Preparation of tagged glucose as a key intermediate in the synthesis of branched oligosaccharides

Article abstract of DOI:10.1016/j.tetlet.2011.01.149

Tagged and activated d-glucose was introduced as a building block for branched oligosaccharides. This building block was the oligosaccharide branching point at which selectively the C-2 followed by the C-3 position can be glycosylated. After the activation of the C-1 position by oxidation of a thiophenyl group, the newly formed tagged oligosaccharide can be used as a glycoside donor. Synthetic procedures for the preparation of phthalimide-based tag molecules as well as tagged monosaccharides are presented.

Full text of DOI:10.1016/j.tetlet.2011.01.149

Tetrahedron Letters 52 (2011) 1835–1838
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Preparation of tagged glucose as a key intermediate in the synthesis
of branched oligosaccharides
Sunil K. Upadhyay a, Branko S. Jursic a,b,
⇑
a Department of Chemistry, University of New Orleans, New Orleans, LA 70148, United States
b Stepharm, PO Box 24220, New Orleans, LA 70184, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Tagged and activated D-glucose was introduced as a building block for branched oligosaccharides. This
Received 13 December 2010
Revised 28 January 2011
Accepted 31 January 2011
building block was the oligosaccharide branching point at which selectively the C-2 followed by the
C-3 position can be glycosylated. After the activation of the C-1 position by oxidation of a thiophenyl
group, the newly formed tagged oligosaccharide can be used as a glycoside donor. Synthetic procedures
for the preparation of phthalimide-based tag molecules as well as tagged monosaccharides are presented.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
in the development of modern sequencing methods6 such as the
development of protocols toward solution-phase and solid-phase
Glycoproteins, glycolipids, and glycophospholipids (glycoconju-
gates) are components of membranes.1 The oligosaccharide resi-
dues are responsible for intercellular recognition and interaction
and they also act as receptors for proteins, hormones, and viruses
and govern or manipulate immune reactions.2 These significant
activities have stimulated an interest in oligosaccharides and gly-
coconjugates.3 In contrast to peptides and nucleotides, in which
the information content is determined solely by the number and
sequence of different monomer units, the information content of
oligosaccharides is also determined by the site of coupling, the
oligosaccharide synthesis. However, more advances in the oligo-
saccharide synthesis are necessary to drive the development of
diagnostic tests, vaccines and carbohydrate therapeutics.7 The syn-
thesis of carbohydrates has been pursued for more than a century,
and many oligosaccharides can now be synthesized, albeit with
considerable effort.8 This tremendously hampers the exploration
of oligosaccharide biological studies. One can argue that a fully
automated oligosaccharide synthesis process must be developed.9
However, for an efficient automated synthesis a large number of
oligosaccharide building blocks (monomers) and the methodology
to connect them into the oligomers is a necessity. Applying poly-
mer supported syntheses in the preparation of oligosaccharides
has its drawbacks. For oligosaccharide synthesis, the main problem
is associated with use of an excess of the expensive carbohydrate
building blocks that are required to drive the heterogeneous
reaction to completion, and it is very difficult to monitor the
sugar–sugar coupling process by normal characterization methods
such as TLC, NMR and mass spectrometry. Furthermore, the heter-
ogeneous nature of the insoluble polymers and restricted reaction
conditions often result in non-linear reaction kinetics, unequal
distribution, difficult access to the reaction sites, improper solva-
tion, and inefficient coupling rates to name a few.10 To eliminate
some of these problems, new synthetic methodology with TAG
molecules11 must be developed that would take into account the
advantages of both polymer supported reactions (such as easy
purification) and all solution chemistry (such as easy reaction
monitoring and reaction intermediate characterization). Because
the preparation of branched oligosaccharides is a difficult task,
we have employed the use of a tagged glucose that can be used
as a middle building block for various branched oligosaccharides
(Fig. 1). With such an oligosaccharide building block, other saccha-
ride units can be added in positions 1, 2, and 3 selectively.12
configuration of glycosidic linkage (a or b), and by the occurrence
of branching. Thus, polymers made by carbohydrates can carry
considerably more information per building block than proteins
and nucleic acids. Therefore, the chemical synthesis of oligosaccha-
rides is more complicated than that of other biopolymers.4 Due to
the fact that they are not under direct genetic control, glycoconju-
gates are typically heterogeneous. A major source of branched
oligosaccharides is still isolated from natural resources. Unfortu-
nately, the amplification methods—such as the polymerase chain
reaction (PCR) for nucleic acids or bacterial expression systems
for protein production—do not exist for glycoconjugates. Therefore,
the overall progress in glycobiology has suffered from a lack of
tools that would quickly provide a diverse library of oligosaccha-
rides in a similar fashion to ones that are currently available for
nucleic acids and amino acids. Classical all liquid carbohydrate
synthetic methodologies5 are relatively complicated and time-
consuming, therefore oligosaccharide synthesis has been mostly
performed in specialized laboratories. There are some advances
⇑
Corresponding author. Tel.: +1 504 858 0515.
E-mail address: bsjstepharm@ymail.com (B.S. Jursic).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.01.149

1836
S. K. Upadhyay, B. S. Jursic / Tetrahedron Letters 52 (2011) 1835–1838
After a highly efficient method for the preparation of large
TAG Pr otec tion
O
O
quantities of tag reagent 4 was developed our attention was turned
to the preparation of tagged and activated monosaccharide. The
accomplished reaction pathway was shown in Scheme 2 on glu-
O
OH
HO
OH
1
3
cose as a monosaccharide. Starting with D-glucose (5) all hydroxyl
groups were protected with an acetyl group.17 The anomeric acetyl
group of 6 was substituted by thiophenol in methylene dichloride
using borontrifloride acidic catalyst to get compound 7 in 96%
yield.18,23 Acetyl protection groups were removed in almost quan-
titative yields with sodium methoxide in dry methanol to give
2
Figure 1. Proposed tagged glucose as a synthone for preparation of branched
oligosaccharides.
compound 8.19 Same compound was also prepared from
D-glucose
and thiophenyl in aqueous media by using 2-chloro-1,3-dimethy-
2. Results and discussion
limidazolinium chloride as catalyst.20
There are several classic methods for benzaldehyde protection
of monosaccharide.19 It is now common to use dimethyl acetals
of benzaldehyde derivatives in DMF and camphorsulfonic acid as
the catalyst.21 A similar procedure to introduce our tag molecules
with dimethyl acetals of aldehyde 4 was not successful. The main
product of the reaction was the free aldehyde 4. To avoid this prob-
lem, a study preparation of tagged monosaccharide 1 from 8 and
benzyl dibromides 3 in pyridine as reaction media was used.
Although there is some literature evidence that supports this syn-
thetic approach22 in our case isolated yields were around 40% at
best. Therefore, this approach was not applicable to large scale
preparation of tagged monosaccharides. Considering that in the
process of tagging 8 with dimethyl acetal of 4, the corresponding
aldehyde 4 was detected, we developed a method of monosaccha-
ride tagging with the aldehyde 4 (Scheme 2).25 To simplify and
make the reaction more economic, p-toluenesulfonic acid monohy-
drate was used as an acid catalyst with a minimal amount of DMF
to make the benzene solution homogeneous. The reaction was car-
ried with benzene reflux and a Soxhlet extraction apparatus filled
with anhydrous calcium chloride for the removal of water from
azeotropically distilled benzene–water mixture. The reaction was
completed after 3–4 h with isolated yield of tagged monosaccha-
ride between 80% and 90%. The prepared tagged glucose corre-
sponded to the properties that were initially sought after.
The tag attachment should give the tagged saccharide desirable
physical properties that saccharide by itself does not have, such as
solubility in low polarity organic solvents, easy purification, and
easy reaction monitoring and structure elucidation. In addition
the tag attachment should be stable under reaction conditions
required to prepare oligosaccharides, and at the same time easily
removed once the final reaction is completed and the product is
purified. Several variations of these types of tag molecules were
explored and phthalimide derivatives of p-toluidine (Fig. 2) have
proven to be the best in compliance with these required condi-
tions. These compounds are crystalline and are easily purified by
crystallization; they are stable in acidic media which is commonly
required for glycosylation13 but not stable in a base and can be re-
moved from newly formed oligosaccharide at the same time when
ester groups (OH protection) of oligosaccharides are hydrolyzed.
Additionally, the remaining part of the tag (p-aminobenzyl group)
can easily be removed upon neutralization of the reaction mixture.
The thiophenyl group in position 1 is also a protection group that
upon oxidation becomes a good leaving group and the whole mol-
ecule is now a good glycoside donor.14
We selected three tag molecules for our studies. Preparation of
each starts from the readily available anhydrides and p-toluidine.
The large scale microwave-assisted preparation procedures for
compound 215 as well as for their radical dibenzyl bromination16
are well developed. These dibromides were easily hydrolyzed into
the corresponding aldehyde 4 (Scheme 1).24
To demonstrate the efficiency of our tag approach in the prepa-
ration of oligosaccharides, the simplicity of the reaction conditions,
O
a
=
=
N
CH
:
TAG
TAG
TAG
O
TAG
O
O
b
N
CH
:
O
O
SC6H5
O
HO
O
N
O
OH
=
CH
c
:
1
Figure 2. Phthalimide-based tag molecules.
pyridine-water/reflux
85-90%
NBS/benzene/MW
88-92%
R
CHO
R
CH3
R
CHBr2
4
2
3
O
N
O
O
O
O
N
O
; b: R =
; c: R =
N
a
: R =
Scheme 1. Preparation of phthalimide-based tag molecules.

S. K. Upadhyay, B. S. Jursic / Tetrahedron Letters 52 (2011) 1835–1838
1837
HO
HO
HO
AcO
AcO
AcO
AcO
AcO
O
O
i
O
ii
OAc
SC6H5
83%
AcO
96%
OH
5
D-glucose ( )
OAc
OH
OAc
7
6
iii
v
93%
TAG
88%
O
HO
HO
HO
iv
~80%
O
O
O
SC6H5
SC6H5
HO
1a, 1b, and 1c
OH
OH
8
O
O
i = (CH3CO)2O/pyridine;
O
ii = C6H5SH/CH2Cl2/BF3O(C2H5)2;
iii = CH3ONa/CH3OH;
N
a
: R =
N
b
N
O
;
: R =
c
; : R =
iv = 4a, 4b, or 4c/p-CH3C4H4SO3H/DMF-benzene;
v = C6H5SH + 2-chloro-1,3-dimethylimidazolinium chloride
in (C2H5)3N/H2O/CH3CN
O
O
Scheme 2. Preparation of activated and tagged glucose.
O
O
N
O
BzO
O
O
O
i
SC6H5
N
O
BzO
O
+
O
O
BzO
OH
O
O
BzO
BzO
BzO
OBz
OC(=NH)CCl3
ii
HO
SC6H5
10
9
O
1a
OH
BzO
O
iii
HO
N
O
O
HO
O
O
O
SC6H5
O
BzO
O
SC6H5
O
O
O
N
CHO
+
OH
OH
HO
HO
HO
OBz
BzO
OH
BzO
11
O
12
4a
(90-98%)
Scheme 3. Glycosylation, oligosaccharide deprotection, and tag molecule removal. Reagents and conditions: (i) (CH3)3SiOSO2CF3, CH2Cl2/(0 °C to room temperature in 2 h),
75%; (ii) NaOCH3/CH3OH/CH2Cl2 (room temperature), yield 93%; (iii) CF3COOH/CH2Cl2 (room temperature), yield 95%.
and the recyclability of tag aldehydes, we are presenting, as exam-
ple preparations, the synthesis of compounds 10, 11, and 12
(Scheme 3). All reactions were performed in a common organic sol-
vent (dichloromethane). The glycosylation was selectively per-
formed with 9 (see Ref. 26) in the C-3 position of the tagged
monosaccharide and our isolated yield was 75% using standard
glycosylation procedures.26 Product 10 was purified by filtration
of the reaction mixture through a short silica gel column using
dichloromethane–ethyl acetate (3/1) as an eluent. Selective benzo-
ate hydrolysis was started at 0 °C with sodium methoxide in a
dichloromethane–methanol solution. The solution was allowed to
stir at room temperature for 4 h, was evaporated and mixed with
dichloromethane–ethyl acetate and filtered through short column
of silica gel. The tag molecule was removed with trifluroacetic acid
in dichloromethane. The formed precipitate was an oligosaccharide
solution containing the tag aldehyde.
Acknowledgment
We thank the National Science Foundation for financial support
(CHE-0611902).
References and notes
1. Cooper, G. M.; Housman, R. E. The Cell: A Molecular Approach, 5th ed.; ASM
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6. Ashline, D.; Singh, S.; Hanneman, A.; Reinhold, V. Anal. Chem. 2005, 77, 6250–
6262.
In conclusion, we can state that we have developed synthetic
procedures for the preparation of tag reagents based on phthali-
mide of p-toluidine. All procedures are simple and materials can
be obtained in large quantities. Tagging monosaccharides with
these tag molecules is simple, and requires refluxing the DMF/ben-
zene solution of the corresponding tag aldehyde and monosaccha-
ride under Soxhlet extraction apparatus reaction conditions.
Tagged monosaccharides can be glycosylated first in position 3,
then 2, and finally 1 with the same or different saccharide building
blocks. In the last step of the synthesis, the tag molecule can be
selectively deprotected, isolated as an aldehyde, and reused in
new oligosaccharide syntheses.
7. Magnani, J. L.; Ernst, B. Nat. Rev. Drug Disc. 2009, 8, 661–667; Cipolla, L.; Araújo,
A. C.; Bini, D.; Gabrielli, L.; Russo, L.; Shaikh, N. Expert Opin. Drug Discovery
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9. (a) Seeberger, P. H. Chem. Soc. Rev. 2007, 37, 19–28; (b) Seeberger, P. H.; Haase,
W.-C. Chem. Rev. 2000, 100, 4349–4394; (c) Plate, O. J.; Palmacci, E. R.;
Seeberger, P. H. Science 2001, 291, 1523–1527.
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11. (a) Pathak, A. K.; Yerneni, C. K.; Young, Z.; Pathak, V. Org. Lett. 2008, 10, 145–
148; (b) Huang, J.-Y.; Li, A.; Li, J. R. Carbohydr. Polym. 2011, 83, 297–302.
12. (a) Li, A.; Kong, F. Carbohydr. Res. 2004, 339, 2499–2506; (b) Crich, D. Acc. Chem.
Res. 2010, 43, 1144–1153.
13. (a) Robina, I.; Carmona, A. T.; Moreno-Vargas, A. J. Curr. Org. Synth. 2008, 5, 33–
60; (b) Carmona, A. T.; Moreno-Vargas, A. J.; Robina, I. Curr. Org. Synth. 2008, 5,
81–116.

1838
S. K. Upadhyay, B. S. Jursic / Tetrahedron Letters 52 (2011) 1835–1838
14. (a) Crich, D.; Lim, L. B. L. Org. React. 2004, 64, 115–251; (b) France, R. R.;
Compton, R. G.; Davis, B. G.; Fairbanks, A. J.; Rees, N. V.; Wadhawan, J. D. Org.
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separated by filtration, washed with water (3 Â 15 ml) and dried to give 0.61 g
(91%) product. 1H NMR (CDCl3, 400 MHz) d 10.45 (1H, s), 8.48 (2H, s), 8.15 (2H,
m), 8,05 (2H, d, J = 8.4 Hz), 7.78 (2H, d, J = 8.4 Hz) and 7.75 (2H, m) ppm.
13C NMR d 191.5, 166.7, 137.6, 136.0, 136.5, 130.7, 130.6, 129.9, 127.2, 126.9,
and 126.0 ppm.
16. Upadhyay, S. K.; Jursic, B. S. Synth. Commun. in press.
17. Wolfrom, M. L.; Thompson, A. Methods Carbohydr. Chem. 1963, 2, 211–
215.
25. Typical procedure for preparation of tagged monosaccharide 1. Preparation of 2-
[4-(7,8-dihydroxy-6-phenylsulfanyl-hexahydro-pyrano[3,2-d][1,3]dioxin-2-
yl)-phenyl]-benzo[f]isoindole-1,3-dione (1b). Into N,N-dimethylformamide
18. Agnihotri, G.; Tiwari, P.; Misra, A. K. Carbohydr. Res. 2005, 340, 1393–1396.
19. Lindhorst, T. K. Essentials of Carbohydrate Chemistry and Biochemistry, 2nd ed.;
Wiley-VCH: Weinheim, Germany, 2003.
20. Tanaka, T.; Matsumoto, T.; Noguchi, M.; Kobayashi, A.; Shoda, S.-I. Chem. Lett.
2009, 38, 458–459.
(5 ml) solution of aldehyde 4b (222 mg, 1.59 mmol) and saccharide 9
(200 mg, 1.65 mmol), benzene (30 ml) solution of p-toluenesulfonic acid
monohydrate (15 mg) was added. Resulting mixture refluxed at reduced
pressure (17 mmHg) for 4 h with condenser equipped with anhydrous calcium
chloride filed Dean–Stark’ apparatus. Reaction mixture was diluted with
dichloromethane (100 ml) and washed saturated sodium bicarbonate solution
(3 Â 30 ml) and evaporated. Solid residue was dissolved in dichloromethane
(5 ml) filtered through short column of silica gel. Silica gel was washed with
ethyl acetate/dichloromethane (3 Â 20 ml, 1:4 volume ratio) and combined
filtrates are evaporated to give 335 mg (82%) of white solid product. 1H NMR
(CDCl3, 400 MHz) d 8.52 (s, 2H), 8.15 (m, 2H), 7.68 (m, 4H), 7.62 (dd, 2H), 7.5–
7.45 (m, 4H), 7.28 (m, 3H), 5.6 (s, 1H), 4.62 (d, 1H), 4.4 (dd, 1H), 3.8 (m, 1H), and
3.58–3.40 (m, 2H) ppm. 13C NMR d 168.2, 138.0, 136.1, 134.2, 131.0, 129.6,
129.5, 129.4, 129.0, 128.1, 126.5, 126.0, 102.0, 89.5, 80.9, 75.3, 72.5, 71.1, and
68.2 ppm.
21. For instance, see Ellervik, U. Tetrahedron Lett. 2003, 44, 2279–2281.
22. Rajanbabu, T. V. J. Org. Chem. 1988, 53, 4522–4530.
23. Penta-O-acetyl-D-glucopyranose (6) was prepared from D-glucose and acetic
anhydride in pyridine by following literature procedure. Phenyl tetra-O-acetyl-
1-thio-b-
glucose
D
-glucopyranoside (7) was prepared from both 7 and directly from
by following literature Phenyl-1-thio-b-
procedure.18
D
-
-
D
glucopyranoside (8) was prepared directly from
D
-glucose20 and also by
CH3ONa/CH3OH hydrolysis of all ester groups protected saccharide 7.
24. Typical procedure for preparation of benzaldehyde 4. Preparation of 4-(1,3-dioxo-
1H-benzo[f]isoindol-2(3H)-yl)benzaldehyde (4b). Dibromide 3b (1 g
2.25 mmol) was refluxed in pyridine (15 ml) for 2 h and then poured over
crushed ice (ꢀ100 g) and stirred for 30 min. Formed white precipitate was
26. Schmidt, R. R.; Kinzy, W. Carbohydr. Chem. Biochem. 1994, 50, 21–124.