Total syntheses of tumor-related antigens N3: Probing the feasibility limits of the glycal assembly method

Article abstract of DOI:10.1021/ja0022730

The total syntheses of two octasaccharide antigens isolated from human milk, 1 and 2, and their corresponding allyl glycosides, 3 and 4, have been achieved by utilizing the glycal method. Convergent assembly of the core hexasaccharides and concurrent intr

Full text of DOI:10.1021/ja0022730

J. Am. Chem. Soc. 2001, 123, 35-48
35
Total Syntheses of Tumor-Related Antigens N3: Probing the
Feasibility Limits of the Glycal Assembly Method
Hyunjin M. Kim,†,‡,§ In Jong Kim,†,| and Samuel J. Danishefsky*,†,‡
Contribution from the Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer
Research, 1275 York AVe., New York, New York 10021, and Department of Chemistry,
Columbia UniVersity, HaVemeyer Hall, New York, New York 10027
ReceiVed June 23, 2000
Abstract: The total syntheses of two octasaccharide antigens isolated from human milk, 1 and 2, and their
corresponding allyl glycosides, 3 and 4, have been achieved by utilizing the glycal method. Convergent assembly
of the core hexasaccharides and concurrent introduction of two R-L-fucosyl moieties at the late stage of the
syntheses provided these complex carbohydrates in a concise manner. With synthetic material obtained, biological
evaluations of these antigens as potential gastrointestinal cancer immunotherapeutic agents have been initiated.
Background
Human milk contains numerous oligosaccharides. Their
compositions are largely dependent on the blood type of the
lactating mother. These oligosaccharides can be classified as
either “acidic” or “neutral,” depending on the presence or
absence of sialic acid moieties.1 Due to the difficulties associated
with the isolation of homogeneous carbohydrate entities in
decent quantity from such sources, and due to the intricacies of
the determination of function in the complex arena of glyco-
biology, the specific roles of such carbohydrates are not yet
known, though progress is certainly being realized.2 For instance,
it has been found that some of these entities present in milk,
which survive the digestive process of the baby, are similar to
tumor-associated antigens.3 Given the roles of carbohydrates
in inter- and intracellular signaling, such oligosaccharide
constellations may have a role in infant growth and development.
Herein we describe research related to two such milk-derived
antigens1,4 known as N3 major and N3 minor. These substances
isolated from human milk are found in “neutral” oligosaccharide
fractions. The compounds are the octasaccharides, difucosyl-
lacto-N-hexaose (1) and difucosyllacto-N-neohexaose (2) (Figure
1). The major N3 component, which will henceforth be called
“N3 major”, is difucosyllacto-N-hexaose (1). Inspection of its
structure reveals the presence of Lea and Lex moieties branching
from a central lactose framework. By contrast, the minor
component of N3 (compound 2) displays two Lex subunits
projecting from the lactose core. These two octasaccharides will
be collectively described as N3 antigens.
Figure 1.
Although the Lea trisaccharide moiety is not known per se
to be a tumor-associated antigen, Lex has been so recognized,
and it is indeed closely related to various cancers. In fact, the
Lex antigen is often referred to as an oncofetal antigen due to
its transient expression during ontogeny and its reappearance
in tumors.5 Increased expression of Lex antigen has been
observed in most common human cancers including those of
leukocyte, stomach, liver, lung, and colon origin. It is interesting
to note that the cell surface expression of Lex antigen occurs
with a higher frequency in early gastric cancer than in advanced
stage disease.6 In addition, Lex is present in 81% of the cases
of histologically classified undifferentiated gastric cancer and
67% of differentiated gastric cancer. Since the N3 antigens are
† Sloan-Kettering Institute for Cancer Research.
‡ Columbia University.
§ Present address: Division of Chemistry and Chemical Engineering,
California Institute of Technology, 1201 E. California Blvd (Mail Code:
164-30), Pasadena, CA 91125.
| Present address: Kanazawa University, Faculty of Pharmaceutical
Sciences, Takara-machi 13-1, Kanazawa, 920, Japan.
(1) Kobata, A. Methods Enzymol. 1972, 28, 262.
(2) (a) Reddy, G. P.; Bush, C. A. Anal. Biochem. 1991, 198, 278. (b)
Kunz, C.; Rudloff, S.; Hintelmann, A.; Pohlentz, G.; Egge, H. J. Chro-
matogr. B 1996, 685, 211. (c) Cancilla, M. T.; Penn, S. G.; Carroll, J. A.;
Lebrilla, C. B. J. Am. Chem. Soc. 1996, 118, 6736.
(3) Sabharwal, H.; Nilsson, B.; Gro¨nberg, G.; Chester, M. A.; Dakour,
J.; Sjo¨blad, S.; Lundblad, A. Arch. Biochem. Biophys. 1988, 265, 390.
(4) Kobata, A.; Ginsburg, V. Arch. Biochem. Biophys. 1972, 150, 273.
(5) Brockhausen, I., Kuhns, W., Eds. Glycoproteins and Human Disease;
R. G. Landes Co.: Austin, TX, 1997.
(6) Dohi; et al. Gastroenterol. Jpn. 1989, 24, 238.
10.1021/ja0022730 CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/14/2000

36 J. Am. Chem. Soc., Vol. 123, No. 1, 2001
Kim et al.
composed of either Lea and Lex or two Lex antigens, it would
not be too surprising for them to be overexpressed in various
tumors. As it turns out, the N3 antigens have been particularly
associated with gastrointestinal (GI) cancer cells.
A recently filed patent claims the use of the N3 antigen as a
probe for gastrointestinal cancer.7 In the patent, antibody titers
to N3 antigens in the sera of GI cancer patients were measured
and it was found that the titers are higher for the GI cancer
sera than for the normal sera. Using a cutoff titer of 1:80, 93%
of normal sera fell below this titer, while 87% of early stage,
58% of intermediate stage, and 75% late stage carcinoma sera
had titers above this dilution. It is of particular interest that the
percentage of patients having anti-N3 titer over 1:80 was greater
in the early stage than in the later stages. Thus, it was envisioned
that measurement of anti-N3 antibody titers could be a useful
detection method for early stage GI cancer. This finding and
claim argued to us the case for initiating studies of N3 antigens
in cancer vaccinology.
However, the supply of N3 antigens is quite limited due to
the difficulties associated with isolation of complex carbohy-
drates. Even though the antigens are commercially available at
rather high prices ($278.00/0.1 mg for N3 major and $901.00/
0.1 mg for N3 minor),8 they can be obtained only as anomeric
mixtures of “free reducing end” carbohydrates. Their utility is
limited for subsequent biological studies. Development of viable
syntheses of N3 antigens can perhaps alleviate the above-
mentioned problems.
Earlier, our laboratory has focused on the synthesis of allyl
and pentenyl glycosides of various tumor-associated antigens.
Such glycosides have proven to be appropriately versatile
substrates for subsequent biological studies.9 Accordingly,
glycosides 3 and 4 (Figure 2) were identified as end points of
this study even though they are not the naturally isolated species
of N3 antigens.
A total synthesis venture aimed at the N3 antigens was of
interest, not only for potential medical reasons. It was also
anticipated that the synthesis would be challenging and would
help to probe the perimeters of feasibility of the glycal assembly
logic of oligosaccharide total synthesis. As will be chronicled
in detail, our expectations of the difficulties and learning
opportunities were surpassed in reality. Indeed, as will be
described, a major reassessment of synthetic design was
necessary to reach our goal. However, eventually, the objectives
were met. In this paper, we describe (vide infra) the total
syntheses of N3 allyl glycosides 3 and 4 as well as the natural
products 1 and 2.
Figure 2.
derived (cf. 8) from the initial coupling of 6 with 5 or 7. We
were aware, through earlier experiences, of the reactivity
differences of hydroxyl groups on lactal 6 (C6′ > C3′ > C2′ >
C4′) and planned to join the Lea trisaccharide 5 first on C3′-
hydroxyl of 6, followed by coupling of Lex 7 on the C6′-
hydroxyl of the resultant pentasaccharide. In this way, we hoped
to avoid the late stage installation of a trisaccharide on the less
reactive C3′-hydroxyl group of the pentasaccharide. To pursue
the approach we favored, the C6′-hydroxyl group of the original
lactal 6 would have to be equipped with a suitable protecting
group P* that could be differentiated from other existing
masking units in the system and unmasked at a propitious time.
The synthesis of 12 (corresponding to a specific form of 6 in
Scheme 1) commenced with the known lactal derivative 1010
which was treated with TBAF in THF (Scheme 2). Treatment
of this product with sodium methoxide in methanol led to the
lactal tetraol 11 in 88% yield. We came to favor protecting the
6′-hydroxyl group as a levulinate ester. The C6′-alcohol could
be exposed in a variety of ways without unmasking other
protective arrangements (for example triisopropyl silyl ethers).
Accordingly, the C6′-OH of 11 was selectively levulinoylated
to provide 12. From previous synthetic studies directed toward
KH-1,11 we were convinced that the glycosidation would occur
selectively on the C3′-OH in the presence of unmasked C2′-
and C4′-hydroxyl groups. Accordingly, no further protection
on the triol 12 was performed.
Turning to the synthesis of Lea trisaccharide donor 20 (5 in
Scheme 1), we started with protection of 6-O-triisopropylsilyl-
glucal (13). Treatment of this compound with triphenylsilyl
chloride in the presence of Et3N and DMAP afforded 14 in 72%
yield. The remaining hydroxyl group of 14 was then glycosy-
lated with fluorofucosyl donor 1512 according to the Mukaiyama
protocol.13 The resultant crude disaccharide was not purified.
Removal of its triphenylsilyl function afforded 16 in 64% in
over 2 steps. The reaction between 16 and the epoxide derivative
of 17 through mediation by anhydrous zinc chloride provided
Preliminary Synthetic Studies
The initial strategy for reaching N3 major reflected earlier
realizations of glycal assembly methodology in reaching Lex
and various other carbohydrate antigens.9 Thus, we charted a
synthesis of N3 major by anticipating late stage mergers of
donors derived from glycals 5 and 7 (Scheme 1) in some
undefined sequence with appropriate glycal acceptors. The first
acceptor type to be employed was formulated as 6. The second
glycal acceptor would be a differentiated pentasaccharide glycal
(7) Anderson, B. E.; Grove, M.; Davis, L. E. U.S. Pat. 5376531, 1994.
(8) BioCarb Chemicals, 1997 catalogue.
(10) Danishefsky, S. J.; Behar, V.; Randolph, J. T.; Lloyd, K. O. J. Am.
Chem. Soc. 1995, 117, 570.
(9) (a) Seeberger, P. H.; Bilodeau, M. T.; Danishefsky, S. J. Aldrichim.
Acta 1997, 30, 75. (b) Danishefsky, S. J.; Bilodeau, M. T. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1380. (c) Danishefsky, S. J.; Allen, J. R. Angew.
Chem., Int. Ed. Engl. 2000, 39, 836. (d) For the current state of the art in
carbohydrate synthesis, see: Khan, S. H., O’Neil, R. A., Eds. Modern
Methods in Carbohydrate Synthesis; Harwood Academic: Amsterdam,
1996.
(11) (a) Deshpande, P. P.; Kim, H. M.; Zatorski, A.; Park, T.-K.;
Ragupathi, G.; Livingston, P. O.; Live, D.; Danishefsky, S. J. J. Am. Chem.
Soc. 1998, 120, 1600. (b) Deshpande, P. P.; Danishefsky, S. J. Nature, 1997,
387, 164. (c) Kim, H. M. Ph.D. Thesis, Columbia University, 1999.
(12) Nicolaou, K. C.; Hummel, C. W.; Bockovich, N. J.; Wong, C.-H.
J. Chem. Soc., Chem. Commun. 1991, 10, 870.
(13) Mukaiyama, T.; Murai, Y.; Shoda, S. Chem. Lett. 1981, 431.

Tumor-Related Antigens N3
J. Am. Chem. Soc., Vol. 123, No. 1, 2001 37
Scheme 1
Scheme 2a
a Reagents: (a) (i) TBAF, THF; (ii) NaOMe, MeOH (88%); (b) levulinic acid, 2-chloro-1-methylpyridinium iodide, Et3N, dioxane (64%); (c)
Ph3SiCl, Et3N, DMAP, CH2Cl2 (72%); (d) (i) 15, AgClO4, SnCl2, di-tert-butylpyridine, 4 Å MS, Et2O; (ii) K2CO3, MeOH, THF (64% over two
steps); (e) (i) DMDO, CH2Cl2; (ii) 16, ZnCl2, THF (76%); (f) TBSOTf, Et3N, CH2Cl2 (94%); (g) I(sym-coll)2ClO4, PhSO2NH2, 4 Å MS, CH2Cl2
(89%).
Scheme 3a
a Reagents: (a) (i) (Bu3Sn)2O, benzene; (ii) 20, AgBF4, 4 Å MS, THF (45%); (b) H2NNH2, AcOH, pyridine (93%).
the trisaccharide glycal 18. The newly generated hydroxyl
group of glycal 18 was protected as its TBS ether, and the
resulting glycal 19 was, in turn, transformed to iodosulfonamide
donor 20 by treatment with I(sym-coll)2ClO4 and benzene-
sulfonamide.
The acceptor lactal 12 was converted to its tributylstannyl
ether derivative and combined with 20 in the presence of AgBF4
(Scheme 3). As shown in our synthesis of the KH-1 antigen,
the formation of stannyl ether resulted in selective activation
of C3′-OH so that the subsequent glycosidation would occur
only at the activated position.11 The well-documented predilec-
tion of the iodosulfonamide glycosylative rearrangement reaction
to afford â-glycosidic linkages was another consideration
favoring this route.14 Indeed, the desired pentasaccharide 21 was
obtained in 45% yield as the sole recognized product in the
azaglycosylation. However, the low reactivity of the Lea donor
20 necessitated elevated temperature (45 °C), long reaction times
(100 h), and a large excess (10 equiv) of a stannyl ether

38 J. Am. Chem. Soc., Vol. 123, No. 1, 2001
Kim et al.
Scheme 4a
a Reagents: (a) AgClO4, SnCl2, di-tert-butylpyridine, 4 Å MS, Et2O (55%); (b) p-methoxybenzyl chloride, NaH, DMF (83%); (c) I(sym-coll)2ClO4,
PhSO2NH2, 4 Å MS, CH2Cl2 (88%); (d) EtSH, LHMDS, DMF (95%); (e) (i) EtSH, ZnCl2, THF; (ii) Ac2O, DMAP, pyridine, CH2Cl2 (23%).
Scheme 5a
a Reagents: (a) MeOTf, di-tert-butylpyridine, 4 Å MS, Et2O, CH2Cl2 (52% + 18% R isomer); (b) Ac2O, DMAP, pyridine, CH2Cl2 (58%); (c)
DDQ, di-tert-butylpyridine, CH2Cl2, H2O (57%); (d) 28, MeOTf, di-tert-butylpyridine, 4 Å MS, CH2Cl2, Et2O (96%).
derivative of 12 to achieve a moderate coupling yield. For the
moment, we accepted this limitation as the synthesis was
explored further. The levulinate ester of 21 was cleanly cleaved
through hydrazinolysis to provide pentasaccharide C6′-OH
acceptor 22.15
25. The treatment of 25 with I(sym-coll)2ClO4 and benzene-
sulfonamide gave iodosulfonamide 26 in 88% yield. Subse-
quently, 26 was cleanly converted to donor 27 by the action of
basic ethanethiolate. Similarly, the required terminal galactal
donor 28 was obtained in a two-step sequence from 6-O-TIPS
galactal 3,4-cyclic carbonate (17) (see Scheme 4).
The stage was now set for the anticipated merger of the Lex
moiety with the pentasaccharide 22 at its C6′ hydroxyl group
(see asterisk). Unfortunately, attempted coupling protocols with
various trisaccharide Lex donors were not successful. Inadequate
reactivities of these bulky Lex trisaccharide donors were
perceived to be an obstacle to glycosidation. Thus, it was
deemed necessary to devise more activated and less bulky
donors that could be converted to Lex at later stage. The concept
of using a fully developed trisaccharide donor was set aside.
Instead, the disaccharide thioglycoside donor 27 was synthesized
as shown in Scheme 4. Thus, following published procedures,
6-O-TIPS glucal (13) was selectively fucosylated at the C3
alcohol to give disaccharide 24 in 55% yield.16 The remaining
C4-OH of 24 was protected as its PMB ether to yield the glycal
In the event, coupling between pentasaccharide 22 and thio-
donor 27 provided the desired heptasaccharide 29 in 52% yield
along with its R-linked isomer (as a 3:1 ratio in favor of the
desired â-anomer, Scheme 5). The remaining task for the
completion of the N3 major synthesis was to install the last
remaining galactose moiety. Before proceeding to this goal, we
sought to mask the two remaining hydroxyl groups to prevent
the glycosidation on the undesired hydroxyl group. After many
experiments, effective conditions to acetylate both hydroxyl
groups were found, and the diacetylated heptasaccharide 30 was
obtained in 58% yield. The PMB ether of 30 was cleaved by
the action of DDQ. The reaction was buffered with di-tert-
butylpyridine to minimize the decomposition of glycal double
bond. The hydroxyl group (see asterisk) of heptasaccharide 31
was coupled with thioglycoside 28 in the presence of MeOTf
(14) Griffith, D. A.; Danishefsky, S. J. J. Am. Chem. Soc. 1990, 112,
5811.
(15) (a) Veeneman, G. H.; Van Der Marel, G. A.; Van Den Elst, H.;
Van Boom, J. H. Tetrahedron 1991, 47, 1547. (b) Van Boom, J. H.; Burgers,
P. M. J. Tetrahedron Lett. 1983, 24, 245.
(16) Danishefsky, S. J.; Gervay, J.; Peterson, J. M.; McDonald, F. E.;
Koseki, K.; Griffith, D. A.; Oriyama, T.; Marsden, S. P. J. Am. Chem. Soc.
1995, 117, 1940.

Tumor-Related Antigens N3
J. Am. Chem. Soc., Vol. 123, No. 1, 2001 39
Scheme 6a
a Reagents: (a) (i) TBAF, THF; (ii) Ac2O, DMAP, Et3N, CH2Cl2; (iii) NaOMe, MeOH; (iv) Na, NH3; (v) Ac2O, DMAP, pyridine (43%); (b)
NaOMe, MeOH.
Scheme 7
to afford an octasaccharide. The structure of the oligosaccharide
was tentatively assigned as 32.
provided two products in a nonconstant ratio (Scheme 6). One,
as indicated by mass spectrometric analysis, was a heptasac-
charide glycal lacking one of the terminal galactose units. The
precise structure of this heptamer could not be fully determined
by spectroscopic methods. On other occasions, an octasaccharide
glycal, which was tentatively assigned as peracetyl N3 major
glycal 33, was obtained in modest yield of 43%. However,
A carefully optimized five-step deprotection sequence on the
octasaccharide 32 was conducted. It commenced with treatment
of 32 with TBAF followed by acetylation of the crude product,
for purposes of purification. Subsequent, sequential deacylation
of the resulting product, Birch reduction, and peracetylation

40 J. Am. Chem. Soc., Vol. 123, No. 1, 2001
Kim et al.
Scheme 8a
a Reagents: (a) levulinic acid, 2-chloro-1-methylpyridinium iodide, Et3N, dioxane (91%); (b) BnBr, NaH, DMF (90%); (c) I(sym-coll)2ClO4,
PhSO2NH2, 4 Å MS, CH2Cl2 (94%); (d) EtSH, LHMDS, CH2Cl2 (85%).
attempted “per-deacetylation” with NaOMe in MeOH failed to
produce the desired product. At best, we could identify a
compound with an extra mass of 42 (corresponding to an extra
acetate group). Unfortunately, several attempts to remove this
“extra” acetyl group were not successful. Since per-deacylation
of 33, were it present, should have been accomplished under
these conditions, failure to achieve this expected end point
engendered concerns about the actual structure of the synthetic
octasaccharide. Thus, while the gross molecular features of these
molecules were verifiable by mass spectrometric analysis, we
could not vouch for the integrity of all of the linkages of the
presumed 33. Given this impasse, a new and different approach
was in order.
subunits. However, the strategy of simultaneous difucosylation
could still be appropriate. As in the case required for N3 minor,
retrosynthetic “deletion” of two fucosyl moieties from the goal
octasaccharide glycal, 9, takes one back to hexasaccharide, 39
bearing Lea and Lex disaccharide precursors linked to the C3′
and C6′ positions of the central lactose unit. Consecutive
stepwise glycosidation on C3′ with 35, followed by reaction
on C6′ of 37 with 36, would provide the hexasaccharide 39.
With the three disaccharide building blocks, 34-36, we could
hope to obtain each of the N3 antigens.
Synthesis of N3 Minor
The synthesis of N3 minor focused first on the preparation
of a central lactal acceptor, 43, corresponding to the generalized
34 in Scheme 7. The program commenced with lactal 42,
previously employed in our Globo-H synthesis (Scheme 8).17
A levulinate ester group was again selected for the protection
of C6′-hydroxyl, and the desired central acceptor lactal 43 was
obtained by conversion of 42 to its levulinate ester (91% yield).
The synthesis of the des-fucosyl Lex precursor, which was
designated as 36 in Scheme 7, started from lactal 44.11 The
remaining C2′-hydroxyl group of 44 was protected as its benzyl
ether. Lactal 45 was then treated with I(sym-coll)2ClO4, in the
presence of benzenesulfonamide to yield 46. When this com-
pound was treated with basic ethanethiolate, the desired disac-
charide 47 featuring an anomeric thioethyl donor was obtained
in 85% yield. The menu of building blocks necessary for N3
minor was now in hand.
The assembly process was launched via coupling between
thioglycoside 47 and the central lactal acceptor 43 (Scheme 9).
With methyl triflate (MeOTf) as the promoter, tetrasaccharide
48 was obtained in 80% yield. The levulinate group of 48 was
then unmasked to free the C6′-hydroxyl group for the next
coupling. Again, thioglycoside 47 was joined to tetrasaccharide
acceptor 49 through the agency of MeOTf to give hexasaccha-
ride 50 in 91% yield. Following treatment with acetic acid
buffered TBAF, hexasaccharide 50 was successfully converted
to hexasaccharide acceptor 51 with two cleanly identified
hydroxyl groups ready for the crucial bis-fucosylation reaction.
Indeed, triol 51 was successfully fucosylated with 4 equiv
of fluorofucosyl donor 2316,18 under promotion by Sn(OTf)2.19
Modification of the Synthetic Routes to N3 Major and N3
Minor
The synthetic program directed toward N3 was redesigned
with the difficulties described above in mind. The more
symmetric N3 minor, rather than N3 major, was chosen as the
target to test the new departures. The corresponding octasac-
charide 41 became the primary synthetic goal since its glycal
double bond would provide functionality to gain access to
various end products including the desired allyl glycoside as
well as the natural product itself (Scheme 7). A simultaneous
2-fold fucosylation strategy, of the type which was successful
in the case of our KH-1 antigen synthesis,11 was to be applied
to the case at hand. In the retrosynthetic vein, the elimination
of two fucose moieties would leave a hexasaccharide (cf. 40)
with two identical lactose moieties each linked to C3′- and C6′-
hydroxyl groups on the central lactose unit. Provisions for future
fucosylation sites would be anticipated through the use of special
protecting groups (R) allowing for exposure of the free
hydroxylic acceptor sites in an optimal setting. Initially, the
installation of the two flanking lactose units 36 on the central
lactose 34 was slated to be carried out stepwise. We were not
unaware of the fact that glycosidations tend to be much more
difficult when larger entities are to be merged. Since the C6′
position of lactose type 34 was expected to be more reactive, it
seemed that once the synthesis had progressed to the tetrasac-
charide stage, it would be easier to install the appropriate
disaccharide at C6′ rather than at C3′. Thus, the glycosidation
at C3′ was to be carried out first on the lactal 34 with the C6′
position masked Via a suitable protecting group, P*.
(17) Park, T.-K.; Kim, I. J.; Hu, S.; Bilodeau, M. T.; Randolph, J. T.;
Kwon, O.; Danishefsky, S. J. J. Am. Chem. Soc. 1996, 118, 11488.
(18) Danishefsky, S. J.; Gervay, J.; Peterson, J. M.; McDonald, F. E.;
Koseki, K.; Oriyama, T.; Griffith, D. A.; Wong, C.-H.; Dumas, D. P. J.
Am. Chem. Soc. 1992, 114, 8329.
The synthesis of N3 major posed a somewhat more difficult
challenge in that it requires installation of both Lea and Lex

Tumor-Related Antigens N3
J. Am. Chem. Soc., Vol. 123, No. 1, 2001 41
Scheme 9a
a Reagents: (a) MeOTf, di-tert-butylpyridine, 4 Å MS, Et2O, CH2Cl2 (80%); (b) H2NNH2, pyridine, AcOH (87%); (c) 47, MeOTf, di-tert-
butylpyridine, 4 Å MS, Et2O, CH2Cl2 (91%); (d) TBAF, AcOH (96%).
Scheme 10a
a Reagents: (a) 2.5 equiv. of 23, Sn(OTf)2, di-tert-butylpyridine, 4 Å MS, THF, toluene (30% for 52 + 55% for 53).
1
This donor was shown to have a strong tendency for favoring
the formation of R-linked glycosides, possibly due to the
anchimeric stabilization of the oxonium transition state by the
C4-benzoyl moiety. Unexpectedly, the major product turned out
to be nonasaccharide 52 wherein the axial hydroxyl had also
suffered fucosylation. With more careful control of equivalency
of the fucosyl donor, the difucosylated octasaccharide was
obtained in acceptable yield. However, reliable high-yielding
methodology to accomplish bis fucosylation proved to be quite
difficult.
Fucosylation on the seemingly sterically hindered axial C4′-
hydroxyl group of the central lactal was unexpected and certainly
unwelcome since it lowered the efficiency of the reaction.
Moreover, there was a structure assignment problem in deter-
mining which two of three potential acceptor sites had been
fucosylated. As is not uncommon with complex carbohydrates,
structural determination of minute quantities of samples with
sole reliance on NMR may not always be fully persuasive.
As a potential solution to this problem, we examined the
possibility of protection of the C4′-hydroxyl group. Fortunately,
acetylation was successfully carried out on tetrasaccharide 48
to give 54 in 92% yield (Scheme 11). The levulinate group of
54 was again selectively removed with buffered hydrazine
solution without the removal of the newly installed acetyl group.
Hexasaccharide 56 was obtained in 71% yield by MeOTf
mediated coupling between the tetrasaccharide 55 and thiodonor
47. The two triethylsilyl groups of 56 were conveniently
removed by treatment with TBAF buffered with AcOH. The
resulting diol 57 was then bis-fucosylated with the fucosyl
fluoride 23 in the presence of Sn(OTf)2 to give the octasaccha-
ride 58. A cardinal goal of the program had been achieved. With
this material in hand, we were in a position to pursue the
syntheses of a variety of N3 minor congeners, including allyl
glycoside 4 as well as naturally isolated 2.
Upon comparison of the H NMR spectrum of the octasac-
charide 58 with that of 53, it was evident that they were not
identical. Thus, one of two fucose moieties in 53 was on the
axial C4′-hydroxyl group. It will be of interest to determine the
reason for the unexpectedly enhanced reactivity of the axial C4′-
hydroxyl group in 51. For the moment this finding was not
followed up.
In the planning stage, a protecting group strategy had been
devised to ensure that global deprotection of the fully protected
N3 minor octasaccharide could be conducted concisely. Thus,
all of the protecting groups of 58 could be removed by
dissolving metal conditions. In the event, removal of the benzyl
ether groups by Birch reduction, followed by peracetylation,
provided the peracetyl octaglycal 59 in 43% overall yield
(Scheme 12). Dimethyldioxirane oxidation20a,b of the peracetyl
octacyclic glycal 59, followed by opening of the resulting
epoxide with allyl alcohol under solvolytic conditions, provided
the peracetyl allyl glycoside 60 in 48% yield along with its
R-mannose type isomer (an 11:9 ratio favoring 60). In the
synthesis of the final allyl glycoside, the serious stereochemical
problem associated with late stage glycal epoxidation in the
presence of resident acetate functions had been noted earlier.
A solution to this difficulty using n-pentenyl glycosides has
recently been described but not yet applied to the case at
hand.20c,d Finally, removal of the acetyl esters of 60 by treatment
with NaOMe in MeOH produced the allyl glycoside 4. The
structure of 4 was rigorously proven by 2 D NMR experiments
including HMBC and HMQC performed by Dr. David Live.21
Thus, the synthesis of N3 minor allyl glycoside 4 was
completed. As a further proof of the structure, allyl glycoside
(20) (a) Halcomb, R. L.; Danishefsky, S. J. J. Am. Chem. Soc. 1989,
111, 6661. (b) Preparation of DMDO: Murray, R. W.; Jeyaraman, R. J. J.
Org. Chem. 1985, 50, 2847. (c) Allen, J. R.; Ragupathi, G.; Livingston, P.
O.; Danishefsky, S. J. J. Am. Chem. Soc. 1999, 10875, 5. (d) Allen, J. R.;
Allen, J. G.; Zhang, X. F.; Williams, L. J.; Zatorski, A.; Danishefsky, S. J.
Chem.-Eur. J. 2000, 6, 1366.
(19) (a) Lubineau, A.; Malleron, A. Tetrahedron Lett. 1985, 26, 1713.
(b) Lubineau, A.; Le Gallic, J.; Malleron, A. Tetrahedron Lett. 1987, 28,
5041. (c) Ito, Y.; Ogawa, T. Tetrahedron Lett. 1987, 28, 6221.
(21) Structural Biology NMR Resource, Department of Biochemistry,
Medical School, University of Minnesota.

42 J. Am. Chem. Soc., Vol. 123, No. 1, 2001
Kim et al.
Scheme 11a
a Reagents: (a) Ac2O, pyridine, DMAP, CH2Cl2 (92%); (b) H2NNH2, pyridine, AcOH (96%); (c) 47, MeOTf, di-tert-butylpyridine, 4 Å MS,
Et2O, CH2Cl2 (71%); (d) TBAF, AcOH, THF (quantitative); (e) 23, Sn(OTf)2, di-tert-butylpyridine, 4 Å MS, THF, toluene (70-90%).
Scheme 12a
a Reagents: (a) (i) Na, NH3, THF; (ii) Ac2O, pyridine, DMAP (43% over two steps); (b) (i) DMDO, CH2Cl2; (ii) allyl alcohol (48% over two
steps + 37% R-mannose type isomer); (c) NaOMe, MeOH (88%); (d) PdCl2, wet MeOH (quantitative).
4 was treated with PdCl2 in wet MeOH to provide free reducing
For the synthesis of N3 major, the first issue to be addressed
was that of building a disaccharide precursor that would
eventually become the Lea trisaccharide. To that end, a dif-
ferently protected glucal starting material had to be assembled.
Provision for several stringent criteria would be necessary. First,
the glycal had to be equipped, eventually to pave the way for
a free hydroxyl at C3 position to achieve the 1,3 disaccharide
linkage which is present in Lea. Second, this glycal had to have
robust and easily cleavable protecting groups at the C6 and C4
positions, respectively. The protecting group at the C4 position
was to be removed at the propitious time, exposing a unique,
free hydroxyl group to serve as a fucosylation site.
1
end carbohydrate 2.22 The H NMR spectra of synthetic N3
minor 2 was identical with the available spectra of the isolated
compound, thus providing additional confirmation as to comple-
tion of the N3 minor synthesis.23
After we accomplished the syntheses of N3 minor octasac-
charide 2, as well as its more useful congener, allyl glycoside
4, N3 major became our next focus of research. The plan called
for reaching a fully protected N3 major octasaccharide glycal
intermediate, which would be convertible to the N3 major allyl
glycoside. From the synthetic studies on N3 minor series, we
had already confirmed the validity of a general strategy, which
anticipates a bis-fucosylation reaction.
In a high-yielding three-step sequence, 6-O-benzylglucal (61)
was converted to 6-O-benzyl-4-O-TES glucal (62) (Scheme 13).
The previously utilized 6-O-benzyl-3,4-cyclocarbonate galactal
(63) was oxidized with dimethyldioxirane, and the resulting
(22) (a) Guibe´, F. Tetrahedron, 1999, 53, 13509. (b) Qian, X.; Hindsgaul,
O.; Li, H.; Palcic, M. M. J. Am. Chem. Soc. 1998, 120, 2184.
(23) Haeuw-Fievre, S.; Wieruszeski, J.-M.; Plancke, Y.; Michalski, J.-
C.; Montreuil, J.; Strecker, G. Eur. J. Biochem. 1993, 215, 361.

Tumor-Related Antigens N3
J. Am. Chem. Soc., Vol. 123, No. 1, 2001 43
Scheme 13a
a Reagents: (a) (i) trimethylacetyl chloride, DMAP, pyridine, CH2Cl2 (88%); (ii) TESOTf, Et3N, CH2Cl2 (98%); (iii) DIBAL-H, CH2Cl2 (97%);
(b) (i) DMDO, CH2Cl2; (ii) 62, ZnCl2, THF (47%); (c) BnBr, NaH, DMF (82%); (d) I(sym-coll)2ClO4, PhSO2NH2, 4 Å MS, CH2Cl2 (90%).
Scheme 14a
a Reagents: (a) (i) (Bu3Sn)2O, benzene; (ii) 66, AgBF4, 4 Å MS, THF (37% 67 + 30% 68).
Scheme 15a
a Reagents: (a) (i) Ac2O, DMAP, pyridine, CH2Cl2; (ii) H2NNH2, pyridine, AcOH (73% over two steps); (b) 47, MeOTf, di-tert-butylpyridine,
4 Å MS, Et2O, CH2Cl2 (75%); (c) TBAF, AcOH (93%); (d) 23, Sn(OTf)2, di-tert-butylpyridine, 4 Å MS, THF, toluene (76%).
epoxide was coupled with 62, to yield 1,3-linked disaccharide
64. The newly generated C2′-hydroxyl group was then benzy-
lated to give disaccharide glycal 65. Treatment of 65 with I(sym-
coll)2ClO4 in the presence of benzenesulfonamide provided
iodosulfonamide 66 in 90% yield, thus completing the synthesis
of the necessary pre-Lea precursor donor.
of the donor must reflect anomeric “onium” ion character. Even
though the coupling reaction to produce the tetrasaccharide 67
was far from optimal, we were able to obtain sufficient amounts,
through the “direct rollover” reaction, to enable completion of
our objective.
As before, the remaining hydroxyl group of 67 was acetylated,
and the levulinate group was then removed by treatment with
hydrazine to provide the tetrasaccharide acceptor 69 (Scheme
15). The 4 +2 glycosidic coupling between 47 and 69 afforded
the hexasaccharide 70 in 75% yield. This time, no other
anomeric isomer was obtained in this thioglycoside mediated
glycosidation reaction. The two remaining TES groups of 70
were simultaneously removed by the treatment with TBAF
buffered with AcOH to gave the diol 71, which was in turn
fucosylated with the fluorofucosyl donor 23 thus affording,
unambiguously, the octasaccharide 72. The initial goal of
synthesizing fully protected octasaccharide glycal 72 had been
reached.
The direct use of a 1,2-iodosulfonamide carbohydrate as an
azaglycosylation donor constitutes an alternative to the thiogly-
coside coupling.11a Indeed, in the synthesis of KH-1 antigen,
we were able to obtain high yields of â-linked tetrasaccharides
and hexasaccharide by this method. In the case at hand, the
reaction between the stannyl ether derived from 43 and the
iodosulfonamide 66 gave the tetrasaccharide 67 in 37% yield.
Much to our surprise, R-linked anomeric isomer 68 was
obtained in 30% yield as well (Scheme 14). During our study
of glycal chemistry using the direct aza glycosylation reaction,9b,c
we obtained R-linked glycosides on the rarest occasions. Such
a result clearly does not fit our simple model which presupposes
a 1,2-aziridine-based intermediate to account for the â-azagly-
cosidation phenomenon. In the case in hand, the aziridine (or
aziridinium) must be short-lived, and a significant population
The concluding phase of the synthesis of N3 major allyl
glycoside commenced with the global deprotection of 72 under
dissolving metal conditions, followed by peracetylation (Scheme

44 J. Am. Chem. Soc., Vol. 123, No. 1, 2001
Kim et al.
Scheme 16a
a Reagents: (a) (i) Na, NH3, THF; (ii) Ac2O, pyridine, DMAP (58% over two steps); (iii) DMDO, CH2Cl2; (iv) allyl alcohol; (v) NaOMe, MeOH
(40% over 3 steps + 15% of R-mannose type isomer); (b) PdCl2, wet MeOH (quantitative).
1,4-dioxane were added levulinic acid (0.604 mL, 5.78 mmol) and Et3N
(4.03 mL, 28.9 mmol). The yellow reaction mixture was stirred at RT
(room temperature) overnight with exclusion of light. Then, the reaction
mixture was transferred into a separatory funnel containing 200 mL of
saturated NaHCO3(aq). The aqueous layer was extracted with EtOAc
(3 × 100 mL), and the combined organic layers were washed with
saturated NaHCO3(aq) (3 × 100 mL) and brine (1 × 100 mL), dried
over Na2SO4, and concentrated with rotary evaporator. Upon purification
by column chromatography with 55% EtOAc in hexanes, levulinate
16). This deprotection sequence provided the peracetyl N3 major
glycal in 58% overall yield. After a sequence consisting of
DMDO oxidation,20 solvolysis of the epoxide with allyl alcohol,
and removal of the acetates with NaOMe, N3 major allylgly-
coside 3 was obtained along with the R-mannose type isomer.
As discussed above, this result reflects a lack of stereospecificity
in the epoxidation of the terminal glycal in the peracetate series.
After the work described here, a solution to the problem was
devised in the context of the use of n-pentenyl glycoside.20c,d
In any case, the synthesis of N3 major allyl glycoside had now
been achieved. Each stereochemical outcome of the glycosida-
tion reactions along the way to 3 was confirmed by HMQC
NMR analysis, and high-resolution mass spectrometry further
served to confirm the molecular formula of 3. Definite proof
of the structure of synthetic N3 major was obtained at the stage
of the free reducing end carbohydrate 1. The allyl glycoside 3
was converted to free reducing end N3 major with PdCl2 in
wet MeOH.22 The 1H NMR spectrum of this synthetic material
matched with the published spectrum of isolated N3 major
fromthe natural source, thus further confirming the synthesis
of N3 major.24
43 (2.90 g, 89%) was obtained as colorless oil: [R]23 ) +14.5° (c
D
0.49, CHCl3); FTIR (CHCl3 film) 3455, 3088, 3064, 3031, 2922, 2870,
1736, 1718, 1649, 1496, 1454, 1404, 1365, 1312, 1247, 1211, 1157,
1
1133, 1074, 912, 826, 738, 700 cm-1; H NMR (500 MHz, CDCl3) δ
7.36-7.15 (m, 15H), 6.45 (d, 1H, J ) 6.3 Hz), 4.91-4.87 (m, 2H),
4.63-4.60 (m, 3H), 4.56 (d, 1H, J ) 7.6 Hz), 4.53 (s, 2H), 4.38-4.31
(m, 2H), 4.24-4.19 (m, 2H), 4.12 (t, 1H, J ) 4.0 Hz), 3.90-3.86 (m,
2H), 3.71 (dd, 1H, J ) 10.7, 3.8 Hz), 3.58-3.52 (m, 2H), 3.47-3.44
(m, 1H), 2.72-2.60 (m, 2H), 2.53-2.42 (m, 4H), 2.22 (s, 3H); 13C
NMR (125 MHz,CDCl3) δ 206.5, 172.7, 144.5, 138.5, 138.2, 137.7,
128.5, 128.4, 128.3, 127.9, 127.7, 127.6, 127.6, 127.5, 102.3, 99.5,
78.6, 75.7, 74.6, 73.3, 73.1, 72.8, 72.0, 71.5, 70.1, 68.1, 67.8, 62.8,
37.8, 29.7, 27.8; LRMS (EI) (m/e) calcd for C38H44O11Na [M + Na]+
699.3, found 699.4.
The binding of synthetic N3 to the anti-major N3 monoclonal
antibodies was examined by ELISA. As expected, major N3-
KLH conjugates derived from the allyl glycoside 3 were found
to bind the anti-major N3 monoclonal antibodies tightly at the
concentration of 25 µg/mL. Surprisingly, minor N3-KLH
conjugates made from the allyl glycoside 4 bound the antibodies
as well, at the same concentration. In addition, KLH conjugates
of Lex trisaccharide was synthesized and found to bind the same
antibodies equally well. Apparently this particular monoclonal
is focusing on Lewisx moieties. Further experiments following
up on these points are in progress.
Lactal 45. To a solution of azeotropically dried lactal 44 (0.875 g,
1.39 mmol) in 10 mL of DMF was added benzyl bromide (0.253 mL,
2.09 mmol). The solution was then cooled to 0 °C and treated with
NaH of (0.0835 g, 2.09 mmol, 60% dispersion in oil) at 0 °C. The
reaction mixture was stirred at 0 °C for 5 min and at RT thereafter.
After 4 h, the reaction mixture was diluted with EtOAc (100 mL) and
poured into a separatory funnel containing cold saturated NH4Cl(aq).
The organic layer was separated, washed with saturated NH4Cl(aq) (3
× 60 mL), H2O (3 × 60 mL), and brine (1 × 60 mL), and dried over
Na2SO4. Purification of the resulting oil by column chromatography
with 15% EtOAc in hexanes yielded benzyl protected lactal 45 (0.932
g, 93%) as colorless oil: [R]23 ) -12.5° (c 0.17, CHCl3); FTIR
D
(CHCl3 film) 3064, 3031, 2953, 2875, 1811, 1647, 1497, 1454, 1410,
1368, 1299, 1247, 1208, 1169, 1097, 1038, 961, 938, 873, 848, 813,
Conclusions
1
742, 698 cm-1; H NMR (500 MHz, CDCl3) δ 7.35-7.23 (m, 15H),
The syntheses of the N3 major and minor allyl glycosides
have been accomplished. Three disaccharide building blocks
sufficed to assemble these systems. The syntheses successfully
applied the late stage concurrent multifucosylation reaction, thus
providing convergent and efficient routes to these structurally
complex octasaccharides. In addition, overall protecting group
strategies employed in these syntheses minimized the number
of steps in the final deprotection reactions, thus providing further
conciseness. The biological evaluations of these synthetic
antigens are in progress, and the results will be disclosed in
due course.
6.35 (d, 1H, J ) 6.3 Hz), 4.75-4.70 (m, 4H), 4.62-4.78 (m, 6H),
4.21 (m, 1H), 4.15 (t, 1H, J ) 3.9 Hz), 3.94-3.89 (m, 2H), 3.85 (m,
1H), 3.69 (s, 1H), 3.67 (d, 1H, J ) 3.2 Hz), 3.63 (dd, 1H, J ) 11.0,
3.0 Hz), 3.55 (t, 1H, J ) 5.5 Hz), 0.92 (t, 9H, J ) 8.0 Hz), 0.58 (q,
6H, J ) 7.9 Hz); 13C NMR (125 MHz, CDCl3) δ 153.8, 143.1, 138.0,
137.4, 137.0, 128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.6, 102.5,
99.1, 77.2, 77.1, 75.2, 74.9, 74.2, 73.73, 73.70, 73.3, 69.9, 67.9, 67.8,
65.1, 6.8, 4.8; HRMS (FAB) (m/e) calcd for C40H50O10SiNa [M + Na]+
741.3071, found 741.3042.
Iodosulfonamide 46. Azeotropically dried lactal 45 (0.640 g, 0.891
mmol), benzene sulfonamide (0.357 g, 2.23 mmol), and 4 Å molecular
sieves (1 g) were suspended in 17 mL of CH2Cl2, and the resulting
suspension was stirred at RT for 10 min. Then, the suspension was
cooled to 0 °C and treated with I(sym-coll)2ClO4 (0.700 g, 1.49 mmol).
The reaction mixture was stirred with exclusion of light at 0 °C for 1
h. Then, the reaction mixture was diluted with EtOAc (30 mL) and
filtered through a pad of silica gel. The clear yellow filtrate was washed
Experimental Section
Acceptor 43. To a solution of lactal 42 (2.79 g, 4.92 mmol) and
2-chloro-1-methylpyridinium iodide (2.54 g, 9.94 mmol) in 80 mL of
(24) Dua, V. K.; Goso, K.; Dube, V. E., Bush, C. A. J. Chromatogr.
1985, 328, 259.

Tumor-Related Antigens N3
J. Am. Chem. Soc., Vol. 123, No. 1, 2001 45
with saturated Na2S2O3(aq) (3 × 100 mL), saturated CuSO4(aq) (3 ×
100 mL), and brine (3 × 100 mL) and dried over Na2SO4. Resulted
crude product was purified by column chromatography with 20%
EtOAc in hexanes to yield the iodosulfonamide 46 (0.883 g, 99%) as
allyl glycoside 4 (0.0027 g, 88%) was obtained from 60 and so was
the mannose type isomer (0.0015 g, 64%) from the isomer of 60.
4: mp 197 °C (decomp); [R]23D ) -115.2° (c 0.07, MeOH); FTIR
(KBr) 3332, 1650, 1378, 1145, 1071 cm-1; 1H NMR (500 MHz, D2O)
δ 6.04-5.97 (m, 1H), 5.41 (br d, 1H, J ) 17.1 Hz), 5.31 (br d, 1H, J
) 10.3 Hz), 5.15 (d, 1H, J ) 3.6 Hz), 5.13 (d, 1H, J ) 3.7 hz), 4.73
(d, 1H, J ) 8.2 Hz), 4.66 (d, 1H, J ) 7.0 Hz), 4.56 (d, 1H, J ) 8.0
Hz), 4.50-4.40 (m, 4H), 4.26 (dd, 1H, J ) 12.4, 6.3 Hz), 4.17 (br s,
1H), 4.04-3.87 (m, 15H), 3.86-3.79 (m, 4H), 3.77-3.60 (m, 16H),
3.52 (t, 2H, J ) 8.4 Hz), 3.37 (t, 1H, J ) 8.4 Hz), 2.08 (s, 3H), 2.05
(s, 3H), 1.20 (d, 6H, J ) 6.3 Hz); 13C NMR (125 MHz, D2O) δ 175.1,
174.7, 133.6, 119.1, 103.4, 102.9, 102.2, 102.1, 101.3, 101.2, 99.00,
98.97, 82.2, 79.4, 75.7, 75.5, 75.3, 75.24, 75.20, 75.1, 75.0, 74.8, 73.7,
73.4, 73.2, 72.8, 72.3, 71.4, 71.0, 70.2, 69.6, 68.7, 68.1, 61.9, 60.4,
60.1, 60.0, 56.3, 56.0, 49.2, 22.9, 22.6, 15.7 (2C); HRMS (FAB) (m/e)
calcd for C55H92N2O39Na [M + Na]+ 1427.5174, found 1427.5250.
a white foam: [R]23 ) -16.0° (c 0.3, CHCl3); FTIR (CHCl3 film)
D
3349, 3254, 3062, 3030, 2952, 2874, 1804, 1552, 1448, 1337, 1245,
1159, 10919, 1035, 907, 848, 813, 754, 697 cm-1; 1H NMR (500 MHz,
CDCl3) δ 7.02-7.88 (m, 3H), 7.56-7.22 (m, 17H), 5.83 (br s, 1H),
5.32 (t, 1H, J ) 8.8 Hz), 5.06 (s, 1H), 4.86 (d, 1H, J ) 8.3 Hz), 4.82
(s, 1H), 4.66 (d, 1H, J ) 5.6 Hz), 4.61 (d, 1H, J ) 11.4 Hz), 4.57-
4.51 (m, 4H), 4.41 (d, 1H, J ) 11.6 Hz), 4.34 (dd, 1H, J ) 8.2, 2.5
Hz), 4.06 (br s, 2H), 3.81 (br s, 1H), 3.68-3.61 (m, 5H), 3.16 (br s,
1H), 0.94 (t, 9H, J ) 7.9 Hz), 0.64 (m, 6H); 13C NMR (125 MHz,
CDCl3) δ 154.0, 141.2, 138.0, 137.3, 136.6, 132.4, 128.63, 128.59,
128.5, 128.3, 128.0, 127.9, 127.7, 127.4, 98.5, 75.07, 74.7, 73.8, 73.4,
73.33, 73.30, 68.6, 68.3, 67.9, 7.0, 4.9; HRMS (FAB) (m/e) calcd for
C46H56NO12SISiNa [M + Na]+ 1024.2235, found 1024.2262.
Mannose type isomer: mp 235-240 °C (decomp.); [R]23
)
D
-326.7° (c 0.03, MeOH); FTIR (KBr) 3306, 1644, 1556, 1376, 1068
Thioglycoside 47. A solution of azeotropically dried iodosulfonamide
46 (1.29 g, 1.29 mmol) in 43 mL of DMF was cooled to -40 °C.
Then, the solution was treated with ethanethiol (0.147 mL, 1.92 mmol)
and followed by dropwise addition of LHMDS (2.57 mL, 1 M in THF)
at -40 °C. After being stirred at -40 °C for 3 min, the cooling bath
was removed, and the reaction mixture was kept stirred at RT for 1 h.
Then, the reaction mixture was transferred directly to a separatory funnel
containing cold saturated NH4Cl(aq) (ca. 100 mL) and was diluted with
100 mL of EtOAc. The organic layer was separated, washed in sequence
with saturated NH4Cl(aq) (3 × 100 mL), H2O (3 × 100 mL), and brine
(1 × 100 mL), and dried over Na2SO4. The oil obtained upon
concentration with rotary evaporator was purified by column chroma-
tography with 20% EtOAc in hexanes to yield thioglycoside 47 (1.09
1
cm-1; H NMR (500 MHz, D2O) δ 6.05-5.97 (m, 1H), 5.40 (br d,
1H, J ) 17.3 Hz), 5.31 (br d, 1H, J ) 10.4 Hz), 5.16 (d, 1H, J ) 3.8
Hz), 4.97 (br s, 1H), 4.74 (d, 1H, J ) 8.1 Hz), 4.73 (d, 1H, J ) 8.1
Hz), 4.50 (d, 1H, J ) 7.6 Hz), 4.49 (d, 1H, J ) 7.6 Hz), 4.43 (d, 1H,
J ) 7.9 Hz), 4.28 (dd, 1H, J ) 12.8, 5.4 Hz), 4.16-4.10 (m, 3H),
4.05-3.87 (m, 18H), 3.85-3.80 (m, 4H), 3.77-3.69 (m, 8H), 3.67 (d,
1H, J ) 3.2 Hz), 3.65-3.58 (m, 5H), 3.55-3.52 (m, 2H), 2.10 (s,
3H), 2.06 (s, 3H), 1.21 (d, 6H, J ) 6.5 Hz); 13C NMR (125 MHz,
D2O) δ 174.4, 174.1, 132.9, 118.1, 103.0, 102.2, 101.4 (2C), 100.7,
98.5, 98.3, 98.2, 76.5, 74.9, 74.8, 74.7, 74.6, 74.4, 73.6, 73.0, 72.7,
72.12, 72.11, 71.6, 71.1, 70.71, 70.67, 69.5, 69.3, 68.89, 68.86, 68.04,
68.00, 67.95, 67.4, 66.38, 66.35, 61.2, 59.7, 59.5, 59.3, 55.6, 55.3, 22.0,
21.9, 14.99, 14.96; LRMS (EI) (m/e) calcd for C55H92N2O39Na [M +
Na]+ 1427.5, found 1427.8.
N3 minor antigen 2. To a solution of 4 (0.0017 g, 0.0012 mmol) in
wet MeOH (1 mL) was added PdCl2 (0.001 g, 0.006 mmol) at RT.
The mixture was stirred for 4 h. After removal of PdCl2 by passing the
reaction mixture through Lichroprep RP-18 pad, it was subjected to
gel filtration to provide 2 (0.0017 g, quantitative) as a 1:1 (R:â) mixture
of anomers: mp 180 °C (decomp); [R]23D ) -40.6° (c 0.12, MeOH);
FTIR (KBr) 3264, 1643, 1205, 1149, 1070 cm-1; 1H NMR (500 MHz,
D2O) δ 5.24 (d, 1/2H, J ) 3.7 Hz), 5.15 (d, 1H, J ) 4.0 Hz), 5.13 (d,
1H, J ) 4.0 Hz), 4.73 (d, 1H, J ) 8.2 Hz), 4.69 (d, 1H, J ) 8.0 Hz),
4.66 (d, 1/2H, J ) 7.2 Hz), 4.49 (d, 1H, J ) 7.3 Hz), 4.48 (d, 1H, J
) 7.4 Hz), 4.45 (d, 1H, J ) 7.9 Hz), 4.16 (d, 1H, J ) 3.2 Hz), 4.04-
3.81 (m, 22H), 3.79-3.58 (m, 18 and 1/2H), 3.53 (d, 1H, J ) 7.9 Hz),
3.51 (d, 1H, J ) 8.0 Hz), 3.31 (t, 1/2H, J ) 8.5 Hz), 2.08 (s, 3H), 2.05
(s, 3H), 1.20 (d, 6H, J ) 6.6 Hz); LRMS (EI) (m/e) calcd for
C52H88N2O39Na [M + Na]+ 1387.5, found 1387.8.
6-O-Benzyl-4-O-TES Glucal (62). To a solution of 6-benzylglucal
(60) (2.62 g, 11.1 mmol) and catalytic amount of DMAP in CH2Cl2
(200 mL) was added pyridine (2.69 mL, 33.3 mmol). The reaction
mixture was cooled to 0 °C and was treated with trimethylacetyl
chloride (2.05 mL, 16.6 mmol). The reaction mixture was stirred for
24 h at RT. The reaction mixture was concentrated with rotary
evaporator and redissolved in EtOAc (100 mL). The resulting solution
was washed with saturated NaHCO3(aq) (4 × 100 mL), saturated
CuSO4(aq) (2 × 100 mL), and brine (1 × 100 mL) and dried over
Na2SO4. Chromatographic purification of crude product with 10%
EtOAc in hexanes yielded pivaloate (2.57 g, 72%) as a colorless oil:
[R]23D ) +1.58° (c 2.79, CHCl3); FTIR (CHCl3 film) 3471, 3067, 3031,
2971, 2873, 1724, 1649.1, 1480, 1456, 1396, 1366, 1283, 1236, 1162,
1101, 1058, 953, 850, 827, 743, 699 cm-1; 1H NMR (500 MHz, CDCl3)
δ 7.35-7.27 (m, 5H), 6.46 (dd, 1H, J ) 6.1, 1.2 Hz), 5.22 (dt, 1H, J
) 6.3, 1.8 Hz), 4.68 (dd, 1H, J ) 6.1, 2.6 Hz), 4.61 (dd, 2H, J ) 16.0,
12.1 Hz), 4.00 (m, 1H), 3.94 (dd, 1H, J ) 9.5, 6.4 Hz), 3.81 (d, 2H,
J ) 4.1 Hz), 3.41 (br s, 1H), 1.21 (s, 9H); 13C NMR (125 MHz, CDCl3)
δ 180.1, 146.2, 137.8, 128.4, 127.7, 98.9, 77.3, 73.6, 73.1, 68.7, 68.2,
38.8, 27.0; LRMS (EI) (m/e) calcd for C18H24O5Na [M + Na]+ 343.4,
found 343.1.
g, 90%) as colorless oil: [R]23 ) -41.7° (c 0.92, CHCl3); FTIR
D
(CHCl3 film) 3310, 3063, 3031, 2953, 2927, 2874, 1809, 1454, 13701,
1330, 1162, 1093, 1036, 739, 698, 590, 553 cm-1; 1H NMR (500 MHz,
CDCl3) δ 7.90 (m, 2H), 7.48-7.25 (m, 18H), 5.75 (d, 1H, J ) 9.7
Hz), 4.83 (dd, 1H, J ) 7.9, 1.6 Hz), 4.74-4.61 (m, 5H), 4.56 (s, 2H),
4.49 (s, 2H), 3.96 (m, 2H), 3.85 (m, 2H), 3.81 (m, 2H), 3.72 (d, 2H,
J ) 6.9 Hz), 3.64 (t, 1H, J ) 4.7 Hz), 3.36 (m, 1H), 2.47 (m, 2H),
1.12 (t, 3H, J ) 7.5 Hz), 0.87 (t, 9H, J ) 7.9 Hz), 0.50 (m, 6H); 13C
NMR (125 MHz, CDCl3) δ 153.9, 140.7, 138.1, 137.5, 136.6, 132.4,
128.8, 128.6, 128.5, 128.34, 128.30, 128.0, 127.9, 127.7, 127.6, 99.4,
82.8, 76.9, 76.5, 76.2, 76.0, 74.0, 73.8, 73.7, 73.3, 71.6, 70.5, 69.9,
67.8, 57.4, 25.8, 14.7, 6.8, 4.4; HRMS (FAB) (m/e) calcd for C48H61-
NO12S2SiNa [M + Na]+ 958.3302, found 958.3292.
N3 minor Allyl Glycoside 4. To a deep blue solution of sodium
(0.0560 g, 2.43 mmol) in liquid ammonia (ca. 7 mL) under argon at
-78 °C was added fully protected octasaccharide 58 (0.0460 g, 0.0150
mmol) in 2 mL of anhydrous THF. After being stirred for 45 min, the
reaction mixture was quenched by addition of 3 mL of anhydrous
MeOH at -78 °C. Most of ammonia was removed by a stream of argon,
and the solution was diluted with 10 mL of MeOH and stirred for
overnight at RT. Then, the reaction mixture was treated with Dowex
50-X8 ion-exchange resin (0.525 g), stirred for 15 min, filtered, washed
with MeOH (30 mL) and ammoniacal MeOH (20 mL), and concentrated
to dryness. The crude material and catalytic amount of DMAP was
suspended in DMF, THF, and Et3N (1 mL of each). Then, the reaction
mixture was treated with 0.3 mL of acetic anhydride, stirred for 18 h
at RT, and concentrated to dryness with rotary evaporator. Flash column
chromatography of the crude material with 3% MeOH in CH2Cl2
provided peracetylated octasaccharide 59 (0.0145 g, 43%). A solution
of the peracetylated octasaccharide 59 (0.0100 g, 0.00500 mmol) in
CH2Cl2 (1 mL) was treated with dimethyldioxirane (0.15 mL, 0.075
M solution in acetone) at 0 °C and stirred for 45 min. Then, most of
the volatiles were removed by a stream of argon until ca. 0.3 mL of
the solution was left in the flask. It was treated with 3 mL of ally
alcohol. After further removal of the volatiles by a stream of argon to
ca. 2 mL of volume, the solution was stirred at RT for overnight and
concentrated to dryness. Flash column chromatography of the crude
material with 2% MeOH in CH2Cl2 gave two compounds [mannose
type isomer, higher Rf value, 0.0038 g (37%); 60, 0.0049 g (48%)].
These compounds were deacetylated according to the procedure
described in reference (NaOMe, MeOH). Upon deacetylation, N3 minor
To a solution of pivaloate (2.44 g, 7.62 mmol) in CH2Cl2 (120 mL)
was added Et3N (4.25 mL, 30.5 mmol). The reaction mixture was cooled
to 0 °C and was treated with TESOTf (2.44 mL, 10.7 mmol). The

46 J. Am. Chem. Soc., Vol. 123, No. 1, 2001
Kim et al.
reaction mixture was stirred at 0 °C for 2 h. Then, the reaction mixture
was concentrated with rotary evaporator and redissolved in EtOAc (100
mL). The organic solution was washed with saturated NaHCO3(aq) (3
× 100 mL) and brine (1 × 100 mL) and dried over Na2SO4.
Chromatographic purification with 3% EtOAc in hexanes afforded TES
protected glucal (3.18 g, 96%) a colorless oil: [R]23D ) -45.5° (c 2.73,
CHCl3); FTIR (CHCl3 film) 3068, 3031, 2957, 2912, 2877, 1728, 1651,
1480, 1457, 1414, 1395, 1366, 1313, 1279, 1240, 1206, 1151, 1125,
mL), dried over Na2SO4, and concentrated to dryness. Flash column
chromatography of crude material afforded 65 (0.370 g, 82%) as
colorless oil: [R]23 ) -26.3° (c 1.35, CHCl3); FTIR (CHCl3 film)
D
1
2952, 2874, 1811, 1647, 1454, 1367, 1244, 1098, 739, 698 cm-1; H
NMR (500 MHz, CDCl3) δ 7.37-7.25 (m, 15H), 6.46 (d, 1H, J ) 6.3
Hz), 4.82-4.79 (m, 2H), 4.75-4.70 (m, 3H), 4.61 (d, 1H, J ) 11.3
Hz), 4.58-4.53 (m, 4H), 4.14-4.10 (m, 2H), 3.99-3.95 (m, 2H), 3.72-
3.68 (m, 3H), 3.62 (dd, 1H, J ) 11.0, 2.5 Hz), 3.49 (t, 1H, J ) 4.9
Hz), 0.93-0.90 (m, 9H), 0.63-0.58 (m, 6H); 13C NMR (125 MHz,
CDCl3) δ 153.9, 145.3, 138.0, 137.4, 136.9, 128.48, 128.45, 128.3,
128.1, 127.92, 127.89, 127.8 (2C), 127.5, 97.1, 96.5, 78.5, 77.2, 76.78,
74.0, 73.8, 73.7, 73.3, 72.1, 69.9, 68.5, 68.0, 67.9, 6.8, 4.7; LRMS
(EI) (m/e) calcd for C40H50O10SiNa [M + Na]+ 741.3, found 741.5.
1
1098, 1011, 959, 875, 828, 776, 742, 698 cm-1; H NMR (500 MHz,
CDCl3) δ 7.34-7.26 (m, 5H), 6.43 (d, 1H, J ) 6.1 Hz), 5.12 (t, 1H,
J ) 3.8 Hz), 4.69 (dd, 1H, J ) 6.1, 3.3 Hz), 4.59 (dd, 2H, J ) 31.5,
12.1 Hz), 4.05 (m, 2H), 3.76 (dd, 1H, J ) 10.5, 5.5 Hz), 3.68 (dd, 1H,
J ) 10.7, 2.8 Hz), 1.18 (s, 9H), 0.92 (t, 9H, J ) 8.0 Hz), 0.60 (dd, 6H,
J ) 16.0, 7.8 Hz); 13C NMR (125 MHz, CDCl3) δ 177.9, 145.7, 137.8,
128.3, 127.7, 127.6, 98.6, 78.2, 73.5, 71.6, 68.2, 67.3, 38.7, 27.1, 6.7,
4.8; LRMS (EI) (m/e) calcd for C24H38O5SiNH4 [M + NH4]+ 452.3,
found 452.2.
Iodosulfonamide 66. A mixture of 65 (0.357 g, 0.496 mmol),
benzenesulfonamide (0.156 g, 0.992 mmol), and freshly activated 4 Å
molecular sieves (0.45 g) in CH2Cl2 (10 mL) was stirred for 15 min at
RT, cooled to 0 °C, treated with I(sym-coll)2ClO4 (0.465 g), and stirred
for 1 h at 0 °C. The reaction was diluted with EtOAc (150 mL), filtered
through a pad of silica gel, and washed with saturated Na2S2O3(aq) (2
× 50 mL), saturated CuSO4(aq) (2 × 50 mL), H2O (1 × 50 mL), and
brine (1 × 50 mL). The organic layer was dried over Na2SO4 and
concentrated to dryness. Flash column chromatography of crude material
with 25% EtOAc in hexanes gave 66 (0.445 g, 90%) as a white foam:
To the solution of azeotropically dried TES glucal (3.18 g, 7.32
mmol) in 150 mL of CH2Cl2 was added DIBAl-H (14.6 mL, 1 M
solution in toluene) at -78 °C. After being stirred at -78 °C for 1 h,
the reaction mixture was quenched by dropwise addition of H2O (10
mL). The reaction mixture was then warmed to RT and was treated
with saturated potassium sodium tartarate aqueous solution (300 mL).
The resulting mixture was stirred vigorously at RT overnight. Then,
the organic layer was separated, and the aqueous layer was extracted
with CH2Cl2 (3 × 300 mL). All of the organic layers were combined
and dried over Na2SO4. The crude product was purified by column
chromatography with the gradient of 8% EtOAc in hexanes-10%
EtOAc in hexanes to afford of 3-hydroxyglucal 62 (1.81 g,70%) as
colorless oil: [R]23D +21.5° (c 1.59, CHCl3); FTIR (CHCl3 film) 3455,
3065, 3031, 2953, 2876, 1950, 1877, 1809, 1649, 1496, 1455, 1414,
[R]23 -24.0° (c 1.47, CHCl3); FTIR (neat) 3261, 2952, 2876, 1809,
D
1454, 1330, 1165, 1095, 739, 699 cm-1; 1H NMR (500 MHz, CDCl3)
δ 7.89 (d, 2H, J ) 8.3 Hz), 7.39-7.23 (m, 18H), 5.83 (br s, 1H), 5.47
(t, 1H, J ) 8.0 Hz), 4.94 (d, 1H, J ) 11.4 Hz), 4.88 (br s, 1H), 4.82-
4.77 (m, 2H), 4.65 (d, 1H, J ) 11.4 Hz), 4.56-4.48 (m, 5H), 4.02(dt,
1H, J ) 6.6, 1.2 Hz), 3.84-3.74 (m, 5H), 3.62 (d, 2H, J ) 6.7 Hz),
3.28 (d, 1H, J ) 9.8 Hz), 0.86 (t, 9H, J ) 8.0 Hz), 0.53 (q, 9H, J )
7.9 Hz); 13C NMR (125 MHz, CDCl3) δ 153.7, 140.9, 138.3, 137.2,
136.7, 132.6, 128.7, 128.6, 128.5, 128.3, 128.2 (2C), 128.0, 127.9,
127.8, 127.5, 127.4, 74.0, 73.73, 73.66, 73.3, 70.1, 69.4, 67.4, 67.3,
30.1, 6.7, 4.6; LRMS (ESI) (m/e) calcd for C46H57NO12SSiNa [M +
Na]+ 1024.9, found 1024.3.
1365, 1322, 1236, 1116, 1043, 1017, 973, 874, 777, 741, 698 cm-1
;
1H NMR (500 MHz, CDCl3) δ 7.33-7.24 (m, 5H), 6.36 (dd, 1H, J )
6.0, 1.0 Hz), 4.68 (dd, 1H, J ) 6.0, 2.6 Hz), 4.57 (dd, 2H, J ) 27.9,
12.1 Hz), 4.09 (d, 1H, J ) 5.2 Hz), 3.89 (m, 1H), 3.78-3.68 (m, 3H),
2.12 (br s, 1H), 0.93 (t, 9H, J ) 8.0 Hz), 0.63 (m, 6H); 13C NMR (125
MHz, CDCl3) δ 144.3, 137.7, 128.2, 127.6, 127.5, 102.7, 78.0, 73.4,
71.5, 70.0, 69.0, 6.7, 4.9; LRMS (EI) (m/e) calcd for C19H30O4SiNa
[M + Na]+ 373.2, found 373.2.
Tetrasaccharide 67. To a solution of disaccharide acceptor 43 (0.804
g, 1.19 mmol) in anhydrous benzene (90 mL) was added bis(tributyltin)
oxide (0.347 mL, 0.653 mmol). The resulting solution was heated at
reflux overnight with azeotropic elimination of H2O via a Dean-Stark
trap. Removal of volatiles by N2 stream provided the stannyl ether of
43 as oil. To a mixture of 66 (0.470 g, 0.475 mmlo) and 1 g of 4 Å
molecular sieves was added the solution of the stannyl ether in dry
THF (10 mL) via cannula. The reaction mixture was stirred at RT for
15 min, cooled to 0 °C, and treated with the solution of silver
tetrafluoroborate (0.243 g, 1.25 mmol) in THF (2 mL). Then, the
reaction was heated at 45-50 °C for 36 h. After the completion of
reaction, it was filtered through a pad of Celite and washed with EtOAc
(150 mL). The organic layer was washed with saturated NaHCO3(aq)
(3 × 50 mL) and brine (1 × 50 mL), dried over Na2SO4, and
concentrated to dryness. Flash column chromatography of crude material
with 35-50% EtOAc in hexanes afforded 67 (higher Rf value, 0.269
g, 37%) with R-isomer 68 (0.225 g, 30%) as a white foam. Also
recovered was 0.4 g of acceptor 43.
Disaccharide 64. A solution of 63 (2.204 g, 8.404 mmol) in CH2-
Cl2 (20 mL) was treated with dimethyldioxirane (130 mL, 0.07 M
solution in acetone) at 0 °C and stirred for 1 h. Most of the volatiles
were removed by a stream of N2. Then, the residue was treated with
20 mL of benzene, concentrated with a stream of N2, and treated with
62 (1.477 g, 4.214 mmol) in THF (20 mL). Most of the solvent was
removed by a stream of N2. The mixture as a form of thick oil was
cooled to 0 °C, treated with ZnCl2 (4.4 mL, 1.0 M solution in Et2O),
and kept stirred at RT for 4 h. The reaction mixture was diluted with
EtOAc (300 mL) and poured into H2O (100 mL). The organic layer
was separated, washed with H2O (2 × 100 mL), saturated NaHCO3-
(aq) (2 × 100 mL), and brine (1 × 100 mL), dried over Na2SO4, and
concentrated to dryness. Flash column chromatography of the crude
material with 33% EtOAc in hexanes afforded 64 (1.24 g, 47%) as a
colorless oil: [R]23 ) -24.4° (c 0.98, CHCl3); FTIR (CHCl3 film)
D
67: [R]23 ) -28.2° (c 0.64, CHCl3); FTIR (CHCl3 film) 3514,
3454, 3064, 3031, 2953, 2912, 2878, 1951, 1806, 1648, 1496, 1455,
1413, 1369, 1245, 1074, 912, 869, 833, 740, 699 cm-1; 1H NMR (400
MHz, CDCl3) δ 7.40-7.27 (m, 10H), 6.44 (d, 1H, J ) 6.2 Hz), 4.79-
4.75 (m, 2H), 4.65-4.52 (m, 5H), 4.47 (d, 1H, J ) 7.1 Hz), 4.20-
4.18 (m, 1H), 4.08-4.05 (m, 1H), 3.98-3.93 (m, 2H), 3.72-3.66 (m,
4H), 3.63-3.59 (m, 1H), 2.73 (d, 1H, J ) 2.7 Hz), 0.91 (t, 9H, J )
7.9 Hz), 0.57 (q, 6H, J ) 7.9 Hz); 13C NMR (100 MHz, CDCl3) δ
154.0, 145.5, 137.81, 137.3, 128.4, 128.3, 127.9, 127.8, 127.74, 127.65,
97.6, 96.6, 78.1, 78.0, 74.6, 73.7, 73.3, 72.7, 71.5, 70.6, 68.3, 68.0,
67.9, 6.7, 4.7; HRMS (FAB) (m/e) calcd for C33H44O10SiNa [M + Na]+
651.2601, found 651.2597.
Benzyl-Protected Disaccharide 65. To a solution of 64 (0.392 g,
0.623 mmol) and BnBr (0.0890 mL, 0.748 mmol) in anhydrous DMF
(8 mL) was added NaH (0.0370 g, 0.935 mmol, 60% dispersion in oil)
at 0 °C. The mixture was stirred at 0 °C for 1.5 h, poured into cold
water (100 mL), and extracted with EtOAc (3 × 50 mL). The combined
organic layer was washed with H2O (3 × 70 mL) and brine (1 × 70
D
1
2874, 1809, 1736, 1718, 1454, 1367, 1094, 740, 699 cm-1; H NMR
(500 MHz, CDCl3) δ 7.73 (d, 2H, J ) 7.5 Hz), 7.43-7.25 (m, 33H),
6.42 (d, 1H, J ) 6.2 Hz), 4.93 (d, 1H, J ) 9.4 Hz), 4.89 (dd, 1H, J )
6.0, 4.3 Hz), 4.82 (t, 2H, J ) 6.0 Hz), 4.77 (dd, 1H, J ) 7.8, 1.6 Hz),
4.73-4.66 (m, 3H), 4.64-4.59 (m, 2H), 4.57-4.53 (m, 3H), 4.51-
4.45 (m, 4H), 4.39 (d, 1H, J ) 11.2 Hz), 4.29-4.26 (m, 1H), 4.25-
4.18 (m, 2H), 4.12-4.09 (m, 1H), 3.92-3.87 (m, 2H), 3.85-3.82 (m,
2H), 3.81-3.76 (m, 2H), 3.74-3.71 (m, 1H), 3.65-3.62 (m, 3H), 3.60-
3.56 (m, 1H), 3.53-3.46 (m, 2H), 3.43-3.41 (m, 2H), 2.64 (br s, 1H),
2.60-2.53 (m, 2H), 2.40-2.34 (m, 2H), 2.08 (s, 3H), 0.89 (t, 9H, J )
7.9 Hz), 0.55 (q, 6H, J ) 7.9 Hz); 13C NMR (125 MHz, CDCl3) δ
206.3, 172.4, 153.8, 144.4, 140.7, 138.6, 138.5, 138.0, 137.8, 137.4,
137.0, 132.7, 129.0, 128.51, 128.47, 128.4, 128.33, 128.28, 128.1, 128.0,
127.9, 127.83, 127.79, 127.73, 127.71, 127.63, 127.62, 127.5, 127.0,
102.2, 100.9, 99.5, 99.2, 79.9, 78.6, 78.3, 78.1, 75.5, 74.3, 74.2, 73.7,
73.3, 73.0, 72.8, 71.8, 71.5, 70.1, 70.0, 69.9, 69.2, 68.4, 67.8, 67.5,

Tumor-Related Antigens N3
J. Am. Chem. Soc., Vol. 123, No. 1, 2001 47
63.4, 56.9, 37.8, 29.7, 27.7, 6.7, 4.5; HRMS (FAB) (m/e) calcd for
C84H99NO23SSiNa [M + Na]+ 1572.5953, found 1572.5996.
68: [R]23D ) +33.1° (c 0.48, CHCl3); FTIR (CHCl3 film) 3502, 2875,
4.49-4.40 (m, 4H), 4.43-4.41 (m, 4H), 4.29-4.25 (m, 2H), 4.22 (d,
1H, J ) 7.0 Hz), 4.12 (d, 1H, J ) 5.8 Hz), 4.05-4.03 (m, 1H), 3.88-
3.82 (m, 2H), 3.74-3.71 (m, 2H), 3.70-3.63 (m, 6H), 3.62-3.53 (m,
9H), 3.43 (t, 1H, J ) 6.0 Hz), 3.37 (d, 1H, J ) 7.3 Hz), 3.35 (d, 1H,
J ) 7.5 Hz), 3.32-3.27 (m, 3H), 3.20 (br d, 1H, J ) 8.0 Hz), 2.75 (d,
1H, J ) 8.3 Hz), 2.02 (s, 3H), 0.87-0.82 (m, 18H), 0.53-0.37 (m,
12H); 13C NMR (125 MHz, CDCl3) δ 169.7, 153.9, 153.7, 144.4, 142.3,
141.6, 138.7, 138.5, 138.1, 138.0, 137.5, 137.32, 137.25, 137.1, 132.3,
131.9, 128.8, 128.5, 128.43, 128.41, 128.33, 128.31, 128.27, 128.22,
128.1, 128.01, 127.96, 127.93, 127.86, 127.8, 127.74, 127.68, 127.66,
127.64, 127.60, 127.57, 127.54, 127.48, 127.4, 127.1, 127.0, 102.1,
101.9, 101.5, 100.2, 100.0, 99.8, 80.0, 79.09, 79.07, 78.8, 78.7, 77.9,
77.8, 77.1, 76.14, 76.05, 75.1, 74.8, 74.6, 74.3, 73.9, 73.8, 73.7, 73.4,
73.3, 73.23, 73.20, 73.17, 71.6, 70.61, 70.56, 70.3, 70.0, 69.8, 67.94,
67.86, 67.52, 67.46, 60.1, 58.2, 20.8, 7.0, 6.8, 4.9, 4.6; HRMS (FAB)
(m/e) calcd for C127H150N2O34S2Si2Na [M + Na]+ 2389.8846, found
2389.8948.
1
1809, 1738, 1718, 1453, 1366, 1093, 738, 699 cm-1; H NMR (500
MHz, CDCl3) δ 7.93 (d, 2H, J ) 7.9 Hz), 7.49-7.24 (m, 31H), 7.15
(d, 2H, J ) 6.6 Hz), 6.48 (d, 1H, J ) 6.3 Hz), 5.97 (d, 1H, J ) 10.1
Hz), 5.01 (d, 1H, J ) 7.4 Hz), 4.95-4.93 (m, 2H), 4.74 (d, 1H, J )
3.3 Hz), 4.65-4.55 (m, 5H), 4.49-4.46 (m, 3H), 4.42 (d, 1H, J )
10.0 Hz), 4.37 (d, 1H, J ) 10.7 Hz), 4.34-4.21 (m, 7H), 4.11-4.08
(m, 2H), 3.97 (d, 1H, J ) 9.1 Hz), 3.84 (dd, 1H, J ) 10.5, 6.7 Hz),
3.79-3.74 (m, 3H), 3.65 (d, 1H, J ) 10.5, 3.8 Hz), 3.61 (br s, 1H),
3.55-3.52 (m, 4H), 3.46-3.39 (m, 3H), 3.34 (d, 1H, J ) 10.2 Hz),
3.18 (d, 1H, J ) 5.3 Hz), 2.71-2.59 (m, 2H), 2.53-2.42 (m, 2H),
2.17 (bs, 1H), 2.10 (s, 3H), 0.87 (t, 9H, J ) 7.9 Hz), 0.55 (q, 6H, J )
7.9 Hz); 13C NMR (125 MHz, CDCl3) δ 206.4, 172.6, 153.9, 144.5,
140.4, 138.5, 137.9, 137.7, 137.64, 137.55, 137.2, 133.1, 129.3, 128.9,
128.4, 128.34, 128.33, 128.30, 128.22, 128.17, 127.9, 127.8, 127.71,
127.69, 127.61, 127.56, 127.5, 127.3, 102.7, 100.4, 99.3, 93.2, 80.2,
79.2, 76.6, 75.7, 75.6, 75.3, 74.9, 74.3, 73.4, 73.2, 73.1, 72.9, 71.7,
71.2, 71.0, 70.8, 70.0, 69.9, 68.3, 67.8, 67.7, 64.1, 62.9, 57.6, 37.8,
29.7, 27.8, 7.0, 5.2; LRMS (EI) (m/e) calcd for C84H99NO23SSiNa [M
+ Na]+ 1572.6, found 1572.7.
Hexa Acceptor 71. To a solution of 70 (0.0790 g, 0.0330 mmol) in
THF (5.4 mL) was dropwise added AcOH (0.011 mL, 0.20 mmol) and
TBAF (0.2 mL, 1 M in THF) successively. The reaction mixture was
stirred at RT for 17 h, diluted with EtOAc (150 mL), washed with
saturated NaHCO3(aq) (2 × 70 mL), H2O (2 × 50 mL), and brine (1
× 50 mL), dried over Na2SO4, and concentrated to dryness. Flash
column chromatography of crude material with 45% EtOAc in hexanes
Tetrasaccharide Acceptor 69. To a solution of 67 (0.281 g, 0.181
mmol), a catalytic amount of DMAP, and Et3N (0.303 mL, 2.17 mmol)
in CH2Cl2 (15 mL) was added acetic anhydride (0.205 mL, 2.17 mmol)
at 0 °C. The reaction mixture was stirred at RT for 40 h, diluted with
EtOAc (50 mL), washed with saturated NaHCO3(aq) (3 × 30 mL) and
brine (1 × 30 mL), dried over Na2SO4, and concentrated to dryness.
Flash column chromatography of crude material with 33% EtOAc in
hexanes afforded acetylated tetrasaccharide (0.254 g, 88%) as a white
foam. It was dissolved in CH2Cl2 (20 mL) and sequentially treated with
pyridine (0.670 mL) and 1 M NH2NH2 in pyridine-AcOH (3:2) (0.670
mL) at RT. The reaction mixture was stirred for 4 h, diluted with EtOAc
(300 mL), washed with saturated NaHCO3(aq) (3 × 100 mL), saturated
CuSO4(aq) (3 × 100 mL), and brine (1 × 50 mL), dried over Na2SO4,
and concentrated to dryness. Flash column chromatography of crude
afforded 71 (0.0644 g, 93%) as a white foam: [R]23 ) -14.8° (c
D
0.97, CHCl3); FTIR (CHCl3 film) 3477, 2872, 1808, 1746, 1454, 1371,
1329, 1161, 1098, 738, 699 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.90
(bd, 2H, J ) 7.6 Hz), 7.87 (bd, 2H, J ) 7.1 Hz), 7.54-7.47 (m, 3H),
7.42 (bd, 2H, J ) 7.3 Hz), 7.39-7.17 (m, 44H), 7.14 (bd, 2H, J ) 7.5
Hz), 6.30 (d, 1H, J ) 6.2 Hz), 5.43 (d, 1H, J ) 7.4 Hz), 5.19 (d, 1H,
J ) 3.5 Hz), 4.93 (bs, 1H), 4.86 (dd, 1H, J ) 6.1, 3.1 Hz), 4.81 (d,
1H, J ) 8.7 Hz), 4.77 (d, 1H, J ) 12.1 Hz), 4.73 (d, 1H, J ) 8.0 Hz),
4.69 (br s, 2H), 4.66 (br d, 1H, J ) 8.7 Hz), 4.63-4.61 (m, 5H), 4.57
(d, 1H, J ) 5.3 Hz), 4.54-4.52 (m, 4H), 4.50 (bs, 1H), 4.47-4.44 (m,
4H), 4.40 (d, 1H, J ) 7.9 Hz), 4.34-4.29 (m, 3H), 4.23-4.19 (m,
2H), 4.10 (bs, 1H), 3.98 (d, 1H, J ) 12.2 Hz), 3.94 (t, 1H, J ) 6.9
Hz), 3.85 (d, 1H, J ) 10.4 Hz), 3.82-3.81 (m, 2H), 3.73-3.68 (m,
4H), 3.64-3.59 (m, 4H), 3.57-3.53 (m, 2H), 3.51-3.49 (m, 3H), 3.47-
3.38 (m, 7H), 3.35-3.26 (m, 2H), 3.12 (dd, 1H, J ) 10.0, 7.9 Hz),
2.85 (bd, 1H, J ) 8.3 Hz), 2.13 (s, 3H); 13C NMR (125 MHz, CDCl3)
δ 169.8, 153.6, 153.5, 144.5, 142.3, 142.1, 139.0, 138.5, 138.1, 137.7,
137.2, 137.1, 137.0, 136.5, 132.4, 131.8, 128.7, 128.53, 128.49, 128.47,
128.4, 128.30, 128.26, 128.2, 128.1, 128.0, 127.83, 127.78, 127.7,
127.63, 127.58, 127.55, 127.48, 127.46, 127.1, 102.0, 101.6, 101.3,
100.3, 99.9, 99.3, 76.54, 76.50, 76.3, 75.7, 75.5, 74.2, 74.0, 73.9, 73.7,
73.64, 73.61, 73.42, 73.39, 73.21, 73.18, 73.1, 72.89, 72.86, 72.7, 70.9,
70.2, 69.9, 69.4, 68.7, 68.1, 68.0, 67.9, 67.8, 67.6, 59.3, 58.5, 20.9;
HRMS (FAB) (m/e) calcd for C115H122N2O34S2Na [M + Na]+ 2161.7142,
found 2161.7218.
material gave 69 (0.194 g, 83%) as a white foam: [R]23 ) -8.9° (c
D
0.38, CHCl3); FTIR (CHCl3 film) 3351, 3030, 2875, 1809, 1746, 1649,
1
1454, 1369, 1240, 1094, 738 cm-1; H NMR (500 MHz, CDCl3) δ
7.75 (d, 2H, J ) 8.0 Hz), 7.45 (t, 1H, J ) 8.0 Hz), 7.37-7.22 (m,
32H), 6.42 (d, 1H, J ) 6.2 Hz), 5.19 (d, 1H, J ) 3.3 Hz), 4.87 (dd,
1H, J ) 6.0, 3.3 Hz), 4.79 (d, 1H, J ) 11.6 Hz), 4.77 (d, 1H, J ) 7.1
Hz), 4.71 (d, 1H, J ) 7.7 Hz), 4.69 (d, 1H, J ) 11.6 Hz), 4.63-4.48
(m, 11H), 4.44 (d, 1H, J ) 12.0 Hz), 4.35 (d, 1H, J ) 11.6 Hz), 4.16-
4.11 (m, 3H), 3.86 (t, 1H, J ) 4.2 Hz), 3.79-3.67 (m, 5H), 3.65-3.55
(m, 6H), 3.52-3.49 (m, 1H), 3.44-3.40 (m, 2H), 3.35-3.31 (m, 1H),
2.75 (bs, 1H), 2.01 (s, 3H), 0.86 (t, 9H, J ) 7.9 Hz), 0.51 (q, 6H, J )
7.9 Hz); 13C NMR (125 MHz, CDCl3) δ 171.8, 153.7, 144.6, 141.4,
138.6, 138.4, 138.1, 137.8, 137.2, 137.1, 132.3, 128.8, 128.5, 128.4,
128.32, 128.25, 128.00, 127.95, 127.8, 127.74, 127.65, 127.6, 127.5,
127.4, 127.3, 127.0, 102.4, 101.5, 100.0, 99.6, 79.5, 79.3, 78.9, 78.0,
77.8, 75.9, 74.5, 74.4, 73.8, 73.6, 73.40, 73.36, 73.2, 73.1, 72.4, 70.7,
70.4, 70.1, 69.8, 69.6, 67.7, 67.4, 60.2, 58.3, 20.7, 6.7, 4.6; HRMS
(FAB) (m/e) calcd for C81H95NO22SSiNa [M + Na]+ 1516.5787, found
1516.5733.
Hexasaccharide 70. A mixture of thiodonor 47 (0.201 g, 0.215
mmol), tetrasaccharide acceptor 69 (0.168 g, 0.112 mmol), di-tert-
butylpyridine (0.097 mL, 0.43 mmol), and freshly activated 4 Å
molecular sieves (1.5 g) in Et2O-CH2Cl2 (2:1, 8.1 mL) was stirred at
RT for 15 min, cooled to -40 °C, treated with MeOTf (0.0970 mL,
0.859 mmol), and slowly allowed to warm to RT for 6 h. Then, it was
quenched by addition of Et3N (2 mL), stirred for 15 min, diluted with
EtOAc (300 mL), washed with saturated NaHCO3(aq) (2 × 70 mL),
saturated CuSO4(aq) (2 × 70 mL), and brine (1 × 70 mL), dried over
Na2SO4, and concentrated to dryness. Flash column chromatography
of crude material provided hexasaccharide 70 (0.199 g, 75%) as a white
foam: [R]23D ) -7.0° (c 0.37, CHCl3); FTIR (CHCl3 film) 2874, 1811,
1747, 1454, 1370, 1092, 738, 698 cm-1; 1H NMR (500 MHz, CDCl3)
δ 7.85 (d, 2H, J ) 7.6 Hz), 7.74 (d, 2H, J ) 7.6 Hz), 7.45-7.13 (m,
51H), 6.36 (d, 1H, J ) 6.0 Hz), 5.15 (d, 1H, J ) 3.5 Hz), 4.85-4.77
(m, 4H), 4.72-4.66 (m, 4H), 4.65-4.56 (m, 7H), 4.55-4.50 (m, 5H),
Fully Protected N3 Major Glycal 72. A mixture of 71 (0 0352 g,
0.0160 mmol), fucosyl floride 23 (0.0370 g, 0.0820 mmol), di-tert-
butylpyridine (0.026 mL, 0.16 mmol), and 4 Å molecular sieves (0.57
g) in toluene (2.8 mL) was stirred at RT for 15 min, cooled to -15 °C,
treated with the solution of Sn(OTf)2 (0.0240 g, 0.0576 mmol) in THF
(0.28 mL) via cannula, and stirred at -7 °C for 12 h, 0 °C for 24 h,
and RT for 5 h. Then, the reaction mixture was quenched by addition
of Et3N (2 mL) and stirred for 10 min It was poured into a separatory
funnel containing EtOAc (150 mL), washed with saturated NaHCO3-
(aq) (2 × 50 mL) and brine (1 × 50 mL), dried over Na2SO4, and
concentrated to dryness. Flash column chromatography of crude material
with 35% EtOAc in hexanes gave 72 (0.0375 g, 76%) as white foam:
[R]23D ) -67.1° (c 0.83, CHCl3); FTIR (CHCl3 film) 2872, 1811, 1747,
1720, 1453, 1271, 1160, 1094, 911 cm-1; 1H NMR (500 MHz, CDCl3)
δ 8.06-8.03 (m, 4H), 7.69 (d, 2H, J ) 7.7 Hz), 7.56 (d, 4H, J ) 7.4
Hz), 7.45 (t, 4H, J ) 7.7 Hz), 7.40-7.16 (m, 71H), 6.94 (d, 1H, J )
3.5 Hz), 6.38 (d, 1H, J ) 6.2 Hz), 5.58 (d, 1H, J ) 2.6 Hz), 5.48 (d,
1H, J ) 2.8 Hz), 5.19 (d, 1H, J ) 3.3 Hz), 5.12 (d, 1H, J ) 3.4 Hz),
5.00 (bs, 1H), 4.98 (d, 1H, J ) 6.8 Hz), 4.89-4.78 (m, 6H), 4.74-
4.66 (m, 6H), 4.63-4.57 (m, 7H), 4.54-4.43 (m, 12H), 4.33-4.25
(m, 5H), 4.21-4.15 (m, 3H), 4.11 (t, 1H, J ) 4.8 Hz), 4.04 (bs, 1H),
4.01-3.91 (m, 3H), 3.84-3.76 (m, 6H), 3.73-3.60 (m, 10H), 3.53-

48 J. Am. Chem. Soc., Vol. 123, No. 1, 2001
Kim et al.
3.40 (m, 5H), 3.33-3.28 (m, 1H), 3.35-3.20 (m, 2H), 2.89-2.81 (m,
2H), 1.95 (s, 3H), 1.02 (t, 6H, J ) 6.0 Hz); 13C NMR (125 MHz,
CDCl3) δ 169.5, 166.1, 166.0, 153.6, 144.3, 142.6, 141.4, 138.8, 138.7,
138.3, 138.2, 138.10, 138.06, 137.9, 137.7, 137.6, 137.4, 137.3, 137.2,
133.0, 132.9, 132,3, 131.6, 130.02, 129.97, 129.8, 129.0, 128.8, 128.5,
128.44, 128.40, 128.36, 128.31, 128.27, 128.23, 128.15, 128.1, 127.98,
127.96, 127.90, 127.89, 127.86, 127.82, 127.75, 127.72, 127.69, 127.65,
127.62, 127.58, 127.4, 127.34, 127.29, 126.9, 101.91, 101.87, 101.0,
100.0, 99.64, 99.55, 99.5, 97.2, 79.9, 79.5, 78.5, 78.4, 77.9, 77.4, 77.2,
76.9, 76.4, 75.9, 75.6, 75.0, 74.8, 74.7, 74.5, 74.4, 74.1, 73.9, 73.73,
73.71, 73.6, 73.5, 73.4, 73.2, 72.9, 72.0, 71.8, 71.4, 71.1, 71.0, 70.5,
70.41, 70.37, 69.5, 69.2, 68.3, 67.8, 67.6, 67.3, 66.5, 66.0, 65.5, 59.0,
58.3, 20.7, 16.31, 16.28; LRMS (EI) (m/e) calcd for C169H174N2O44S2-
Na [M + 2Na]2+ 1522.5 for z ) 2, found 1522.6.
101.0, 100.2, 99.4, 97.0, 96.4, 80.2, 77.4, 74.3, 73.8, 73.6, 73.3, 73.23,
73.20, 73.1, 72.8, 71.8, 71.7, 71.2, 70.8, 70.7, 70.5, 70.33, 70.29, 69.4,
69.1, 68.9, 68.2, 67.6, 67.5, 67.0, 66.2, 66.1, 65.2, 65.1, 60.0, 59.9,
58.4, 58.1, 58.0, 54.3, 54.0, 20.9, 20.7, 13.8, 13.7; HRMS (FAB) (m/
e) calcd for C55H92N2O39Na [M + Na]+ 1427.5128, found 1427.5175.
Mannose type isomer (from A): mp 226 °C (decomp); [R]23
)
D
-21.4° (c 0.07, MeOH); FTIR (KBr) 3280, 1642, 1376, 1070 cm-1
;
1H NMR (500 MHz, D2O) δ 6.05-5.97 (m, 1H), 5.39 (bd, 1H, J )
17.2 Hz), 5.31 (bd, 1H, J ) 10.3 Hz), 5.15 (d, 1H, J ) 3.7 Hz), 5.05
(d, 1H, J ) 3.4 Hz), 4.96 (bs, 1H), 4.72 (d, 1H, J ) 7.8 Hz), 4.71 (d,
1H, J ) 8.2 Hz), 4.53 (d, 1H, J ) 7.6 Hz), 4.48 (d, 1H, J ) 7.7 Hz),
4.42 (d, 1H, J ) 7.9 Hz), 4.27 (dd, 1H, J ) 12.8, 5.4 Hz), 4.15-4.08
(m, 4H), 4.04-3.49 (m, 41H), 2.09 (s, 3H), 2.06 (s, 3H), 1.20 (d, 6H,
J ) 6.4 Hz); 13C NMR (125 MHz, D2O) δ 175.1, 174.8, 133.6, 118.8,
103.7, 103.2, 103.0, 102.1, 101.4, 99.2, 98.9, 93.4, 82.3, 77.2, 76.3,
75.62, 75.58, 75.4, 75.3, 75.2, 74.3, 73.8, 72.8, 72.7, 72.5, 72.32, 72.27,
71.9, 71.4, 70.9, 70.2, 70.0, 69.61, 69.57, 69.5, 68.8, 68.7, 68.7, 68.2,
68.1, 67.2, 67.1, 62.0, 61.9, 60.4, 60.2, 60.0, 56.2, 56.0, 22.8, 22.7,
15.73, 15.71; HRMS (FAB) (m/e) calcd for C55H92N2O39Na [M + Na]+
1427.5175, found 1427.5161.
N3 Major Allyl Glycoside 3. To a deep blue solution of sodium
(0.070 g) in liquid ammonia (ca. 7 mL) under argon at -78 °C was
added fully protected octasaccharide 72 (0.058 g, 0.019 mmol) in
anhydrous THF (3 mL). After being stirred for 45 min, the reaction
mixture was quenched by addition of anhydrous MeOH (3 mL) at -78
°C. Most of the ammonia was removed by a stream of argon, and the
solution was diluted with MeOH (6 mL) and stirred for overnight at
RT. It was treated with Dowex 50-X8 ion-exchange resin (0.60 g),
stirred for 15 min, filtered, washed with MeOH (20 mL) and
ammoniacal MeOH (30 mL), and concentrated to dryness. A crude
material and catalytic amount of DMAP was suspended in pyridine (1
mL), treated with acetic anhydride (0.5 mL), stirred at RT for 18 h,
and concentrated to dryness. Flash column chromatography of crude
material with 50% CH2Cl2 in EtOAc-100% EtOAc provided peracety-
lated octasaccharide (0.024 g, 58%). A solution of peracetylated
octasaccharide (0.018 g, 0.0083 mmol) in CH2Cl2 (1 mL) was treated
with 3,3′-dimethyldioxirane (0.3 mL, 0.75 M solution) at 0 °C and
stirred for 45 min. Then, most of the volatiles were removed by a stream
of argon until ca. 0.3 mL of mixture was left. It was treated with ally
alcohol (5 mL). After further removal of volatiles being left by a stream
of argon, it was stirred at RT overnight and concentrated to dryness.
Flash column chromatography of the crude material with ethyl acetate
gave two compounds [A, higher Rf value (C6H6-acetone), 0.0034 g
(18%); B, 0.0083 g (45%)]. These compounds were deacetylated
according to the procedure described in reference (NaOMe, MeOH).
Upon deacetylation, mannose type isomer (0.0026 g, 84%) was obtained
from A (0.005 g), and so was 3 (2.7 mg, 88%) from B (0.0049 g).
3 (from B): mp 225 °C (decomp); [R]23D ) -96.0° (c 0.05, MeOH);
FTIR (KBr) 3352, 1644, 1375, 1206, 1149, 1071 cm-1; 1H NMR (500
MHz, D2O) δ 6.05-5.97 (m, 1H), 5.41 (dd, 1H, J ) 17.3, 1.4 Hz),
5.30 (bd, 1H, J ) 10.4 Hz), 5.13 (d, 1H, J ) 3.9 Hz), 5.06 (d, 1H, J
) 3.9 Hz), 4.72 (d, 1H, J ) 8.2 Hz), 4.66 (d, 1H, J ) 7.5 Hz), 4.56
(d, 1H, J ) 8.0 Hz), 4.53 (d, 1H, J ) 7.6 Hz), 4.48 (d, 1H, J ) 7.8
Hz), 4.45 (d, 1H, J ) 8.0 Hz), 4.42 (dd, 1H, J ) 12.7, 5.5 Hz), 4.26
(d, 1H, J ) 12.7, 6.4 Hz), 4.17 (d, 1H, J ) 3.1 Hz), 4.11 (t, 1H, J )
9.8 Hz), 4.04-3.50 (m, 41H), 3.37 (t, 1H, J ) 8.8 Hz), 2.08 (s, 3H),
2.06 (s, 3H), 1.21 (d, 3H, J ) 6.3 Hz), 1.20 (d, 3H, J ) 6.3 Hz); 13C
NMR (125 MHz, D2O) δ 173.1, 172.7, 131.7, 117.1, 101.5, 101.3,
N3 Major Antigen 1. To a solution of 3 (0.0015 g, 0.0011 mmol)
in MeOH (1 mL) was added PdCl2 (0.001 g, 0.006 mmol) at RT. The
mixture was stirred for 4 h. After removal of PdCl2 by passing it through
Lichroprep RP-18 pad, it was subjected to gel filtration to give 1 (0.0015
g, quantitative) as a 1:1 (R:â) mixture of anomers: mp 175 °C
(decomp); [R]23D ) -46.2° (c 0.15, MeOH); FTIR (neat) 3318, 1644,
1207, 1149, 1072 cm-1; 1H NMR (500 MHz, D2O) δ 5.25 (d, 1/2H, J
) 3.5 Hz), 5.13 (d, 1H, J ) 3.9 Hz), 5.06 (d, 1H, J ) 3.7 Hz), 4.72
(d, 1H, J ) 8.0 Hz), 4.69 (d, 1H, J ) 8.0 Hz), 4.67 (d, 1/2H, J ) 7.0
Hz), 4.53 (d, 1H, J ) 7.6 Hz), 4.48 (d, 1H, J ) 7.8 Hz), 4.45 (d, 1H,
J ) 7.9 Hz), 4.17 (d, 1H, J ) 2.9 Hz), 4.11 (d, 1H, J ) 9.6 Hz),
4.04-3.50 (m, 41 and 1/2H), 3.32 (t, 1/2H, J ) 10.0 Hz), 2.08 (s,
3H), 2.06 (s, 3H), 1.21 (d, 3H, J ) 6.3 Hz), 1.20 (d, 3H, J ) 6.3 Hz);
LRMS (EI) (m/e) calcd for C52H88N2O34Na [M + Na]+ 1387.5, found
1387.6.
Acknowledgment. This work was supported by the National
Institutes of Health (Grant No. CA28824). H.M.K. gratefully
acknowledges the U.S. Army for a Department of Defense
Breast Cancer Predoctoral/Graduate Fellowship (No. DAMD
17-97-1-7119). We thank Dr. George Sukenick for providing
mass spectral and NMR analyses (SKI Core Grant No. CA-
08748).
Supporting Information Available: Experimental proce-
dures for general methods for 11, 12, 14, 16, 19-22, 25-32,
48, and 54-58 and NMR spectra (1H for 1-4, 43, 45-48, 54-
58, 62, and 64-72, 13C for 3, 4, 43, 45-48, 54-58, 62, 64,
and 66-72, and HMQC for 4) (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
JA0022730