Versatile set of orthogonal protecting groups for the preparation of highly branched oligosaccharides

Article abstract of DOI:10.1021/ol101951u

A new set of orthogonal protecting groups has been developed based on the use of a diethylisopropylsilyl (DEIPS), methylnaphthyl (Nap), allyl ether, and levulinoyl (Lev) ester. The protecting groups are ideally suited for the preparation of highly branche

Full text of DOI:10.1021/ol101951u

ORGANIC
LETTERS
2010
Vol. 12, No. 20
4636-4639
Versatile Set of Orthogonal Protecting
Groups for the Preparation of Highly
Branched Oligosaccharides
Thomas J. Boltje,† Chunxia Li,†,‡ and Geert-Jan Boons*,†
Complex Carbohydrate Research Center, UniVersity of Georgia, 315 RiVerbend Road,
Athens, Georgia 30602, and School of Medicine and Pharmacy, Ocean UniVersity of
China, 5 Yushan Road, Qingdao 266003, China
gjboons@ccrc.uga.edu
Received August 19, 2010
ABSTRACT
A new set of orthogonal protecting groups has been developed based on the use of a diethylisopropylsilyl (DEIPS), methylnaphthyl (Nap), allyl
ether, and levulinoyl (Lev) ester. The protecting groups are ideally suited for the preparation of highly branched oligosaccharides and their
usefulness has been demonstrated by the chemical synthesis of a ꢀ-D-Man-(1f4)-D-Man disaccharide, which is appropriately protected for
making a range of part-structures of the unusual core region of the lipopolysaccharide of Francisella tularensis.
F. tularensis is a gram-negative bacterium that can cause
tularemia (rabbit fever) in animals and humans1 and has been
classified by the CDC as a top-priority (Class A) bioterrorism
agent.2 Common to all class-A agents, tularemia transmits
easily, has the capacity to inflict substantial morbidity and
mortality on a large number of people and can induce
widespread panic. To prevent tularemia, an attenuated life
vaccine strain (LVS) was developed in the 1950s, but was
not licensed for use as a human vaccine because the nature
of its attenuation was not known and may not be stable.
Diagnoses are based on time-consuming culture, serology
or sophisticated molecular techniques. Therefore improved
vaccine candidates and rapid diagnostic tests are needed for
this pathogen.
The structure of the lipopolysaccharide (LPS) of F.
tularensis has been determined (Figure 1)3 and it was
established that it has an unusual core structure. The core is
linked to the lipid A region by only one 3-deoxy-D-manno-
2-octulosonic acid (KDO) moiety instead of the usual two
KDO residues. It does not contain heptosyl residues but
contains two mannosyl moieties. One of the mannosides is
ꢀ-linked to another mannoside, and this disaccharide frag-
ment is further substituted at C-2, C-2′ and C-3′ by a
ꢀ-glucoside, an R-galactosamine and a R-glucoside, respec-
tively. It has been proposed that the lipopolysaccharide of
F. tularensis is an attractive candidate for vaccine and
diagnostic test development.4 However, isolation of saccha-
rides from a Class A bioterrorism agent is highly undesirable.
Furthermore, it is difficult to conjugate short oligosaccharides
† University of Georgia.
‡ Ocean University of China.
(1) Elkins, K. L.; Cowley, S. C.; Collazo, C. M. Expert. ReV. Vaccines
2004, 2, 735.
(2) Dennis, D. T.; Inglesby, T. V.; Henderson, D. A.; Bartlett, J. G.;
Ascher, M. S.; Eitzen, E.; Fine, A. D.; Friedlander, A. M.; Hauer, J.; Layton,
M.; Lillibridge, S. R.; McDade, J. E.; Osterholm, M. T.; O’Toole, T.; Parker,
G.; Perl, T. M.; Russell, P. K.; Tonat, K. J. Am. Med. Assoc. 2001, 285,
2763.
(3) Vinogradov, E.; Perry, M. B.; Conlan, J. W. Eur. J. Biochem. 2002,
269, 6112.
(4) Conlan, J. W.; Shen, H.; Webb, A.; Perry, M. B. Vaccine 2002, 20,
3465.
10.1021/ol101951u  2010 American Chemical Society
Published on Web 09/16/2010

of a 4-methoxybenzyl- or methylnaphthyl (Nap) ether using
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ).11-15 The ether
can be present on the glycosyl donor or acceptor, the latter
being referred to as reverse tethering. In the second step,
the glycosyl donor is activated and the glycosyl acceptor is
forced to attack from the same face as the C-2′ tether leading
to the introduction of a 1,2-cis-glycoside with concomitant
loss of the C-2′ protecting group.
Monosaccharide building blocks 1-4 were prepared (see
Supporting Information) to explore the utility of IAD for
the synthesis of an orthogonal protected ꢀ-D-Man-(1f4)-
R-D-Man disaccharide (Scheme 1). It was envisaged that
Scheme 1
.
Intramolecular Aglycon Delivery using Acetal
Tethering
Figure 1. Target molecule and synthetic strategy. (A) Highly
branched hexasaccharide fragment isolated from F. tularensis LVS
lipopolysaccharide, target ꢀ-D-Man-(1f4)-R-D-Man disaccharide
highlighted in red. (B) Intramolecular aglycon delivery through
acetal tethering. (C) 4,6-Benzylidene mediated R-triflate formation
followed by SN2-like displacement.
to carrier proteins without destroying vital immunological
domains. Synthetic chemistry can address these issues since
it makes it possible to incorporate an artificial linker for
controlled conjugation to proteins. In addition, substructures
can be prepared to determine the minimal epitope required
to elicit protective immune responses.
Herein we report the chemical synthesis of a ꢀ-D-Man-
(1f4)-R-D-Man disaccharide that is functionalized with a
set of orthogonal protecting groups at C-1, C-2, C-2′ and
C-3′. The orthogonal protecting groups make it possible to
selectively introduce glycosides for the synthesis of a library
of F. tularensis oligosaccharides.5-8
To this end, two ꢀ-mannosylation strategies were explored
as well as a variety of orthogonal protecting group combina-
tions. ꢀ-Mannosides, which are an important class of 1,2-
cis glycosides, are difficult to introduce due to the axial C-2
substituent, which sterically blocks incoming nucleophiles
from the ꢀ-face and the ∆-anomeric effect, which provides
additional stabilization of the R-anomer.9,10 An elegant
methodology for the construction of ꢀ-mannosidic linkages
is based on intramolecular aglycon delivery (IAD), which
usually gives absolute ꢀ-anomeric selectivity. In this two-
step protocol, the glycosyl donor and acceptor are tethered
through a mixed acetal by for example oxidative coupling
compound 1, which is equipped with a methylnapthyl ether,
would provide a useful starting material to make tethered
derivative 5, which upon IAD should provide disaccharide
6. The resulting free hydroxyl at C-2′ of 6 can then
immediately be used for the introduction of the GalN of
the core region of F. Tularensis. It was expected that the
allyloxycarbonyl (Alloc), levulinoyl ester (Lev)16 and the
2-(trimethylsilyl)ethyl ether (SE)17 would provide an attrac-
tive set of orthogonal protecting groups for further glyco-
sylations.
Thus, a mixture of 1 and 3 in CH2Cl2 in the presence of
molecular sieves was treated with DDQ to afford mixed
acetal 5 in a moderate yield of 51% as a 10/1 mixture of
diastereoisomers (Scheme 1). The yield was significantly
improved when reverse tethering was employed using 2 and
4 to give 5 in 72% yield. Presumably, a higher yield is
obtained due to the higher nucleophilicity of the C-2′ alcohol.
Next, mixed acetal 5 was activated with methyl triflate in
the presence of 2,6-di-tert-butyl-4-methylpyridine (DTBMP)
(5) Wong, C. H.; Ye, X. S.; Zhang, Z. Y. J. Am. Chem. Soc. 1998, 120,
(11) Jung, K. H.; Muller, M.; Schmidt, R. R. Chem. ReV. 2000, 100,
7137
.
4423.
(6) Zhu, T.; Boons, G. J. Tetrahedron: Asymmetry 2000, 11, 199
(7) Markad, S. D.; Schmidt, R. R. Eur. J. Org. Chem. 2009, 5002
(8) Wang, C. C.; Lee, J. C.; Luo, S. Y.; Kulkarni, S. S.; Huang, Y. W.;
Lee, C. C.; Chang, K. L.; Hung, S. C. Nature 2007, 446, 896
.
(12) Lee, Y. J.; Ishiwata, A.; Ito, Y. J. Am. Chem. Soc. 2008, 130, 6330
(13) Boltje, T. J.; Buskas, T.; Boons, G. J. Nat. Chem. 2009, 1, 611
(14) Cumpstey, I. Carbohydr. Res. 2008, 343, 1553
(15) Zhu, X. M.; Schmidt, R. R. Angew. Chem., Int. Ed. 2009, 48, 1900
(16) van Boom, J. H.; Burgers, P. M. J. Tetrahedron Lett. 1976, 52,
.
.
.
.
.
.
(9) Gridley, J. J.; Osborn, H. M. I. J. Chem. Soc., Perkin Trans. 1 2000,
1471
.
4875.
(10) Cai, F.; Wu, B. L.; Crich, D. AdV. Carbohydr. Chem. Biochem.
2009, 62, 251
(17) Jansson, K.; Frejd, T.; Kihlberg, J.; Magnusson, G. Tetrahedron
Lett. 1986, 27, 753.
.
Org. Lett., Vol. 12, No. 20, 2010
4637

in 1,2-dichloroethane (DCE) at 40 °C. Despite the relatively
high reaction temperature, the glycosylation was rather
sluggish, and after a reaction time of 16 h, disaccharide 6
was isolated in a disappointing yield of 26%. The low yield
was due to the formation of several byproducts including
C-2′ methylated disaccharide. To address the problem of
methylation, the methyl triflate promoted glycosylation was
performed in the presence of triethyl silane. Under these
conditions, the intermediate naphthylic cation is trapped
providing a C-2′ Nap ether instead of a hydroxyl. Unfortu-
nately, these modified reaction conditions did not improve
the yield of ꢀ-mannoside 6. Dimethyl(thiomethyl)sulfonium
triflate (DMTST)18 is also commonly employed as a thio-
philic promoter for IAD.19 However, the use of this promoter
in the absence of a base did not lead to product formation.
Nevertheless, the application of DMTST in combination with
the base DTBMP gave 6 in a somewhat improved yield of
31%. Other promoter systems such as N-iodosuccinimide
(NIS) could not be employed because of incompatibility with
the Alloc function. Although other orthogonal protecting
group pairs could be examined, attention was focused on an
alternative approach for ꢀ-mannoside synthesis.
Table 1. Stereoselective ꢀ-Mannosylations
Crich and co-workers have pioneered an attractive ap-
proach for the introduction of ꢀ-mannosides by in situ
formation of an R-anomeric triflate because of a strong
endoanomeric effect.20,21 An SN2 like-displacement of the
R-triflate by a sugar hydroxyl then results in the formation
of a ꢀ-mannoside. A prerequisite of ꢀ-mannoside formation
is that the donor is protected by a 4,6-O-benzylidene acetal.
It has been proposed that this protecting group opposes
oxacarbenium formation (SN1 glycosylation) due to the
torsional strain engendered by the half chair or boat
conformation of this intermediate and a destabilizing elec-
tronic effect caused by placing the O-6 dipole antiparallel
to the oxacarbenium ion.22
Glycosyl donors 7-12 were therefore prepared (see
Supporting Information) and examined in glycosylations with
glycosyl acceptor 13 using trifluoromethanesulfonic anhy-
dride (Tf2O), 1-benzenesulfinylpiperidine (BSP) as the
promoter system (Table 1).23 The 4,6-diol of the glycosyl
donors is protected as a benzylidene- or p-methoxyben-
zylidene acetal and the C-2 and C-3 hydroxyls by different
sets of orthogonal protecting groups. The latter was deemed
important because previous studies have indicated that steric
and electronic features of the C-2 and C-3 protecting groups
can influence the ꢀ-anomeric selectivity of mannosylations.25
It was anticipated that the Nap,24 TBS, Lev and allyl would
provide an attractive set of orthogonal protecting groups.
Glycosyl donor 8 has a similar structure as 7; however, the
bulky TBS group is used for the protection of the C-2
a Isolated yields of the pure ꢀ anomer. b BSP/Tf2O promotor system.
c p-NO2-C6H4SCl/AgOTf promotor system. d ꢀ/R ratio was determined by
intergration of key signals in the 1H NMR of the reaction mixture purified
by size exclusion chromotography. The ꢀ-anomeric configuration was
confirmed by the C1′-H1′ heteronuclear coupling constant (158-162 Hz)
and the chemical shift of H-5′ (∼3 ppm). e ꢀ/R ratio was the same regardless
of the promotor system used.
hydroxyl and the Nap ether for the C-3 position. Low
temperature (-78 °C) activation of glycosyl donor 7 was
achieved with BSP/Tf2O in the presence of DTBMP in
CH2Cl2.23 Addition of the acceptor and slow warming to -35
°C afforded the mannoside in a disappointing yield of 31%
as a 1/1.5 mixture of ꢀ/R anomers. It has been observed that
a bulky TBS groups at C-3 gives poor ꢀ/R ratios due to a
so-called buttressing effect.25 Indeed, when donor 8 was used,
the stereoselectivity improved dramatically to ꢀ/R>20/1,
however the yield was still moderate (40%). Attempts to
remove the TBS ether using TBAF led to partial Lev
cleavage and buffering with AcOH made the desilylation
impractically slow. Hence, glycosyl donors carrying more
labile silyl protecting groups were evaluated.
(18) Fugedi, P.; Garegg, P. J. Carbohydr. Res. 1986, 149, C9.
(19) Krog-Jensen, C.; Oscarson, S. J. Org. Chem. 1998, 63, 1780.
(20) Crich, D.; Sun, S. X. Tetrahedron 1998, 54, 8321
.
(21) Crich, D.; Sun, S. X. J. Am. Chem. Soc. 1997, 119, 11217
.
(22) Jensen, H. H.; Nordstrom, L. U.; Bols, M. J. Am. Chem. Soc. 2004,
126, 9205.
(23) Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015.
The triisopropylsiloxymethyl (TOM) ether was selected
since it is less sterically demanding than the TBS ether and
can thus be used as a C-3 protecting group and is also readily
(24) Gaunt, M. J.; Yu, J. Q.; Spencer, J. B. J. Org. Chem. 1998, 63,
4172.
(25) Crich, D.; Jayalath, P. Org. Lett. 2005, 7, 2277.
4638
Org. Lett., Vol. 12, No. 20, 2010

Table 2. Selective Removal of the Orthogonal Protecting Groups
entry
product
conditions
yield
1
2
3
4
20: R ) H, R1 ) Lev, R2 ) DEIPS, R3 ) Nap
21: R ) Allyl, R1 ) H, R2 ) DEIPS, R3 ) Nap
22: R ) Allyl, R1 ) Lev, R2 ) H, R3 ) Nap
23: R ) Allyl, R1 ) Lev, R2 ) DEIPS, R3 ) H
PdCl2, NaOAc, AcOH, H2O
NH2NH2•AcOHTol/EtOH
TBAF, AcOH, THF
72%
99%
98%
93%
DDQ, CH2Cl2, H2O
cleaved using TBAF buffered with AcOH.26 Glycosyl donor
9 and its p-methoxybenzylidene derivative 10 showed good
ꢀ-selectivity and moderate yields (53 and 34%, respectively).
In each case, the activation of the donor was exceptionally
clean but when the glycosyl acceptor was added some
byproduct formation was observed. Sulfoxide promoter
systems such as BSP/Tf2O are known to generate electro-
philic byproducts and since glycosyl acceptor 13 is relatively
unreactive, it was assumed that it partly reacted with these
electrophilic byproducts thus lowering the overall yield.27
This assumption was supported by the fact that all the
glycosyl acceptor was consumed even though it was used
in excess. Benzenesulfenyl triflate (generated in situ from
benzenesulfenyl chloride and silver trifluoromethane sul-
fonate) is known to be a powerful activator of thioglycosides
that leads only to an inert disulfide as byproduct when
successful. Thus, commercially available p-NO2C6H4SCl was
used in combination with AgOTf in the presence of DTBMP
for activation of 10.28 Indeed, when 10 was activated with
these reagents, and reacted with 13, disaccharide 17 was
obtained in a much-improved yield of 63%. Although
removability of the TOM was not an issue the stereoselec-
tivity was moderate. Hence, the diethylisopropylsilyl ether
(DEIPS) was evaluated as an orthogonal protecting group
since it has a similar structure to the TBS ether but can more
easily be removed.29,30 Glycosyl donor 11 and it p-meth-
oxybenzylidene derivative 12 showed excellent stereoselec-
tivity (ꢀ/R>20/1). The yields with BSP/Tf2O were moderate
(41 and 40%, respectively) but could be improved to 73%
using p-NO2C6H4SCl/AgOTf. Disaccharide 18 was expected
to be an excellent candidate for the preparation of the core
region of F. tularensis.
the selective removal of the temporary protecting groups
(Table 2). The anomeric allyl ether of 18 could be removed
using PdCl2 in high yield without affecting the other
protecting groups. It is to be expected that lactol 20 can easily
be converted into a trichloroacetimidate and used as a
glycosyl donor in an ensuing glycosylation. The Lev ester
was removed in near quantitative yield using hydrazinium
acetate in a mixture of toluene and ethanol to afford 21.
DEIPS removal was performed under buffered conditions
(TBAF/AcOH) to prevent the aforementioned Lev cleavage
and gave 22 in 98% yield. Finally, the Nap ether was cleaved
by DDQ oxidation in wet CH2Cl2 to provide compound 23
in high yield (93%).
In conclusion, the disaccharide ꢀ-D-Man-(1f4)-D-Man,
modified with four orthogonal protecting groups, was
prepared in high yield with excellent anomeric selectivity.
It was found that the protecting group pattern was critical
for achieving high ꢀ-anomeric selectivity and the best results
were achieved with a mannosyl donor having a C-2 DEIPS,
and a C-3 Nap ether and an acceptor modified with an
anomeric allyl ether and a C-2 Lev ester. High yields of
disaccharide were obtained when p-NO2C6H4SCl/AgOTf was
used as the promoter system for activating an anomeric
thioglycoside. Each temporary protecting group could be
removed in high yield without affecting the other protecting
groups. The new set of orthogonal protecting groups is
expected to be suited for the preparation of a library of F.
tularensis inner-core oligosaccharides and will facilitate the
preparation of other highly branched oligosaccharides.
Acknowledgment. This research was supported by the
National Institute of General Medicine (NIGMS) of the
National Institutes of Health (Grant No 2R01GM065248).
Having established conditions for a high yielding and
stereoselective ꢀ-mannosylation, attention was focused on
(26) Codee, J. D. C.; Krock, L.; Castagner, B.; Seeberger, P. H.
Chem.-Eur. J. 2008, 14, 3987.
1
Supporting Information Available: H and 13C NMR
(27) Codee, J. D. C.; van den Bos, L. J.; Litjens, R.; Overkleeft, H. S.;
van Boeckel, C. A. A.; van Boom, J. H.; van der Marel, G. A. Tetrahedron
2004, 60, 1057.
spectra and experimental procedures for the preparation of
compounds 1-23. This material is available free of charge
via the Internet at http://pubs.acs.org.
(28) Crich, D.; Cai, F.; Yang, F. Carbohydr. Res. 2008, 343, 1858.
(29) Zhu, T.; Boons, G. J. J. Am. Chem. Soc. 2000, 122, 10222
.
(30) Toshima, K.; Mukaiyama, S.; Kinoshita, M.; Tatsuta, K. Tetrahe-
dron Lett. 1989, 30, 6413
.
OL101951U
Org. Lett., Vol. 12, No. 20, 2010
4639