Syntheses of LewisX and dimeric LewisX: Construction of branched oligosaccharides by a combination of preactivation and reactivity based chemoselective one-pot glycosylations

Article abstract of DOI:10.1021/jo701694k

(Chemical Equation Presented) Two asymmetrically branched oligosaccharides, Lewis^X and dimeric Lewis^X, were assembled in one pot with high yields and exclusive regio- and stereoselectivities. p-Tolyl thioglycosides were utilized as t

Full text of DOI:10.1021/jo701694k

Syntheses of LewisX and Dimeric LewisX:
Construction of Branched Oligosaccharides by a
Combination of Preactivation and Reactivity
Based Chemoselective One-Pot Glycosylations
promising target for carbohydrate based anticancer vaccine
studies.6 With their biological significance and structural and
stereochemical complexities, Lewis antigens have served as
targets for the development of new synthetic methodologies,7,8
including automated solid-phase synthesis,9 automated parallel
synthesis in solution,7 soluble polymer supported synthesis,10
and reactivity based chemoselective glycosylation.11,12
Recently, we have developed a preactivation based chemose-
lective one-pot glycosylation method, where a thioglycosyl
donor is activated in the absence of an acceptor.13 Upon
completion of the activation, addition of a thioglycosyl acceptor
will lead to the formation of a disaccharide containing a thioaryl
aglycon, ready for the next round of preactivation and glyco-
sylation. Multiple glycosylations can be performed in a single
reaction flask without intermediate oligosaccharide purifications,
thus significantly expediting the glycoassembly process. We
have demonstrated that this is a powerful methodology, which
has been successfully applied in syntheses of linear oligosac-
charides, including hyaluronic acid oligosaccharides,14 heparin
trisaccharides,15 chitotetraose,16 and GloboH.17 Herein, we report
the application of the preactivation based methodology to one-
pot construction of branched oligosaccharides.
Adeline Miermont,†,‡ Youlin Zeng,†,‡ Yuqing Jing,†
Xin-shan Ye,§ and Xuefei Huang*,†
Department of Chemistry, The UniVersity of Toledo, 2801 West
Bancroft Street, MS 602, Toledo, Ohio 43606,§The State Key
Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking UniVersity, Xue Yuan Rd. 38,
Beijing 100083, China
xuefei.huang@utoledo.edu
ReceiVed August 2, 2007
Two asymmetrically branched oligosaccharides, LewisX and
dimeric LewisX, were assembled in one pot with high yields
and exclusive regio- and stereoselectivities. p-Tolyl thiogly-
cosides were utilized as the sole type of building blocks,
thus simplifying the overall synthetic design. The reactivity-
independent nature of the preactivation based method allows
modular assembly of the dimeric LewisX octasaccharide
without the need for tedious protective group manipulation
to achieve exact anomeric reactivities.
It is a challenging task to assemble Lewis antigens 1 and 2
in one pot. It is well-known that the 4-hydroxyl group of
glucosamine derivatives has very low nucleophilicity.18 In
addition, fucosylation on 3-OH needs to be carried out with
high R selectivity for efficient one-pot synthesis. The in situ
anomerization procedure for introducing R fucosyl linkage19,20
(6) Danishefsky, S. J.; Allen, J. R. Angew. Chem., Int. Ed. 2000, 39,
836-863.
(7) Tanaka, H.; Matoba, N.; Tsukamoto, H.; Takimoto, H.; Yamada, H.;
Takahashi, T. Synlett 2005, 824-828 and references cited therein.
(8) An excellent review on Lewis family oligosaccharide and sphingolipid
syntheses: Vankara, Y. D.; Schmidt, R. R. Chem. Soc. ReV. 2000, 29, 201-
216 and references cited therein.
The Lewis family of oligosaccharides, as represented by
LewisX pentasaccharide 1 and dimeric LewisX octasaccharide
2, is involved in a wide array of biological events, such as
modulation of the immune system toward a Th2 response1 and
bacterial and viral infection.2,3 In addition, they are known to
be overexpressed on tumor cell surface,4,5 thus providing a
(9) Love, K. R.; Seeberger, P. H. Angew. Chem., Int. Ed. 2004, 43, 602-
605.
† The University of Toledo.
(10) Zhu, T.; Boons, G.-J. Chem. Eur. J. 2001, 7, 2382-2389.
(11) Mong, K.-K. T.; Wong, C.-H. Angew. Chem., Int. Ed. 2002, 41,
4087-4090.
‡ These authors have contributed equally to this work.
§ Peking University.
(1) Okano, M.; Nishizaki, K.; Da’dara, A.; Thomas, P.; Carter, M.; Harn,
D. A. J. In Vaccine AdjuVants; Hackett, C. J., Harn, D. A. J., Eds.; Humana
Press Inc.: Totowa, NJ, 2006; pp 177-191.
(12) Zhang, Z.; Niikura, K.; Huang, X.; Wong, C.-H. Can. J. Chem.
2002, 121, 1051-1054.
(13) Huang, X.; Huang, L.; Wang, H.; Ye, X.-S. Angew. Chem., Int.
Ed. 2004, 42, 5221-5224.
(2) de Witte, L.; Nabatov, A.; Pion, M.; Fluitsma, D.; de Jong, M. A.
W. P.; de Gruijl, T.; Piguet, V.; van Kooyk, Y.; Geijtenbeek, T. B. H. Nat.
Med. 2007, 13, 367-371.
(14) Huang, L.; Huang, X. Chem. Eur. J. 2007, 13, 529-540.
(15) Huang, L.; Teumelsan, N.; Huang, X. Chem. Eur. J. 2006, 12,
5246-5252.
(3) Chmiela, M.; Wadstrom, T.; Folkesson, H.; Malecka, I. P.; Czk-
wianianc, T.; Rechcin´ski, T.; Rudnicka, W. Immunol. Lett. 1998, 61, 119-
125.
(16) Huang, L.; Wang, Z.; Li, X.; Ye, X.-S.; Huang, X. Carbohydr. Res.
2006, 341, 1669-1679.
(4) Holmes, E. H.; Hakomori, S.; Ostrander, G. K. J. Biol. Chem. 1987,
262, 15649-15658.
(5) Fukushi, Y.; Hakomori, S.; Nudelman, E.; Cochran, N. J. Biol. Chem.
1984, 259, 4681-4685.
(17) Wang, Z.; Zhou, L.; El-Boubbou, K.; Ye, X.-S.; Huang, X. J. Org.
Chem. 2007, 72, 6409-6420.
(18) Crich, D.; Dudkin, V. J. Am. Chem. Soc. 2001, 123, 6819-6825.
(19) Shao, N.; Guo, Z. W. Pol. J. Chem. 2005, 79, 297-307.
10.1021/jo701694k CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/16/2007
8958
J. Org. Chem. 2007, 72, 8958-8961

TABLE 1. One-Pot Synthesis of Oligosaccharides 10-12 and 14
of p-TolSCl chemoselectively activated 5 leading to trisaccharide
10. Lactoside 7 was then added followed by AgOTf and
p-TolSCl producing the fully protected LewisX pentasaccharide
11 with 40-60% yield in just 4 h for this four-component one-
pot synthesis.
protocol
donor
B1
B2
B3
Pdt
yield (%)
1a
2b
3c
4d
A
B
B
B
3
3
4
6
6
6
5
5
5
7
7
11
10
12
14
40-60
68
71
44-61
10
13
a Donor:B1:B2:B3 ) 1.1:1.0:1.1:0.8. b Donor:B1:B2 ) 1.1:1.0:1.1. c Do-
nor:B1:B2 ) 1.4:1.0:1.2. d Donor:B1:B2 ) 1.1:1.0:0.7.
In a separate experiment, the one-pot reaction was stopped
prior to addition of the lactoside acceptor 7, from which
trisaccharide 10 was isolated as the sole trisaccharide in an
excellent 68% yield (Table 1, entry 2). Trisaccharide 10 was
characterized by extensive NMR experiments. Correlations of
the anomeric carbon of the Gal unit (99.8 ppm) with H4 of GlcN
(4.25 ppm) and the anomeric carbon of Fuc (98.0 ppm) with
the H3 of GlcN (4.68 ppm) were observed in its gHMBC
spectrum, confirming that galactosylation occurred exclusively
on the C4-OH of diol 6. The anomeric configurations of newly
formed glycosyl linkages were established by coupling constants
of anomeric protons (Gal 4.99 ppm, 3JH1,H2 ) 7.8 Hz indicating
â linkage; Fuc 4.64 ppm, 3JH1,H2 ) 4.2 Hz suggesting R linkage).
Recently, an elegant synthesis of LewisX pentasaccharide9
was reported with use of the automated solid-phase synthesis
method pioneered by Seeberger and co-workers.32 The glycoas-
sembly process on the carbohydrate synthesizer took 18 h with
10-15 equiv of each glycosyl building block with a 12.7%
overall yield. As a comparison, through one-pot synthesis,
LewisX was assembled rapidly with higher yield without
resorting to the usage of a large excess of building blocks, which
can be very tedious to prepare. Furthermore, the progress of
the one-pot synthesis can be easily followed by TLC allowing
convenient reaction monitoring and the intermediate oligosac-
charide can be readily characterized as demonstrated by trisac-
charide 10. This highlights that our one-pot method comple-
ments well the existing automated solid-phase synthesis method.
is not applicable since the reaction condition cannot be extended
to â glycosylations. Furthermore, the rate of glycosylation with
the in situ anomerization procedure is low requiring room
temperature overnight, which is undesirable for multiple se-
quential glycosylations in one pot. To overcome these difficul-
ties, we designed building blocks 3-8 with glucosamine diol 6
serving as a key compound. Keeping the C3 hydroxyl group of
6 unprotected reduces the steric hindrance to the C4 hydroxyl
group thus increasing its nucleophilicity. In addition, this allows
fucosylation on C3-OH immediately following â-glycosylation
of C4-OH without the need to remove the C3 protective group.
The N-Phth moiety in 6 is crucial to ensure exclusive regiose-
lectivity for â-galactosylation of 4-OH, as smaller Troc,21 azido,
or acetamido groups22 on C2 led to regioisomers. Diol 8 was
examined initially as the lactoside acceptor with its axial 4-OH
assumedtobemuchlessreactivethantheequatorial3-OH.10,12,23,24
However, glycosylation of 8 produced two regioisomeric
oligosaccharides in similar quantities.25 The lack of regioselec-
tivity led to the use of lactoside acceptor 7 for our synthesis.26
Preactivation of galactoside 3 in dichloromethane (DCM) at
-78 °C by p-TolSOTf,13 formed in situ through reaction of
p-TolSCl and AgOTf, was followed by addition of acceptor 6
and a sterically hindered base, tri-tert-butylpyrimidine (TTBP)27
(Table 1, entry 1). TLC analysis indicated that acceptor 6
disappeared within just a few minutes. Fucosyl donor 5 was
then added to the reaction mixture as a solution in diethyl ether,
a solvent known to favor the formation of thermodynamically
more stable axial product.28-30 Because the armed fucosyl donor
5 has high anomeric reactivity,31 addition of another equivalent
(20) Vermeer, H. J.; Van Dijk, C. M.; Kamerling, J. P.; Vliegenthart, J.
F. G. Eur. J. Org. Chem. 2001, 193-203 and references cited therein.
(21) Koeller, K. M.; Wong, C.-H. Chem. Eur. J. 2000, 6, 1243-1251.
(22) Ellervik, U.; Magnusson, G. J. Org. Chem. 1998, 63, 9314-9322.
(23) Lahmann, M.; Bu¨low, L.; Teodorovic, P.; Gyba¨ck, H.; Oscarson,
S. Glycoconj. J. 2004, 21, 251-256.
(24) Andersen, S. M.; Ling, C.-C.; Zhang, P.; Townson, K.; Willison,
H. J.; Bundle, D. R. Org. Biomol. Chem. 2004, 2, 1199-1212.
(25) Jing, Y. Fluorous Thiols in Oligosaccharide Synthesis and Iterative
One-pot Synthesis of Tumor-associated Carbohydrate Antigen LewisX
Pentasaccharide: M.S. Thesis, University of Toledo, 2005.
(26) Aly, M. R. E.; Castro-Palomino, J. C.; Ibrahim, E.-S. I.; El-Ashry,
E.-S. H.; Schmidt, R. R. Eur. J. Org. Chem. 1998, 2305-2316.
(27) Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis 2001, 323-
326.
(28) Chiba, H.; Funasaka, S.; Mukaiyama, T. Bull. Chem. Soc. Jpn. 2003,
76, 1629-1644.
J. Org. Chem, Vol. 72, No. 23, 2007 8959

SCHEME 1
a complex mix of compounds. Instead, Staudinger reduction of
the azide moiety in 16 by trimethylphosphine under basic
condition gave the amine,17 which underwent smooth hydro-
genolysis with Pd(OH)2 to produce the desired fully deprotected
LewisX 1 as an acetate salt.
For dimeric LewisX deprotection, removal of benzoyl and
Phth from 14 and selective acetylation were performed under
the same conditions as those for LewisX 11. Subsequent
Staudinger reduction of 17 under basic condition, however, led
to partial removal of one acetamido moiety. As an alternative,
compound 17 was treated with trimethylphosphine in aqueous
THF without any base to give a free amine, which was
hydrogenated to produce dimeric LewisX octasaccharide 2 in
35% overall yield for the four steps (Scheme 1b).
In conclusion, we have demonstrated that branched oligosac-
charides can be constructed by using the combination of
preactivation and reactivity based one-pot synthesis with
exclusive regio- and stereoselectivities. High synthetic efficiency
was achieved without requiring a large excess of building blocks.
A single type of glycosyl donors, i.e., p-tolyl thioglycosides,
was used for all glycosylations, thus significantly simplifying
overall synthetic design. With its anomeric reactivity indepen-
dent nature, the preactivation based chemoselective glycosyla-
tion method presents a powerful strategy for modular synthesis
of complex oligosaccharides.
For a highly convergent synthesis of dimeric LewisX, we
designed three modules, trisaccharide donor 10, bifunctional
trisaccharide acceptor 13, and lactoside 7. To simplify the overall
synthetic design without relying on selective activation of
different types of glycosyl donors,7 the same aglycon leaving
group p-STol was used for 10 and 13. If the traditional armed-
disarmed chemoselective glycosylation approach31,33 were to be
applied, the acceptor must be less reactive than the glycosyl
donor. This would require manipulations of the protective groups
to significantly decrease the relative anomeric reactivity of the
trisaccharide acceptor 13, which can be difficult due to the large
number of protective groups present. Because donor activation
and addition of acceptor occur at two distinct steps with the
preactivation based method, anomeric reactivities of glycosyl
donor and acceptor are independent of each other.13 This would
allow the direct glycosylation of trisaccharide 13 by trisaccharide
10, even though 10 is less reactive.
Diol 13 was prepared by hydrazine acetate treatment of
trisaccharide 12, which was obtained by one-pot sequential
reactions of galactoside 4 with glucosamine 5 and fucose 6 in
a similar fashion as the formation of 10 (Table 1, entry 3). With
all the necessary building blocks in hand, the synthesis of
dimeric LewisX octasaccharide was carried out in one pot (Table
1, entry 4). The trisaccharide donor 10 was preactivated by
p-TolSCl/AgOTf, followed by addition of acceptor 13. After
complete consumption of 13 was confirmed by TLC analysis,
the lactose acceptor 7 was added followed by another equivalent
of p-TolSCl/AgOTf. The desired fully protected dimeric LewisX
octasaccharide 14 was successfully acquired in 44-61% yield.
Glycosylation on 3-OH of trisaccharide 13 was confirmed by
1H NMR analysis of the p-nitrobenzoate derivative of 14
(octasaccharide 15) with its H2d proton appearing at 5.25 ppm
as a triplet (3J ) 9.0 Hz).
Experimental Section
3-Azidopropyl (2,3-di-O-benzoyl-4,6-O-benzylidene-â-D-ga-
lactopyranosyl)-(1f4)-[(2,3,4-tri-O-benzyl-R-L-fucopyranosyl)-
(1f3)]-(6-O-benzyl-2-deoxy-2-N-phthalimido-â-D-glucopyranosyl)-
(1f3)-(2,4,6-tri-O-benzyl-â-D-galactopyranosyl)-(1f4)-2,3,6-tri-
O-benzyl-â-D-glucopyranoside (11): Galactose 3 (100 mg, 0.172
mmol) was dissolved in DCM (3 mL) and stirred at -78 °C with
freshly activated molecular sieves MS 4 Å (100 mg) for 30 min.
Silver triflate (173 mg, 0.67 mmol) dissolved in acetonitrile (0.3
mL) was added to the reaction mixture. Five minutes later, orange-
colored p-TolSCl (27 µL, 0.172 mmol) was added directly into the
reaction mixture. This needs to be performed quite quickly to
prevent the p-TolSCl from freezing inside the syringe tip or on the
flask wall. The yellow color of the solution quickly dissipated within
1 min, indicating the complete consumption of p-TolSCl, and the
complete activation of galactose donor 3 was confirmed by TLC
analysis. The glucosamine acceptor 4 (78 mg, 0.155 mmol) along
with TTBP (43 mg, 0.172 mmol) dissolved in DCM (2 mL) was
then added dropwise to the reaction mixture. This was stirred for
20 min at which point the glucosamine acceptor 4 was completely
consumed. The fucose donor 5 (93 mg, 0.172 mmol) and TTBP
(43 mg, 0.172 mmol) dissolved in Et2O (2 mL) were added to the
mixture. After 10 min, p-TolSCl (27 µL, 0.172 mmol) was added
into the reaction mixture, which was stirred for an additional 45
min. When complete consumption of the fucose donor 5 was
confirmed by TLC analysis, the lactose acceptor 6 (116 mg, 0.120
mmol) and TTBP (43 mg, 0.172 mmol) were dissolved in DCM
(2 mL) and added to the reaction mixture. This was stirred for 10
min at -78 °C and then silver triflate (44 mg, 0.172 mmol) was
added. After 5 min, p-TolSCl (27 µL, 0.172 mmol) was added into
the reaction mixture, which was stirred for 45 min. The mixture
was then filtered through Celite and the Celite was washed with
DCM until no organic compounds were present in the filtrate. The
filtrate was extracted with a saturated solution of NaHCO3. The
organic layer was then dried over Na2SO4 and concentrated to
dryness. The residue was purified by silica gel column chroma-
tography (hexanes/DCM/EtOAc, 5:3:2). The desired product was
obtained in 40-60% yield as a white solid. [R]D25 -44 (c 1.5, CH2-
Cl2); 1H NMR (600 MHz, CDCl3) δ 8.04-6.76 (m, 69H), 5.85 (t,
Deprotection of LewisX pentasaccharide 11 was performed
by removal of benzoyl and Phth with ethylenediamine, followed
by selective acetylation of the free amine leading to pentasac-
charide 16 (Scheme 1a). Attempts to simultaneously reduce the
azido moiety and benzyl groups in 16 through catalytic
hydrogenation with Pd/C or Pd(OH)2 under atmospheric pressure
or high-pressure (100-250 psi) hydrogen gas failed to yield
1
any desired product. H NMR of the reaction mixture showed
(29) Adinolfi, M.; Barone, G.; Iadonisi, A.; Schiattarella, M. Tetrahedron
Lett. 2002, 43, 5573-5577.
(30) Wulff, G.; Rohle, G. Angew. Chem., Int. Ed. 1974, 13, 157-170.
(31) Koeller, K. M.; Wong, C.-H. Chem. ReV. 2000, 100, 4465-4493.
(32) Seeberger, P. H. Chem. Commun. 2003, 1115-1121.
(33) Mootoo, D. R.; Konradsson, P.; Udodong, U.; Fraser-Reid, B. J.
Am. Chem. Soc. 1988, 110, 5583-5584.
8960 J. Org. Chem., Vol. 72, No. 23, 2007

3
3
1H, J ) 9.0 Hz), 5.56 (s, 1H), 5.19 (d, 1H, J ) 7.8 Hz), 5.17
(dd, 1H, 3J ) 4.8, 13.8 Hz), 5.12 (d, 1H, 3J ) 12 Hz), 5.02 (d, 1H,
by silica gel column chromatography (hexanes/EtOAc, 1:1f1:3)
to afford the pentasaccharide 16 as a white solid. A mixture of
16 (100 mg, 0.052 mmol), a 1 M solution of PMe3 in THF
(0.360 mL, 7 equiv), and 0.1 M NaOH (0.5 mL) in THF was stirred
at 60 °C overnight. The mixture was then concentrated and the
resulting residue was dissolved in DCM and extracted twice with
H2O. The organic layer was dried over Na2SO4 and concentrated
to dryness. The crude product was purified by silica gel column
chromatography (DCM f DCM/MeOH 8:1). The desired product
bearing a terminal free amine was obtained as an off-white solid.
Finally a mixture of this product (80 mg, 0.042 mmol) and Pd-
(OH)2 (80 mg) in DCM/MeOH/H2O/AcOH (1 mL:1 mL:2 mL:2
mL) was stirred at room temperature under atmospheric pressure
H2 for 24 h. The mixture was filtered through Celite, concentrated,
and extracted with DCM (3 times) and EtOAc (3 times). The water
layer was then lyophilized to afford a white solid, which was
purified by Sephadex G-10 size exclusion column. The pure LewisX
3J ) 8.4 Hz), 4.88 (m, 2H), 4.81 (t, 2H, J ) 10.2 Hz), 4.75 (d,
3
1H, 3J ) 10.8 Hz), 4.61 (d, 1H, 3J ) 4.8 Hz), 4.54-4.49 (m, 4H),
3
4.44-4.29 (m, 6H), 4.27 (d, 1H, J ) 11.4 Hz), 4.19-4.06 (m,
7H), 4.03-3.99 (m, 2H), 3.96 (s, 1H), 3.89 (dd, 1H, 3J ) 2.4, 10.2
3
Hz), 3.84-3.80 (m, 2H), 3.79 (s, 1H), 3.76 (d, 1H, J ) 4.8 Hz),
3.62 (d, 1H, 3J ) 10.2 Hz), 3.55 (dd, 1H, 3J ) 4.8, 10.2 Hz), 3.49-
3.43 (m, 2H), 3.41-3.37 (m, 5H), 3.32-3.29 (m, 4H), 3.28 (q,
3
3
1H, J ) 7.8, 9.0 Hz), 3.20 (d, 1H, J ) 10.8 Hz), 3.16 (s, 1H),
3
3
2.86 (d, 1H, J ) 8.4 Hz), 1.80-1.77 (m, 2H), 1.26 (d, 3H, J )
6.6 Hz); 13C NMR (150 MHz, CDCl3) δ 166.3, 164.9, 139.7, 139.7,
139.6, 139.2, 138.8, 138.6, 138.6, 138.5, 138.4, 138.2, 137.8, 130.1,
129.9, 129.5, 129.3, 128.9, 128.8, 128.7, 128.6, 128.5, 128.5, 128.4,
128.4, 128.3, 128.3, 128.3, 128.2, 128.1, 128.1, 128.1, 128.0, 127.9,
127.9, 127.9, 127.9, 127.8, 127.8, 127.7, 127.6, 127.4, 127.3, 127.2,
127.1, 127.0, 126.9, 126.4, 125.9, 103.6, 102.8 (1JC-H ) 163.4 Hz),
100.0 (1JC-H ) 161.3 Hz), 99.9 (1JC-H ) 158.2 Hz), 99.8 (1JC-H
) 164.6 Hz), 97.6 (1JC-H ) 174.1 Hz), 83.1, 82.2, 81.8, 79.2, 78.7,
77.5, 77.2, 77.0, 76.8, 76.2, 75.3, 75.2, 75.0, 74.9, 74.8, 74.1, 73.6,
73.5, 73.2, 73.0, 72.9, 71.6, 66.7, 66.6, 66.5, 48.5, 29.4, 16.5; HRMS
[M + Na]+ m/z calcd for C132H132N4NaO28 2244.8926, found
2244.8960.
pentasaccharide 1 was obtained in acetate form as a solid in 53%
1
yield over four steps. [R]25 -217 (c 0.5, H2O); H NMR (600
D
3
3
MHz, D2O) δ 4.94 (d, 1H, J ) 3.6 Hz), 4.52 (d, 1H, J ) 8.4
Hz), 4.33 (d, 1H, 3J ) 8.4 Hz), 4.28 (d, 1H, 3J ) 8.4 Hz), 4.25 (d,
3
3
1H, J ) 8.4 Hz), 3.97 (d, 1H, J ) 2.4 Hz), 3.80-3.75 (m, 4H),
3
3.73-3.69 (m, 4H), 3.63-3.37 (m, 20H), 3.31 (t, 1H, J ) 7.2
3
3-Aminopropyl â-D-galactopyranosyl-(1f4)-[(R-L-fucopyra-
nosyl)-(1f3)]-(2-N-acetamido-2-deoxy-â-D-glucopyranosyl)-(1f3)-
(â-D-galactopyranosyl)-(1f4)-â-D-glucopyranoside (1): The fully
protected pentasaccharide 11 (200 mg, 0.09 mmol) was mixed with
ethylenediamine (0.5 mL) in n-butanol (5 mL). The reaction was
stirred at 130 °C for 20 h. The mixture was then concentrated and
the residue was dissolved in DCM and extracted with a saturated
solution of NH4Cl. The organic layer was dried over Na2SO4 and
concentrated to dryness. The crude residue was purified by silica
gel column chromatography. The desired product was obtained in
its pure form as an off-white solid. The newly formed compound
was then dissolved in MeOH (5 mL) along with triethylamine (0.1
mL) and acetic anhydride (0.1 mL, 15 equiv). The reaction mixture
was stirred at room temperature for 4 h. It was then concentrated
and the residue was dissolved in DCM and extracted with a
saturated solution of NH4Cl. The organic layer was dried over Na2-
SO4 and concentrated to dryness. The crude residue was purified
Hz), 3.16-3.13 (m, 2H), 2.97 (t, 2H, J ) 7.2 Hz), 1.83 (s, 3H),
0.99 (d, 3H, J ) 6.0 Hz); 13C NMR (150 MHz, D2O) δ 181.7,
3
174.8, 103.0, 102.7, 102.2, 101.9, 98.7, 82.2, 78.4, 75.2, 75.1, 75.0,
74.9, 74.4, 73.1, 72.8, 72.6, 72.0, 71.1, 70.0, 69.3, 68.5, 68.4, 68.0,
67.8, 66.8, 61.6, 61.1, 60.1, 59.7, 56.1, 37.7, 26.8, 23.4, 22.4, 15.4;
HRMS [M + Na]+ m/z calcd for C35H62N2NaO25 933.3539, found
933.3531.
Acknowledgment. We are grateful for financial supports
from the University of Toledo, the National Institutes of Health
(R01-GM-72667), and the Pardee foundation.
Supporting Information Available: Experimental procedures
and selected 1H, 13C, and 2D NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO701694K
J. Org. Chem, Vol. 72, No. 23, 2007 8961