Synthesis of fluorinated mucin core 2 branched oligosaccharides with the potential of novel substrates and enzyme inhibitors for glycosyltransferases and sulfotransferases

Article abstract of DOI:10.1021/jo052626j

Syntheses of fluorinated mucin core 2 tri- and tetrasaccharides modified at the C-3 or C-4 position of the pertinent galactose residue are reported. These compounds were used for the study of sialyltransferases and 3-O- sulfotransferases involved in the b

Full text of DOI:10.1021/jo052626j

Synthesis of Fluorinated Mucin Core 2 Branched Oligosaccharides
with the Potential of Novel Substrates and Enzyme Inhibitors for
Glycosyltransferases and Sulfotransferases
Jie Xia, Jun Xue, Robert D. Locke, E. V. Chandrasekaran, T. Srikrishnan, and
Khushi L. Matta*
Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, New York 14263
khushi.matta@roswellpark.org
ReceiVed December 22, 2005
Syntheses of fluorinated mucin core 2 tri- and tetrasaccharides modified at the C-3 or C-4 position of the
pertinent galactose residue are reported. These compounds were used for the study of sialyltransferases
and 3-O-sulfotransferases involved in the biosynthesis of O-glycans. Our acceptor substrate specificity
studies on three cloned sialyltransferases (Sia-Ts) revealed that a 3- or 4-fluoro substituent in â1,4Gal
resulted in poor acceptors for R2,6(N)Sia-T and R2,3(N)Sia-T, whereas 4-fluoro-Galâ1,3GalNAcR was
a good acceptor for R2,3(O)Sia-T. Uniquely, 4-F-Galâ1,4GlcNAcâ1,6(Galâ1,3)GalNAcR-OBn was an
inhibitor of R2,6(N)Sia-T activity but not R2,3(N)Sia-T activity. Further we found that the activities of
only Gal 3-O-sulfotransferases and not sialyltransferases were adversely affected by a C-3 fluoro substituent
at the other Gal terminal of mucin core 2. The strategy of building branched mucin core 2 structures by
three glycosidation sequence coupling three classes of glycosyl donors with the reactivity-matching
acceptors proved to be successful in syntheses of modified mucin-type core structures of O-glycan. The
relative poor yields of the glycosylations using fluorinated galactosyl donors indicated that the fluorine
modification dramatically decreased the donor reactivity due to electron-withdrawing effect.
Introduction
sialylated and sulfated mucin core 2 oligosaccharides in cancer,
inflammation, and metastasis are yet to be elucidated in details.
A knowledge of the detailed mechanism of enzymatic catalysis
involved in glycosylation or sulfation on the C-3 or C-4 position
of galactose residues of the mucin core 2 structure (Figure 1)
in the formation of sialylated and sulfated O-glycan is needed
for the design of metabolic inhibitors of these enzymes. In turn,
the synthetic capability of these complex oligosaccharides will
aid in the studies directed toward understanding the biosynthetic
mechanism of these enzymes that catalyze the formation of these
natural sialylated and sulfated O-glycans. Consequently, a
tremendous effort has been made by various research groups
The natural mucin ligands GlyCAM-1 and PSGL-1 for cell
adhesion proteins1 and various tumor-associated glycoproteins2
are known to contain the core structure Galâ1f4GlcNAcâ1f
6(Galâ1f3)GalNAcR-1 (Figure 1), which serves as a backbone
for carrying glycan units playing an essential role in many
biological processes.3 However, the physiological roles of
(1) (a) Capon, C.; Laboisse, C. L.; Wieruszrski, J.-M.; Maoret, J. J.;
Auheron, C.; Fournet, B. J. Biol. Chem. 1992, 267, 19248. (b) Capon, C.;
Wieruszeski, J.-M.; Lemoine, J.; Byrd, J. C.; Leffler, H.; Kim, Y. S. J.
Biol. Chem. 1997, 272, 31957.
(2) (a) Hemmerich, S.; Leffer, G.; Rosen, S. D. J. Biol. Chem. 1995,
270, 12035. (b) Wilkins, P.; McEVer, R. P.; Cummings, R. D. J. Biol. Chem.
1996, 271, 18732.
(3) (a) Dwek, R. A. Chem. ReV. 1996, 96, 683. (b) Lee, Y. C.; Lee, T.
R. Acc. Chem. Res. 1995, 28, 321. (c) Varki, A. Glycobiology 1993, 3, 97.
10.1021/jo052626j CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/14/2006
3696
J. Org. Chem. 2006, 71, 3696-3706

Synthesis of Fluorinated Mucin Core 2 Oligosaccharides
cosaminyltransferase during elongation of these mucin core 2
oligasaccharides. This approach may also provide useful
information for devising highly effective inhibitors of these
glycosyltransferases and sulfotransferases as well as metabolic
inhibitors of glycoprotein synthesis. Herein, we first describe
the effective syntheses and biochemical evaluation of fluorinated
core 2 branched oligosaccharide analogues 1-5.
Results and Discussion
Retrosynthesis. Scheme 1 outlines the retrosynthetic analysis
of fluorinated mucin core 2 trisaccharides 1 and 2. Retrosyn-
thetic simplification of trisaccharides is rather straightforward,
furnishing glucosamine donor 8 and C-3 or C-4 fluorinated
disaccharide acceptors (16 and 20) bearing the mucin core 1
structure of Galâ1,3GalNAcR-1. In the forward sense, a highly
regioselective glycosylaion 1 of the primary hydroxyl group in
the presence of the free secondary hydroxyl of the disaccharide
acceptors would be expected to achieve the desired trisaccha-
rides 1 and 2. N-Phthalimido (N-Phth) protected glucosamine
thioglycoside was chosen as a moderate glycosyl donor for
glcosylation 1 because of its potential of enhancing the
regioselectivity toward the primary hydroxyl group of the
disaccharide acceptors. Chemical modulations of galactose
residues in mucin core 1 acceptors are involved in substitution
of 3-OH or 4-OH of the galactose residue with fluorine.
Retrosynthetic cleavage of the 1,3-glycosylic bond between
galactosyl and N-acetylgalactosamine provides C-3 or C-4
fluorinated donors (15, 19) and monosaccharide acceptor 7 as
potential precursors. The hydroxyl group on the C-3 position
of N-acetylgalactosamine is similar to the 4-OH of N-acetyl-
glucosamines which are widely acknowledged to be among the
most difficult alcohols to be glycosylated. On the basis of
variable-temperature NMR studies, Crich et al.11 suggested the
intermolecular hydrogen bonding effects of the amide group
which result in low reactivity toward glycosylation 2 should be
magnified at lower temperatures. Glycosylation 2 carried out
at a high temperature of 65 °C would be expected to achieve
the desired fluorinated mucin core 1 structures by overcoming
the hydrogen bonding effects of the amide group. To couple
with the unreactive N-acetylgalactosamine acceptor 7 more
efficiently, bromides (15, 19) were selected to serve as glycosyl
donors due to the matching reactivities. A sequence of straight-
forward functional group interconversions leads from 3-fluor-
inated donor 19 back to compound 10 and 4-fluorinated donor
15 back to the starting material 6. The fluorination is carried
out on the level of galactose, which minimizes the number of
protecting groups that might interfere. In addition, the overall
number of reaction steps would be limited.
FIGURE 1. The possible positions modified by carbohydrate associ-
ated enzymes.
in developing a variety of synthetic methodologies including
glycosyl bond formation and protection-deprotection strategies.4
Several laboratories reported the use of selectively fluorinated
carbohydrate substrates as probes for studying enzymes involved
in carbohydrate metabolism. Blood group A and B glycotrans-
ferases were shown to act on C-6 fluoro-substituted Gal whereas
a C-3 fluoro substituent on Gal created competitive inhibitors
for both enzymes.5 van Dorst et al.6 synthesized 3-F-Galâ1,-
4GlcNAcâ1,2ManR-O(CH2)7CH3 as a potential substrate probe
for studing N-glycan biosynthesis. Scott and Viola7 found that
3-fluoro and 4-fluoro analogues of D-glucose were higher affinity
substrates than D-glucose for aldolase reductase but 2-fluoro
and as well as 4-fluoro analogues of D-glucitol were inactive
acceptors for sorbitol dehydrogenase. Tai et al.8 synthesized a
fluoro analogue of dolichol diphopho GlcNAc by replacing the
acetamido group with fluorine. This compound was an inactive
acceptor but a competitive inhibitor for GlcNAc transferase.
Polyprenol monophosphomannosyl-dependent R1,6-Mannosyl-
transferase was able to utilize 2-fluoro ManR1,6Man as a
substrate,9 whereas 2,6-difluoro ManR1,6Man was neither an
acceptor nor an inhibitor for this enzyme.10
Our laboratory has been engaged in the synthesis of complex
oligosaccharide molecules, such as mucin core 2 branched
sialylated and sulfated oligosaccharides for biochemical inves-
tigations. Substitution of the 3- or 4-hydroxyl group of the
galactose residue in the sugar chain Galâ1f4GlcNAcâ1f
6(Galâ1f3)GalNAcR- with a fluorine atom would have a
differential effect on the acceptor substrate specificities of R2,3-
sialyltransferase, 3-O-sulfotransferase, and R1,4-N-acetylglu-
(4) (a) Nicolaou, K. C.; Mitchell, H. J. Angew. Chem., Int. Ed. 2001,
40, 1576. (b) Sanders, W. J.; Manning, D. D.; Koeller, K. M.; Kiessling,
L. L. Tetrahedron 1997, 53, 16391. (c) Wong, C.-H. Acc. Chem. Res. 1999,
32, 376. (d) Gege, C.; Vogel, J.; Bendas, G.; Rothe, U.; Schmidt, R. R.
Chem. Eur. J. 2000, 6, 111. (e)Herzner, H.; Reipen, T.; Schultz, M.; Kunz,
H. Chem. ReV. 2000, 100, 4495. (f) Cook, B. N.; Bhakta, S.; Biegel, T.;
Bowman, K. G.; Armstrong, J. I.; Hemmerich, S.; Bertozzi, C. R. J. Am.
Chem. Soc. 2000, 122, 8612. (g) Lergenmuler, M.; Ito, Y.; Ogawa, T.
Tetrahedron 1998, 54, 1381. (h) Kanie, O.; Ito, Y.; Ogawa, T. J. Am. Chem.
Soc. 1994, 116, 12073.
(5) Lowary, T. L.; Hindsgaul, O. Carbohydr. Res. 1993, 249, 163.
(6) van Dorst, J.; van Heusden, C. J.; Tikkanen, J. M.; Kamerling, J. P.
Carbohydr. Res. 1997, 297, 209.
(7) Scott, M. E.; Viola, R. E. Carbohydr. Res. 1998, 313, 247.
(8) Tai, V. W. F.; O’Reilly, M. K.; Imperiali, B. Bioorg. Med. Chem.
2001, 10 (11), 3673.
The retrosynthetic analysis of fluorinated mucin core 2
tetrasaccharides 3-5 is outlined in Scheme 2. The employment
of three classes of glycosylations 1, 2, and 3 to carry out the
desired structural transformations is the most distinguishing
feature of the syntheses of the branched mucin core 2 tetrasac-
charides. Retrosynthetic disconnection of tetrasaccharides 3-5
is most productive, for it furnishes lactosamine-type donors (13,
24, 30) and disaccharide acceptors (12, 20). The N-phthalimido
protected lactosamine thioglycoside serving as a glycosyl donor
was found to be central to the success of the synthesis, which
would minimize the synthetic steps by taking advantage of the
thioglycoside, which can offer efficient temporary protection
(9) Subramamiam, V.; Gurcha, S. S.; Besra, G. S.; Lowary, T. L. Bioorg.
Med. Chem. 2005, 13,1083.
(10) Subramamiam, V.; Gurcha, S. S.; Besra, G. S.; Lowary, T. L.
Tetrahedron: Asymmetry 2005, 16, 553.
(11) Crich, D.; Vinod A. U. J. Org. Chem. 2005, 70 (4), 1291.
J. Org. Chem, Vol. 71, No. 10, 2006 3697

Xia et al.
SCHEME 1. Retrosynthetic Analysis of Fluorinated Mucin Core 2 Trisaccharides 1 and 2
SCHEME 2. Retrosynthetic Analysis of Fluorinated Mucin Core 2 Tetrasaccharide 3-5
of the anomeric center stable to the condition of glycosylation
SCHEME 3a
3. Retrosynthetic disassembly of lactosamine-type donors by
cleavage of the indicated bond provides fluorinated imidate
donors (23, 29) and diol thioglycosides (9, 11) as glycosyl
acceptors. In the synthetic direction, the bulky N-phthalimido
protect group of thioglycosides was expected to serve two
important roles in glycosylation 3. Not only would it block the
neighbor hydroxyl at the C-3 position of the diol thioglycosides,
but it would also enhance the reactivity of the hydroxyl group
at the 4-position of glucosamine by masking the amide group
to disturb its intermolecular hydrogen bonding, which effectively
increases steric bulk around the nucleophilic alcohol.11 Although
N-trichloroethoxycarbonyl (N-Troc) functionality was found to
afford greater glucosamine 4-OH reactivity than the comparable
N-phthalimido protected acceptor in trichloroacetimidate cou-
pling, an unexpected intermolecular aglycon transfer was
observed in trimethylsilyl triflate (TMSOTf)-mediated glyco-
sylation when using N-Troc protected thioglycoside as the
glycosyl acceptor.12 Therefore, the N-phthalimido protecting
group could tune the reactivity of the anomeric center of the
thioglycoside to a suitable extent under the condition of
glycosylation 3 in which the phenylthio functionality is com-
pletely stable. A sequence of straightforward functional group
interconversions leads from 3-fluorinated imidate donors 29 and
19 back to compound 10 and 4-fluorinated imidate donor 23
back to the starting material 6.
a Reagents and conditions: (a) Ac2O/H2SO4, 0-5 °C, 2 h, 58%; (b) HBr/
HOAc, rt, 65%; (c) Hg(CN)2, CH3NO2/benzene, 65 °C, 12 h; (d) 60%
HOAc, 65 to 70 °C, 1.5 h, 28% over two steps; (e) NIS-TfOH/CH2Cl2,
-65 °C, 48%; (f) NH2-NH2H2O/CH3OH (1/5), 90 °C, 6 h; (g) Ac2O-pyridine
(1/1), DMAP, rt, 12 h; (h) 1 M CH3ONa in CH3OH/CH3OH-H2O, rt, 12
h, 75% over three steps.
Synthesis. Syntheses of oligosaccharides 1 and 2 that
commenced with two monosaccharide bromide donors (15 and
19) are illustrated in Schemes 3 and 4. Fluorinated compound
6 was prepared according to the literature method in a good
(12) Xue, J.; Khaja, S. D.; Locke, R. D.; Matta, K. L. Synlett 2004, 861.
3698 J. Org. Chem., Vol. 71, No. 10, 2006

Synthesis of Fluorinated Mucin Core 2 Oligosaccharides
SCHEME 4a
SCHEME 5a
a Reagents and conditions: (a) NH2NH2.HOAc/DMF, 50 °C, 2 h; (b)
CCl3CN/DBU, CH2Cl2, 0 to 25 °C, 80% over two steps; (c) TMSOTf/
CH2Cl2, 4A-MS, -65 to -70 °C, 2 h; (d) Ac2O/pyridine (1/1), DMAP, rt,
12 h, 63% over two steps.
a Reagents and conditions: (a) HBr/HOAc, rt, 2 h, 71%; (b) Hg(CN)2,
CH3NO2/benzene, 65 °C, 12 h; (c) 60% HOAc, 65 to 70 °C, 1.5 h, 47%
over two steps; (d) NIS-TfOH/CH2Cl2, -65 to -60 °C, 40%; (e) NH2-
NH2‚H2O/CH3OH (1/5), 90 °C, 6 h; (f) Ac2O-pyridine (1:1), DMAP, rt,
12 h; (g) 1 M CH3ONa in CH3OH/CH3OH in H2O, rt, 12 h, 70% over
three steps.
SCHEME 6a
yield.13 The conversion of a methyl group into an acetyl group
was achieved by acetolysis of compound 6 with a catalytic
amount of concentrated sulfuric acid in the presence of
anhydrous acetic anhydride in an ice bath for 2 h in 58% yield.
Bromide donor 15 was obtained upon treatment of tetraacetate
14 with 33% HBr-HOAc at room temperature for 2 h.
Disaccharide 16 was then furnished through a Hg(CN)2-
promoted glycosylation in a poor yield of 28% over two steps.
The relatively poor yield of the glycosylation with use of this
fluorinated galactosyl bromide 15 compared with the 70% yield
of reported glycosylation14 with use of fully acetylated galactosyl
bromide as donor indicated that the fluorine modification
dramatically decreased the reactivity of the glycosyl donor due
to its electron-withdrawing effect. Observation in our previous
total synthesis of branched mucin core 2 sialylated and sulfated
oligosaccharides15 suggested that it is a plausible regioselective
glycosylation of the 6-hydroxyl group of disaccharide acceptor
16 in the presence of a secondary 4-hydroxyl group. Coupling
of disaccharide diol 16 with monosaccharide donor 8 at a low
temperature of -65 °C provided trisaccharide 17 as the only
glycosylated product in 48% yield. The stereochemical output
of both the glycosidic and position connectivity of trisaccharide
17 was well established through two-dimensional NMR experi-
ment including DQF-COSY, TOCSY, and ROESY. Structure
1 was then constructed by global deprotection of compound 17
in 75% yield over three steps: (1) removal of the N-Phth group
from the glucosamine sugar residue, (2) complete acetylation
with pyridine-acetic anhydride, and (3) removal of O-acetyl
groups with 0.1 M sodium methoxide in methanol. The structure
of compound 1 was established by a combination of electron-
spray ionization high-resolution mass spectroscopy (ESI-HRMS)
and two-dimensional NMR experiments (DQF-COSY and
ROESY).
a Reagents and conditions: (a) NIS-TfOH/CH2Cl2, 4A-MS, - 65 to -
60 °C, 2 h, 76%; (b) Ac2O/pyridine (1/1), DMAP, rt, 12 h, 80%; (c) NH2-
NH2‚H2O/CH3OH (1/5), 90 °C, 6 h; (d) Ac2O/pyridine (1/1), DMAP, rt,
12 h; (e) 1 M CH3ONa-CH3OH/CH3OH-H2O, rt, 12 h, 45% over three
steps.
76% starting from known compound 10 obtained from 1,2:5,6-
diisopropylidene-R-D-gulose16 over three steps: (1) fluorination
of 1,2:5,6-disopropylidene-R-D-glucose with DAST,17 (2) hy-
drolysis of resultant fluorinated compound in 60% acetic acid,
and (3) acetylation of free hydroxyl groups with acetic anhydride
in pyridine.18 Fluorinated disaccharide diol 20 was then obtained
by a Hg(CN)2-catalyzed glycosylation of bromide donor 19 with
acceptor 7, followed by hydrolysis of the benzylidene group in
60% acetic acid. Compared with the 4-fluorine galactosyl
bromide 15, the 3-fluorine galactosyl bromide 19 exhibited
increased reactivity to furnish disaccharide 20 in an improved
yield. Regioselective glycosylation of the primary hydroxyl of
diol 20 with phenylthio donor 8, followed by full de-protection
of resultant intermediate 21, provided the targeted mucin core
2 trisaccharide 2.
Synthesis of tetrasaccride 3 is outlined in Schemes 5 and 6.
The fluorine-containing disaccharide donor 24 constitutes the
first synthetic objective in the course of the synthesis of target
3. Starting from the same tetraacetate 14 used for synthesis of
Synthesis of compound 2 is outlined in Scheme 4. Fluorinated
monosaccharide bromide 19 was prepared in a good yield of
(16) Meyer, W.; Reckendorf, M. Z. Methods Carbohydr. Res. 1972, 6,
129.
(17) Xia, J.; Alderfer, J. L. Srikrishnan, T.; Chandrasekaran, E. V.; Matta,
K. L. Bioorg. Med. Chem. 2002, 10, 3673.
(13) Card, P. J.; Reddy, C. S. J. Org. Chem. 1983, 48, 4734.
(14) Jain, R. K.; Piskorz, C. F.; Chandrasekaran, E. V.; Matta, K. L.
Carbohydr. Res. 1995, 271, 247.
(15) Xia, J.; Alderfer, J. L.; Matta, K. L. Bioorg. Med. Chem. Lett. 2000,
10, 2485.
(18) Somawardhana, C. W.; Brunngraber, E. G. Carbohydr. Res. 1983,
121, 51.
J. Org. Chem, Vol. 71, No. 10, 2006 3699

Xia et al.
SCHEME 7a
FIGURE 2. ORTEP22 drawing of 23.
bromide 15, monosaccharide imidate 23 was obtained in an
overall yield of 80% over two steps as illustrated in Scheme 5.
The fluorination dramatically changed the physical property of
the trichloroacetimidate 23, which resulted in a well-crystallized
form through a slow evaporation. The absolute configuration
of fluorinated imidate donor was first characterized by X-ray
analysis of compound 23 (Figure 2), which confirmed the
trichloroacetimidate displaying the desired R-configuration. An
attempt to couple acceptor 9 with imidate 23 at -45 to -40 °C
led to a mixture of the â1,4- and â1,3-linkage of disaccharides
that was difficult to separate by column chromatography.
However, when the temperature was lowered to -70 to -65
°C, only â1,4-linkage disaccharide was obtained as the glyco-
sylation product. Therefore, disaccharide 24 was prepared by
coupling of the 4-hydroxyl of diol 9 with imidate 23 in a
regioselective manner, followed by acetylation of the remaining
free hydroxyl functionality in an overall yield of 63%. Substitu-
tion of 4-OH with a fluorine atom that has a strong electron-
withdrawing negative effect seemed to reduce the glycosylation
activity of imidate 23. Regioselective glycosylation of the
6-hydroxyl group of diol 12 with lactosamine phenylthio donor
24 in a temperature-controlled manner provided tetrasacchride
25 in a high yield of 76%. The structure of tetrasaccharide 25
was established through 2D NMR experiments. Target 3 was
obtained from the fully protected tetrasaccharide 26 by a similar
procedure as described for de-protection of compound 17 to
compound 1 in an overall yield of 39% over three steps. The
structure and purity of compound 3 were also established by
tremendous spectra experiments including ESI-HRMS, 19F and
a Reagents and conditions: (a) NIS-TfOH/CH2Cl2, -45 to -40 °C,
65%; (b) Ac2O/pyridine (1/1), DMAP, rt, 12 h, 80%; (c) NH2-NH2‚H2O/
CH3OH (1/5), 90 °C, 6 h; (d) Ac2O/pyridine (1/1), DMAP, rt, 12 h; (e) 1
M CH3ONa-CH3OH/CH3OH-H2O, rt, 12 h, 45% over three steps.
SCHEME 8a
a Reagents and conditions: (a) NH2-NH2‚HOAc/DMF, 50 °C, 2 h; (b)
CCl3CN/DBU, CH2Cl2, 0 to 25 °C, 32% over two steps; (c) TMSOTf/
CH2Cl2, 4A-MS, -65 °C, 2 h, 43%; (d) Ac2O/pyridine (1/1), DMAP, rt,
12 h, 76%.
SCHEME 9a
1
13C NMR, 2D H-1H DQF-COSY, and 2D ROESY.
Synthesis of tetrasacchride 4 is described in Scheme 7.
Regioselective glycosylation of 6-OH of the fluorinated disac-
charide diol 20 with lactosamine donor 13 at low temperature
afforded tetrasaccharide 27 in 65% yield. The resultant com-
pound 27 was subjected to similar procedures as described for
de-protection of the compound 21 to give compound 2, and
afforded targeted tetrasacchride 4 in an overall yield of 45%
over three steps. The structure of compound 4 was fully
established by ESI-HRMS, 2D DQF-COSY, and 2D ROESY.
Synthesis of targeted oligosaccharide 5 is shown in Schemes
8 and 9. Regioselective glycosylation of the 4-hydroxyl group
of acceptor 11 with the imidate donor 29 through a well-
established glycosylation procedure from our laboratory19g
provided a â1,4-linked disaccharide 30 in 32% yield. Coupling
of disaccharide acceptor 12 with disaccharide donor 30 afforded
a â1,6-linked tetrasaccharide 31 in a reasonable yield of 52%.
a Reagents and conditions: (a) NIS-TfOH/CH2Cl2, 4A-MS, -65 to -60
°C, 2 h, 52%; (b) Ac2O/pyridine (1/1), DMAP, rt, 12 h, 45% over two
steps; (b) NH2-NH2‚H2O/CH3OH (1/5), 90 °C, 6 h; (c) Ac2O/pyridine (1/
1), DMAP, rt, 12 h; (d) DDQ in CH2Cl2/MeOH (4/1), rt, 18 h; (e) 1 M
CH3ONa-CH3OH/CH3OH-H2O, rt, 12 h, 45% over four steps.
Target 5 was then obtained by complete de-protection of the
resultant 31 in four steps. The structure and purity of tetrasac-
chride 5 were also established by tremendous spectra experi-
1
ments including ESI-HRMS, 19F and 13C NMR, 2D H-1H
DQF-COSY, and 2D ROESY.
3700 J. Org. Chem., Vol. 71, No. 10, 2006

Synthesis of Fluorinated Mucin Core 2 Oligosaccharides
FIGURE 3. Inhibition of R2,6(N)Sia-T activity: (a) measurement of R2,6(N)Sia-T activity and (b) inhibition of R2,6(N)Sia-T activity by compound
3.
3-O-Sulfo- and Sialyltransferase Activities. Sulfate and
sialyl groups are located at various positions in glycoconjugates.
A sulfated Lewisx determinant was identified as a major
structural motif in mucins produced by human colon carcinoma
cells.1b Previous studies from our group20a,b indicated that two
distinct Gal 3-O-sulfotransferases (Gal3STs) in tumor tissues
and cancer cells exhibited distinct acceptor preferences. Enzymes
from colon cancer cell lines and colon tumor tissues prefer to
sulfate the C-3 position of Gal in the Galâ1,4GlcNAcâ moiety
of the mucin core 2 structure. In contrast, enzymes from breast
cancer cells prefer to act on the Galâ1,3GalNAcR moiety.
Therefore, to determine the structural features of the mucin core
2 that influence Gal3ST activity, assay was undertaken with
compounds 4 and 5 for various sulfotransferases.20c As com-
pared to their activities toward the acceptor Galâ1,4GlcNAcâ1,6-
(3-OMeGalâ1,3)GalNAcR-O-Bn, LS180 Gal3ST, Gal3ST-2,
and Gal3ST-3 exhibited poor activity (12-16%) toward com-
pound 4 and as anticipated, negligible activity (1-2%) toward
compound 5; Gal3ST4 showed only 23% activity with com-
pound 5 as compared to its activity toward 3-OMeGalâ1,-
4GlcNAcâ1,6(Galâ1,3)GalNAcR-OBn. Gal3ST2, Gal3ST3, and
LS180 Gal3ST exhibited very little activity (1-2%) toward
compound 5. Thus it became evident that the sulfotransferases
Gal3ST-2, Gal3ST-3, and LS180 GalST utilizing the Galâ1,-
4GlcNAcâ terminal as acceptor acted poorly when there is a
C-3 fluoro substituent on the â1,3 linked Gal of mucin core 2.
Similarly, the sulfotransferase Gal3ST-4 specific for Galâ1,-
3GalNAc terminal was adversely affected by a C-3 fluoro
substituent on â1,4 linked Gal in mucin core 2.
When sialyltransferases were examined,21 the cloned enzymes
R2,6(N)Sia-T and R2,3(N)Sia-T specific for Galâ1,4GlcNAc
terminal exhibited good activities (78% and 97%, respectively)
toward compound 4; the cloned R2,3(O)Sia-T and prostate
cancer cell LNCaP R2,3(O)Sia-T utilized compound 5 quite
effectively (103% and 92%, respectively). Thus, in contrast to
Gal 3-O-sulfortransferases, sialyltransferases were not affected
by the C-3 fluoro substituent on the other Gal terminal of mucin
core 2 structure. Compound 3 was also a good acceptor for
cloned R2,3(O)Sia-T (102%) but a poor acceptor for cloned
R2,3(N)Sia-T and R2,6(N)Sia-T (8% and 3%, respectively).
When compound 3 was tested as an inhibitor as shown in Figure
3, R2,6(N)Sia-T activity was competitively inhibited by com-
pound 3 (Ki 1.9 mM) whereas R2,3(N)Sia-T activity was not
inhibited at all by compound 3. Thus fluorination of acceptors
resulted in entirely different effects on the ability of sulfo- and
siayltransferases in utilizing these modified compounds as
acceptors. In addition, C-4 fluorination of â1,4 linked Gal in
mucin core 2 created a selective inhibitor, namely compound
3, for R2,6(N)Sia-T, thus enabling a determination of R2,3(N)-
Sia-T activity in the presence of R2,6(N)Sia-T activity.
In summary, fluorine-containing mucin core 2 oligosaccharide
analogues 1-5 in which the fluorine atom located at different
positions of galactose residue were synthesized in a convergent
way for probing carbohydrates-enzyme interaction and carbo-
hydrate-selection interaction.
Experimental Section
Assay of r2,3(N)Sia-T and r2,6(N)Sia-T Activity. Both R2,3-
(N)Sia-T and R2,6(N)Sia-T were assayed with use of the acceptor
Galâ1,4GlcNAcâ1,6(3-OMeGalâ1,3)GalNAcR-OBn.21 For inhibi-
tion studies, an increasing concentration of compound 3 and a 2
mM concentration of the acceptor were taken in the reaction mixture
(RM) run in duplicate. The inhibition constant Ki was determined
by Lineweaver-Burke plot.
2,3,6-Tri-O-acetyl-4-deoxy-4-fluoro-R-D-galactopyranosyl Bro-
mide (15). To a cold (ice-bath) solution of compound 6 (500 mg,
1.55 mmol) in anhydrous acetic anhydride (3.6 mL) was added
dropwise anhydrous acetic anhydride (3.6 mL) containing concen-
trated sulfuric acid (80 µL). The reaction mixture was stirred at
(19) (a) Jain, R. K.; Vig, R.; Locke, R. D.; Rampal, R.; Chandrasekaran,
E. V.; Matta, K. L. J. Am. Chem. Soc. 1994, 116, 12123. (b) Jain, R. K.;
Vig, R.; Locke, R. D.; Mohammad, A.; Matta, K. L. Chem. Commun. 1996,
65. (c) Jain, R. K.; Huang, B.-G.; Chandrasekaran, E. V.; Matta, K. L.
Chem. Commun. 1997, 23. (d) Huang, B.-G.; Jain, R. K.; Locke, R. D.;
Alderfer, J. L.; Tabaczynski, W. A.; Matta, K. L. Tetrahedron Lett. 2000,
41, 6279. (e) Xia, J.; Piskorz, C. F.; Alderfer, J. L.; Locke, R. D.; Matta,
K. L. Tetrahedron Lett. 2000, 41, 2773. (f) Xia, J.; Alderfer, J. L.; Piskorz,
C. F.; Matta, K. L. Chem. Eur. J. 2000, 6, 3442. (g) Xia, J.; Alderfer, J. L.;
Piskorz, C. F.; Matta, K. L. Chem. Eur. J. 2001, 7, 356. (h) Xia, J.; Alderfer,
J. L.; Locke, R. D.; Piskorz, C. F.; Matta, K. L. J. Org. Chem. 2003, 68,
2752. (i) Xia, J.; Alderfer, J. L.; Locke, R. D.; Matta, K. L. Synlett 2003,
1291.
(20) (a) Chandrasekaran, E. V.; Jain, R. K.; Vig, R.; Matta, K. L.
Glycobiology 1997, 7, 753. (b) Chandrasekaran, E. V.; Jain, R. K.; Rhodes,
J. M.; Chawda, R.; Piskorz, C.; Matta, K. L. Glycoconj. J. 1999, 16, 523.
(c) Chandrasekaran, E. V.; Lakhaman, S. S.; Chawda, R.; Piskorz, C. F.;
Neelamegham, S. N.; Matta, K. L. J. Biol. Chem. 2004, 279, 10032.
(21) Chandrasekaran, E. V.; Xue, J.; Xia, J.; Chawda, R.; Piskorz, C.;
Locke, R. D.; Neelamegham, S.; Matta, K. L. Biochemistry. 2005, 44, 15619.
(22) Johnson, C. K. ORTEPII Report ORNL-5138, 1976, Oak Ridge
National Laboratory, Oak Ridge, TN.
J. Org. Chem, Vol. 71, No. 10, 2006 3701

Xia et al.
the same temperature for 2 h, quenched with sodium bicarbonate
solution, and extracted with dichloromethane (3 × 20 mL). The
organic phase was dried with Na2SO4 and concentrated to a crude
residue that was passed through a column of silica gel eluted by
hexane-ethyl acetate (4:1) to give pure compound 7 in quantitative
yield. To a cold (ice-bath) solution of compound 14 (1.65 g, 47
mmol) in a mixture of dichloromethane (48 mL)-anhydrous acetic
anhydride (1.4 mL) was added dropwise 33% HBr-HOAc (12 mL)
with stirring at 0 to 25 °C for 2.5 h. The reaction mixture was
quenched with saturated sodium bicarbonate solution and extracted
with dichloromethane (3 × 100 mL). The organic phase was washed
with saturated NaHCO3 solution and brine, dried (Na2SO4), and
concentrated to a crude residue that was passed through a short
column of silica gel eluted with hexane-ethyl acetate (4:1) to give
pure compound 15 (1.0 g) as a syrup: Rf 0.80 (hexane/ethyl acetate
4.28-4.17 (m, 3 H), 4.10-4.00 (m, 3 H), 4.00-3.70 (m, 5 H),
2.16 (s, 3 H, Ac), 2.15 (s, 3 H, Ac), 2.08 (s, 3 H, Ac), 2.06 (s, 3
H, Ac), 2.05 (s, 3 H, Ac), 1.88 (s, 3 H, Ac), 1.86 (s, 3 H, Ac); 13
C
NMR (CDCl3, 100.6 MHz) δ 170.5 (CdO), 170.2 (CdO), 169.7
(CdO), 169.5 (CdO), 168.2 (CdO), 134.4, 134.3, 128.8, 128.7,
128.7, 128.5, 128.4, 101.1 (3JC-F ) 10.9 Hz), 99.0, 97.2, 88.6 (1JC-F
) 194.0 Hz), 78.2, 72.2, 70.8, 70.7, 70.5, 70.4, 69.4, 69.2, 68.7,
66.8, 62.2, 61.6, 61.5, 54.9, 47.9, 23.5 (NAc), 21.0 (Ac), 20.9 (Ac),
20.86 (Ac), 20.85 (Ac), 20.64 (Ac), 20.62 (Ac). Anal. Calcd for
C47H55O21N2F: C, 56.28; H, 5.53; F, 1.89. Found: C, 56.55; H,
5.73; F, 1.58.
Benzyl[(â-D-galactopyranosyl)-(1f4)-(2-deoxy-2-acetamido-
â-D-glucopranosyl)]-(1f6)-[(4-deoxy-4-fluoro-â-D-galactopyra-
nosyl)]-2-deoxy-2-acetamido-D-r-galactopyranoside (1). A so-
lution of compound 17 (57 mg, 0.056 mmol) in dry methanol (10
mL) was treated with NH2-NH2‚H2O (2 mL) at 90 °C for 6 h.
The reaction mixture was concentrated to a crude residue and
coevaporated with dry toluene, dried, and acetylated with anhydrous
acetic anhydride and dry pyridine (1:1, 6 mL) in the presence of
DMAP (5 mg) overnight at room temperature, then concentrated
to a crude residue that was purified by a short column of silica gel
eluted with dichloromethane-methanol (80:1) to give a pure
compound (48.3 mg) that was treated with 1 M sodium methoxide-
methanol solution (102 µL) in a mixture of methanol-water (2
mL, 1:1) overnight at room temperature. The reaction mixture was
concentrated to a crude residue and purified by a short column of
silica gel eluted with i-C3H9OH/HOAc/H2O (3:1:1) to give pure
compound 1 (30 mg, 75%) as an amorphous solid: Rf 0.5 (i-C3H9-
OH/HOAc/H2O 3:1:1); 1H NMR (D2O, DQF-COSY, and ROESY,
400 MHz) δ 7.45-7.20 (m, 5 H, ArH), 4.99 (d, 1 H, J1,2 ) 3.2
Hz, HA-1), 4.84 (dd, 1 H, J ) 2.8 Hz, JH-F ) 78 Hz, HB-4), 4.72
(d, 1 H, Jgem ) 12.4 Hz, PhCHAO, ABq), 4.56 (d, 1 H, J1,2 ) 8.4
1
2:1); H NMR (CDCl3, 400 MHz) δ 6.60 (d, 1 H, J ) 2.3 Hz,
H-1), 5.50 (m, 1 H), 5.12-4.96 (m, 2 H, H-3), 4.43 (dd, 1 H),
4.23 (dd, 1 H), 4.15 (dd, 1 H), 2.14 (s, 6 H, Ac), 2.07 (s, 3 H, Ac);
13C NMR (CDCl3, 100.6 MHz) δ 171.5 (CdO), 170.2 (CdO),
1
169.7 (CdO), 88.7 (C-1), 86.2 (d, JC-F ) 192.6 Hz, C-4), 71.5
3
2
2
(d, JC-F ) 5.4 Hz), 68.9 (d, JC-F ) 20.1 Hz), 67.1 (d, JC-F
)
17.3 Hz), 61.0, 20.87 (Ac), 20.78 (Ac), 20.70 (Ac); 19F NMR
(CDCl3, 323.6 MHz) δ -172.00 ppm.
Benzyl(2,3,6-tri-O-acetyl-4-deoxy-4-fluoro-â-D-galactopyrano-
syl)-2-deoxy-2-acetamido-D-r-galactopyranoside (16). A mixture
of compound 719f (684 mg, 1.71 mmol) and powdered Hg(CN)2 in
benzene/nitromethane (1:1, 100 mL) was heated until 50 mL of
solvent had been distilled off. The temperature was then adjusted
to 70 to 80 °C. Bromide donor 15 (951 mg, 2.57 mmol) was added
and the stirring was continued for 12 h at the same temperature.
The mixture was then diluted with benzene and washed with
saturated NaHCO3 aqueous solution, 10% KI, and water, and dried
(Na2SO4), and concentrated. The crude residue was taken up in
60% aqueous acetic acid and stirred for 1.5 h at 60-65 °C. The
solution was then concentrated under reduced pressure to a crude
residue that was applied to a column of silica gel and eluted with
dichloromethane-methanol (60:1) to give pure compound 16 (286
mg, 28%) as an amorphous solid: 1H NMR (CD3OD, 400 MHz)
δ 7.38-7.35 (m, 5 H, ArH), 5.53 (t, 1 H), 5.20-5.16 (m, 2 H),
4.95 (d, 1 H, J ) 3.4 Hz), 4.73 (d, 1 H, Jgem ) 12.4 Hz, PhCHAO,
Hz, HC-1), 4.54 (d, 1 H, J1,2 ) 7.6 Hz, HB-1), 4.53 (d, 1 H, J
gem
) 12.7 Hz, PhCHBO, ABq), 4.36 (dd, 1 H, J ) 3.2 Hz, J ) 9.6
Hz, HA-2), 4.21 (d, 1 H, J ) 2.8 Hz, HA-4), 4.15 (dd, 1 H, HA-5),
4.07 (dd, 1 H, HA-6b), 4.05 (dd, 1 H, J ) 2.8 Hz, J ) 9.6 Hz,
HA-3), 4.94 (dd, 1 H, HC-3), 3.85-3.66 (m, 5 H, HC-6b, HB-2,
HB-3, HA-6a, HC-6a), 3.64-3.41 (m, 6 H, HB-6b, HB-6a, HC-5, HB-
2, HB-5, HC-4), 2.31 (s, 3 H, Ac), 2.21 (s, 3 H, Ac); 13C NMR
(D2O, 100.6 MHz) δ 175.7 (CdO), 175.5 (CdO), 138.1, 130.1,
130.0, 129.7, 105.6, 102.8, 97.5, 90.7 (d, 1JC-F ) 178.3 Hz), 78.4,
ABq), 4.68 (dd, 1 H, J ) 3.4 Hz, J ) 9.4 Hz), 4.67 (d, 1 H, J1,2
)
2
2
77.1, 75.2, 74.7 (d, JC-F ) 17.1 Hz), 72.5 (d, JC-F ) 18.9 Hz),
71.8, 71.2, 70.9, 70.7, 70.1, 62.0, 61.2, 61.10, 56.8, 49.8, 23.5
(NAc), 23.2 (NAc). ESI-HRMS calcd for C29H43O15N2FNa (m/z):
[MNa+] 701.2540, found 701.2551.
8.7 Hz), 4.56-4.53 (m, 1 H), 4.46 (d, 1 H, Jgem ) 11.4 Hz,
PhCHBO, ABq), 4.18-4.08 (m, 2 H), 3.90-3.78 (m, 3 H), 3.58-
3.37 (m, 2 H), 2.16 (s, 3 H, Ac), 2.12 (s, 3 H, Ac), 2.03 (s, 3 H,
Ac), 1.95 (s, 3 H, Ac); 13C NMR (CD3OD, 100.6 MHz) δ 170.9
(CdO), 170.6 (CdO), 170.3 (CdO), 164.1 (CdO), 137.0, 128.6,
128.5, 128.3, 101.0, 97.3, 88.5 (d, 1JC-F ) 193.8 Hz, CB-4), 77.5,
76.9, 70.4, 70.1, 70.0, 69.9, 69.8, 69.1, 67.3, 67.1, 61.9, 61.4, 48.0,
22.8 (NAc), 20.5 (2Ac), 20.4 (Ac). Anal. Calcd for C27H36O12NF‚
H2O: C, 53.72; H, 6.34; F, 3.15. Found: C, 54.00; H, 6.10; F,
3.21.
2,3,6-Tri-O-acetyl-4-deoxy-4-fluorogalactosyl Trichloroace-
timidate (23). Compound 14 (440 mg, 1.26 mmol) was treated
with hydrazine acetic acid (150 mg, 1.66 mmol) in dry DMF (4
mL) at 50 °C for 2 h. The reaction mixture was concentrated to a
crude residue, which was redissolved in ethyl acetate (100 mL).
The organic phase was washed with saturated sodium bicarbonate
solution (3 × 100 mL), dried with Na2SO4, and concentrated to a
crude residue that was treated with CCl3CN (0.76 mL) and DBU
(38 µL) in dry dichloromethane (5 mL) at 0-25 °C for 2 h. The
reaction mixture was concentrated to a crude residue that was passed
through a short column of silica gel eluted with hexanes-ethyl
acetate (2:1) to provide pure compound 23 in 80% yield as a white
needle solid: 1H NMR (CDCl3, 400 MHz) δ 8.96 (s, 1 H, NH),
6.62 (d, 1 H, J ) 3.2 Hz, H-1), 5.45-5.30 (m, 2 H), 5.04 (dd, 1 H,
Benzyl[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-â-D-gluco-
pranosyl)]-(1f6)-[(2,3,6-tri-O-acetyl-4-deoxy-4-fluoro-â-D-galac-
topyranosyl)]-2-deoxy-2-acetamido-D-r-galactopyranoside (17).
A solution of compound 16 (80 mg, 0.133 mmol), compound 8
(74 mg, 0.140 mmol), and NIS (40 mg, 0.178 mmol) in dry
dichloromethane (6 mL) containing 4 Å MS (2.0 g) was stirred for
1.5 h at -65°C. TfOH (27 uL) in dry dichloromethane (0.5 mL)
was added and the solution was stirred for 1 h at the same
temperature under N2 atmosphere. The reaction was quenched with
saturated sodium bicarbonate solution. The solids were filtered off
and the organic layer was washed with saturated 10% Na2S2O3,
dried with Na2SO4, and concentrated. The crude residue was passed
through a short column of silica gel eluted with dichloromethane/
methanol (80:1) to give pure compound 17 (65 mg, 48%) as an
amorphous solid: Rf 0.38 (CH2Cl2/MeOH 30:1); 1H NMR (CDCl3,
400 MHz) δ 7.72-7.56 (m, 4 H, ArH), 7.36-7.29 (m, 5 H, ArH),
5.86-5.81 (m, 1 H), 5.49 (t, 1 H), 5.43 (d, 1 H), 5.49-5.31 (m, 1
H), 5.24-5.17 (m, 2 H), 4.60-4.53 (m, 3 H), 4.50-4.30 (m, 5 H),
2
J ) 3.2 Hz, J ) 1.8 Hz, JH-F ) 50.6 Hz, H-4), 4.38-4.23 (m, 3
H), 2.15 (s, 3 H, Ac), 2.06 (s, 3 H, Ac), 2.03 (s, 3 H, Ac); 13C
NMR (CDCl3, 100.6 MHz) δ 170.5 (CdO), 170.4 (CdO), 170.0
(CdO), 93.6 (C-1), 86.5 (d, 1JC-F ) 195.6 Hz, C-4), 69.5 (d, 2JC-F
) 18.1 Hz), 68.3 (d, 2JC-F ) 17.4 Hz), 66.9, 61.6, 61.5, 20.9 (Ac),
20.8 (Ac), 20.6 (Ac); 19F NMR (CDCl3, 323.6 MHz) δ -172.00
ppm.
Phenyl(2,3,6-tri-O-acetyl-4-deoxy-4-fluoro-â-D-galactopyra-
nosyl)-(1f4)-2-deoxy-6-O-pivaloyl-3-O-acetyl-2-phthalimido-1-
thio-â-D-glucopyranoside (24). A solution of compound 917 (178
3702 J. Org. Chem., Vol. 71, No. 10, 2006

Synthesis of Fluorinated Mucin Core 2 Oligosaccharides
mg, 0.367 mmol) and compound 23 (174 mg, 0.385 mmol) in dry
dichloromethane (6 mL) containing 4 Å MS (2.0 g) was stirred for
1.5-2 h at -65 to -70 °C. TMSOTf (14 µL) in dry dichlo-
romethane (0.5 mL) was added and stirred for 1 h under N2
atmosphere. Another portion of donor 23 (174 mg) was added and
the solution was stirred for another 1.5 h. The reaction was
quenched with saturated sodium bicarbonate solution. The solids
were filtered off and the organic layer was washed with saturated
sodium bicarbonate, dried with Na2SO4, and concentrated. The
crude residue was passed through a short column of silica gel eluted
with hexanes-ethyl acetate (1.5:1) to give a disaccharide (120 mg,
41%), which was further treated with anhydrous anhydride-
pyridine (1:1, 6 mL) in the presence of DMAP (4 mg) overnight at
room temperature. The reaction mixture was concentrated to a crude
residue, which was purified by a short column of silica gel eluted
with hexanes-ethyl acetate (2:1) to give pure compound 24 (80
mg, 63%) as an amorphous solid: 1H NMR (CDCl3, 400 MHz,
DQF-COSY, TOCSY, and ROESY) δ 7.92-7.84 (m, 2 H, ArH),
7.76-7.72 (m, 2 H, ArH), 7.44-7.36 (m, 2 H, ArH), 7.32-7.20
(m, 3 H, ArH), 5.76-5.71 (m, 2 H, J1,2 ) 10.2 Hz, HA-3, HA-1),
5.17 (dd, 1 H, HB-2), 4.87 (qd, 1 H, J2,3 ) 10.3 Hz, J3,4 ) 2.8 Hz,
3JH-F ) 27.6 Hz, HB-3), 4.79 (dd, 1 H, J3,4 ) 3.3 Hz, 2JH-F ) 50.0
Hz, HB-4), 4.53 (dd, 1 H, J ) 14.3 Hz, HA-6b), 4.51 (d, 1 H, J1,2
) 7.9 Hz, HB-1), 4.31-4.26 (m, 1 H, HB-6b), 4.23 (t, 1 H, J )
10.1 Hz, HA-2), 4.17-4.12 (m, 3 H, HB-6a, HA-6a, HA-5), 3.87-
3.68 (m, 2 H, HA-4, HB-5), 2.08 (s, 6 H, 2Ac), 2.06 (s, 3 H, Ac),
1.87 (s, 3 H, Ac), 1.20 (s, 9 H, t-Bu); 13C NMR (CDCl3, 100.6
MHz) δ 177.9 (CdO), 174.4 (CdO), 174.3 (CdO), 170.4 (CdO),
169.9 (CdO), 169.0 (CdO), 168.9 (CdO), 134.6, 134.4, 133.4,
70.9, 70.8, 69,6, 69.5, 69.5, 69.2, 69.0, 68.7, 68.4, 67.0, 62.1, 61.4,
61.2, 55.3, 47.8, 27.4, 23.4 (NAc), 20.87 (2Ac), 20.83 (2Ac), 20.78
(Ac), 20.75 (Ac), 20.71 (Ac), 20.68 (Ac). Anal. Calcd for
C62H77O30N2F: C, 55.18; H, 5.75; N, 2.09. Found: C, 55.31; H,
5.58; N, 2.04.
Benzyl[(4-deoxy-4-fluoro-â-D-galactopyranosyl)-(1f4)-(2-
deoxy-2-acetamido-â-D-gluco-ppranosyl)]-(1f6)-[(â-D-galacto-
pyranosyl)]-2-deoxy-2-acetamido-D-r-galactopyranoside (3). A
solution of compound 25 (82 mg, 0.06 mmol) in dry methanol (10
mL) was treated with NH2-NH2‚H2O (2 mL) at 90 °C for 6 h.
The reaction mixture was concentrated to a crude residue and
coevaporated with dry toluene, dried, and acetylated with anhydrous
acetic anhydride-dry pyridine (1:1, 6 mL) in the presence of DMAP
(5 mg) overnight at room temperature, then it was concentrated to
a crude residue, which was purified by a short column of silica gel
eluted with dichloromethane-methanol (80:1) to give a pure
compound that was treated with 1 M sodium methoxide-methanol
solution in a mixture of methanol-water (2 mL, 1:1) overnight at
room temperature. The reaction mixture was then concentrated to
a crude residue, which was purified by a short column of silica gel
eluted with i-C3H9OH/HOAc/H2O (3:1:1) to give pure compound
1
3 (20 mg, 39%) as an amorphous solid: H NMR (D2O, DQF-
COSY, TOCSY, and ROESY, 400 MHz) δ 7.50-7.00 (m, 5 H,
ArH), 5.00 (d, 1 H, J1,2 ) 3.2 Hz, HA-1), 4.87 (dd, 1 H, J ) 2.8
Hz, 2JH-F ) 52.0 Hz, HD-4), 4.73 (d, 1 H, Jgem ) 12.3 Hz, PhCHAO,
ABq), 4.59 (d, 1 H, J1,2 ) 7.6 Hz, HD-1), 4.58 (d, 1 H, J1,2 ) 8.0
Hz, HC-1), 4.53 (d, 1 H, Jgem ) 12.4 Hz, PhCHBO, ABq), 4.46 (d,
1 H, J1,2 ) 7.8 Hz, HB-1), 4.34 (dd, 1 H, HA-2), 4.25 (d, 1 H, J )
2.8 Hz, HA-4), 4.16 (dd, 1 H, HA-5), 4.10 (dd, 1 H, HA-6b), 4.08
(dd, 1 H, HA-3), 4.02 (dd, 1 H), 3.92-3.80 (m, 6 H, HC-4), 3.81
(dd, 1 H, HD-3), 3.80-3.50 (m, 10 H, HD-2, HA-6a, HC-3, HD-2,
HC-2), 2.31 (s, 3 H, Ac), 2.29 (s, 3 H, Ac); 13C NMR (D2O, 100.6
MHz) δ 175.8 (CdO), 175.6 (CdO), 129.6, 129.4, 129.3, 105.9,
1
129.1, 128.5, 123.9, 123.7, 100.8(CB-1), 85.5 (d, JC-F ) 187.1
Hz, CB-4), 83.2 (CA-1), 77.2, 76.6, 71.6 (d, 2JC-F ) 17.1 Hz), 71.1
2
3
(d, JC-F ) 18.4 Hz), 69.2, 62.5, 61.2 (d, JC-F ) 5.1 Hz), 54.0,
31.7, 27.4 (3CH3), 23.1 (Ac), 20.8 (Ac), 20.7 (Ac), 20.6 (Ac). Anal.
Calcd for C39H44O15NSF: C, 57.27; H, 5.42; F, 2.32. Found: C,
57.30; H, 5.42,; F, 2.41.
1
2
104.1, 102.8, 97.8, 79.8 (d, JC-F ) 178.5 Hz), 76.1 (d, JC-F
)
2
50.6 Hz), 73.9, 72.1 (d, JC-F ) 50.9 Hz), 71.5, 71.2, 70.2, 70.0,
62.2, 61.2, 56.2, 49.9, 23.5 (NAc), 23.2 (NAc); 19F NMR (CDCl3,
376.4 MHz) δ -172.95 ppm; ESI-HRMS calcd for C35H53O20N2-
FNa (m/z) [MNa+] 863.3068, found 863.3035.
Benzyl[(2,3,6-tri-O-acetyl-4-deoxy-4-fluoro-â-D-galactopyra-
nosyl)-(1f4)-(2-deoxy-6-O-pivaloyl-3-O-acetyl-2-phthalimido-
â-D-glucopranosyl)]-(1f6)-[(2,3,4,6-tetra-O-acetyl-â-D-galacto-
pyranosyl)]-2-deoxy-2-acetamido-D-r-galactopyranoside (25). A
solution of compound 24 (62 mg, 0.074 mmol), compound 12 (45
mg, 0.070 mmol), and NIS (50 mg, 0.222 mmol) in dry dichlo-
romethane (5 mL) containing 4 Å MS (2.0 g) was stirred for 1.5-2
h at -65 to -70 °C. TfOH (14 µL) in dry dichloromethane (0.5
mL) was added and the solution was stirred for 1 h at the same
temperature under N2 atmosphere. The reaction was quenched with
saturated sodium bicarbonate solution. The solids were filtered off
and the organic layer was washed with saturated 10% Na2S2O3,
dried with Na2SO4, and concentrated. The crude residue was passed
through a short column of silica gel eluted with hexanes-ethyl
acetate (1:4) to give pure compound 25 (72 mg, 76%) as an
amorphous solid: 1H NMR (CDCl3, 600 MHz, DQF-COSY,
TOCSY, and ROESY) δ 7.88-7.84 (m, 4 H, ArH), 7.40-7.28 (m,
3 H, ArH), 7.20-6.84 (m, 2 H, ArH), 5.75-5.71 (m, 1 H, HC-3),
5.41 (d, 1 H, J1,2 ) 8.3 Hz, HC-1), 5.32 (d, 1 H, J ) 9.3 Hz, NHAc),
5.29 (d, 1 H, J ) 3.0 Hz, HB-4), 5.21-5.10 (m, 2 H, HD-2, HB-2),
1
1,2:5,6-Diisopropyl-3-deoxy-3-fluoro-r-D-galactose (10). H
NMR (CDCl3, 400 MHz) δ 5.92 (d, 1 H, J ) 4.0 Hz, H-1), 4.80
(dd, 1 H, J ) 4.3 Hz, J ) 3.3 Hz, JH-F ) 67.7 Hz, H-3), 4.75 (d,
1 H, J ) 3.2 Hz, H-4), 4.34 (dd, 1 H, J ) 6.8 Hz, J ) 7.3 Hz),
4.15-4.06 (m, 2 H), 3.83 (dd, 1 H), 1.55 (s, 3 H, CH3), 1.46 (s, 3
H, CH3), 1.38 (s, 3 H, CH3), 1.36 (s, 3 H, CH3); 13C NMR (CDCl3,
4
100.6 MHz) δ 114.0 (d, ketal carbon), 110.3 (d, JC-F ) 2.8 Hz,
3
1
ketal carbon), 105.2 (d, JC-F ) 2.1 Hz, C-1), 94.4 (d, JC-F
)
181.7 Hz, C-3), 85.1-84.6 (m, 2C), 75.2-75.1 (m, 1C), 65.8, 27.3
(CH3), 26.7 (CH3), 26.6 (CH3), 25.4 (CH3); ESI-HRMS calcd for
C12H19O5FNa (m/z) [MNa+] 285.1109, found 285.1110.
2,4,6-Tri-O-acetyl-3-deoxy-3-fluoro-r-D-galactopyranosyl Bro-
mide (19). Compound 19 (yield 71%) was prepared from compound
18 according to the procedure for the preparation of compound
15. Rf 0.36 (hexane/ethyl acetate 4:1); 1H NMR (CDCl3, 400 MHz)
δ 6.68 (t, 1 H, H-1), 5.68 (m, 1 H), 5.12-4.96 (m, 2 H, H-3), 4.43
(t, 1 H), 4.22 (dd, 1H), 4.10 (dd, 1 H), 2.16 (s, 6 H, Ac), 2.07 (s,
3 H, Ac); 13C NMR (CDCl3, 100.6 MHz) δ 170.4 (CdO), 170.1
(CdO), 169.7 (CdO), 87.7 (d, 3JC-F ) 8.0 Hz, C-1), 86.2 (d, 1JC-F
3
4.88 (qd, 1 H, J2,3 ) 10.2 Hz, J3,4 ) 2.6 Hz, JH-F ) 28.0 Hz,
HD-3), 4.80 (dd, 1 H, J ) 2.1 Hz, 2JH-F ) 55.6 Hz, HD-4), 4.60 (d,
1 H, J ) 3.8 Hz, HA-1), 4.43 (ddd, 1 H, HA-2), 4.39 (d, 1 H, J1,2
) 7.7 Hz, HD-1), 4.32-4.27 (m, 2 H, HA-5, HB-1), 4.24-4.14 (m,
4 H, PhCHAO, HD-6b, HC-2, HD-6a), 4.08-3.90 (m, 5 H, HB-6b,
HB-6a, HA-5), 3.91-3.50 (m, 9 H, HC-4, PhCHBO, HA-4, HA-6b,
HD-5, HA-6a, HB-5, HA-3), 2.13 (s, 3 H, Ac), 2.09 (s, 6 H, 2Ac),
2.08 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.02 (s, 3 H, Ac), 1.96 (s, 3
H, Ac), 1.88 (s, 3 H, Ac), 1.87 (s, 3 H, Ac), 1.26 (s, 9 H, t-Bu);
13C NMR (CDCl3, 100.6 MHz) δ 181.0 (CdO), 178.1 (CdO),
174.5 (CdO), 170.6 (CdO), 170.5 (CdO), 170.4 (CdO), 170.3
(CdO), 170.6 (Cd), 169.5 (CdO), 169.4 (CdO), 169.1 (CdO),
167.8 (CdO), 137.0, 134.7, 134.3, 128.6, 128.8, 128.7, 128.5, 123.9,
123.5, 101.6, 100.8, 98.5, 97.2, 85.5, 78.5, 71.1, 71.0, 71.0, 70.9,
) 192.6 Hz, C-3), 71.4 (d, 3JC-F ) 5.4 Hz, H-4), 68.9 (d, 2JC-F
)
2
20.1 Hz), 67.1 (d, JC-F ) 17.3 Hz), 61.0, 20.9 (Ac), 20.8 (Ac),
20.7 (Ac); 19F NMR (CDCl3, 323.6 MHz) δ -172.00 ppm
Benzyl(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-â-D-galactopyrano-
syl)-2-deoxy-2-acetamido-D-r-galactopyranoside (20). Compound
20 was prepared according to the procedure for the preparation of
compound 16 with a yield of 47% over two steps. Rf 0.32
1
(dichloromethane-methanol: 20:1). H NMR (CDCl3, 400 MHz)
δ 7.38-7.35 (m, 5 H, ArH), 5.53 (t, 1 H), 5.20-5.16 (m, 2 H),
4.95 (d, 1 H, J ) 3.4 Hz), 4.73 (d, 1 H, Jgem ) 12.4 Hz, PhCHAO,
ABq), 4.68 (dd, 1 H, J ) 3.4 Hz, J ) 9.4 Hz), 4.67 (d, 1 H, J1,2
)
8.7 Hz), 4.56-4.53 (m, 1 H), 4.46 (d, 1 H, Jgem ) 11.4 Hz,
J. Org. Chem, Vol. 71, No. 10, 2006 3703

Xia et al.
PhCHBO, ABq), 4.18-4.08 (m, 3 H), 3.90-3.78 (m, 5 H), 3.58-
3.37 (m, 2 H), 2.16 (s, 3 H, Ac), 2.12 (s, 3 H, Ac), 2.03 (s, 3 H,
Ac), 1.95 (s, 3 H, Ac); 13C NMR (CDCl3 + CD3OD, 100.6 MHz)
δ 170.9 (CdO), 170.7 (CdO), 170.3 (CdO), 164.2 (CdO), 137.0,
stirred for 1 h at the same temperature under N2 atmosphere. The
reaction was quenched with saturated sodium bicarbonate solution.
The solids were filtered off and the organic layer was washed with
saturated 10% Na2S2O3, dried with Na2SO4, and concentrated. The
crude residue was passed through a short column of silica gel eluted
with dichloromethane-methanol (60:1) to give pure compound 27
(64 mg, 26%) as an amorphous solid: Rf 0.15 (CH2Cl2/MeOH 40:
1) 1H NMR (CDCl3, 600 MHz, DQF-COSY, TOCSY, and ROESY)
δ 7.88-7.28 (m, 4 H, ArH), 7.20-6.84 (m, 5 H, ArH), 5.75-5.72
(m, 1 H), 5.41 (d, 1 H), 5.32 (d, 1 H, J ) 9.3 Hz, NHAc), 5.29 (d,
1 H, J ) 3.0 Hz), 5.21-5.08 (m, 2 H), 4.88 (qd, 1 H), 4.80 (dd, 1
H), 4.61 (d, 1 H, J ) 3.8 Hz, HA-1), 4.45 (ddd, 1 H, HA-2), 4.40
(d, 1 H, J1,2 ) 7.7 Hz, HD-1), 4.32-4.25 (m, 2 H), 4.24-4.14 (m,
4 H), 4.09-3.90 (m, 5 H), 3.91-3.51 (m, 9 H), 2.13 (s, 3 H, Ac),
2.09 (s, 6 H, 2Ac), 2.08 (s, 6 H, 2Ac), 2.04 (s, 3 H, Ac), 2.02 (s,
3 H, Ac), 1.95 (s, 3 H, Ac), 1.89 (s, 3 H, Ac), 1.88 (s, 3 H, Ac);
13C NMR (CDCl3, 100.6 MHz) δ 181.0 (CdO), 178.1 (CdO),
174.5 (CdO), 170.6 (CdO), 170.45 (CdO), 170.41 (CdO), 170.38
(CdO), 170.6 (Cd), 169.5 (CdO), 169.4 (CdO), 169.1 (CdO),
137.0, 134.7, 134.3, 128.7, 128.8, 128.7, 128.5, 123.9, 123.5, 101.6,
100.8, 98.5, 97.3, 85.6, 78.5, 71.1, 71.08, 71.03, 70.93, 70.90, 70.8,
69,6, 69.54, 69.51, 69.2, 69.0, 68.7, 68.4, 67.0, 62.2, 61.4, 61.3,
61.2, 55.3, 47.8, 23.4 (NAc), 20.88 (Ac), 20.84 (Ac), 20.79
(Ac), 20.76 (Ac), 20.72 (Ac), 20.69 (Ac). Anal. Calcd for
C59H71O30N2F: C, 54.19; H, 5.47; F, 1.45. Found: C, 54.25; H,
5.38; F, 1.31.
3
128.6, 128.5, 128.3, 101.0 (d, JC-F ) 11.6 Hz, CB-1), 97.4 (CA-
1
1), 88.5 (d, JC-F ) 193.8 Hz, CB-3), 77.5, 76.9, 70.4, 70.0, 69.9,
69.9, 69.1, 67.2, 67.1, 61.9, 48.1, 22.8 (NAc), 20.6 (NAc), 20.5
(NAc). Anal. Calcd for C27H36O12NF‚H2O: C, 53.72; H, 6.34; N,
2.33. Found: C, 53.43; H, 5.89; N, 2.12.
Benzyl[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-â-D-gluco-
pranosyl)]-(1f6)-[(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-â-D-galac-
topyranosyl)]-2-deoxy-2-acetamido-D-r-galactopyranoside (21).
A solution of compound 20 (50 mg, 0.073 mmol), compound 8
(45 mg, 0.085 mmol), and NIS (58 mg, 0.256 mmol) in dry
dichloromethane (10 mL) containing 4 Å MS (1.5 g) was stirred
for 1.0 h at -65 °C. TfOH (16 µL) in dry dichloromethane (0.5
mL) was added and the solution was stirred for 1 h at the same
temperature under N2 atmosphere. The reaction was quenched with
saturated sodium bicarbonate solution. The solids were filtered off
and the organic layer was washed with saturated 10% Na2S2O3,
dried with Na2SO4, and concentrated. The crude residue was passed
through a short column of silica gel eluted with dichloromethane-
methanol (100:1) to give pure compound 21 (32 mg, 40%) as an
amorphous solid: 1H NMR (CDCl3, 400 MHz) δ 7.72-7.56 (m, 4
H, ArH), 7.36-7.29 (m, 3 H, ArH), 7.20-7.12 (m, 2 H, ArH),
5.83 (m, 1 H, HC-3), 5.48 (dd, 1 H, HB-4), 5.43 (d, 1 H, J1,2 ) 8.6
Hz, HC-1), 5.36 (m, 1 H), 5.31 (d, 1 H, J ) 7.6 Hz, HB-1), 5.24-
5.17 (m, 2 H, NHAc, HC-4), 4.54 (d, 1 H, J ) 3.2 Hz, HA-1), 4.51
(dq, 1 H, HB-3), 4.50-4.30 (m, 2 H, HB-2, HA-2), 4.28-4.17 (m,
4 H, HC-6b, HC-2, PhCHAO, HC-6a), 4.10-4.00 (m, 2 H, HA-5),
4.00-3.70 (m, 5 H, HA-4, HC-5, HA-6b, HA-6a,), 3.60 (dd, 1 H,
HA-3), 3.59 (m, 1 H), 2.16 (s, 3 H, Ac), 2.15 (s, 3 H, Ac), 2.08 (s,
3 H, Ac), 2.05 (s, 3 H, Ac), 2.04 (s, 3 H, Ac), 1.88 (s, 3 H, Ac),
1.85 (s, 3 H, Ac); 13C NMR (CDCl3, 100.6 MHz) δ 170.5 (CdO),
170.3 (CdO), 169.8 (CdO), 169.5 (CdO), 168.2 (CdO), 134.5,
134.4, 128.8, 128.8, 128.7, 128.6, 128.5, 101.1 (3JC-F ) 10.9 Hz),
99.0, 97.2, 88.7 (1JC-F ) 194.9 Hz), 78.2, 72.2, 70.9, 70.7, 70.5,
70.4, 69.4, 69.3, 68.8, 66.8, 62.3, 61.6, 61.6, 54.9, 47.9, 23.5 (NAc),
21.0 (Ac), 20.9 (Ac), 20.86 (Ac), 20.83 (Ac), 20.65 (Ac), 20.62
(Ac). Anal. Calcd for C47H55O21N2F‚2H2O: C, 54.32; H, 5.72.
Found: C, 54.74; H, 5.73.
Benzyl[(â-D-galactopyranosyl)-(1f4)-(2-deoxy-2-acetamido-
â-D-glucopranosyl)]-(1f6)-[(3-deoxy-3-fluoro-â-D-galactopyra-
nosyl)]-2-deoxy-2-acetamido-D-r-galactopyranoside (4). Com-
pound 4 (28 mg, 80%) was obtained from compound 27 according
to procedure described for compound 17. Rf 0.5 (i-C3H9OH/HOAc/
H2O 3:1:1); 1H NMR (D2O, DQF-COSY and ROESY, 400 MHz)
δ 7.50-7.20 (m, 5 H, ArH), 4.99 (d, 1 H, J1,2 ) 3.4 Hz, HA-1),
4.72 (d, 1 H, Jgem ) 12.6 Hz, PhCHAO, ABq), 4.61 (dq, 1 H, HB-
3), 4.58 (d, 1 H, J1,2 ) 7.6 Hz, HB-1), 4.53 (d, 1 H, Jgem ) 12.4
Hz, PhCHBO, ABq), 4.49 (d, 1 H, J1,2 ) 8.6 Hz, HC-1), 4.48 (d, 1
H, J1,2 ) 7.6 Hz, HD-1), 4.35 (dd, 1 H, HA-2), 4.23 (d, 1 H, J )
2.8 Hz, HA-4), 4.20 (dd, 1 H, HB-4), 4.15 (dd, 1 H, HA-5), 4.07
(dd, 1 H, HA-6b), 4.04 (dd, 1 H, HA-3), 4.01 (dd, 1 H), 3.94 (d, 1
H, J ) 2.7 Hz, HD-4), 3.90-3.50 (m, 15 H, HB-2, HD-2), 2.31 (s,
3 H, Ac), 2.21 (s, 3 H, Ac); 13C NMR (D2O, 100.6 MHz) δ 175.7
3
(CdO), 175.5 (CdO), 138.2, 130.1, 129.9, 129.7, 105.1 (d, JC-F
Benzyl[(2-deoxy-2-acetadimido-â-D-glucopranosyl)]-(1f6)-(3-
deoxy-3-fluoro-â-D-galactopyranosyl)-2-deoxy-2-acetamido-D-r-
galactopyranoside (2). Compound 2 was obtained from compound
21 according to the procedure described for compound 17 in 75%
yield. 1H NMR (D2O, DQF-COSY and ROESY, 400 MHz) δ 7.60-
7.20 (m, 5 H, ArH), 4.98 (d, 1 H, J ) 3.4 Hz, HA-1), 4.76 (d, 1 H,
Jgem ) 12.6 Hz, PhCHAO, ABq), 4.55 (d, 1 H, J ) 8.4 Hz, HC-1),
4.54 (dq, 1 H, HB-3), 4.51 (d, 1 H, Jgem ) 12.3 Hz, PhCHBO, ABq),
4.48 (d, 1 H, J ) 7.6 Hz, HB-1), 4.33 (dd, 1 H, HA-2), 4.23 (d, 1
H, J ) 2.8 Hz, HA-4), 4.20 (dd, 1 H, HB-4), 4.15 (dd, 1 H, HA-5),
4.07 (dd, 1 H, HA-6b), 4.03 (dd, 1 H, HA-3), 3.94 (dd, 1 H, HC-3),
3.86-3.60 (m, 7 H, HB-2, HC-6b, HC-2, HA-6a, HB-5, HB-6b, HB-
6a), 3.60-3.40 (m, 3 H, HC-5, HC-6a, HC-4), 1.96 (s, 3 H, Ac),
1.93 (s, 3 H, Ac); 13C NMR (CD3COOD, 100.6 MHz) δ 175.3
1
) 11.3 Hz), 104.2, 102.7, 97.6, 94.1 (d, JC-F ) 183.4 Hz), 79.9,
78.5, 76.6, 76.1, 75.0, 74.9, 73.9, 73.8, 72.3, 70.9, 70.8, 70.7, 70.6
2
2
(d, JC-F ) 18.6 Hz), 69.9 (d, JC-F ) 20.4 Hz), 62.3, 61.9, 61.8,
61.4, 56.4, 49.8, 23.5 (NAc), 23.2 (NAc); ESI-HRMS calcd for
C35H53O20N2FNa (m/z) [MNa+] 863.3068, found 863.3037.
2,4,6-Tri-O-acetyl-3-deoxy-3-fluorogalactosyl Trichloroace-
timidate (29). A solution of compound 18 (2.34 g, 6.94 mmol) in
DMF (8 mL) was treated with hydrazine acetic acid (922 mg) at
50 °C for 2 h. The reaction mixture was concentrated to a crude
residue, which was redissolved in ethyl acetate (100 mL) and
washed with saturated sodium bicarbonate solution (3 × 100 mL)
and the organic phase was dried with Na2SO4 and concentrated to
a crude residue that was treated with CCl3CN (2 mL) and DBU
(200 µL) in dry dichloromethane (10 mL) at 0-25 °C for 2 h. The
reaction mixture was concentrated to a crude residue, which was
passed through a short column of silica gel eluted with hexanes-
ethyl acetate (2:1) to provide pure compound 29 (984 mg) in 32%
as an amorphous solid: 1H NMR (CDCl3, 400 MHz) δ 8.95 (s, 1
H, NH), 6.70 (d, 1 H, J ) 3.1 Hz, H-1), 5.45-5.30 (m, 2 H), 5.04
(m, 1 H), 4.38-4.23 (m, 3 H), 2.15 (s, 3 H, Ac), 2.06 (s, 3 H, Ac),
2.03 (s, 3 H, Ac); 13C NMR (CDCl3, 100.6 MHz) δ 170.5 (CdO),
(CdO), 175.1 (CdO), 138.1, 129.9, 129.9, 129.6, 105.0 (3JC-F
)
12.2 Hz), 102.7, 97.5, 94.0 (1JC-F ) 183.9 Hz), 80.6, 78.4, 77.1,
75.1, 74.9, 71.2, 70.8, 70.7, 70.6, 70.5, 70.4, 69.9, 67.9, 61.9, 61.8,
56.8, 49.7, 23.5 (NAc), 23.1 (NAc); 19F NMR (CDCl3, 376.4 MHz)
δ -175.4 ppm; ESI-HRMS calcd for C29H43O15N2FNa (m/z)
[MNa+] 701.2540, found 701.2543.
Benzyl[(3,4,6-tri-O-acetyl-D-â-galactosyl)-(1f4)-2-deoxy-2-
phthalimido-â-D-glucopranosyl)]-(1f6)-[(2,4,6-tri-O-acetyl-3-
deoxy-3-fluoro-â-D-galactopyranosyl)]-2-deoxy-2-acetamido-D-
r-galactopyranoside (27). A solution of compound 20 (124 mg,
0.183 mmol), compound 13 (148 mg, 0.192 mmol), and NIS (130
mg, 0.57 mmol) in dry dichloromethane (5-6 mL) containing 4 Å
MS (3.0 g) was stirred for 1.5 h at -65 °C then TfOH (37 µL) in
dry dichloromethane (0.5 mL) was added and the solution was
1
170.4 (CdO), 170.1 (CdO), 93.6 (C-1), 86.4 (d, JC-F ) 195.6
2
2
Hz, C-3), 69.5 (d, JC-F ) 18.2 Hz), 68.3 (d, JC-F ) 17.8 Hz),
66.9, 61.6, 61.5, 20.9 (Ac), 20.8 (Ac), 20.6 (Ac); 19F NMR (CDCl3,
323.6 MHz) δ -172.15 ppm.
Phenyl(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-â-D-galactopyra-
nosyl)-(1f4)-2-deoxy-6-O-naphthylmethyl (NAP)-3-O-acetyl-2-
3704 J. Org. Chem., Vol. 71, No. 10, 2006

Synthesis of Fluorinated Mucin Core 2 Oligosaccharides
phthalimido-1-thio-â-D-glucopyranoside (30). A solution of
compound 1119f (368 mg, 0.77 mmol) and compound 29 (354 mg,
0.808 mmol) in dry dichloromethane (10 mL) containing 4 Å MS
(4.0 g) was stirred for 1.5-2 h at -45 °C. TMSOTf (38 µL) in
dry dichloromethane (0.5 mL) was added and the solution was
stirred for 1 h under N2 atmosphere. The reaction was quenched
with sodium bicarbonate solution. The solids were filtered off and
the organic layer was washed with sodium bicarbonate, dried with
Na2SO4, and concentrated. The crude residue was passed through
a short column of silica gel eluted with hexanes-ethyl acetate (2:
1) to give a disaccharide (275 mg, 43%), which was further treated
with anhydrous anhydride-pyridine (1:1, 6 mL) in the presence
of DMAP (5 mg) overnight at room temperature. The reaction
mixture was concentrated to a crude residue that was purified by a
short column of silica gel eluted with hexanes-ethyl acetate (2:1)
to give pure compound 30 (210 mg, 76%) as an amorphous solid:
Rf 0.16 (hexane/ethyl acetate 2:1); 1H NMR (CDCl3, 400 MHz) δ
7.92-7.80 (m, 9 H, ArH), 7.60-7.40 (m, 5 H, ArH), 7.28-7.20
(m, 2 H, ArH), 5.74-5.68 (m, 2 H), 5.29-5.28 (m, 1 H), 5.04-
4.99 (m, 1 H), 4.95 (d, 1 H, Jgem ) 13.1 Hz, ArCHAO, ABq), 4.66
(d, 1 H, Jgem ) 13.0 Hz, ArCHBO, ABq), 4.35-4.29 (m, 2 H),
4.13-3.94 (m, 4 H), 3.86-3.85 (m, 2 H), 3.70 (dt, 1 H), 3.47-
3.44 (m, 1 H), 2.08 (s, 3 H, Ac), 2.04 (s, 3 H, Ac), 1.99 (s, 3 H,
Ac), 1.84 (s, 3 H, Ac); 13C NMR (CDCl3, 100.6 MHz) δ 170.5
(CdO), 170.1 (CdO), 170.1 (CdO), 168.8 (CdO), 135.5, 134.6,
134.4, 133.2, 129.1, 128.5, 128.4, 128.02, 127.9, 127.2, 126.7,
Benzyl[(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-â-D-galactopyran-
osyl)-(1f4)-(2-deoxy-6-O-naphthylmethyl-3-O-acetyl-2-acet-
adimido-â-D-glucopranosyl)]-(1f6)-[(2,3,4,6-tetra-O-acetyl-â-
D-galactopyranosyl)]-2-deoxy-2-acetamido-D-r-galactopyranoside
(32). A solution of compound 31 (141 mg, 0.10 mmol) in dry
methanol (10 mL) was treated with NH2-NH2‚H2O (2 mL) at 85-
90 °C for 6 h. The reaction mixture was concentrated to a crude
residue and coevaporated with dry toluene, dried, and acetylated
with anhydrous acetic anhydride and dry pyridine (1:1, 8 mL) in
the presence of DMAP (5 mg) overnight at room temperature, then
concentrated to a crude residue, which was purified by a short
column of silica gel eluted with dichloromethane-methanol (80:
1) to give pure compound 32 (103 mg, 93%) as an amorphous
solid: Rf 0.16 (CH2Cl2/MeOH 30:1); 1H NMR (CDCl3, DQF-
COSY, and ROESY, 400 MHz) δ 7.88-7.81 (m, 4 H, ArH), 7.54-
7.48 (m, 3 H, ArH), 7.37-7.31 (m, 5 H, ArH), 5.64 (d, 1 H, J )
9.9 Hz, NHAc), 5.61 (d, 1 H, J ) 9.5 Hz, NHAc), 5.33 (d, 2 H, J
) 3.4 Hz, HB-4, HA-4), 5.23 (dd, 1 H, HD-4), 5.09 (dd, 1 H, J )
7.7 Hz, J ) 8.5 Hz, HB-2), 5.04-4.90 (m, 4 H, HC-3, HA-1, HD-2,
HB-3), 4.71 (d, 1 H, Jgem ) 11.4 Hz, PhCHAO, ABq), 4.58 (d, 1 H,
J1,2 ) 7.6 Hz, HD-1), 4.54 (d, 1 H, J1,2 ) 8.4 Hz, HB-1), 4.54 (dd,
1 H, HA-2), 4.43 (d, 1 H, J ) 8.4 Hz, HC-1), 4.39 (d, 1 H, Jgem
)
11.0 Hz, PhCHBO, ABq), 4.20-3.76 (m, 14 H, HC-2, HA-3, HD-
3), 3.50-3.38 (m, 4 H, HC-5, HD-5), 2.11 (s, 6 H, 2Ac), 2.09 (s, 3
H, Ac), 2.06 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.00 (s, 3 H, Ac), 1.98
(s, 3 H, Ac), 1.97 (s, 3 H, Ac), 1.95 (s, 6 H, 2Ac), 1.86 (s, 3 H,
Ac); 13C NMR (CDCl3, 100.6 MHz) δ 171.1 (CdO), 170.6 (Cd
O), 170.5 (CdO), 170.4 (CdO), 170.3 (CdO), 170.2 (CdO), 170.1
(CdO), 170.0 (CdO), 169.9 (CdO), 169.8 (CdO), 169.1 (CdO),
137.1, 135.5, 133.4, 133.3, 130.2, 129.6, 129.3, 128.9, 128.8, 128.7,
128.6, 128.5, 128.0, 127.9, 127.4, 126.8, 126.6, 126.5, 101.7,
3
126.5, 126.3, 123.9, 123.7, 99.9 (d, JC-F ) 10.7 Hz, CB-1), 88.9
(d, 1JC-F ) 193.9 Hz, CB-3), 88.5 (CA -1), 79.1, 75.5, 73.9, 72.1,
2
2
70.5, 70.2 (d, J
) 19.4 Hz), 67.5, 66.7 (d, JC-F ) 16.7 Hz),
C-F
61.0, 54.2, 20.9 (Ac), 20.8 (Ac), 20.7 (Ac), 20.7 (Ac). Anal. Calcd
for C45H44O14NFS: C, 61.84; H, 5.07; F, 2.17. Found: C, 61.77;
H, 5.12; F, 1.95.
3
1
100.92, 99.8 (d, JC-F ) 11.4 Hz), 97.1, 88.7 (d, JC-F ) 195.0
Hz), 74.8, 74.7, 73.9, 73.4, 72.8, 71.1, 70.9, 69.9, 69.8, 69.2, 68.9,
68.9, 67.4, 67.0, 66.8, 66.7, 61.4, 61.2, 61.1, 53.8, 49.1, 23.5 (NAc),
23.5 (NAc), 21.0 (Ac), 20.87 (Ac), 20.86 (Ac), 20.81(Ac), 20.78
(Ac), 20.74 (Ac). Anal. Calcd for C62H77O27N2F: C, 57.22; H, 5.96;
F, 1.46. Found: C, 56.90; H, 5.85; F, 1.30.
Benzyl[(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-â-D-galactopyranosyl)-
(1f4)-(2-deoxy-6-O-naphthylmethyl-3-O-acetyl-2-phthalimido-
â-D-glucopranosyl)]-(1f6)-[(2,3,4,6-tetra-O-acetyl-â-D-
galactopyranosyl)]-2-deoxy-2-acetamido-D-r-galactopyranoside
(31). A solution of compound 1217 (150 mg, 0.234 mmol),
compound 30 (216 mg, 0.246 mmol), and NIS (166 mg, 0.738
mmol) in dry dichloromethane (6 mL) containing 4 Å MS (3.0 g)
was stirred for 1.5-2 h at -65 °C. TfOH (47 µL) in dry
dichloromethane (0.5 mL) was added and the solution was stirred
for 1 h at the same temperature under N2 atmosphere. The reaction
was quenched with saturated sodium bicarbonate solution. The
solids were filtered off and the organic layer was washed with
saturated 10% Na2S2O3, dried with Na2SO4, and concentrated. The
crude residue was passed through a short column of silica gel eluted
with dichloromethane-methanol (60:1) to give pure compound 31
(170 mg, 52%) as an amorphous solid: Rf 0.47 (CH2Cl2/MeOH
Benzyl[(3-deoxy-3-fluoro-â-D-galactopyranosyl)-(1f4)-2-deoxy-
2-acetadimido-â-D-glucopranosyl)]-(1f6)-(â-D-galactopyrano-
syl)-2-deoxy-2-acetamido-D-r-galactopyranoside (5). To a solu-
tion of compound 32 (78 mg, 0.06 mmol) in a mixture of
dichloromethane-methanol (10 mL, 4:1) was added 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone (DDQ) (87 mg, 0.38 mmol), and the
reaction mixture was concentrated and taken up in dichloromethane
(10 mL), which was washed with saturated NaHCO3 (2 × 10 mL),
dried (Na2SO4), and concentrated. The crude residue was applied
to a column of silica gel eluted with hexanes/ethyl acetate (2:1) to
give a pure compound that was treated with 1 M sodium
methoxide-methanol solution (20 µL) in a mixture of methanol-
water (0.5 mL, 1:1) overnight at room temperature. The reaction
mixture was concentrated to a crude residue and purified by a short
column of silica gel eluted with i-C3H9OH/HOAc/H2O (3:1:1) to
give pure compound 5 (38 mg, 75%) as an amorphous solid: Rf
0.5 (i-C3H9OH/HOAc/H2O 3:1:1); 1H NMR (D2O, DQF-COSY and
ROESY, 400 MHz) δ 7.50-7.20 (m, 5 H, ArH), 4.99 (d, 1 H, J1,2
) 3.4 Hz, HA-1), 4.73 (d, 1 H, Jgem )12.6 Hz, PhCHAO, ABq),
1
30:1); H NMR (CDCl3, DQF-COSY, TOCSY, and ROESY, 600
MHz) δ 7.80-7.48 (m, 11 H, ArH), 7.36-7.28 (m, 3H, ArH),
7.20-7.12 (m, 2 H, ArH), 5.71-5.65 (m, 1 H, HC-3), 5.41 (d, 1
H, J1,2 ) 7.8 Hz, HC-1), 5.37 (d, 1 H, J ) 10.4 Hz, NHAc), 5.29
(d, 1 H, J ) 2.8 Hz, HB-4), 5.20-4.87 (m, 5 H, HD-3, HB-2,
PhCHAO, HD-2, HB-3), 4.62 (d, 1 H, Jgem ) 12.3 Hz, PhCHBO,
ABq), 4.59 (d, 1 H, J1,2 ) 2.1 Hz, HA-1), 4.45 (ddd, 1 H, HA-2),
4.39 (d, 1 H, J1,2 ) 7.6 Hz, HB-1), 4.34-4.24 (m, 3 H, HC-2), 4.18
(d, 1 H, J1,2 ) 7.6 Hz, HD-1), 4.12-3.52 (m, 16 H), 3.36 (t, 1 H),
2.12 (s, 3 H, Ac), 2.08 (s, 3 H, Ac), 2.07 (s, 3 H, Ac), 2.02 (s, 3
H, Ac), 2.00 (s, 3 H, Ac), 1.98 (s, 3 H, Ac), 1.96 (s, 3 H, Ac), 1.88
(s, 3 H, Ac), 1.83 (s, 3 H, Ac); 13C NMR (CDCl3, 100.6 MHz) δ
170.5 (CdO), 170.4 (CdO), 170.2 (CdO), 170.1 (CdO), 169.6
2
4.61 (dq, 1 H, J ) 2.8 Hz, J ) 9.6 Hz, JH-F )68.0 Hz, HD-3),
4.58 (d, 1 H, J1,2 ) 7.6 Hz, HD-1), 4.55 (d, 1 H, J1,2 ) 8.6 Hz,
HC-1), 4.52 (d, 1 H, Jgem ) 12.5 Hz, PhCHBO), 4.46 (d, 1 H, J1,2
) 7.6 Hz, HB-1), 4.33 (dd, 1 H, HA-2), 4.24 (d, 1 H, J ) 2.8 Hz,
HA-4), 4.03 (d, 1 H, J ) 3.2 Hz, HD-4), 4.16 (m, 1 H, HA-5), 4.07
(dd, 1 H, HA-6b), 4.04 (dd, 1 H, HA-3), 4.00 (dd, 1 H, HC-3), 3.91
(d, 1 H, J ) 2.8 Hz, HB-4), 3.91-3.72 (m, 9 H), 3.64 (m, 2 H,
HC-5, HB-3), 3.52 (dd, 1 H, HB-2), 2.31 (s, 3 H, Ac), 2.28 (s, 3 H,
Ac); 13C NMR (D2O, 100.6 MHz) δ 175.8 (CdO), 175.5 (CdO),
138.2, 130.1, 129.9, 129.7, 105.9, 103.4 (d, 3JC-F ) 12.7 Hz), 102.7,
97.6, 94.1 (d, 1JC-F ) 183.9 Hz), 79.8, 78.3, 76.2 (d, 2JC-F ) 17.8
(CdO), 168.9 (CdO), 137.1, 134.6, 134.3, 128.9, 128.8, 128.7,
3
128.6, 128.1, 128.0, 126.9, 126.8, 126.6, 102.5, 99.9 (d, JC-F
)
10.3 Hz), 98.8, 97.3, 88.9 (d, 1JC-F ) 193.4 Hz), 75.3, 75.7, 74.7,
74.1, 71.2, 71.1, 70.9, 69.9, 69.8, 69.6, 69.1, 68.8, 68.6, 67.4, 67.1,
61.6, 61.2, 55.9, 47.9, 23.6 (NAc), 20.99 (Ac), 20.96 (Ac), 20.91
(Ac), 20.87 (Ac), 20.82 (Ac), 20.80 (Ac). Anal. Calcd for
C68H77O28N2F: C, 58.78; H, 5.59; F, 1.37. Found: C, 57.37; H,
5.41; F 1.40.
3
Hz), 75.3 (d, JC-F ) 6.8 Hz), 73.8, 73.7, 71.9, 71.1, 70.9, 70.8,
J. Org. Chem, Vol. 71, No. 10, 2006 3705

Xia et al.
70.7, 70.1, 69.9, 68.2, 67.9, 62.3, 61.99, 61.97, 61.3, 56.4, 49.8,
23.6 (NAc), 23.3 (NAc); ESI-HRMS calcd for C35H53O20N2FNa
(m/z) [MNa+] 863.3068, found 863.3088.
Supporting Information Available: General experimental
methods, X-ray crystallographic data (including structure factors),
1D or 2D NMR spectra of key compounds, and ESI-HRMS of
compounds 1-5 and 10. This material is available free of charge
via the Internet at http://pubs.acs.org.
Acknowledgment. This work was supported by Grant Nos.
CA35329, CA63218, and, in part, P30CA16056, all awarded
by the National Cancer Institute.
JO052626J
3706 J. Org. Chem., Vol. 71, No. 10, 2006