for the glyco head group of compounds 1–5b, 1–5c, 14c, 19a, 19b, 20b, 21a, and 21b

H-6⁰(J_6,6

)

Bz_o

Bz_m,p

AcCH₃

OH

0

Compound H-1

H-2 (J_2,3

)

H-3 (J_3,4

)

H-4 (J_4,5

)

H-5 (J_5,6

)

H-6 (J_5,6

)

and

(J_1,2

)

(J_4,OH)

sugar

moiety

1b Glc^I

4.54 d

(8.0)

4.47 d

(8.0)

4.90 dd (9.6)

5.10 dd (10.5)

5.18 br t (9.2)

4.95 dd (3.4)

3.79 br t

(9.6)

5.35 br d

3.54 m

3.86 m

4.47 br d 4.09 m

2.15, 2.11,

2.06, 2.04

(6H), 2.03, 1.96

Gal^II

4.09 m

3.85 m

4.09 m

3.85 m

1c Glc^I

4.29 d

(7.8)

4.34 d

(7.8)

3.29 m

3.57 m

3.38 m

Gal^II

3.57 m 3.48 m 3.83 br d 3.63 m 3.79 dd

(3.5)

3.70 (11.9)

(7.2)

(4.4)

2b Glc^I

4.58 d

(8.0)

5.40 d

(4.2)

4.83 br t (9.5)

5.26 br t (9.0)

3.99 br t

(9.0)

5.04 br t

(9.7)

3.66 m

3.95 m

4.46 dd

(2.4)

4.23 m

(3.0)

4.23 m (11.9)

2.13, 2.09,

2.04, 2.01,

2.00, 1.99 (6H)

Glc^II

4.86 dd (9.3)

5.35 br t (9.8)

4.03 dd

(12.5)

2c Glc^I

4.26 d

(7.9)

5.10 d

(3.8)

3.28 m (9.7)

3.46 dd (9.9)

3.61 br t (9.1)

3.62 br t (9.6)

3.54 br t

(8.9)

3.26 br t

(9.1)

3.31 m

3.66 m

3.83 m

Glc^II

3.69 m

3.82 m

3b Glc^I

4.52 d

(8.3)

4.49 d

(8.1)

4.92 m (9.5)

4.92 m (9.9)

5.15 br t (9.3)

5.13 br t (9.5)

3.76 br t

3.55 m

4.48 (4.9) 4.08 dd

(12.0)

2.11, 2.07,

2.015, 2.011,

2.0 (6H), 1.97

Glc^II

5.05 br t

3.64 m

(3.9)

4.36 dd

4.03 br d

(12.2)

3c Glc^I

4.29 d

(7.8)

4.39 d

(7.9)

3.29 dd (8.8)

3.26 dd (8.5)

3.53 br t (9.1)

3.36 br t (9.1)

3.57 m

3.37 m

3.85 m

Glc^II

3.38 br t

(9.4)

3.34 m

3.87 br d 3.67 br d

(10.8)

4b Glc^I

4.58 d

(8.0)

5.17 d

(4.1)

4.94 br t (8.9)

5.10 dd (10.4)

5.19 br t (10.0)

5.33 dd (3.2)

5.12 m

3.58 m (4.0) 3.74 dd

3.58 m (10.8)

2.132, 2.128,

2.05, 2.04, 2.03,

2.00, 1.98

Gal^II

5.45 br t

(2.6)

4.22 m

4.08 br d 4.08 br d

4c Glc^I

4.30 d

(7.9)

4.87 d

(3.0)

3.24 dd (9.1)

3.73 m

3.38 br t (8.6)

3.91 m

3.47 m

(2.9)

3.85 br t

4.05 dd

3.73 m

3.63 m (10.6)

3.73 m

Gal^II

3.73 m

5b Glc^I

4.59 d

(7.0)

5.12 d

(3.8)

4.94 dd (9.7)

4.86 dd (10.3)

5.20 br t (9.3)

5.44 br t (9.6)

5.07 br t

(9.4)

5.05 br t

(9.7)

3.65 m

(4.6)

4.07 m (4.4) 4.26 dd

3.75 dd

3.65 m (10.9)

4.07 m (12.6)

2.12, 2.09, 2.05,

2.03, 2.02, 2.00

(6H)

Glc^II

(continued on next page)

226

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

Table 3 (continued)

H-6⁰(J_6,6

)

Bz_o

Bz_m,p

AcCH₃

OH

0

Compound H-1

H-2 (J_2,3

)

H-3 (J_3,4

)

H-4 (J_4,5

)

H-5 (J_5,6

)

H-6 (J_5,6

)

and

(J_1,2

)

(J_4,OH)

sugar

moiety

5c Glc^I

4.30 d

(7.9)

4.82 d

(3.4)

3.24 br t (9.1)

3.37 dd (10.0)

3.38 br t (9.1)

3.62 br t (9.9)

3.50 m

3.32 m

3.41 m

(3.5)

3.64 m

(2.3)

4.03 dd

3.64 m (10.8)

Glc^II

3.78 dd

(5.3)

3.69 dd

(11.4)

14c Glc^I

4.78 d

(3.5)

4.31 d

(7.8)

3.40 dd (10.0)

3.26 m

3.63 br t (9.5)

3.38 m

3.47 br t

(9.8)

3.32 m

3.70 m

4.08 br d 3.79 dd

(10.3)

Glc^II

3.26 m

(2.3)

3.85 dd

(4.9)

3.69 m (11.8)

19a Glc I

4.63 d

(7.8)

4.88 d

(7.8)

5.32 dd (10.0)

5.43 dd (9.8)

5.56 t (9.8)

5.65 t (9.5)

5.08 t (9.6) 3.79 m

3.97 br d 3.79 m

7.95 (2H) d

(7.3)

7.90 (4H) d

(8.1)

7.48 m,

7.35 m

2.15, 1.94, 1.92

Glc II

5.34 t (9.7) 3.87 ddd

(4.9)

4.37 dd

(2.5)

4.22 dd

(12.5)

7.86 (2H) d

(8.1)

19b Glc I

4.26 d

(7.8)

4.31 d

(7.6)

3.26 m

3.47 m

3.36 m

3.47 m

(1.8)

3.36 m

(2.5)

4.12 dd

(4.5)

3.85 dd

(5.3)

3.78 dd

(11.4)

3.68 dd

(12.1)

Glc II

20b

4.81 d

(7.3)

5.44 t (9.7)

5.41 t (9.7)

3.95 m

3.60 m

4.00 br d 3.89 br d

7.96 (4H) br d 7.50 m,

7.36 m

n.d.

21a Glc I

Glc II

5.03 d

(8.0)

4.61 d

(7.6)

4.63 d

(7.4)

5.42–5.24 m

5.54 br t

3.74–3.62 3.57 m

m

4.19 m

3.90–3.74 m 7.95 (2H) d

(7.4),

7.6–7.4

2.12, 1.89, 1.85 3.2 m

(8H) m,

7.3 (8H)

m,

7.23 (4H)

m

5.04 br t

3.90–3.74 3.90–3.74 3.90–3.74 m 7.85 (4H) d

m

(7.0),

4.19 m (12.5) 7.84 (2H) d

(6.8),

Glc III

5.63 br t

5.42–5.24 3.90–3.74 4.33 dd

(4.7)

m

7.68 (2H) d

(7.4)

21b Glc I

Glc II

4.27 d

(7.8)

4.32 d

(7.7)

4.33 d

(7.6)

3.26 m

3.5–3.34 m

3.5–3.34 m 3.5–3.34 m 4.11 br dd 3.77 m

3.5–3.34 m 3.5–3.34 m 4.14 br dd 3.77 m

Glc III

3.5–3.34 m 3.26 m

3.85 dd

(2.0)

3.69 m (11.9)

Table 4

¹³C NMR data for the glyco head group of compounds 1–5b, 1–5c, 14c, 19a, 19b, 20b, 21a, and 21b

Compound

and sugar

moiety

C1

C2

C3

C4

C5

C6

Ac C@O

Ac CH₃

Bz C@O

Bz_p

Bz_ip,o,m

1b Glc^I

100.8

101.1

71.6

69.0

72.9

71.0

76.3

66.5

72.5

70.6

62.0 170.3 (2C), 170.1,

60.7 170.0, 169.7, 169.4,

169.0

20.81, 20.76,

20.65, 20.58,

20.46

Gal^II

1c Glc^I

104.1

104.6

74.1

76.5

75.6

74.3

80.7

69.8

75.8

72.0

61.8

62.2

Gal^II

2b Glc^I

100.5

95.5

72.2

70.0

75.5

69.1

72.8

68.0

72.0

68.5

62.9 170.51, 170.50,

61.5 170.4, 170.2, 169.9,

169.5, 169.4

20.9, 20.8,

20.7

Glc^II

2c Glc^I

104.2

102.6

73.9

73.5

74.0

76.9

81.0

70.97 74.5

76.0

61.5

62.5

Glc^II

3b Glc^I

100.86 71.57 72.58 76.5

72.54 61.9 170.4, 170.2, 170.17,

20.8, 20.64,

20.61, 20.5

Glc^II

100.76 71.57 72.9

67.8

71.9

61.5 169.7, 169.4, 169.2,

169.0

3c Glc^I

104.05 74.1

104.13 74.1

75.6

75.8

80.6

77.2

70.6

77.4

61.7

61.9

Glc^II

4b Glc^I

108.8

96.6

71.3

67.97 67.4

73.0

68.9

68.04 66.4

72.6

66.2 170.6, 170.4, 170.3,

61.7 170.2, 169.8, 169.3,

169.2

20.8, 20.71,

20.66, 20.6

Gal^II

4c Glc^I

104.7

99.6

74.5

70.1

77.3

70.5

71.3

75.7

71.6

66.4

62.4

Gal^II

5b Glc^I

100.8

96.0

71.3

70.6

72.9

69.9

69.2

68.4

72.5

67.4

66.5 170.6, 170.29,

61.8 170.27, 170.0, 169.6,

169.4, 169.2

20.7, 20.6

Glc^II

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

227

Table 4 (continued)

Compound

and sugar

moiety

C1

C2

C3

C4

C5

C6

Ac C@O

Ac CH₃

Bz C@O

Bz_p

Bz_ip,o,m

5c Glc^I

104.7

99.3

74.4

77.2

73.2

74.9

70.4

71.2

75.6

72.8

66.4

62.3

Glc^II

14c Glc^I

99.8

103.9

73.0

74.3

74.5

77.3

70.7

70.9

71.9

77.0

69.0

62.4

Glc^II

19a Glc I

Glc II

101.1

71.6

71.5

72.9

73

69.4

68.3

73.5

72.2

68.3 170.7, 169.7, 169.4

61.9

20.8, 20.6,

20.5

165.7 (2C), 165.0,

164.9

133.4, 133.3,

133.2, 133.1

129.8– 128.3

19b Glc I

Glc II

104.4

104.2

74.3

70.6

70.9

77.1

76.1

77

69.4

62.4

20b

101.2

71.4

77.3

70.1

75.8

62.3

167.6, 165.1

133.6, 133.2

130.0, 129.7,

129.4, 128.8,

128.5, 128.3

21a Glc I

101.1

71.4

76.8

69.9

74.9

69

170.7, 169.6, 169.4

20.8, 20.5,

20.4

166.8, 166.2,

165.6, 165.15,

165.06, 164.9

133.56, 133.5,

133.4, 133.2,

133.1, 133.0

129.9– 128.3

Glc II

Glc III

72.7

73

69

68.9

74.7

72.2

67.7

61.8

101.14 71.4

21b Glc I

Glc II

Glc III

103.9

103.6

73.5

75.2

75.6

69.7

76.2

69

68.9

61

Table 5

¹H NMR data for the lipid (archaeol) moiety of compounds 1–5b, 1–5c, 14c, 19a, 19b, 20b, 21a, and 21b (Glyc1–3 are protons on glycerol carbons sn-1–3; Phy1–2 are protons on

C1, C1⁰, C2, or C2⁰; CH, CH₂, and CH₃represent combined proton signals from these groups of the isoprenoid chains)

Compound Glyc1

Glyc2

Glyc3

Phy 1

Phy 1⁰

Phy 2, 2⁰

CH

CH₂

CH₃

1b

1c

2b

2c

3b

3c

4b

4c

3.86 m, 3.54 m

3.54 m 3.54 m

3.63 m 3.63 m, 3.57 m

3.53 m 3.42 m

3.61 m 3.51 m

3.55 m 3.40 m

3.54 m 3.41 m 1.52 m, 1.30 m 1.60 m, 1.30 m 1.39–1.03 0.85 m

3.63 m 3.57 m 1.57 m, 1.38 m 1.59 m, 1.31 m 1.40–1.02 0.85 m

3.53 m 3.42 m 1.60 m, 1.37 m 1.55 m, 1.40 m 1.41–1.01 0.85 m

3.61 m 3.48 m 1.61 m, 1.31 m 1.55 m, 1.38 m 1.40–1.01 0.85 m

3.55 m 3.40 m 1.58 m, 1.37 m 1.61 m, 1.31 m 1.40–1.06 0.78 m

3.60 m 3.49 m 1.56 m, 1.38 m 1.59 m, 1.31 m 1.37–1.01 0.82 m

3.58 m 3.43 m 1.55 m, 1.32 m 1.60 m, 1.30 m 1.37–1.01 0.86 m

3.92 m, 3.63 m

3.89 m, 3.53 m

3.92 m, 3.61 m

3.87 m, 3.55 m

3.88 m, 3.60 m

3.86 m, 3.58 m

3.91 m, 3.63 m

3.85 m, 3.56 m

3.92 m, 3.58 m

3.73 m, 3.46 m

3.79 m 3.37 m

3.60 m 3.55 m, 3.49 m

3.58 m 3.43 m

3.63 m 3.55 br d (10.3), 3.47 m 3.63 m 3.47 m 1.52 m, 1.33 m 1.55 m, 1.30 m 1.40–1.02 0.85 m

3.56 m 3.43 m

3.63 m 3.52 m, 3.44 m

3.63 m 3.52 m

3.37 m 3.37 m

5b

5c

3.56 m 3.43 m 1.56 m, 1.40 m 1.52 m, 1.28 m 1.37–1.03 0.85 m

3.60 m 3.44 m 1.51 m, 1.38 m 1.58 m, 1.32 m 1.40–1.01 0.86 m

3.62 m 3.52 m 1.58 m, 1.32 m 1.58 m, 1.31 m 1.40–1.00 0.84 m

3.28 m 3.26 m 1.48 m, 1.23 m 1.50 m, 1.33 m 1.40–1.00 0.85 m, 0.74 d (6.4)

14c

19a

19b

20b

21a

21b

3.90 dd (3.7, 10.3), 3.56 m 3.61 m 3.58 m

3.50 m 3.44 m 1.47 m, 1.31 m 1.54 m, 1.2 m

1.40–1.00 0.85 m

3.95 m 3.60 m

3.74 m 3.66 m

3.72 m, 3.45 m

3.49 m 3.40 m

3.37 m 3.34 m

3.49 m 3.50 m

3.37 m 3.35 m 1.51 m, 1.25 m 1.50 m, 1.34 m 1.40–1.00 0.85 m, 0.75 d (6.4)

3.2 m

1.48 m, 1.32 m 1.50 m, 1.36 m 1.4–1.0

0.85 m, 0.71 d (6.8)

0.8 m

3.39 m 3.32 m 1.42 m, 1.22 m 1.34 m, 1.20 m 1.4–1.0

Table 6

¹³C NMR data for the lipid (archaeol) moiety of compounds 1–5b, 1–5c, 14c, 19a, 19b, 20b, 21a, and 21b

Compound Glyc1 Glyc2 Glyc3 Phy

1

Phy

1⁰

CH

CH₂

CH₃

1b

1c

70.4

70

77.8

70.5

71.1

70

70.8

69.1

69.4

32.7, 29.9, 29.8, 27.9

33.5, 30.6, 30.5, 28.7

39.3, 37.40, 37.36, 37.2, 37.1, 36.5, 24.7, 24.4, 24.3

40.1, 38.2, 38.1, 38.0, 37.7, 37.4, 25.5, 25.2, 25.1

22.7, 22.6, 19.70, 19.65

23.15, 23.05, 20.28, 20.24,

20.20

2b

2c

3b

70.47 77.8

70.51 70.1

69.3

69.4

69.1

32.8, 29.9, 29.8, 27.9

33.5, 30.6, 30.5, 28.7

32.8, 29.9, 29.8, 27.9

39.3, 37.44, 37.40, 37.36, 37.3, 37.1, 36.6, 24.8, 24.5, 24.3 22.7, 22.6, 19.73, 19.69

69.8

70.4

78.6

77.8

71.1

70.5

71

70.1

40.1, 38.1, 38.0, 37.6, 37.3

23.1, 23.0, 20.2

39.3, 37.51, 37.45, 37.42, 37.37, 37.3, 37.2, 37.1, 36.6,

24.8, 24.4, 24.3

22.7, 22.6, 19.71, 19.66

3c

4b

4c

69.8

70.2

69.5

78.6

78.1

78.8

71.1

70.1

71.1

70.8

71.3

70.8

69.4

69.2

70.3

33.5, 30.6, 30.5, 28.7

32.8, 30.0, 29.9, 28.0

33.6, 30.7, 30.6, 30.4,

28.7

40.0, 38.1, 38.0, 37.7, 37.4, 25.5, 25.2

39.6, 37.5, 37.4, 37.3, 37.2, 36.6, 24.8, 24.5, 24.4

40.2, 38.19, 38.17, 38.15, 38.0, 37.8, 37.4, 25.6, 25.22,

25.17

23.2, 23.1, 20.2

19.74, 19.69

23.2, 23.1, 20.30, 20.27,

20.2

5b

5c

14c

70.1

70.2

71.1

78

78.6

78.4

70.6

71

68.1

70.1

70.7

70.2

69.2

69.4

69.8

32.8, 30.0, 29.0, 28.0

33.5, 30.6, 30.5, 28.7

33.4, 30.6, 30.4, 30.3,

28.6

39.4, 37.5, 37.4, 37.3, 37.2, 36.6, 24.8, 24.5, 24.4

40.1, 38.2, 38.1, 38.0, 37.7, 37.3, 25.5, 25.13, 25.09

40.0, 38.08, 38.03, 37.9, 37.6, 37.3, 25.4, 25.1, 25.0

22.7, 22.6, 19.74, 19.70

23.1, 23.0, 20.2

23.1, 23.0, 20.2, 20.15,

20.14

19a

70.3

77.7

69.1

70.5

69.9

32.8, 29.9, 29.7, 28.0

39.4, 37.6, 37.5, 37.4, 37.0, 36.6, 24.8, 24.5, 24.4, 24.3

22.7, 22.6, 19.7, 19.6, 19.5

(continued on next page)

228

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

Table 6 (continued)

Compound Glyc1 Glyc2 Glyc3 Phy

1

Phy

1⁰

CH

CH₂

CH₃

19b

20b

70.0

70.6

77.2

77.9

69.4

69.1

71.2

70.2

70.7

33.5, 30.6, 30.5, 28.7

40.1, 38.13, 38.09, 38.0, 37.7, 37.3, 25.5, 25.14, 25.08

23.1, 23.0, 20.3, 20.23,

20.16

70.07 32.8, 29.9, 29.7, 28.0

39.4, 37.5, 37.42, 37.39, 37.3, 36.9, 36.5, 24.8, 24.5, 24.4, 22.7, 22.6, 19.8, 19.7, 19.5

24.3

21a

21b

70.1

70.5

77.6

78.1

69.6

69.7

70.8

70.5

69.9

70.5

32.8, 29.9, 29.7, 28.0

33.1, 30.3, 30.2, 30.0

39.3, 37.55, 37.45, 37.4, 37.3, 37.0, 36.6, 24.8, 24.5, 24.4

39.7, 37.7, 37.6, 37.2, 36.9, 25.1, 24.8, 24.4

22.7, 22.6, 19.7, 19.6, 19.5

22.9, 22.8, 20.0

Table 7

M.S. and optical rotation data for compounds 1–5b, 1–5c, 14c, 19a, 19b, 20b, 21a, and 21b (nd.: not determined)

Compound

Formula

MW

HRMS

calcd and obsd

Optical rotation

([ ]_D)

Concentration

(c for [a]_D, g/mL)

a

1b

C₆₉H₁₂₂O₂₀

1271.86

976.78

1288.8873 (M+NH₄)⁺

1288.8965

ꢁ4.6

ꢁ0.5

23.1

20.8

ꢁ8.6

nd.

0.128

1c

C

₅₅H₁₀₈O₁₃

₆₉H₁₂₂O₂₀

₅₅H₁₀₈O₁₃

₆₉H₁₂₂O₂₀

₅₅H₁₀₈O₁₃

₆₉H₁₂₂O₂₀

₅₅H₁₀₈O₁₃

₆₉H₁₂₂O₂₀

₅₅H₁₀₈O₁₃

₈₉H₁₃₀O₂₀

₅₅H₁₀₈O₁₃

994.8133 (M+NH₄)⁺

994.8214

0.043

0.277

0.101

0.465

nd.

2b

1271.86

976.78

1288.8873 (M+NH₄)⁺

1288.8864

2c

994.8133 (M+NH₄)⁺

994.8197

3b

1271.86

976.78

1288.8873 (M+NH₄)⁺

1288.8981

3c

994.8133 (M+NH₄)⁺

994.8155

4b

1271.86

976.78

1288.8873 (M+NH₄)⁺

1288.9000

23.5

nd.

0.0541

nd.

4c

994.8133 (M+NH₄)⁺

994.8249

5b

1271.86

976.78

1288.8873 (M+NH₄)⁺

1288.8969

31.4

16.3

9.1

0.167

0.024

0.066

0.020

0.122

0.210

0.112

0.264

5c

994.8133 (M+NH₄)⁺

994.8138

14c

19a

19b

20b

21a

21b

976.78

994.8133 (M+NH₄)⁺

994.8214

1518.92

976.78

1536.9499 (M+NH₄)⁺

1536.9601

38.0

ꢁ22.9

15.6

43.8

ꢁ21.5

994.8133 (M+NH₄)⁺

994.8226

C₆₃H₁₀₆O₁₀

1022.78

1889.02

1138.83

1040.8129 (M+NH₄)⁺

1040.8226

C

₁₀₉H₁₄₈O₂₇

₆₁H₁₁₈O₁₈

1907.0551 (M+NH₄)⁺

1907.0796

1161.8215 (M+Na)⁺

1161.8072

5. Arakawa, K.; Eguchi, T.; Kakinuma, K. Bull. Chem. Soc. Jpn. 2001, 74, 347–356.

6. Wolfrom, M. L.; Thompson, A. Methods Carbohydr. Chem. 1963, 2, 211–215.

7. Pozsgay, V.; Jennings, H. J. Tetrahedron Lett. 1987, 28, 1375–1376.

8. Tropper, F. D.; Andersson, F. O.; Grand-Maître, C.; Roy, R. Synthesis 1991, 734–

736.

phy1

phy1'

glyc3

glyc2

O

9. Kanie, O.; Ito, Y.; Ogawa, T. J. Am. Chem. Soc. 1994, 116, 12073–12074.

10. Zemplen, G.; Pacsu, E. Berichte Deut. Chem. Ges. 1929, 62, 1613–1614.

11. (a) Whitﬁeld, D. M.; Douglas, S. P.; Tang, T.-H.; Csizmadia, I. G.; Pang, H. Y. S.;

Moolten, F. L.; Krepinsky, J. J. Can. J. Chem. 1994, 72, 2225–2238; (b) Nukada, T.;

Bérces, A.; Whitﬁeld, D. M. J. Org. Chem. 1999, 64, 9030–9045; (c) Bérces, A.;

Whitﬁeld, D. M.; Nukada, T.; do Santos, I. Z.; Obuchowska, A.; Krepinsky, J. J.

Can. J. Chem. 2004, 82, 1157–1171.

HO

glyc1

Scheme 7. Structure of archaeol with labels indicated which are used in the NMR

Tables 5 and 6; glyc as in glycerol and phy as in phytanyl.

12. For example: Deng, S.; Yu, B.; Xie, J.; Hui, Y. J. Org. Chem. 1999, 64, 7265–7266.

13. Tatai, J.; Fugedi, P. Org. Lett. 2007, 9, 4647–4650.

14. Prakash, G. K. S.; Mathew, T.; Marinez, E. R.; Esteves, P. M.; Rasul, G.; Olah, G. A.

J. Org. Chem. 2006, 71, 3952–3958.

15. Jayaprakash, K. N.; Radhakrishnan, K. V.; Fraser-Reid, B. Tetrahedron Lett. 2002,

43, 6953–6955.

16. Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H. Tetrahedron Lett. 1990, 31,

1331–1334.

Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.carres.2009.10.011.

References

17. Borén, H. B.; Eklind, K.; Garegg, P. J.; Lindberg, B.; Piloti, A. Acta Chem. Scand.

1972, 26, 4143–4146.

1. Sprott, G. D.; Dicaire, C. J.; Whitﬁeld, D. M. Glycobiology 2008, 18, 559–565.

2. Whitﬁeld, D. M.; Eichler, E.; Sprott, G. D. Carbohydr. Res. 2008, 343, 2349–

2360.

18. (a) Wulff, G.; Röhle, G. Angew. Chem., Int. Ed. 1974, 13, 157–170; (b) Crich, D. J.

Carbohydr. Chem. 2002, 21, 667–690; (c) Crich, D.; Sun, S. J. Am. Chem. Soc. 1997,

119, 11217–11223.

3. Sprott, G. D.; Brisson, J.-R.; Dicaire, C. J.; Pelletier, A. K.; Deschatelets, L. A.;

Krishnan, L.; Patel, G. B. Biochem. Biophys. Acta 1999, 1440, 275–288.

4. Kates, M. Biochem. Soc. Symp. 1992, 58, 51–72.

19. (a) Matsumoto, K.; Sakai, H.; Ohta, K.; Kameda, H.; Sugawara, F.; Abe, M.;

Sakaguchi, K. Chem. Phys. Lipids 2005, 133, 203–214; (b) Jarrell, H. C.; Wand, A.

J.; Giziewicz, J. B.; Smith, I. C. Biochim. Biophys. Acta 1987, 897, 69–82.

D. M. Whitﬁeld et al. / Carbohydrate Research 345 (2010) 214–229

229

20. Nukada, T.; Bérces, A.; Zgierski, M. Z.; Whitﬁeld, D. M. J. Am. Chem. Soc. 1998,

28. Whitﬁeld, D. M. Adv. Carbohydr. Chem. Biochem. 2009, 62, 83–159.

29. Schmidt, R. R.; Stumpp, M. Liebigs Ann. Chem. 1983, 1249–1256.

30. Cross, G. G.; Whitﬁeld, D. M. Synlett 1998, 487–488.

31. Lefeber, D. J.; Arevalo, E. A.; Kamerling, J. P.; Vliegenthart, J. F. G. Can. J. Chem.

2002, 80, 76–81.

120, 13291–13295.

21. (a) Tokimoto, H.; Fujimoto, Y.; Fukase, K.; Kusumoto, S. Tetrahedron: Asymmetry

2005, 16, 441–447; (b) Houdier, S.; Vottero, P. J. A. Carbohydr. Res. 1992, 232,

349–352; (c) Fréchet, J. M.; Schuerch, C. J. Am. Chem. Soc. 1972, 94, 604–609.

22. Nukada, T.; Bérces, A.; Wang, L. J.; Zgierski, M. Z.; Whitﬁeld, D. M. Carbohydr.

Res. 2005, 340, 841–852.

32. Kim, S. K.; Ragupathi, G.; Cappello, S.; Kagan, E.; Livingstone, P. O. Vaccine 2001,

19, 530–537.

23. (a) Ray, A. K.; Roy, N. Carbohydr. Res. 1990, 196, 95–100; (b) Koto, S.;

Morishima, N.; Kihara, Y.; Suzuki, H.; Kosugi, S.; Zen, S. Bull. Chem. Soc. Jpn.

1983, 56, 188–191.

24. (a) Konradsson, P.; Mootoo, D. R.; McDevitt, R. E.; Fraser-Reid, B. J. Chem. Soc.,

Chem. Commun. 1990, 270–272; (b) Konradsson, P.; Udodong, U. E.; Fraser-

Reid, B. Tetrahedron Lett. 1990, 31, 4313–4316.

25. (a) Ito, Y.; Ogawa, T. Tetrahedron Lett. 1988, 29, 1061–1064; (b) Kim, K. S.; Jeon,

H. B. Chem. Record 2008, 8, 46–65.

26. Fukase, K.; Nakai, Y.; Kanoh, T.; Kusumoto, S. Synlett 1998, 84–86.

27. (a) Duron, S. G.; Polat, T.; Wong, C.-H. Org. Lett. 2004, 6, 839–841; (b) Huang, X.;

Huang, L.; Wang, H.; Ye, X.-S. Angew. Chem., Int. Ed. 2001, 116, 5221–5224; (c)

Crich, D.; Feng Cai, F.; Yang, F. Carbohydr. Res. 2008, 343, 1858–1862.

33. Krishnan, L.; Dicaire, C. J.; Patel, G. B.; Sprott, G. D. Infect. Immun. 2000, 68, 54–

63.

34. Sprott, G. D.; Tolson, D. L.; Patel, G. B. FEMS Microbiol. Lett. 1997, 154, 17–22.

35. Sprott, G. D.; Cote, J. P.; Jarrell, H. C. Glycobiology 2009, 19, 267–276.

36. Eichler, E.; Yan, F.; Sealy, J.; Whitﬁeld, D. M. Tetrahedron 2001, 57, 6679–6693.

37. Sprott, G. D.; Larocque, S.; Cadotte, N.; Dicaire, C. J.; McGee, M.; Brisson, J. R.

Biochim. Biophys. Acta 2003, 1633, 179–188.

38. Sprott, G. D.; Patel, G. B.; Krishnan, L. Methods Enzymol. 2003, 373, 155–172.

39. Sprott, G. D.; Dicaire, C. J.; Gurnani, K.; Sad, S.; Krishnan, L. Infect. Immun. 2004,

72, 5235–5246.

40. Krishnan, L.; Sad, S.; Patel, G. B.; Sprott, G. D. J. Immunol. 2000, 165, 5177–

5185.

Article Doi

Development of new glycosylation methodologies for the synthesis of archaeal-derived glycolipid adjuvants

DOI: 10.1016/j.carres.2009.10.011

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