From solution phase to "on-column" chemistry: Trichloroacetimidate-based glycosylation promoted by perchloric acid-silica

Article abstract of DOI:10.1021/jo051390g

Activation of ester-protected glycosyl trichloroacetimidate donors by perchloric acid immobilized on silica afforded 1,2-trans disaccharides in 60-90% yields. Applying this approach to one-pot sequential glycosylation resulted in efficient syntheses of th

Full text of DOI:10.1021/jo051390g

the precise detail of carbohydrate action remains to be
delineated in many, perhaps most cases.2 The require-
ment for rapid, straightforward syntheses of oligosac-
charides and glycoconjugates is therefore obvious. Of the
various synthetic strategies developed to date, glycoside
syntheses based on glycosyl trichloroacetimidates are
particularly popular,8 second only to thioglycoside-based
reactions.9 Glycosyl imidate donor species were first
reported by Sinay¨ in 1976,10 with Schmidt and co-workers
subsequently introducing the corresponding trichloro-
acetimidates in 1980.11 Recently Yu and co-workers
explored the application of N-substituted glycosyl tri-
fluoroacetimidates12 and reported particularly good re-
activity for sialic acid glycosylation reactions. Demchenko
and co-workers13 noted that thioimidates are versatile
donors for 1,2-cis and 1,2-trans glycosylation reactions.
A number of acids have been exploited as promoters
for trichloroacetimidate-based reactions, including
TMSOTf,14 BF3.Et2O,15 and TBSOTf.16 Noting recent
reports of the use of perchloric acid immobilized on silica
gel as an acid catalyst for various reactions in carbohy-
drate chemistry, including acetylation of reducing sug-
ars17 and Ferrier rearrangement18 of glycals, we were
drawn to explore the application of this reagent system.19
Herein we describe the use of HClO4-silica as an efficient
promoter for conventional and two-step/one-pot solution
phase glycosylation reactions using glycosyl trichloro-
acetimidate donors. In addition, we demonstrate the use
of silica-perchloric acid in solid-phase, “on-column”
glycosylation reactions with both sugar and amino acid
alcohol acceptors.
From Solution Phase to “On-Column”
Chemistry: Trichloroacetimidate-Based
Glycosylation Promoted by Perchloric
Acid-Silica
Balaram Mukhopadhyay,* Sarah V. Maurer,
Nadine Rudolph, Renate M. van Well,
David A. Russell, and Robert A. Field*
Centre for Carbohydrate Chemistry, School of Chemical
Sciences and Pharmacy, University of East Anglia,
Norwich NR4 7TJ, United Kingdom
r.a.field@uea.ac.uk
Received July 6, 2005
Activation of ester-protected glycosyl trichloroacetimidate
donors by perchloric acid immobilized on silica afforded 1,2-
trans disaccharides in 60-90% yields. Applying this ap-
proach to one-pot sequential glycosylation resulted in effi-
cient syntheses of the N-linked glycan trimannoside and LeX
and LeA trisaccharides in very good yield (76%, 62%, and
59% yields, respectively). Solution phase reactions were also
translated to a solid phase format; priming the top of a
standard silica chromatography column with perchloric acid
immobilized on silica facilitated “on-column” glycosylation
with subsequent “in situ” purification of products. Coupling
yields from this approach were comparable to those obtained
from the corresponding solution-phase disaccharide cou-
plings. A series of glycosylated amino acids were also
synthesized in high yield with use of the on-column ap-
proach.
Initial experiments with 2,3,4,6-tetra-O-acetyl-R-D-
glucopyranosyl trichloroacetimidate 1 (1.3 mmol) as
(5) (a) Emilsson, G. M.; Breaker, R. R. Cell. Mol. Life Sci. 2002, 59,
596-607. (b) Breaker, R. R. Nat. Biotechnol. 1997, 15, 427-431. (c)
Breaker, R. R. Science 2000, 290, 2095-2096.
(6) Vacquier, V. D.; Moy, G. W. Dev. Biol. 1997, 192, 125-135.
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(8) Schmidt, R. R.; Jung, K.-H. Carbohydrates in Chemistry and
Biology; Ernst, B., Hart, G. W., Sinay¨, P., Eds.; Wiley-VCH: Weinheim,
Germany, 2000, pp 5-60 and references therein.
(9) Oscarson, S. Glycoscience: Chemistry and Biology I; Fraser-Reid,
B., Tatsuta, K., Thiem, J., Eds.; Springer-Verlag: Berlin, Germany,
2001; pp 643-670 and references therein.
(10) (a) Pougny, J.-R.; Sinay¨, P. Tetrahedron Lett. 1976, 4073-4076.
(b) Pougny, J.-R.; Jacquinet, J.-C.; Nassr, M.; Duchet, D.; Milat, M.-
L.; Sinay¨, P. J. Am. Chem. Soc. 1977, 99, 6762-6763.
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19, 731-733.
Over the last twenty years or so, study of the biological
function of carbohydrates and glycoconjugates, previously
a largely neglected dimension of biological complexity,
has become one of the fastest growing areas in biochem-
istry and cell biology.1,2 It is now clearly understood that
carbohydrates are actively involved in biological events
involving cell-cell interactions,3 inflammation,4 signal
transduction,5 and fertility and development.6,7 However,
(12) (a) Yu, B.; Tao, H. Tetrahedron Lett. 2001, 2405-2407. (b) Yu,
B.; Tao, H. J. Org. Chem. 2002, 67, 9099-9102.
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references therein. (b) Demchenko, A. V.; Pornsuriyasak, P.; De Meo,
C.; Malysheva, N. N. Angew. Chem., Int. Ed. 2004, 43, 3069-3072.
(14) Schaubach, R.; Hemberger, J.; Kinzy, W. Liebigs Ann. Chem.
1991, 607-614.
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Zimmermann, P.; Bommer, R.; Ba¨r, T.; Schmidt, R. R. J. Carbohydr.
Chem. 1988, 7, 435-452.
(1) (a) Varki, A. Glycobiology 1993, 3, 97-130 and references
therein. (b) Essentials of Glycobiology; Varki, A., Cummings, R., Esko,
J., Freeze, H., Hart, G., Martin, J., Eds.; Cold Spring Harbor Labora-
tory Press: Cold Spring Harbor, NY, 1993.
(2) (a) Feizi, T. Glycoconj. J. 2000, 17, 553-565. (b) Le Poole, I. C.;
Gerberi, M. A. T.; Kast W. M. Curr. Opin. Oncol. 2002, 14, 641-648.
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496.
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Res. 2005, 340, 1075-1080. (b) Mukhopadhyay, B.; Collet, B.; Field,
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10.1021/jo051390g CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/01/2005
J. Org. Chem. 2005, 70, 9059-9062
9059

TABLE 1. Glycosylation Reactions of a Range of
Trichloroacetimidate Donors and Sugar Alcohol
Acceptors Promoted by HClO4-Silica
D-mannose, bromoethyl R-D-mannopyranoside 25 was
prepared by a Fischer-type glycosylation with use of
bromoethanol and HClO4-silica. Ortho-esterification of
the bromoethyl glycoside 25 with trimethyl ortho-
benzoate20a and HClO4-silica gave the corresponding 2,3:
4,6-di-ortho ester derivative, which was rearranged by
the addition of H2O to afford the required 2,4-di-O-
benzoyl-R-D-mannopyranoside 26 along with 2,6-di-O-
benzoyl-R-D-mannopyranoside 27 in 84% yield (26:27 1.5:
1.0) (Scheme 1). With the monosaccharide diol acceptor
26 in hand, glycosylation was performed with an excess
of 2,3,4,6-tetra-O-acetyl-R-D-mannopyranosyl trichloro-
acetimidate 3 (3.0 equiv with respect to the acceptor),
using HClO4-silica as the promoter. The desired tri-
mannoside derivative 2822 was obtained in 76% yield
(Scheme 1).
Success in the one-pot trimannoside synthesis led us
to explore the use of HClO4-silica for the synthesis of
more complex oligosaccharides, such as LeX and LeA
trisaccharide derivatives. Here the relative reactivity of
the 3- and 4-OH groups23 of a partially protected N-
acetylglucosamine building block 29 offers scope for one-
pot sequential glycosylation,24 so limiting the number of
protecting group manipulations and removing rounds of
chromatographic purification. The acceptor diol 29 was
synthesized by protecting the primary hydroxyl group of
known allyl 2-deoxy-2-(2,2,2-trichloroethoxycarbonylami-
no)-R-D-glucopyranoside 2925 with a tert-butyldiphenyl-
silyl (TBDPS) group, which leaves the 3- and 4-OH
groups free for glycosylation with fucosyl and galactosyl
residues, in either order, to achieve the target LeX/LeA
trisaccharides (Scheme 2). The TBDPS group not only
serves the purpose of selective protection of the primary
hydroxyl group but also provides sufficient lipophilicity
so that the diol acceptor is soluble in organic solvent (e.g.,
CH2Cl2). Accordingly, acceptor 29 was reacted with
fucosyl trichloroacetimidate donor 30,26 chosen for its
known ability to give R-fucopyranosides in high yield,27
in the presence of HClO4-silica in DCE-Et2O (2:1).28 The
exclusive product was the 3-O-R-fucosylated disaccharide
31, which was formed with 1,2-cis stereoselectivity. The
stereo- and regioselectivity of the reaction was confirmed
by isolating a sample of disaccharide from the reaction
donor provided 72% of the desired 1,2-trans disaccharide
9 when reacted at -10 °C for ca. 1 h in dichloroethane
(10 mL) with methyl 2,3,4-tri-O-benzyl-R-D-glucopyrano-
side 6 (1.0 mmol) in the presence of HClO4-silica (50 mg)
(Table 1). The only other notable components at the end
of the reaction period were unreacted acceptor and
hydrolyzed donor. Encouraged by this result, a series of
ester-protected glycosyl tricholoroacetimidate donors were
synthesized and used for glycosylation reactions with a
series of primary and secondary alcohol acceptor sub-
strates. Results of these experiments are summarized in
Table 1; yields quoted are for the isolated products
formed in reactions conducted by undergraduate students
with no prior experience in carbohydrate chemistry. The
good-to-excellent yields obtained reflect the robustness
of the HClO4-silica glycosylation procedure. Due to the
greater reactivity of primary alcohols, reactions with
primary acceptor 6 gave better yields (72-94%) than
reactions employing secondary alcohol acceptors 7 and
8 (55-93%). Excellent glycosylation yields from reactions
employing the rhamnosyl trichloroacetimidate 4 and
arabinosyl trichloroacetimidate 5 showed that the strat-
egy is also effective for deoxy and pentose sugars,
respectively.
The success of HClO4-silica as a promoter for disac-
charide syntheses prompted further studies toward trisac-
charide syntheses. 3,6-Di-O-(R-D-mannopyranosyl)-R-D-
mannopyranoside, the N-linked glycan core trimannoside,
is the natural ligand for the lectin concanavalin A.20
There are several reported approaches for the synthesis
of this type of trisaccharide.21 Our approach was to
establish a dimannosylation strategy using HClO4-silica
as promoter, while also investigating this reagent system
in all other steps in the synthesis. Starting from free
1
mixture prior to addition of the second donor. H NMR
spectroscopic analysis (δH 5.26 ppm, J ) 3.2 Hz, H-1Fuc)
(20) Naismith, J. H.; Field, R. A. J. Biol. Chem. 1996, 271, 972-
976 and references therein.
(21) (a) Oscarson, S.; Tide´n, A.-K. Carbohydr. Res. 1993, 247, 323-
328. (b) Yu, H. N.; Ling, C.-C.; Bundle, D. R. J. Chem. Soc., Perkin
Trans. 1 2001, 832-837. (c) Ratner, D. M.; Plante, O. J.; Seeberger,
P. H. Eur. J. Org. Chem. 2002, 5, 826-833.
(22) Karamanska, R.; Mukhopadhyay, B.; Russell, D. A.; Field, R.
A. Chem. Commun. 2005, 3334-3336.
(23) Gallo-Rodriguez, C.; Gil-Libarona, M. A.; Mendoza, V. M.; de
Lederkremer, R. M. Tetrahedron 2002, 58, 9373-9380.
(24) (a) Tanaka, H.; Matoba, N.; Tsukamoto, H.; Takimoto, H.;
Yamada, H.; Takahashi, T. Synlett 2005, 5, 824-828. (b) Tanaka, H.;
Adachi, M.; Tsukamoto, H.; Ikeda, T.; Yamada, H.; Takahashi, T. Org.
Lett. 2004, 4, 4213-4216.
(25) Kusomoto, S.; Yoshimura, H.; Imoto, M.; Shimamoto, T.; Shiba,
T. Tetrahedron Lett. 1985, 26, 909-912.
(26) Manzoni, L.; Castelli, R. Org. Lett. 2004, 6, 4195-4198.
(27) Windmu¨ller, R.; Schmidt, R. R. Tetrahedron Lett. 1994, 35,
7927-7930.
(28) When CH2Cl2 was used as solvent at -20 °C, an R/â mixture
of the disaccharide was obtained in a ratio of 2:1. However, the same
reaction in DCE-Et2O (2:1) at -40 °C afforded only R-glycoside. The
result is consistent with solvent effects on glycosylation reactions:
Demchenko, A.; Stauch, T.; Boons, G.-J. Synlett 1997, 818-820.
9060 J. Org. Chem., Vol. 70, No. 22, 2005

SCHEME 1. HClO4-Silica-Promoted Trimannoside Synthesisa
a Reagents and conditions: (a) 2-bromoethanol, HClO4-silica, CH3CN, 45%; (b) (i) trimethyl orthobenzoate, HClO4-silica, CH3CN, (ii)
H2O, 84% (26:27 1.5:1.0); (c) HClO4-silica, CH2Cl2, 76%.
SCHEME 2. One-Pot Sequential Glycosylation Reactions, Promoted by Perchloric Acid-Silica, for the
Synthesis of LeX and LeA Trisaccharide Derivatives
proved consistent with formation of the 1,2-cis-R-fucoside
linkage; a downfield shift of the H-3 signal of the
GlcNHTroc moiety (from 3.98 to 4.38 ppm) confirmed the
(1f3)-fucosyl linkage. After completion of disaccharide
formation, as judged by TLC, galactosyl trichloroacetimi-
date donor 2 was added to the reaction mixture to afford
the protected LeX trisaccharide derivative 32 in 62%
isolated yield (Scheme 2).
In a similar fashion, reaction of diol acceptor 29 with
galactosyl trichloroacetimidate 2, followed by the fucosyl
trichloroacetimidate 30, gave the LeA trisaccharide de-
rivative 34 in 59% isolated yield (Scheme 2). The re-
giospecificity of these reactions is clearly apparent by
comparison of the anomeric carbon region of 13C NMR
spectra of the LeX derivative 32 with that of the LeA
derivative 34 (see Figure 1 in the Supporting Informa-
tion). Although the Rf for both LeX and LeA derivatives
are the same (Rf 0.25, 2:1 n-hexanes-ethyl acetate), only
single trisaccharide derivatives were isolated in both sets
of reactions. It is important to note that when similar
reactions were performed in the presence of TMSOTf as
a promoter, the first step (i.e., the formation of the
disaccharides) was successful but subsequent addition of
a second glycosyl donor to the reaction only gave rise to
a mixture of compounds, dominated by hemiacetal arising
from hydrolysis of the second donor substrate. It would
appear that TMSOTf-mediated activation of trichloro-
acetimidates is more moisture sensitive than HClO4-
silica based glycosylation reactions.
Since the above reactions essentially involve adding
silica to a reaction, we next considered adding the
reaction to silica. That is, a chromatography column filled
with flash silica gel was topped with a band of perchloric
acid-silica, with a view to conducting the reactions “on-
column”, followed by “in situ” purification of the products.
Useful results were obtained when a mixture of glucosyl
trichloroacetimidate donor 1 and glucosyl acceptor 6 was
employed. These two reactants were charged onto the
column in dry CH2Cl and left for 30 min. The column
2
was then eluted with 2:1 n-hexanes-ethyl acetate and
the desired disaccharide 9 was obtained in 64% yield, a
yield comparable to that obtained in the corresponding
solution phase reaction. Further representative experi-
ments from Table 1 were therefore repeated with the “on-
column” approach, again with yields comparable to those
obtained from solution phase chemistry (see Table 2 in
the Supporting Information).
The activity of many proteins is highly dependent on
their glycosylation state.29 Synthesis of homogeneous
glycopeptides, and hence glycosylated amino acid build-
ing blocks, is therefore topical.30,31 We therefore focused
our attention on the synthesis of O-glycosylated amino
(29) Sears, P.; Wong, C.-H. Cell. Mol. Life Sci. 1998, 54, 223-252.
J. Org. Chem, Vol. 70, No. 22, 2005 9061

TABLE 2. “On-Column” Synthesis of Glycosylated
Amino Acids
column” glycosylation, and subsequent “in situ” separa-
tion, provides a novel and robust method for glycoside
synthesis. Studies to explore the compatibility of the
procedures described with other glycosylation building
blocks are ongoing.
Experimental Section
Preparation of HClO4 Immobilized on Silica. Immobi-
lized perchloric acid on silica was prepared essentially as
described previously18a except that it was dried for 2 h at 110
°C instead of 6 h. HClO4 (0.3 mmol, as a 70% aqueous solution)
was added to a slurry of silica gel (5 g, 200 mesh) in Et2O (15
mL) and the solvent was removed under reduced pressure. The
resulting powder was kept at 110 °C for 2 h and used directly
in reactions.
General Procedure for Solution Phase Glycosylation
Reactions. A mixture of glycosyl acceptor (1 mmol), glycosyl
trichloroacetimidate (1.3 mmol), and 4 Å MS (1 g) in dry DCE
(10 mL) was cooled to -10 °C. HClO4-silica (50 mg) was added
and the mixture was allowed to stir until TLC (n-hexanes-
EtOAc 2:1) showed complete disappearance of the starting
materials. The mixture was filtered through a pad of Celite and
the filtrate was evaporated in vacuo. The crude product was
purified by flash chromatography to afford compounds 9-23,
the structures of which were confirmed by 1H and 13C NMR
spectroscopy and mass spectrometry (Supporting Information).
General Procedure for “On-Column” Glycosylation Re-
actions. A 45 × 1.5 cm2 glass column was dry-packed with flash
silica gel (20 g) and HClO4-silica (5 g) on top. Then a mixture
of donor (1.3 mmol) and acceptor (1 mmol) in dry CH2Cl2 (1 mL)
was carefully charged onto the column, which was allowed to
stand at room temperature for 30 min. The column was then
eluted with 1:1 n-hexanes-EtOAc to afford glycosylated amino
acid followed by unreacted amino acid derivative and the
corresponding hemiacetal of the donor.
acids using the “on-column” glycosylation approach. A
mixture of Fmoc-threonine benzyl ester 37 or Fmoc-
serine benzyl ester 38 and a range of “disarmed” glycosyl
trichloroacetimidate donors in dry CH2Cl2 were charged
onto columns. After 10 min, the columns were eluted with
1:1 n-hexanes-EtOAc, giving the 1,2-trans-linked O-
glycosides in good yield (Table 2). Unreacted amino acid
derivative and the corresponding hemiacetal of the donor
were isolated as byproducts; 1,2-cis-glycosides were not
isolable in any case.
In summary, perchloric acid-silica serves as a conve-
nient and effective promoter for trichloroacetimidate-
based glycosylation reactions with ester-protected gly-
cosyl donors. One- and two-step glycosylation reactions
in solution give very good yields of the requisite glycoside
products. In addition, use of perchloric acid-silica for “on-
Acknowledgment. We acknowledge Dr. Alan H.
Haines for helpful discussions during the course of this
work, Mr. Colin MacDonald for assistance with NMR
spectroscopy, and the EPSRC Mass Spectrometry Ser-
vice Centre, Swansea for invaluable support. This work
was supported by the UK Engineering and Physical
Science Research Council and the European Union
through a Marie Curie Intra-European Fellowship
(R.M.v.W.).
Supporting Information Available: Literature refer-
ences for the synthesis of glycosyl donors and acceptors (1-
8); references for known disaccharides (9-13); references for
known glycosylated amino acids (39-45); specific rotations and
1H and 13C NMR and high-resolution mass spectral data for
all new compounds, along with copies of NMR spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(30) (a) Davis, B. G. Chem. Rev. 2002, 102, 579-601. (b) MacMillan,
D.; Daines, A. M. Curr. Med. Chem. 2003, 10, 2733-2773.
(31) Reviewed in: Pratt, M. R.; Bertozzi, C. R. Chem. Soc. Rev. 2005,
34, 58-68.
JO051390G
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