Total synthesis and proof of structure of a human breast tumor (globo-H) antigen

Article abstract of DOI:10.1021/ja962048b

The total synthesis of the Hakomori MBr1 antigen, heavily expressed on human breast tumors, is related. The construction involved the assembly of four glycals: (17 (twice), 18, 20, and 26) and an L-fucose derivative, 34. The sensitivity of the stereochemi

Full text of DOI:10.1021/ja962048b

11488
J. Am. Chem. Soc. 1996, 118, 11488-11500
Total Synthesis and Proof of Structure of a Human Breast
Tumor (Globo-H) Antigen
Tae Kyo Park,† In Jong Kim,† Shuanghua Hu,† Mark T. Bilodeau,†
John T. Randolph,† Ohyun Kwon,†,‡ and Samuel J. Danishefsky*,†,‡
Contribution from the Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer
Research, 1275 York AVenue, New York, New York 10021, and Department of Chemistry,
Columbia UniVersity, HaVemeyer Hall, New York, New York 10027
ReceiVed June 18, 1996X
Abstract: The total synthesis of the Hakomori MBr1 antigen, heavily expressed on human breast tumors, is related.
The construction involved the assembly of four glycals: (17 (twice), 18, 20, and 26) and an L-fucose derivative, 34.
The sensitivity of the stereochemistry of sulfonamido galactosylation by a terminal galactose ring as a function of
the state of protection status of its C4 alcohol was exploited in a key step. (See the formation of compound 51.)
The synthesis served to confirm the Hakomori assignment of structure, and paves the way for immunoconjugation.
(See compound 64.)
Background
In this regard, our attentions were drawn to a novel structure
which seems to be associated with human breast cancer. Traces
of the glycosphingolipid, 1 were isolated by Hakomori6,7 and
associates by processing human breast cancer cell line, MCF-
7. The structure was assigned by spectroscopic measurements
in combination with chemical and enzymatic degradative
mapping. Another advance which facilitated study of this breast
tumor antigen was its immunocharacterization via monoclonal
antibody MBr1 by Colnaghi and associates.8 This antibody had
been obtained from mice immunized with intact MCF-7 cell
lines. Thus, the isolation of 1 from these cell lines and its
binding to MBr1 were taken to implicate this glycolipid as a
breast tumor antigen.
One need hardly dwell on the advantages which might accrue
if the enormous powers of the human immune system could be
mobilized to do battle against cancer.1 It could be imagined
that such a recruitment would be helpful in combating the onset
of the formation of the cancerous cells, in attacking existing
areas of cellular transformation, and in resisting metastasis.2 This
concept has often been discussed in the context of futuristic
hopes and has been supported by some apparent tantalizing early
successes.3 However, at the present writing, no broadly based
clinical application of active immunity against cancer through
vaccination has been demonstrated.
Among the novel constellations, generated by transformed
cells are anomalous glycosidic ensembles.4 The degree to which
particular carbohydrate patterns are indeed specific to cancer
cells, or even to cancers of particular tissues or organs, remains
to be properly and fully sorted out through the techniques of
immunohistology. However, there is an emerging perception
that certain consensus patterns are, minimally, overexpressed
in transformed cells as glycoprotein or glycolipid conjugates.5
Whether such accumulation arises from the coincidental abun-
dance of particular glycosyl transferases, or whether it reflects
the purposeful biosynthesis of these anomalous constellations
to perform a function in the transformed cell, awaits clarification
and may indeed vary, on a case to case basis.
The degree of specificity of antigen 1 to breast tumors has
not been established. The criterion of isolation and character-
ization of 1 has, at this writing, only been met by the Hakomori
experiment using the transformed (MCF-7) mammary cells.7
By the criteria of immunohistology, as evidenced by binding
of whole tissue to MBr1, the antigen seems to be present, to a
lesser extent in normal mammary and other teratocarcinoma
cells. However, caution is appropriate in drawing such a
conclusion. As shown by our mapping studies at the end of
this paper, and by earlier, as well as continuing investigations
by Russo and co-workers,9,10 truncated versions of 1 are also
found to bind to MBr1. Hence, the criterion of immunohistol-
ogy, as based on binding of MBr1 to define the presence of 1
as a total structural entity in a particular tissue, is lacking in
rigor by the standards of chemistry.
† Sloan-Kettering Institute for Cancer Research.
‡ Columbia University.
X Abstract published in AdVance ACS Abstracts, October 15, 1996.
(1) Dranoff, G.; Jaffee, E.; Lazenby, A.; Golumbek, P.; Levitsky, H.;
Brose, K.; Jackson, V.; Hamada, H.; Pardoll, D.; Mulligan, R. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 3539. Tao. M.-H.; Levy, R. Nature 1993, 362,
755. Boon. T. Int. J. Cancer 1993, 54, 177. Livingston. P. O. Curr. Opin.
Immunol. 1992, 4, 2.
While such uncertainties remain to be resolved, the clear
association of compound 1 with mammary and other epithelial
cancers11 rendered it an important synthetic target. Aside from
the inherent challenge to the field of complex oligosaccharide
synthesis posed by this structure, there were additional incentives
(2) Feizi, T. Curr. Opin. Struct. Biol. 1993, 3, 701.
(3) Maclean, G. D.; Bowen-Yacyshyn, M. B.; Samuel, J.; Meikle, A.;
Stuart, G.; Nation, J.; Poppema, S.; Jerry, M.; Kaganty, R.; Wont, T.;
Longecker, B. M. J. Immunol. 1992, 11, 292. Fung, P. Y. S.; Made, M.;
Kogany, R. R.; Lonencer, B. M. Cancer Res. 1990, 50, 4308. Livingston,
P. O.; Ritter, G.; Srivastava, P.; Padavan, M.; Calves, M. J.; Oettgen, H.
F.; Old, L. J. Cancer Res. 1989, 49, 7045. Haraday, Y.; Sakatsume, M.;
Nores, G. A.; Hakomori, S.; Taniguchi, M. Jpn. J. Cancer Res. 1989, 80,
988.
(6) Kannagi, R.; Levery, S. B.; Ishijamik, F.; Hakomori, S.; Schevinsky,
L. H.; Knowles, B. B.; Solter, D. J. Biol. Chem. 1983, 258, 8934.
(7) Bremer, E. G.; Levery, S. B.; Sonnino, S.; Ghidoni, R.; Canevari,
S.; Kannagi, R.; Hakomori, S. J. Biol. Chem. 1984, 259, 14773.
(8) Menard, S.; Tagliabue, E.; Canevari, S.; Fossati, G.; Colnaghi, M. I.
Cancer Res. 1983, 43, 1295.
(4) Hakomori, S. Cancer Res. 1985, 45, 2405. Feizi, T. Cancer SurVeys
1985, 4, 245. Lloyd, K. O. Am. J. Clin. Pathol. 1987, 87, 129. Lloyd, K.
O. Cancer Biol. 1991, 2, 421.
(9) Lay, L.; Nicotra, F.; Panza, L.; Russo, G.; Adobati, E. HelV. Chim.
Acta 1994, 77, 509.
(10) Lay, L.; Panza, L.; Russo, G.; Colombo, D.; Ronchetti, F.; Adobati,
E.; Canevari, S. HelV. Chim. Acta 1995, 78, 533.
(5) Hakomori, S. Cancer Cells 1991, 3, 461.
S0002-7863(96)02048-3 CCC: $12.00 © 1996 American Chemical Society

Human Breast Tumor (Globo-H) Antigen
J. Am. Chem. Soc., Vol. 118, No. 46, 1996 11489
Scheme 1
Scheme 2
arising from the possibility of constructing a carbohydrate-based
antitumor vaccine. Conceivably, an immunofostering conjugate
of fully synthetic 1 might serve to stimulate antibody formation
in patients. Through such studies, the prospects for magnifying
active immunity against cancer could be evaluated experimen-
tally. Clearly, such an undertaking would require access to
substantial amounts of the carbohydrate domain in a form where
it could be connected to an immunotriggering domain. Scheme
1 provides the structure of 1 and the governing paradigm for
creating synthetically derived carbohydrate-based antitumor
vaccines.
3 would have to be distinguished as the unique acceptor site.
With hexasaccharide glycal 4 thus produced, provision for
introduction of the ceramide via its glycal linkage would be
available.16,17 Alternatively, the terminal glycal linkage of the
hexasaccharide could be exploited to lead, through a spacer
sector, to suitable immunoconjugates in keeping with the
biological goal of the program18 (see Scheme 2).
Keeping in mind the realities of the availability of carbohy-
drate building blocks, it seemed clear from the outset that the
DEF sector would be fashioned from the suitable melding of
two galactal units to provide eventually the Gal-Gal glycal 5,
which upon fucosylation at C2′ would lead to 2.
As to the ABC domain, we initially hoped to exploit the ready
accessibility of lactal 6.19 In the early planning, it was
conjectured that it could prove possible to differentiate the
hydroxyl groups of 6 such as to identify the unique axial
hydroxyl at C4′ as the acceptor site (see asterisk in structures 6
and 7). We further envisioned the generation of galactal (8)
wherein C3 would be distinguished via a unique blocking group
which would identify this center as the acceptor site.
Coupling of 7 with an R-galactosyl donor derived from 8
followed by deprotection of the unique P* blocking group would
lead to the previously discussed 3 for coupling with an aza-
glycal donor derived from 2 (Scheme 3).
In this paper, we describe (i) the total syntheses of the MBr1
antigen,12 (ii) a chemically rigorous proof of its structure, and
(iii) the establishment of a linker domain for purposes of
fashioning a functional vaccine.
Synthetic Planning
In studying structure 1 as to its amenability to chemical
synthesis on a decent scale, the interior network of four
galactose/galactosamine residues is quickly recognized.
A
principal retrosynthetic disconnection between ring C and D
seemed attractive. Hence, a DEF glycal (cf. structure type 2)
emerges as a subgoal. In a similar vein glycal type 3 can serve
as an ABC acceptor equivalent. System 2 would be converted
to an “azaglycosylation donor”13 upon suitable activation of its
glycal linkage. For instance, we hoped to apply our own
methodology wherein a 2â-iodo sulfonamide derived from a
type 2 (DEF) glycal would function as the donor for â-sulfon-
amidoglycosylation.14,15 Of course, the appropriate C3 oxygen
of the galactose residue at the nonreducing end of ABC glycal
It is with the building of an ABC sector corresponding to 3
that our account begins.
Several initiatives were undertaken to evaluate the possibility
of using lactal (6) as our starting material to fashion a usefully
differentiated AB system. In accord with previous reports, it
was a relatively straightforward matter to protect C6 and C6′ as
the di-TIPS derivative (see compound 9). As described in an
earlier finding, we could contain the 3′- and 4′-hydroxyl groups
in the form of a cyclic carbonate.20,21 The hydroxyl groups at
C3 and C2′ were thus exposed for silylation. Cleavage of the
(11) Perrone, F.; Menard, S.; Canevari, S.; Calobrese, M.; Borachi, P.;
Bufalino, R.; Testori, S.; Baldini, M.; Colnaghi, M. I. Eur. J. Cancer 1993,
297, 2113. Canevari, S.; Fossati, G.; Balsari, A.; Sonnino, S.; Colnaghi,
M. I. Cancer Res. 1983, 43, 1301. Hareuveni, M.; Gantier, C.; Kieny, M.-
P.; Wreschner, D.; Chambon, P.; Lather, R. Proc. Natl. Acad. Sci. U.S.A.
1990, 87, 9498.
(12) For a previous communication on the total synthesis, see: Bilodeau,
M. T.; Park, T. K.; Hu, S.; Randolph, J. T.; Danishefsky, S. J.; Livingston,
P. O.; Zhang, S. J. Am. Chem. Soc. 1995, 117, 7840. Dennis, J. Oxford
Glycosystems Glyconews Second, 1992.
(13) Griffith, D. A.; Danishefsky, S. J. J. Am. Chem. Soc. 1990, 112,
5811-5819. Griffith, D. A.; Danishefsky, S. J. J. Am. Chem. Soc. 1991,
113, 5863.
(14) Danishefsky, S. J.; Koseki, K.; Griffith, D. A.; Gervay, J.; Peterson,
J. M.; McDonald, F. E.; Oriyama, T. J. Am. Chem. Soc. 1992, 114, 8331.
(15) Griffith, D. A. Ph.D. Thesis, Yale University, 1992.
(16) Liu, K. K.-C.; Danishefsky, S. J. J. Am. Chem. Soc. 1993, 115,
4933.
(17) Gervay, J.; Peterson, J. M.; Oriyama, T.; Danishefsky, S. J. J. Org.
Chem. 1993, 58, 5465.
(18) Behar, V.; Danishefsky, S. J. Angew. Chem., Int. Ed. Engl. 1994,
33, 1468.
(19) Danishefsky, S. J.; Behar, V.; Randolph, J. T.; Lloyd, K. O. J. Am.
Chem. Soc. 1995, 117, 5701.
(20) Danishefsky, S. J.; McClure, K. F.; Randolph, J. T.; Ruggeri, R. B.
Science 1993, 260, 1307.

11490 J. Am. Chem. Soc., Vol. 118, No. 46, 1996
Park et al.
Scheme 3
Scheme 4a
a Reagents: (a) TBSCl, cat. DMAP, Et3N, DMF, 62%; (b) TIPSCl,
imidazole, DMF, 49%; (c) (n-Bu3Sn)2O, PhH, then BnBr, TBAI, 96%;
(d) NaH, BnBr, DMF, 35%; (e) Ac2O, cat. DMAP, Et3N, CH2Cl2, 56%;
(f) (i) 1,1′-carbonyldiimidazole, cat. DMAP, CH2Cl2, 67%; (ii) TBSCl,
imidazole, DMF, 83%; (g) NaOMe, MeOH, THF, 75%; (h) n-Bu2SnO,
PhH, then PMBCl, TBABr, 28% (with 35% of 11).
The C ring construct was fashioned from D-galactal. It was
possible through stannylation to cleanly introduce a p-methoxy-
benzyl group at C3 (see compound 25). At this stage, 2-fold
benzylation at the C4 and C6 centers gave rise to differentiated
galactal 26. The latter was converted to the â-anomeric fluoride
28 via its R epoxide 27.23 Benzylation of the C2 hydroxyl group
gave 29, which was to function as our R galactosylating agent
for introduction of ring C.
cyclic carbonate in 10 gave rise to 11. We attempted to
selectively protect C3′ with a durable blocking group. Unfor-
tunately, we could only obtain a 28% yield of the p-methoxy-
benzyl ether 12 with 35% recovered 11.
The stage was now set for the fateful coupling between
acceptor 24 and donor 29. Indeed, glycosylation was ac-
complished using Mukaiyama24-Nicolaou25 conditions, as
shown. This reaction gave rise to a 54% yield of 30, as well
as an 18% yield of its â anomer 31. Upon discharge of the
lone p-methoxybenzyl group in the C ring, formation of the
ABC acceptor with its differentiated hydroxyl acceptor site was
accomplished. (See compound 32 and asterisk.)
In another initiative, two tert-butyl dimethyl silyl groups were
introduced at C6 and C6′ of 6. A high-yielding monobenzylation
of compound 13 at C3′ was accomplished to afford 14 in high
yield (Scheme 4). The next preferred site of benzylation was
the C3 hydroxyl. However, the selectivity margins were not
acceptable. At best, a 35% yield of 15 was obtained. Further-
more, the selectivity margin for acetylation of C2′ and C4′ was
poor. We could obtain, at best, a 56% yield of 16. On the
basis of these and other early difficulties of a similar character,
we set aside the idea of using lactal (6) itself to fashion the AB
sector, recognizing full well that several unexplored opportuni-
ties to realize this goal remain open for future study.
Fortunately, another route presented itself. In this route we
would fashion our own lactal subunit corresponding to rings A
and B in formal construct 3. The advantage here would be that
in the assembly process we could already have made provision
for distinguishing the C4′ hydroxyl group corresponding to
structure type 7. The synthesis proceeded as follows. Galactal
was silylated at C6 to provide structure 17. Engagement of the
C3 and C4 hydroxyl groups, as previously described, provided
the cyclic carbonate 18. The R-epoxide 19, obtained by the
treatment of 18 with dimethyldioxirane, was to serve as the B
ring. (See Scheme 5.)
The A ring equivalent was obtained from the dibenzylation
of glucal, giving rise to the 3,6-dibenzyl compound 20.
Coupling of compounds 19 and 20 gave rise to A B intermediate
21. The next subgoal was that of identifying the C4′ hydroxyl
group of the B ring as the site of the R galactosylation. It was
possible to introduce a single benzyl group at C2′ in 21 in 81%
yield, to afford compound 22. Desilyation followed by cleavage
of the cyclic carbonate gave rise to 23. Using stannylidene
methodology,22 two benzyl groups could be introduced on C3′
and C6′, giving rise to compound 24 in which the only non
benzylated hydroxyl group was, in fact, at the axial hydroxyl
at C4′ destined to proVide the linkage site of the B and C rings.
For fashioning of the DEF glycosyl donor corresponding to
2, we began with a compound, described earlier, i.e., the TIPS
cyclic carbonate R epoxide, 19. This donor was, in turn, used
to glycosylate the previously described acceptor 17. No
differentiation of the C3 and C4 hydroxyl groups of 17 was
necessary in this coupling which occurred cleanly at C3, under
the influence of zinc chloride. Compound 33 was obtained in
87% yield.
The stage was now set to distinguish C2′ from C4 in a
fucosylation experiment. We hoped that fucosylation would
occur at the equatorial rather than the axial alcohol. In the event
this supposition proved to be correct. Using the previously
described fucosyl donor 34, under Mukaiyama24-Nicolaou25
conditions, there was obtained a 47% yield of compound 35
accompanied by approximately 8% of monofucosylated product
36 where the L-fucosyl residue had penetrated at C4 of the
galactose. For purposes of advancing on our goal, this
somewhat disappointing attrition in regioselectivity was accepted
in deference to the convenience of the route. Compound 35
was acetylated to provide 37 which was converted to its iodo
sulfonomide 38 in the usual way.14,15
The coupling of acceptor 32 and donor 38 was carefully
studied because it loomed as a key step in the synthesis. This
type of merger, though with much simpler substrates, had been
the cornerstone of our azaglycosidation methodology.13-15
Thus, we were surprised to find that under a variety of
conditions, the union of these two substances could not be
(23) Gordon, D. M.; Danishefsky, S. J. Carbohydr. Res. 1990, 206, 361.
(24) Mukaiyama, T.; Murai, Y.; Shoda, S. Chem. Lett. 1981, 431.
(25) Nicolaou, K. C.; Caulifield, T. J.; Kataoka, H.; Stylianides, N. A.
J. Am. Chem. Soc. 1990, 112, 3693. Nicolaou, K. C.; Hummel, C. W.;
Bockovich, N. J.; Wong. C.-H. J. Chem. Soc., Chem. Commun. 1991, 870.
(21) Danishefsky, S. J.; Gervay, J.; Peterson, J. M.; McDonald, F. E.;
Koseki, K.; Griffith, D. A.; Oriyama, T.; Marsden, S. J. Am. Chem. Soc.
1995, 117, 1940.
(22) David, S.; Hanessian, S. Tetrahetron 1985, 41, 663.

Human Breast Tumor (Globo-H) Antigen
J. Am. Chem. Soc., Vol. 118, No. 46, 1996 11491
Scheme 5a
a Reagents: (a) 1,1′-carbonyldiimidazole, cat. DMAP, CH2Cl2, quant.; (b) 3,3′-dimethyldioxirane, CH2Cl2; (c) (n-Bu3Sn)2O, TBABr, BnBr, PhH,
65%; (d) ZnCl2, THF; (e) BnBr, NaH, DMF; (f) TBAF, THF, then MeOH, MeONa, 93%; (g) (n-Bu3Sn)2O, Bu2SnO, PhH, then TBABr, BnBr,
90%; (h) n-Bu2SnO, TBABr, PMBCl, PhH, 70%; (i) TBAF, THF; (j) AgClO4, SnCl2, di-tert-butylpyridine, 4 Å molecular sieves, Et2O; (k) DDQ,
CH2Cl2, H2O, 86%.
Scheme 6a
further aggravated by what we have termed chiral mismatches,
i.e., situations where the match of particular asymmetric contours
of the donor and acceptor26 influence the feasibility of coupling
and its steric course. In a striking example, Spijker and van
Boeckel27 found out that the â-directing power of a C2 benzoyl
group was so overweighed by the chiral mismatch that it led to
a complete R glycosylation.
In our earlier investigations on sulfonamidoglycosylation,14
we had already explored a variation which might be effective
in otherwise difficult cases. This is shown in the transformation
of 40 + 41 f 42 (Scheme 7). In this two-step protocol, the
acceptor for the first azaglycosidation is a thioethyl function,
giving rise to a structure of the type 42. The latent donor
capacity of such a thioglycoside can be promoted through the
action of methyl triflate.28 In the cases previously studied,13,15
very high preferences for â-glycoside in the second had been
realized (cf. 42 + AH f 43).
In principle, it might have been argued that whether one starts
with 40 or 42, after suitable promotion the actual glycosylating
entity is the same N-sulfonylaziridine 44. However, this need
not necessarily be the case. A possibility deserving of
consideration is that when starting with donor type 42, the active
glycosylating agent is actually a form in which the oxygen atom
of the sulfonamide (rather than the nitrogen) had participated
leading to structure type 45 (status of NH undefined). Such a
a Reagents: (a) ZnCl2, THF, 87%; (b) AgClO4, SnCl2, di-tert-
butylpyridine, Et2O; (c) Ac2O, Py, cat. DMAP, 91%; (d) I(coll)2ClO4,
PhSO2NH2, 4 Å molecular sieves, THF, 82%; (e) 32, (n-Bu3Sn)2O,
PhH, then AgBF4, THF.
achieved in a reasonable way. At best, one could detect traces
of a presumed coupled product 39 by examination of NMR
spectra of crude reaction mixtures. (See Scheme 6.) However,
it did not prove feasible to obtain homogeneous product material
necessary to demonstrate even this level of attainment in a
rigorous way. This failure, in conjunction with other failures
in more complicated cases, demonstrated that the azaglycosi-
dation via “direct rollover” of iodo sulfonamides may not be
reliable with seriously hindered substrates which are encountered
in the context of coupling of oligomers. Difficulties can be
(26) For a striking example of matching effects in glycoside synthesis,
see: Halcomb, R. L.; Boyer, H. H.; Wittman, M. D.; Olson, S. H.; Denhart,
D. J.; Liu, K. K. C.; Danishefksy, S. J. J. Am. Chem. Soc. 1995, 117, 5720
and references cited therein.
(27) Spijker, N. M.; van Boeckel, C. A. A. Angew. Chem., Int. Ed. Engl.
1991, 30, 180.
(28) Lo¨nn, H. Carbohydr. Res. 1985, 134, 105. Lo¨nn, H. J. Carbohydr.
Chem. 1987, 6, 301.

11492 J. Am. Chem. Soc., Vol. 118, No. 46, 1996
Park et al.
Scheme 7
Scheme 9a
a Reagents: (a) I(coll)2ClO4, PhSO2NH2, 4 Å molecular sieves, THF,
50%; (b) 41 from EtSH, LHMDS, and DMF, 79%; (c) 32, MeOTf, 4
Å molecular sieves, Et2O-CH2Cl2 (2:1), 70-85% R-H (R-H:â-H )
10:1); (c) Ac2O, cat. DMAP, Et3N, CH2Cl2.
counterparts of 1,2 “cyclo” structures such as 44 or 45, which
would have assured â-galactoside formation.
It seemed possible that structural features in the terminal
galactose ring, or elsewhere in the DEF ensemble, were
encouraging onium ion formation and disfavoring the participat-
ing form (cf. 44 or 45) of the active glycosyl donor. We
naturally wondered whether the outcome favoring the R:â
preponderance of 5:1 could be materially altered by effecting
small changes in the nature of the donor ring. The area of the
donor entity which might be most readily probed experimentally,
without major restructuring of the synthesis, was the disposition
of the oxygen at C4 in the reducing terminus ring. In an earlier
intermediate, 35, this oxygen atom had appeared as an alcohol.
The possibility that the free hydroxyl group at C4 could be
maintained in the fashioning of donor 49 was explored.
In the event, it was feasible to effect iodosulfonimidation on
intermediate 35 to produce 49 (Scheme 9). This compound was,
in turn, converted to the 1â-ethanethiolate donor 50 bearing a
2R-phenylsulfonamido function by the rearrangement shown.
Coupling of 50 with 32 with methyl triflate gave rise to an 10:1
mixture of 51:52. Acetylation of these two compounds quickly
established that they did indeed correspond to the previously
encountered 39 and 47. Most remarkably, the major product
of the glycosidation Via 50, with the hydroxyl in free form, was
51 corresponding in stereochemistry to the minor product 39,
in the glycosidation of the C4 acetoxy bearing donor 46.
Similarly, the minor product 52, arising from 50, corresponded
to the major product (47) of the C4 acetoxy donor 46. Thus,
indeed, very substantial reversal of the directionality of the
glycosylation had been achieved by the seemingly minor device
of changing the character of the C4 in the donor from acetate
to alcohol. This trend has been verified in simpler substances.
It played a key role in our recently completed total synthesis of
asialo GM1.29
Scheme 8a
a Reagents: (a) 41 from EtSH, LHMDS, and DMF, 92%; (b) 32,
MeOTf, 4 Å molecular sieves, Et2O-CH2Cl2 (2:1), 60% â-H (â-H:
R-H ) 5:1).
system could well display different characteristics of stability,
and stereoselectivity patterns, relative to 44 in difficult glycosi-
dations.
We decided to explore implementation of the two-stage
protocol to the case at hand. The effort began on a favorable
note when compound 38 reacted with lithium ethanethiolate
under the specified conditions to afford compound 46 (Scheme
8). We now studied the all-critical glycosidation of acceptor
32 with donor 46. Indeed, under promotion by methyl triflate,
coupling occurred giving rise to a 5:1 mixture of glycosides.
Naturally, on the basis of previous experience, we assumed that
the major product was the â-glycoside 39, while the minor one
was assigned as 47. At this stage, NMR analysis was
complicated by interferences from the signals of the blocking
group based protons. Our assignment was based solely on
precedent and the perceived mechanistic logic of the reaction.
Following introduction of the ceramide residue and global
deprotection by schemes which will be described below, it was
discovered that the major product of this glycosidation was the
R-glycoside 47 while the minor one was actually the desired
39. Clearly, the C2-based sulfonamide had not provided
â-directionality guidance for the glycosidation reaction. We
took this breakdown to imply a failure in the participation effect
of the sulfonamide resulting in intervention of an onium type
specie (cf. 48) as the active form of the donor, rather than
We first demonstrate that our formulations of 39, 47, 51, and
52 are correct. Treatment of presumed compound 47 with 3,3-
dimethyldioxirane followed by coupling of the epoxide with
azidohydrin 53 under the influence of anhydrous zinc chloride
gave rise, after acetylation, to product 54 (Scheme 10). The
capacity to introduce truncated precursors of the ceramide side
chain in this way had been previously demonstrated in our
laboratory.16,17 Reduction of the azide linkage and palmitoyl-
ation of the resultant amine led to 55. Treatment of the latter
(29) Danishefsky, S. J.; Kwon, O. Unpublished data. See also: Berresi,
F.; Hindsgaul, O. J. Carbohydr. Chem. 1996, 14, 1043. For a review
containing related observations on galactosylation, see: Paulson, H. Angew.
Chem., Int. Ed. Engl. 1982, 21, 155.
(30) Three characteristic R anomeric protons were observed through
proton NMR: δ 5.19 (1H, d, J ) 3.8 Hz), 5.11 (1H, d, J ) 3.6 Hz), and
5.01 (1H, d, J ) 3.6 Hz). This fact shows that the C-D glycosidic linkage
is R.

Human Breast Tumor (Globo-H) Antigen
J. Am. Chem. Soc., Vol. 118, No. 46, 1996 11493
Scheme 10a
a Reagents: (a) (i) 3,3-dimethyldioxirane, 4 Å molecular sieves, CH2Cl2; (ii) 53, ZnCl2, THF, 46%; (b) Ac2O, DMAP, Et3N, CH2Cl2, 96%; (c)
Lindlar’s catalyst, H2, palmitic anhydride, EtOAc, 92%; (d) (i) TBAF, THF; (ii) NaOMe, MeOH; (e) (i) Na, NH3, THF; (ii) Ac2O, Et3N, DMAP,
DMF, THF; (f) NaOMe, MeOH, 68% (over five steps).
Scheme 11a
a Reagents: (a) (i) 3,3-dimethyldioxirane, 4 Å molecular sieves, CH2Cl2; (ii) 53, ZnCl2, THF, 53%; (b) Ac2O, DMAP, Et3N, CH2Cl2, 95%; (c)
Lindlar’s catalyst, H2, palmitic anhydride, EtOAc, 90%; (d) (i) TBAF, THF; (ii) NaOMe, MeOH, 94%; (e) (i) Na, NH3, THF; (ii) Ac2O, Et3N,
DMAP, CH2Cl2, 80%; (f) NaOMe, MeOH, quantitative.
product with TBAF and then with sodium methoxide-methanol
resulted in cleavage of the silyl and the two acetate protecting
groups, as well as the cyclic carbonate. The 12 benzyl and
sulfonamido protecting groups were discharged by reaction of
sodium and ammonia. Exhaustive acetylation followed by
deacetylation with sodium methoxide in methanol afforded
compound 56. This structure differed in its NMR spectrum in
several notable respects from the MBr1 antigen itself.4,30 On
the basis of these data, as well as by virtue of its mass spectrum,
we formulate it to be compound 56, i.e., the C-D glycoside
epimer of the desired compound 1.
In a similar vein, starting with compound 51, reaction with
3,3-dimethyldioxirane afforded the usual 1,2-epoxide derivative.
The latter, on reaction with acceptor 53, moderated by zinc
chloride, gave rise to a 53% yield of the adduct 57 (Scheme
11). This glycosidation was soon followed by reduction of the
azide linkage and palmitoylation of the resultant amine to afford
58. The remaining steps were much the same as those used in
the synthesis of 56. Thus starting with substrate 58, a cleavage
sequence consisting of all silyl groups (TBAF), acyl groups
(sodium methoxide-methanol), and benzyl groups (sodium and
ammonia) followed by peracetylation and per-deacetylation of
the oxygen-based acetates gave rise to the MBr1 antigen 1. The
compound thus synthesized in contrast with 56 binds very nicely
to the MBr1 antigen. Moreover, its structure assignment, on
purely chemical grounds, is in complete accord with its NMR,31
its infrared and mass spectra, and the corresponding spectra of
its precursor structures. The region of the spectrum of synthetic
1 reflecting anomeric resonances is identical with the corre-
sponding spectrum displayed in the Hakomori paper.7 The
structural proposal adVanced by Hakomori for the MBr1 antigen
has thus been fully supported and the total synthesis of the breast
tumor antigen has been accomplished.
It was well to determine the structural specificity involved
in binding of target congeners to this MBr1 antigen. The
(31) Only the chemical shifts and coupling constants of anomeric protons
were reported for the natural MBr-1 antigen (1) (see ref 7). Characterisitc
anomeric protons of MBr-1 antigen (1) were observed at 4.95 (1H, br s),
4.81 (1H, d, J ) 3.7 Hz), 4.48 (1H, d, J ) 8.4 Hz), 4.44 (1H, d, J ) 8.1
Hz), 4.25 (1H, d, J ) 7.6 Hz), 4.16 (1H, d, J ) 7.8 Hz) and is in complete
agreement with reported data.

11494 J. Am. Chem. Soc., Vol. 118, No. 46, 1996
Park et al.
Scheme 12a
a Reagents: (a) TBAF, THF, 94%; (b) (i) Na, NH3, THF; (ii) Ac2O, Et3N, DMAP, THF, DMF, 85%; (c) (i) 3,3-dimethyldioxirane, CH2Cl2; (ii)
allyl alcohol, 66% (+29% of R-manno isomer); (d) NaOMe, MeOH, quantitative.
blood group determinants.19 Thus, the peracetylated version
of glycal 51 (see steps a and b) was treated with dimethyldiox-
Table 1. Inhibition of MAb MBr1 Binding to an MCF-7 Cell Line
by Synthetic Antigens (59-64)
compd
IC50 (mM)a
compd
IC50 (mM)a
irane. The resulting epoxide was coupled with allyl alcohol.
Surprisingly, this sequence led to an approximately 2.5:1 ratio
of R:â epoxides. Deacylation through the action of sodium
methoxide completed the synthesis of the allyl glycoside 64.
As discussed earlier,18,19 oxidative cleavage of the double bond
of such allyl glycosides leads to glycolic aldehyde glycosides
which are eminently suitable for bioconjugations. Reports on
these investigations as well as the immunoperformances of our
synthetic constructs will be forthcoming in future disclosures.
59
60
61
>500b
26
62
63
64
27
200
16
10
a 50% inhibitory concentration. b IC50 not reached.
chemistry by which the targets had been synthesized followed
the principles described above and led to compounds 59-64
as reported earlier.32 The binding data with MBr1 antibody are
summarized in Table 1. These data signify the crucial nature
of the terminal fucose residue. In the absence of this residue,
the structure known otherwise as the SSEA-3 (stage specific
embryonic antigen-3, 59) antigen33 fails to bind to the MBr1
antibody at appropriate concentrations.
The data also indicate that the nature of the glycosidic bond
in rings B and C is not critical. Indeed they point to the fact
that rings A and B may be deleted entirely (see compound 60,
Scheme 12). Interestingly, pentacycle 61, in which only the A
ring of the MBr1 hexasaccharide epitope has been deleted, binds
MBr1 antibody even slightly more avidly than does the full
antigen.
Summary
The synthesis of compound 1 accomplished the goal of proof
of structure and provided the basis for orderly immuno-
conjugation. Moreover it served to underscore the power of
glycal assembly in reaching oligosaccharide ensembles. In
particular we cite the conciseness in reaching systems of the
type 32 and 35 using minimalist protection devices involving
systems derived from the readily obtainable galactal 17 and
glucal 20. We also underscore the remarkable reversal in the
stereochemistry of sulfonamidogalactosylation by fine tuning
of the status of the axial C4-based oxygen of the donor terminus.
Experimental Section
We close by describing how the glycal domain (see com-
pound 51) was converted to allyl glycoside 64, which is a major
target site structure for commencement of the process of
conjugation. The methodology we used here was similar to
that used in the synthesis of our truncated constructs,32 as well
as that used in the fashioning of the reducing end of the Lewis
General. All commercial materials were used without further
purifications unless otherwise noted. The following solvents were
distilled under positive pressure of dry nitrogen immediately before
use: THF from sodium benzophenone ketyl, ether from LiAlH4,
CH2Cl2, toluene and benzene from CaH2. All the reactions were
performed under N2 atmosphere. NMR (1H, 13C) spectra were recorded
on Bruker AMX-400 MHz, Bruker Avance DRX-500 MHz, referenced
to TMS (1H-NMR, δ 0.00) or CDCl3 (13C-NMR, δ 77.0) and CD3OD
(13C-NMR, δ 49.05) peaks unless otherwise stated. LB ) 1.0 Hz was
used before Fourier transformation for all of the 13C-NMR. IR spectra
were recorded with a Perkin-Elmer 1600 series-FTIR spectrometer, and
optical rotations were measured with a JASCO DIP-370 digital
(32) Kim, I. J.; Park, T. K.; Hu, S.; Abrampah, K.; Zhang, S.; Livingston,
P. O.; Danishefsky, S. J. J. Org. Chem. 1995, 60, 7716.
(33) Shevinsky, L. H.; Knowles, B. B.; Damjanov., I.; Solter, D. Cell
1982, 30, 697. Andrews, P. W.; Goodfellow, P. N.; Shevinsky, L. H.;
Bronson, D. L.; Knowles, B. B. Int. J. Cancer 1982, 29, 523. Nunomura,
S.; Ogawa, T. Tetrahedron Lett. 1988, 29, 5681. Park, T. K.; Kim, I. J.;
Danishefsky, S. J. Tetrahedron Lett. 1995, 36, 9089 (see also ref 6).

Human Breast Tumor (Globo-H) Antigen
J. Am. Chem. Soc., Vol. 118, No. 46, 1996 11495
polarimeter using 10 cm pathlength cell. Low- and high-resolution
mass spectral analyses were performed with a JEOL JMS-DX-303 HF
mass spectrometer. Analytical thin-layer chromatography was per-
formed on E. Merck silica gel 60 F254 plates (0.25 mm). Compounds
were visualized by dipping the plates in a cerium sulfate-ammonium
molybdate solution followed by heating. Flash column chromatography
was performed using the indicated solvent on E. Merck silica gel 60
(40-63 µm) or Sigma H-Type silica gel (10-40 µm) for normal phase
and EM Science Lichroprep RP-18 (15-25 µm) for reverse phase.
Melting points are obtained with Electrothermal melting point apparatus
(series no. 9100) and are uncorrected.
(2C), 128.12, 127.84, 127.73, 127.64, 127.57, 127.51, 102.28, 99.74,
78.99, 76.03, 74.64, 74.07, 73.24 (2C), 73.17, 72.64, 70.20, 69.10,
67.79, 62.15.
Synthesis of Disaccharide Acceptor 24. A mixture of triol glycal
23 (2.95 g, 5.1 mmol), Bu2SnO (1.33 g, 1.05 equiv), and (Bu3Sn)2O
(1.69 mL, 0.65 equiv) in dry C6H6 (50 mL) under N2 was refluxed for
5 h with azeotropic removal of water. The reaction mixture was cooled
below boiling and treated with BnBr (2.43 mL, 4.0 molar equiv) and
TBABr (3.29 g, 2.0 equiv). C6H6 (10 mL) was distilled off, and the
reaction mixture was refluxed for 16 h. The reaction mixture was
directly loaded on a silica column and eluted with 15-20% EtOAc-
hexanes to give 24 (3.48 g, 90%) as a clear oil: [R]23D -3.3° (c 0.87,
3,6-Di-O-benzyl-D-glucal (20). Triacetylglucal (from Aldrich) (30.7
g, 111 mmol) was dissolved in absolute methanol (300 mL), treated
with NaOMe (25 wt % in MeOH, 1.5 mL), and stirred for 5 h at room
temperature under N2. The reaction mixture was concentrated in Vacuo
and the residue purified by flash column chromatographed with 5-20%
MeOH in CH2Cl2 to give 16.6 g (100.7%) of white crystalline,
hygroscopic solid.
CHCl3); IR (CHCl3 film) 2867, 1652 1454, 1364, 1097, 736 cm-1
;
1H-NMR (400 MHz, CDCl3) δ 7.35-7.21 (25H, m), 6.45 (1H, d, J )
6.2 Hz), 4.88 (1H, dd, J ) 6.2, 3.9 Hz), 4.83 (1H, d, J ) 10.9 Hz),
4.69 (2H, apparent s), 4.68 (1H, d, J ) 10.9 Hz), 4.59 (2H, apparent
s), 4.55 (1H, d, J ) 7.8 Hz), 4.49 (2H, apparent s), 4.47 (2H, apparent
s), 4.29 (1H, dd, J ) 9.6, 5.8 Hz), 4.18 (1H, t, J ) 4.4 Hz), 4.13 (1H,
m), 3.99 (1H, br s), 3.85 (1H, dd, J ) 10.6, 6.4 Hz), 3.75-3.60 (4H,
m), 3.47-3.41 (2H, m); 13C-NMR (100 MHz, CDCl3) δ 144.43, 138.64,
138.42, 137.99, 137.84, 137.80, 128.40, 128.34, 128.26, 128.23, 128.18,
128.15, 127.82, 127.75, 127.69, 127.67, 127.65, 127.55, 127.51, 127.46,
127.3, 1102.56, 99.56, 80.57, 78.69, 75.72, 75.10, 73.57, 73.32, 73.13,
72.94, 72.28, 71.94, 70.12, 68.90, 67.85, 66.62; LRMS (NH3) 776 ([M
+ NH4]+, 100).
D-Glucal (8.0 g, 54.7 mmol) and (Bu3Sn)2O (30.7 mL, 1.1 molar
equiv) in dry C6H6 (150 mL) were refluxed for 20 h with Dean-Stark
trap. The reaction mixture was cooled below boiling temperature and
treated with BnBr (21 mL) and TBABr (35.3 g). The mixture was
refluxed for 17 h. The reaction mixture was cooled and concentrated,
and the residue was diluted with EtOAc (2 × 200 mL), washed with
H2O (3 × 300 mL) and brine (300 mL), dried over Na2SO4, filtered,
and concentrated to dryness. Careful column chromatography of crude
material with 15-20% EtOAc in hexanes gave 20 (11.68 g, 65%) as
3-O-(4-Methoxybenzyl)-D-galactal (25). A suspension of D-galactal
(3.70 g, 25.3 mmol) and Bu2SnO (6.30 g, 1.0 equiv) in dry C6H6 (150
mL) was heated to reflux for 2 h with azeotropic removal of water.
The reaction mixture was cooled and treated with PMBCl (3.80 mL,
1.1 equiv) and TBABr (9.10 g, 1.1 equiv) and refluxed for 4 h. The
reaction mixture was filtered through a silica column and eluted with
EtOAc-hexanes (4:1). Fractions containing product were concentrated,
and the residue was triturated in hexanes to give 4.50 g (70%) (generally
65-75%) of the product as white crystalline solid: mp (hexanes) 117-
118 °C; [R]23D -23.0° (c 1.1, CHCl3); IR (KBr) 3313 (br), 1645, 1513,
a colorless oil: [R]23 -25.0° (c 5.7, CHCl3); IR (CHCl3 film) 3432,
D
1
1646, 1453, 1234, 1096 cm-1; H-NMR (400 MHz, CDCl3) δ 7.34-
7.25 (m, 10H), 6.37 (1H, dd, J ) 6.1, 1.4 Hz), 4.82 (1H, dd, J ) 6.2,
2.3 Hz), 4.67 (1H, d, J ) 11.8 Hz), 4.61-4.53 (m, 3H), 4.08-4.05
(m, 1H), 3.98-3.94 (m, 2H), 3.81-3.75 (m, 2H), 2.63 (1H, d, J ) 3.1
Hz); 13C-NMR (100 MHz, CDCl3) δ 144.6, 138.3, 137.7, 128.4 (two
peaks), 127.7 (two peaks), 100.0, 76.9, 76.2, 73.6, 70.7, 69.1, 68.8;
HRMS calcd for C20H26NO4 ([M + NH4]+) 344.1862, found 344.1841.
1
1228, 1082, 821 cm-1; H-NMR (400 MHz, CDCl3) δ 7.28 (2H, d, J
Synthesis of Lactal Carbonate 22. 21 was prepared according to
the procedure described in ref 19. To a mixture of lactal carbonate 21
(2.70 g, 4.02 mmol) and BnBr (574 µL, 4.83 mmol) in DMF (30 mL)
was added 60% NaH at 0 °C. After being stirred at 0 °C for 5 min,
the reaction mixture was allowed to warm to room temperature and
stirred for 2 h. Then, it was poured into cold water (70 mL), diluted
with EtOAc (120 mL), washed with H2O (2 × 70 mL) and brine (70
mL), dried over Na2SO4, filtered, and concentrated to dryness. Flash
column chromatography of crude material with 10-12% EtOAc in
hexanes afforded 22 (2.479 g, 81%) as a colorless oil: 1H-NMR (400
MHz, CDCl3) δ 7.35-7.25 (15H, m), 6.41 (1H, d, J ) 6.0 Hz), 4.88
(1H, d, J ) 5.5 Hz), 4.86-4.84 (1H, m), 4.65-4.58 (4H, m), 4.57
(1H, d, J ) 12.2 Hz), 4.46 (1H, d, J ) 11.4 Hz), 4.17-4.13 (2H, m),
4.11-4.09 (1H, m), 3.89 (1H, dd, J ) 11.0, 4.7 Hz), 3.84 (1H, d, J )
5.1 Hz), 3.82 (1H, d, J ) 1.6 Hz), 3.74 (1H, dd, J ) 5.5, 1.7 Hz), 3.68
(1H, dd, J ) 11.0, 2.8 Hz), 3.58 (1H, dd, J ) 5.5, 4.7 Hz), 1.08-1.00
(21H, m); 13C-NMR (100 MHz, CDCl3) δ 154.06, 144.56, 138.55,
138.04, 137.01, 128.51, 128.37, 128.25, 128.13, 127.92, 127.77, 127.69,
127.52, 127.44, 100.28, 99.32, 77.06, 76.52, 75.91, 73.81, 73.73, 73.60,
73.49, 73.37, 71.23, 70.62, 67.93, 61.31, 17.89, 17.85, 11.76; LRMS
(NH3) 778 ([M + NH4]+, 100).
Synthesis of Lactal 23. A solution of TIPS carbonate lactal 22
(4.28 g, 5.62 mmol) in THF (25 mL)-MeOH (5 mL) was treated with
TBAF solution (1.0 M, 6.75 mL, 1.2 equiv). After 6 h, additional
TBAF (4 mL) was added, and the mixture was stirred an additional 3
h. The reaction mixture was concentrated and directly chromatographed
with 4:1 EtOAc-hexanes to obtain 2.20 g of the triol. Remaining
mixtures of cyclic carbonate and mixed carbonate were hydrolyzed in
MeOH with MeONa (25 wt % in MeOH, 1.0 mL) and purified
chromatographically. Total yield was 3.02 g (93%). This material
was directly used for the dibenzylation step: 1H-NMR (400 MHz,
CDCl3) δ 7.35-7.24 (15H, m), 6.43 (1H, d, J ) 6.3 Hz), 4.87 (1H,
dd, J ) 6.3, 3.4 Hz), 4.84 (1H, d, J ) 11.4 Hz), 4.63 (2H, apparent s),
4.61 (1H, d, J ) 11.4 Hz), 4.53-4.47 (3H, m), 4.19-4.16 (3H, m),
3.87-3.84 (2H, m), 3.78-3.66 (3H, m), 3.46 (2H, apparent d, J ) 4.6
Hz), 3.29 (1H, t, J ) 5.5 Hz), 3.08 (1H, br), 2.73 (2H, br); 13C-NMR
(100 MHz, CDCl3) δ 144.70, 138.41, 138.22, 137.83, 128.45, 128.33
) 8.4 Hz), 6.89 (2H, d, J ) 8.4 Hz), 6.44 (1H, dd, J ) 6.4, 1.6 Hz),
4.70 (1H, dt, J ) 6.3, 1.9 Hz), 4.59-4.52 (2H, AB q, J ) 11.4 Hz),
4.20-4.18 (1H, m), 4.09 (1H, m), 4.02-3.97 (1H, m), 3.90-3.82 (2H,
m), 3.81 (3H, s), 2.73 (1H, d, J ) 3.1 Hz, C4-OH), 2.54 (1H, dd, J )
8.2, 4.2 Hz, C6-OH); 13C-NMR (100 MHz, CDCl3) δ 159.46, 145.02,
142.05, 129.46, 113.95, 99.36, 76.12, 70.17, 70.14, 63.65, 62.74, 55.26;
LRMS (NH3) 284 [M + NH4]+, 266 [M]+, 249.
4,6-Di-O-benzyl-3-O-(4-methoxybenzyl)-D-galactal (26). A solu-
tion of 3-O-(4-methoxybenzyl)-D-galactal 25 (2.28 g, 8.56 mmol) and
BnBr (3.75 mL, 3.68 molar equiv; passed through basic alumina) in
DMF (30 mL) under N2 at 0 °C was treated with 60% NaH (1.37 g,
4.0 molar equiv) in two portions. The reaction mixture was stirred
0.5 h at 0 °C and 1 h at room temperature. The reaction mixture was
carefully poured into 50 g of crushed ice, diluted to 100 mL with water,
and then extracted with EtOAc-hexanes (1:1, 3 × 100 mL). Organic
extracts were washed with water (2 × 100 mL), dried over Na2SO4,
and concentrated to dryness. Flash column chromatography of crude
material with 15% EtOAc-hexanes gave 26 (3.58 g, 96%) as a clear
liquid: [R]23 -48.2° (c 0.85, CHCl3); IR (neat) 3030, 2867, 1645,
D
1
1613, 1513 1247, 1092, 821, 736 cm-1; H-NMR (400 MHz, CDCl3)
δ 7.34-7.23 (12H, m), 6.86 (2H, d, J ) 8.4 Hz), 6.35 (1H, d, J ) 6.4
Hz), 4.86 (1H, d, J ) 12.0 Hz), 4.84-4.81 (1H, m), 4.62 (1H, d, J )
12.0 Hz), 4.59-4.51 (2H, AB q, J ) 11.7 Hz), 4.50-4.39 (2H, AB q,
J ) 11.9 Hz), 4.15 (2H, m), 3.91 (1H, m), 3.78 (3H, s), 3.78-3.74
(1H, m), 3.63 (1H, dd, J ) 10.2, 5.0 Hz); 13C-NMR (100 MHz, CDCl3)
δ 159.04, 143.99, 138.30, 137.90, 130.43, 128.95, 128.26, 128.20,
128.03, 127.77, 127.57, 127.56, 113.67, 100.00, 75.58, 73.28, 73.17,
71.13, 70.42, 70.28, 68.35, 55.15; LRMS (NH3) 464 ([M + NH4]+,
100), 326 (18), 309 (48), 253 (17).
Synthesis of Fluoride 29. A solution of galactal 26 (3.20 g, 7.17
mmol) in dry CH2Cl2 (10 mL) under N2 at 0 °C was treated with
dimethyldioxirane (0.09 M, 80 mL), and the mixture was stirred until
all of the glycal was consumed (0.5-1 h; TLC 30% EtOAc in hexanes).
Most of the volatiles were removed at 0 °C with a stream of dry N2.
The residue was dissolved in dry THF (30 mL) under N2 at 0 °C, treated
with TBAF (36 mL, stored over molecular sieves), and then stirred at
ambient temperature for 20 h. The dark brown solution was filtered

11496 J. Am. Chem. Soc., Vol. 118, No. 46, 1996
Park et al.
through a pad of silica (∼4 cm depth) and washed with EtOAc (200
mL). The filtrate was washed with water (3 × 200 mL), dried (MgSO4),
and concentrated. The residue was redissolved in 30% EtOAc in
hexanes (50 mL) and filtered through a short silica column (10 cm
diameter × 4 cm height) and washed with the same solvent system (1
L). The filtrate was concentrated to give 2.59 g of fluorohydrin 28
with >90% purity. The residue was dissolved in dry DMF (30 mL)
under N2 at 0 °C, treated with benzyl bromide (958 µL, 1.5 equiv,
freshly filtered through basic alumina) and finally with NaH (322 mg,
60% dispersion, 1.5 equiv), and stirred for 30 min at 0 °C and 30 min
at room temperature. The reaction was quenched by pouring the
mixture into 100 g of ice, and the mixture was extracted with 1:1
EtOAc-hexanes (150 mL × 2). The organic extracts were washed
with water (150 mL × 2), dried (MgSO4), and concentrated in Vacuo.
Flash column chromatography of residue with 10% EtOAc in hexanes
gave 2.00 g (49%) (generally 35-50%) of the title compound as a
yellowish liquid: [R]23D +15.3° (c 0.85, CHCl3); IR (CHCl3 film) 2916,
1612, 1513, 1248, 1103, 1056, 734 cm-1; 1H-NMR (400 MHz, CDCl3)
δ 7.35-7.24 (17H, m), 6.84 (2H, d, J ) 8.4 Hz), 5.15 (1H, dd, J )
53.2, 7.0 Hz), 4.92 (1H, d, J ) 11.6 Hz), 4.84-4.74 (2H, AB q, J )
11.0 Hz), 4.68-4.61 (2H, AB q, J ) 11.4 Hz), 4.56 (1H, d, J ) 11.6
Hz), 4.48-38 (2H, AB q, J ) 11.8 Hz), 3.96-3.89 (1H, m), 3.86 (1H,
br s), 3.78 (3H, s), 3.65-3.56 (3H, m), 3.51 (1H, dd, J ) 9.8, 2.8 Hz);
13C-NMR (100 MHz, CDCl3) δ 159.22, 138.33, 138.11, 137.62, 130.16,
129.19, 128.40, 128.29, 128.21, 128.04 (2C), 127.90, 127.81, 127.69,
127.59, 113.77, 110.20 (d, J ) 214 Hz), 80.60 (d, J ) 11.3 Hz), 79.00
(d, J ) 20.5 Hz), 74.92, 74.52, 73.59 (d, J ) 5.0 Hz), 73.54, 72.99,
72.70, 68.34, 55.20; LRMS (NH3) 454 ([M + NH4]+, 100).
Synthesis of Trisaccharide 30. Lactal 24 (600 mg, 0.791 mmol,
1.0 equiv) and fluoro sugar 29 (679 mg, 1.5 equiv) were combined in
ether, concentrated, dried in vacuum for 2 h, and then treated with
di-tert-butylpyridine (177 µL, 1.0 equiv) in a glovebag and dissolved
in dry ether (8.5 mL) under a nitrogen atmosphere. In a separate 25
mL flask were placed 4 Å molecular sieves (2.0 g), and then these
were flame-dried under vacuum and cooled to room temperature.
Anhydrous silver perchlorate (163 mg, 1.0 equiv) and SnCl2 (150 mg,
1.0 equiv) were added to the molecular sieves in a glovebag, and the
system was flushed with N2. The salt mixture was placed in a water
bath, and the sugar solution was introduced via a double-tipped needle.
The reaction vessel was wrapped with aluminum foil and stirred for
48 h at room temperature. The reaction mixture was diluted with ether
and filtered through a pad of silica gel, rinsed with ether. The filtrate
(70 mL) was washed with dilute NaHCO3 solution (2 × 50 mL), dried
over Na2SO4, and concentrated to dryness. Flash column chromatog-
raphy with 20% EtOAc in hexanes gave a trisaccharides mixture. The
trisaccharide portion was rechromatographed with 2% ether in meth-
ylene chloride to give 561 mg (54%) of the desired R-product 30 and
183 mg (18%) of â-product 31.
139.07, 139.02, 138.67, 138.54, 138.45, 138.39, 138.00, 137.87, 130.77,
129.10, 128.36, 128.22, 128.17, 128.13, 128.07, 127.96, 127.83, 127.77,
127.70, 127.65, 127.59, 127.55, 127.45, 127.33, 127.25, 127.21, 113.63,
102.75, 102.46, 99.87, 82.09, 81.56, 79.85, 79.77, 76.02, 75.19, 74.85,
74.58, 74.23, 73.64, 73.41 (2C), 73.38, 73.08 (2C), 72.78, 72.48 (2C),
70.67, 69.68, 69.33, 68.68, 67.88, 55.16; HRMS (FAB) calcd for
C82H86O15Na (M + Na)+ 1333.5860, found 1333.5900.
Synthesis of Trisaccharide 32. A solution of PMB trisaccharide
30 (561 mg, 0.428 mmol) in CH2Cl2 (12 mL) at 0 °C was treated with
830 µL of water and DDQ (126 mg, 1.3 equiv) and stirred at 0 °C for
1 h. The reaction mixture was poured into saturated NaHCO3 solution
(50 mL), extracted with EtOAc (2 × 50 mL), washed with saturated
NaHCO3 solution (2 × 50 mL) and water (50 mL), dried over Na2SO4,
and concentrated in Vacuo. The crude material was purified with flash
column chromatography (20% EtOAc in hexanes) to give 437 mg (86%)
of the deprotected trisaccharide 32 as a colorless oil: [R]23D +45.6° (c
1.78, CHCl3); IR (CHCl3 film) 2866, 1648, 1496, 1453, 1365, 1248,
1097, 735 cm-1; 1H-NMR (400 MHz, CDCl3) δ 7.36-7.15 (40H, m),
6.43 (1H, d, J ) 6.2 Hz), 5.09 (1H, d, J ) 3.3 Hz), 4.85 (1H, dd, J )
6.2, 3.6 Hz), 4.83-4.65 (5H, m), 4.61-4.41 (9H, m), 4.29-4.08 (8H,
m), 4.02 (1H, d, J ) 2.6 Hz), 3.97 (1H, d, J ) 2.2 Hz), 3.93 (1H, t, J
) 8.4 Hz), 3.86-3.78 (2H, m), 3.67-3.61 (2H, m), 3.53 (1H, t, J )
9.0 Hz), 3.48-3.42 (1H, m), 3.39-3.36 (1H, m), 3.33 (1H, dd, J )
10.0, 2.7 Hz), 3.25 (1H, dd, J ) 8.5, 4.8 Hz); 13C-NMR (100 MHz,
CDCl3) δ 144.38, 138.78, 138.62, 138.47 (2C), 138.20, 138.00, 137.88
(2C), 128.31, 128.29, 128.23, 128.19, 128.16, 128.05, 127.88, 127.83,
127.62, 127.57, 127.49, 127.45, 127.43, 127.41, 127.37, 127.32, 127.23,
102.68, 99.89, 99.34, 80.82, 78.72, 77.49, 77.10, 75.88, 75.13, 75.03,
74.23, 73.62, 73.05, 73.01 (3C), 72.62, 72.19 (2C), 70.46, 69.66, 68.92,
67.85, 67.74, 67.54; HRMS calcd for C74H78O14Na [M + Na]+
1213.5290, found 1213.5270.
Synthesis of Disaccharide Glycal 33. TIPS galactal carbonate 18
(4.32 g, 3.14 mmol) was dissolved in CH2Cl2 (20 mL), and the mixture
was cooled to 0 °C. It was then treated with dimethyldioxirane (219
mL, ∼3.14 mmol) at 0 °C. The epoxidation was finished (to give 19)
within 20 min, and the reaction mixture was concentrated to dryness
by a stream of N2. The residue was dried azeotropically once with
C6H6 (20 mL) and further dried on high vacuum for 30 min at 0 °C. It
was then dissolved in THF (60 mL) and cooled to -78 °C. To the
above solution was added azeotropically dried TIPS galactal 17 (3.32
g, 10.95 mmol) in THF (20 mL) via cannula and ZnCl2 (26.3 mL, 1.0
M in ether). The reaction mixture was allowed to warm to room
temperature and stirred overnight. After treatment with saturated
NaHCO3 (40 mL), the reaction mixture was concentrated and extracted
with ether (500 mL). The combined organic phase was washed with
brine (300 mL), dried over MgSO4, and concentrated to dryness. The
crude product was purified by flash column chromatography (20%
EtOAc in hexanes) to give 6.20 g of 33 as a white foam (87%): [R]23
D
30: [R]23D +41.8° (c 1.8, CHCl3); IR (CHCl3 film) 2867, 1648, 1513,
1496, 1453, 1364, 1248, 1097, 735 cm-1; 1H-NMR (400 MHz, CDCl3)
δ 7.33-7.12 (42H, m), 6.83 (2H, d, J ) 8.4 Hz), 6.45 (1H, d, J ) 6.0
Hz), 5.03 (1H, d, J ) 2.3 Hz), 4.91-4.76 (6H, m), 4.68-4.40 (12H,
m), 4.23-3.97 (11H, m), 3.86-3.82 (1H, dd, J ) 10.7, 6.2 Hz), 3.76
(3H, s), 3.69-3.64 (2H, m), 3.53 (1H, t, J ) 8.7 Hz), 3.47-3.43 (1H,
m), 3.40-3.36 (1H, m), 3.34-3.31 (1H, dd, J ) 9.9, 2.8 Hz), 3.22
(1H, dd, J ) 8.3, 4.8 Hz); 13C-NMR (100 MHz, CDCl3) δ 158.93,
144.50, 138.98, 138.84, 138.78, 138.64, 138.58, 138.06, 138.02 (2C),
130.82, 129.04, 128.33, 128.24, 128.21, 128.15, 128.08, 128.05, 127.83,
127.81, 127.72, 127.64, 127.58, 127.55, 127.50, 127.44, 127.41, 127.36,
127.33, 127.31, 113.65, 103.02, 100.39, 100.01, 80.93, 78.93, 78.70,
76.53, 76.11, 75.14, 74.84, 74.79, 74.35, 73.91, 73.59, 73.36, 73.15,
73.10, 72.98, 72.15, 72.10, 71.99, 70.55, 69.25, 67.92 (2C), 67.69,
55.19; HRMS calcd for C82H86O15Na (M + Na)+ 1333.5860, found
1333.5890.
-31.4° (c 0.85, CHCl3); IR (CHCl3 film) 3434, 2942, 2866, 1802, 1648,
1
1463, 1383, 1239, 1113, 1032, 882, 787, 685 cm-1; H-NMR (400
MHz) δ 6.54 (1H, dd, J ) 6.4, 1.6 Hz), 4.85 (1H, dd, J ) 6.4, 2.0
Hz), 4.72-4.68 (2H, m), 4.65 (1H, d, J ) 7.2 Hz), 4.55 (1H, m), 4.08
(1H, dd, J ) 9.6, 5.6 Hz), 3.96-3.82 (6H, m), 3.33 (1H, d, J ) 3.2
Hz, OH), 3.27 (1H, d, J ) 2.8 Hz, OH), 1.16-1.04 (42H, m); 13C-
NMR (100 MHz, CDCl3) δ 154.45, 145.75, 99.27, 77.83, 76.59, 74.27,
72.04, 71.62, 70.86, 64.52, 62.57, 61.60, 17.84, 11.78, 11.77; HRMS
(FAB) calcd for C31H59O10Si2 647.3647, found 647.3671 (M + 1).
Synthesis of Trisaccharide Glycal 35. A mixture of disaccharide
33 (2.64 g, 4.08 mmol) and fucosyl fluoride 34 (1.64 g, 3.77 mmol)
was azeotroped with C6H6 three times (3 × 10 mL) and further dried
on high vacuum for 1 h. It was dissolved in THF (20 mL) and treated
with 2,6-di-tert-butylpyridine (2.16 g, 11.31 mmol). The above mixture
was added via cannula to a flask containing AgClO4 (1.56 g, 7.54
mmol), SnCl2 (1.43 g, 7.54 mmol), and 4 Å molecular sieves (4.0 g)
in THF (15 mL) at -40 °C. The reaction mixture was stirred for 30
min at -40 °C and then for 34 h at 5 °C. After treatment with saturated
NaHCO3 solution (40 mL) at 5 °C, the reaction mixture was extracted
with EtOAc (2 × 300 mL). The combined organic layer was washed
with brine (200 mL), dried over MgSO4, and concentrated to dryness.
The crude product was purified by flash column chromatography (17%
EtOAc in hexanes) to give the desired trisaccharide glycal 35 (1.93 g,
47%, based on fluorofucose) and regioisomer 36 (329 mg, 8%) with
500 mg of the recovered disaccharide 33.
31: [R]23D +6.8° (c 0.76, CHCl3); IR (CHCl3 film) 2863, 1648, 1513,
1
1453, 1363, 1247, 1100, 735 cm-1; H-NMR (400 MHz, CDCl3) δ
7.45 (2H, d, J ) 7.45 Hz), 7.34-7.13 (40H, m), 6.83 (2H, d, J ) 8.4
Hz), 6.43 (1H, d, J ) 6.0 Hz), 5.07 (1H, d, J ) 10.7 Hz), 5.00 (1H, d,
J ) 11.6 Hz), 4.98 (1H, d, J ) 7.6 Hz), 4.85 (1H, dd, J ) 6.1, 3.6
Hz), 4.72-4.40 (16H, m), 4.35 (2H, app s), 4.30 (1H, d, J ) 2.2 Hz),
4.27 (1H, m), 4.18 (1H, t, J ) 4.6 Hz), 4.14 (1H, m), 3.85-3.72 (4H,
m), 3.78 (3H, s), 3.65 (1H, dd, J ) 10.6, 3.3 Hz), 3.60-3.53 (2H, m),
3.49-3.43 (5H, m); 13C-NMR (100 MHz, CDCl3) δ 159.01, 144.29,

Human Breast Tumor (Globo-H) Antigen
J. Am. Chem. Soc., Vol. 118, No. 46, 1996 11497
35: [R]23 -53.2° (c 1.30, CHCl3); IR (CHCl3 film) 2941, 2866,
CDCl3) δ 7.91 (2H, d, J ) 7.2 Hz), 7.50 (1H, m), 7.41 (2H, m), 7.38-
7.26 (15H, m), 5.42 (1H, d, J ) 3.1 Hz), 5.10 (1H, d, J ) 3.2 Hz),
5.08 (1H, d, J ) 3.6 Hz), 5.03 (1H, d, J ) 6.3 Hz), 4.98 (1H, d, J )
11.5 Hz), 4.86-4.80 (3H, m), 4.74 (1H, d, J ) 11.8 Hz), 4.66 (2H,
m), 4.62 (2H, dd, J ) 19.0, 10.4 Hz), 4.14-4.07 (3H, m), 4.04-3.90
(3H, m), 3.85 (3H, m), 3.68 (2H, m), 3.60 (1H, m), 3.55-3.45 (2H,
m), 2.43 (2H, m), 2.10 (3H, s), 1.17 (3H, d, J ) 6.4 Hz), 1.09 (18H,
s), 1.08 (3H, s), 1.04 (18H, s), 1.02 (3H, s); 13C-NMR (100 MHz,
CDCl3) δ 169.85, 154.00, 141.55, 138.64, 138.43, 138.35, 132.38,
128.61, 128.47, 128.32, 128.25, 128.17, 127.90, 127.86, 127.57, 127.47,
127.36, 127.34, 101.16, 98.22, 83.37, 80.70, 79.09, 78.97, 77.42, 77.32,
77.00, 76.68, 76.34, 74.84, 73.82, 73.38, 72.96, 72.88, 72.72, 70.53,
68.98, 67.75, 62.77, 61.38, 56.22, 24.21, 20.84, 16.67, 14.34; HRMS
(FAB) calcd for C68H99O17NS2Si2Na 1344.5790 (M + Na)+, found
1344.5800.
D
1808, 1649, 1459, 1365, 1239, 1167, 1106, 1054, 882, 788, 738 cm-1
;
1H-NMR (400 MHz) δ 7.30 (15H, m), 6.35 (1H, dd, J ) 6.4, 1.6 Hz),
5.01 (1H, d, J ) 4.0 Hz), 4.98 (1H, d, J ) 11.6 Hz), 4.88 (1H, d, J )
4.8 Hz), 4.86 (1H, d, J ) 3.7 Hz), 4.83 (2H, d, J ) 9.6 Hz), 4.73 (1H,
d, J ) 11.4 Hz), 4.70 (1H, dd, J ) 3.6, 8.2 Hz), 4.65 (2H, d, J ) 11.7
Hz), 4.53 (1H, m), 4.43 (1H, m), 4.15-3.97 (5H, m), 3.92-3.82 (7H,
m), 3.67 (1H, m), 2.66 (1H, d, J ) 2.4 Hz), 1.13 (3H, d, J ) 6.8 Hz),
1.09-1.00 (42H, m); 13C-NMR (100 MHz, CDCl3) δ 154.00, 145.30,
138.49, 138.30, 138.19, 128.43, 128.29, 128.16, 128.11, 127.87, 127.53,
127.49, 127.27, 98.34, 97.93, 97.81, 78.90, 77.32, 77.03, 76.13, 75.30,
74.75, 74.31, 73.92, 73.22, 72.99, 72.13, 71.09, 67.33, 63.44, 61.70,
17.80, 11.78, 11.72; HRMS (FAB) calcd for C58H86O14Si2Na 1085.5450,
found 1085.5480.
Synthesis of Trisaccharide Glycal 37. Trisaccharide glycal 35
(3.10 g, 2.91 mmol) was dissolved in pyridine (10 mL), and acetic
anhydride (5 mL) and 4-(dimethylamino)pyridine (DMAP, 50 mg) were
added. After being stirred at room temperature for 1.5 h, the mixture
was concentrated in Vacuo and loaded directly on silica gel column.
Elution with 30% EtOAc in hexanes afforded the product (2.93 g, 91%)
Synthesis of Hexasaccharide 47 and 39. To a thoroughly dried
mixture of acceptor 32 (75 mg, 0.063 mmol) and acetyl thioglycoside
46 (176 mg, 2.0 equiv) was added freshly activated 4 Å molecular
sieves (450 mg), and the mixture was placed under nitrogen atmosphere.
The mixture was suspended in dry ether (4 mL), stirred for 10 min,
and then cooled to 0 °C. The mixture was treated with MeOTf (35.5
µL, 5.0 equiv) and stirred for 5 h at 0 °C, for 2 h while warming to
room temperature, and finally for 1 h at room temperature. The reaction
mixture was quenched with Et3N (1 mL), and the mixture was diluted
with ether, filtered through a pad of silica gel, and rinsed with ether.
The filtrate (100 mL) was washed with dilute NaHCO3 (100 mL), dried
(MgSO4), and concentrated in Vacuo. The crude products was purified
with HPLC (21% EtOAc-hexanes, 15 mL/min, 260 nm UV detection)
to give 94.1 mg of R-isomer 47 and ca. 1/5 of â-isomer 39 (judged by
HPLC).
as a colorless oil: [R]23 -54.7° (c 0.89, CHCl3); IR (CHCl3 film)
D
2941, 2865, 1817, 1747, 1652, 1455, 1367, 1232, 1105, 1053, 882,
788, 735, 695 cm-1; 1H-NMR (400 MHz, CDCl3) δ 7.41-7.23 (15H,
m), 6.34 (1H, d, J ) 6.4 Hz), 5.36 (1H, m), 4.99 (2H, m), 4.87-4.70
(6H, m), 4.68 (4H, m), 4.45 (1H, m), 4.07 (2H, m), 3.98 (1H, m),
3.91-3.71 (7H, m), 3.64 (1H, s), 2.09 (3H, s), 1.15-1.05 (42H, m);
13C-NMR (100 MHz, CDCl3) δ 170.07, 153.52, 145.26, 138.59, 138.39,
138.25, 128.49, 128.28, 128.27, 128.19, 127.53, 127.62, 127.57, 127.41,
98.81, 98.74, 98.26, 82.45, 78.95, 7.44, 77.19, 76.21, 75.73, 74.84,
74.81, 73.94, 73.22, 73.00, 71.43, 69.91, 67.27, 65.11, 62.12, 61.45,
20.83, 16.63; HRMS (FAB) calcd for C60H88O15Si2K 1143.5300 (M +
K)+, found 1143.5250.
47: [R]23 +10.9° (CHCl3, c 0.15); IR (CHCl3 film) 2940, 2865,
D
1
1813, 1746, 1651, 1454, 1366, 1095, 735 cm-1; H-NMR (400 MHz,
CDCl3) δ 7.71-7.69 (2H, m), 7.43-7.14 (58H, m), 6.44 (1H, d, J )
6.2 Hz), 5.48 (1H, d, J ) 2.4 Hz), 5.05 (2H, m), 4.95 (1H, d, J ) 11.5
Hz), 4.87-4.58 (21H, m), 4.44 (2H, AB q, J ) 12.2 Hz), 4.35-4.11
(7H, m), 4.10-4.01 (3H, m), 3.95-3.76 (11H, m), 3.72-3.61 (4H,
m), 3.57-3.24 (9H, m), 2.07 (3H, s), 1.08 (3H, d, J ) 6.4 Hz), 1.06-
0.96 (42H, m); 13C-NMR (100 MHz, CDCl3) δ 168.65, 153.44, 144.48,
140.70, 138.94, 138.71, 138.63, 138.53, 138.28, 138.23, 138.05, 138.04,
137.86, 132.67, 129.15, 128.48, 128.28, 128.24, 128.18, 128.11, 128.04,
127.97, 127.89, 127.80, 127.64, 127.60, 127.56, 127.48, 127.44, 127.39,
127.36, 127.33, 127.19, 126.91, 103.18, 102.09, 99.20, 98.56, 98.28,
97.05, 80.89, 78.86, 78.80, 77.37, 76.55, 75.98, 75.87, 75.79, 75.65,
75.44, 75.04, 74.99, 74.85, 74.45, 74.41, 74.02, 73.55, 73.47, 73.16,
73.11, 73.06, 72.82, 72.64, 72.57, 72.22, 71.65, 70.66, 70.18, 70.13,
69.19, 69.07, 68.41, 68.01, 67.87, 67.74, 60.93, 60.33, 53.71, 20.82,
17.98, 17.94, 17.91, 17.78, 16.64, 11.82, 11.79; HRMS (FAB) calcd
for C140H171NO31SSi2Na [M + Na]+ 2473.0990, found 2473.0990.
Synthesis of Iodo Sulfonamide 38. A mixture of trisaccharide
glycal 37 (2.53 g, 2.3 mmol) and PhSO2NH2 (2.16 g, 13.7 mmol) was
azeotroped with C6H6 and further dried on high vacuum for 1 h. It
was dissolved in THF (20 mL), and freshly activated 4 Å molecular
sieves (5.0 g) were added. I(sym-coll)2ClO4 was prepared by stirring
I2 (3.56 g, 13.7 mmol) with Ag(sym-coll)2ClO4 (6.18 g, 13.7 mmol) in
THF (20 mL) at room temperature until the disappearance of the brown
color of I2. It was then added via a thick cannula to the flask containing
glycal 37 and benzensulfonamide at 0 °C. The reaction mixture was
stirred at 0 °C overnight and quenched with saturated Na2S2O3 solution
(50 mL). After filtration and extraction with EtOAc (2 × 200 mL),
the combined organic layer was washed with saturated CuSO4 (100
mL) and brine (100 mL) and dried (MgSO4). Concentration and
purification by silica gel chromatography (20% EtOAc in hexanes)
afforded the iodo sulfonamide 38 (2.59 g, 82%): [R]23D -71.6° (c 1.58,
CHCl3); IR (CHCl3 film) 2942, 2866, 1816, 1747, 1454, 1364, 1347,
1232, 1164, 1097, 822, 790, 733, 688 cm-1 1H-NMR (400 MHz,
;
39: [R]23 +11.3° (CHCl3, c 0.31); IR (CHCl3 film) 2940, 2865,
D
1
1815, 1745, 1650, 1454, 1233, 1161, 1099, 735 cm-1; H-NMR (400
CDCl3) δ 7.96 (2H, d, J ) 7.6 Hz), 7.52 (1H, m), 7.45 (2H, m), 7.41-
7.20 (15H, m), 5.36 (1H, t, J ) 9.0 Hz), 5.04 (3H, m), 4.99 (1H, d, J
) 11.5 Hz), 4.89 (2H, m), 4.79 (2H, dd, J ) 14.4, 12.3 Hz), 4.73 (1H,
m), 4.67 (3H, m), 4.38 (1H, m), 4.15-4.05 (3H, m), 4.08-3.93 (5H,
m), 3.92-3.75 (5H, m), 1.99 (3H, s), 1.24 (3H, d, J ) 6.4 Hz), 1.15-
1.05 (42H, m); 13C-NMR (100 MHz, CDCl3) δ 169.65, 153.51, 141.29,
138.76, 138.60, 138.53, 132.69, 128.74, 128.49, 128.38, 128.18, 128.13,
127.96, 127.89, 127.46, 127.35, 127.28, 101.74, 96.82, 78.50, 77.39,
76.04, 75.81, 75.01, 74.41, 74.09, 73.27, 73.09, 72.33, 71.68, 68.00,
67.38, 60.57, 59.57, 20.87, 16.72; HRMS (FAB) calcd for C66H94O17-
INSSi2K 1426.4460 (M + K)+, found 1426.4420.
MHz, CDCl3) δ 7.82 (2H, m), 7.42-7.07 (58H, m), 6.41 (1H, d, J )
6.0 Hz), 5.30 (1H, d, J ) 2.8 Hz), 5.03 (1H, d, J ) 3.0 Hz), 5.00-
4.94 (3H, m), 4.90-4.71 (9H, m), 4.67-4.61 (3H, m), 4.56 (1H, d, J
) 12.2 Hz), 4.51-4.42 (8H, m), 4.36-4.29 (1H, m), 4.26 (1H, d, J )
12.1 Hz), 4.20 (2H, s), 4.14-3.96 (9H, m), 3.93-3.79 (9H, m), 3.69-
3.37 (10H, m), 3.33-3.23 (3H, m), 2.05 (3H, s), 1.18 (3H, d, J ) 6.5
Hz), 1.12-0.98 (42H, m).
Synthesis of Iodo Sulfonamide 49. A mixture of trisaccharide
glycal 35 (118 mg, 0.11 mmol) and benzenesulfonamide (87 mg, 0.55
mmol) was azeotroped with C6H6 once and further dried on high
vacuum for 1 h. It was dissolved in THF (5 mL), and freshly activated
4 Å molecular sieves (870 mg) was added. I(sym-coll)2ClO4 was
prepared by stirring I2 (57.5 mg, 0.22 mmol) with Ag(sym-coll)2ClO4
(100 mg, 0.22 mmol) in THF (1 mL) at room temperature until the
disappearance of the brown color of I2. It was then added via a thick
cannula to the flask containing glycal 35 and benzensulfonamide at 0
°C. The reaction mixture was stirred at 0 °C for 1 h and quenched
with saturated Na2S2O3 solution (10 mL). After filtration and extraction
with EtOAc (80 mL), the combined organic layer was washed with
saturated CuSO4 (20 mL) and brine (20 mL) and dried (MgSO4).
Concentration and purification by silica gel chromatography (20%
EtOAc in hexanes) afforded 74 mg (50%) of the iodo sulfonamide 49,
Synthesis of Ethylthio Sulfonamide 46. To a solution of EtSH
(550 mg, 8.85 mmol) in DMF (30 mL) was added lithium bis-
(trimethylsilyl)amide (LHMDS, 1.0 M in THF, 3.54 mL, 3.54 mmol)
at -42 °C. After being stirred for 5 min, it was then added via cannula
to a flask containing iodo sulfonamide 38 (2.46 g, 1.77 mmol) in DMF
(20 mL) at -42 °C. The reaction mixture was allowed to warm to
room temperature and stirred for total 3 h. After dilution with diethyl
ether (2 × 400 mL), it was washed with saturated NaHCO3 solution (2
× 200 mL) and brine (200 mL) and dried (MgSO4). Concentration
and purification by silica gel chromatography (20% EtOAc in hexanes)
afforded 46 (2.16 g, 92%) as a white solid (92%): [R]23 -23.8° (c
D
1.57, CHCl3); IR (CHCl3 film) 2941, 2865, 2359, 1809, 1745, 1456,
1363, 1325, 1232, 1159, 1092, 883, 689 cm-1; H-NMR (400 MHz,
which was unstable under a variety of conditions: [R]23 -47.0° (c
1
D

11498 J. Am. Chem. Soc., Vol. 118, No. 46, 1996
Park et al.
1
1.35, CHCl3); IR (CHC3 film) 3263, 2942, 2865, 1817, 1454, 1344,
1164, 1098, 882, 687 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.89 (2H,
d, J ) 7.2 Hz), 7.56 (1H, m), 7.48 (2H, m), 7.41-7.24 (15H, m), 5.62
(1H, d, J ) 7.8 Hz), 5.57 (1H, dd, J ) 7.8, 3.7 Hz), 4.98 (2H, m), 4.89
(2H, m), 4.87-4.72 (4H, m), 4.70-4.63 (4H, m), 4.20 (1H, m), 4.14-
4.03 (4H, m), 4.00-3.92 (4H, m), 3.90-3.75 (4H, m), 3.61 (2H, m),
3.19 (1H, dd, J ) 10.0, 5.9 Hz), 2.69 (1H, d, J ) 5.1 Hz), 1.20 (3H,
d, J ) 6.5 Hz), 1.05-1.01 (42H, m); 13C NMR (100 MHz, CDCl3) δ
154.07, 140.45, 138.71, 138.51, 133.07, 128.99, 128.56, 128.44, 128.24,
128.21, 127.98, 127.58, 127.34, 127.24, 97.79, 96.98, 83.72, 78.61,
77.43, 76.26, 75.03, 74.26, 74.15, 73.00, 72.93, 72.62, 70.95, 67.83,
66.36, 61.5, 60.42, 16.73; HRMS (FAB) calcd for C64H92O16INSSi2K
1384.4360 (M + K)+, found 1384.4320.
52: tR 17.7 min; H-NMR (400 MHz, CDCl3) δ 7.74 (2H, d, J )
7.2 Hz), 7.37-7.15 (58H, m), 6.45 (1H, d, J ) 6.4 Hz), 5.05 (1H, d,
J ) 3.2 Hz), 5.00 (1H, br s), 4.97 (1H, d, J ) 11.6 Hz), 4.89-4.42
(24H, m), 4.34 (1H, m), 4.28-3.59 (28H, m), 3.53 (1H, m), 3.45-
3.26 (5H, m), 1.11 (3H, d, J ) 6.5 Hz), 1.06-0.86 (42H, m).
Synthesis of Azide 54. A mixture of hexasaccharide 47 and freshly
activated 4 Å molecular sieves (200 mg) in CH2Cl2 (1 mL) was treated
with dimethyldioxirane (ca. 0.07 M, 1 mL) at 0 °C. After the
completion of reaction, most of the volatiles were removed by a stream
of N2. The residue was dried on high vacuum for 30 min. Then, a
solution of azidohydrin 53 in 1.3 mL of THF was added to the reaction
mixture by cannula. The reaction mixture was cooled to -30 °C,
treated with 1 M ZnCl2 in Et2O (53 µL), slowly allowed to warm to
room temperature, and stirred overnight. The reaction mixture was
diluted with EtOAc (70 mL), washed with saturated NaHCO3 solution
(2 × 200 mL) and brine, dried over Na2SO4, and concentrated in Vacuo.
The crude material was purified with HPLC (15% EtOAc-hexanes,
15 mL/min, 260 nm UV detection) to give 56 mg (46%) of the coupled
product as a colorless oil. To a solution of coupled product (56 mg,
0.019 mmol), DMAP (2.8 mg, 0.023 mmol), and Et3N (5.4 µL, 0.039
mmol) in CH2Cl2 (1.5 mL) was added Ac2O (2.2 µL, 0.023 mmol) at
0 °C. The reaction mixture was allowed to warm to room temperature
and stirred for 1 h. After the solvent was evaporated, the residue was
diluted with EtOAc (30 mL), washed with dilute NaHCO3 solution (2
× 5 mL) and brine, dried over Na2SO4, and concentrated in Vacuo.
Flash column chromatography (using 10-40 µm of silica gel, SIGMA)
of crude material gave 54.7 mg (96%, 42% over two steps) of the
desired product 54 as a colorless oil: [R]23D 11.6° (c 1.05, CHCl3); IR
(CHCl3 film) 3013, 2925, 2100, 1815, 1750, 1497, 1454, 1366, 1232,
Synthesis of Ethylthio Sulfonamide 50. To a solution of EtSH
(102 mg, 1.7 mmol) in DMF (10 mL) was added lithium bis-
(trimethylsilyl)amide (LHMDS, 1.0 M in THF, 0.81 mL, 0.81 mmol)
at -42 °C. After 5 min of stirring, it was transferred via a cannula to
a flask containing iodo sulfonamide 49 (448 mg, 0.33 mmol) in DMF
(10 mL) at -42 °C. The reaction mixture was allowed to warm to
room temperature and stirred for total 3 h. After dilution with diethyl
ether (200 mL), it was washed with saturated NaHCO3 solution (2 ×
10 mL) and brine (10 mL) and dried (MgSO4). Concentration and
purification by silica gel chromatography (5% EtOAc in CH2Cl2)
afforded 50 (338 mg, 79%) as a white solid: [R]23 -75.2° (c 0.99,
D
CHCl3); IR (CHCl3 film) 3447, 2942, 2866, 1795, 1455, 1382, 1367,
1325, 1161, 1107, 883, 744, 688 cm-1; 1H-NMR (400 MHz, CDCl3) δ
7.89 (2H, d, J ) 6.8 Hz), 7.50 (1H, m), 7.47 (2H, m), 7.40-7.25 (15H,
m), 5.42 (1H, m), 5.00 (1H, d, J ) 3.7 Hz), 4.97 (1H, d, J ) 11.6 Hz),
4.90 (1H, m), 4.83 (1H, d, J ) 11.8 Hz), 4.81 (1H, d, J ) 11.7 Hz),
4.73 (1H, d, J ) 11.8 Hz), 4.63 (4H, m), 4.35 (1H, d, J ) 10.2 Hz),
4.09 (4H, m), 4.01 (1H, dd, J ) 6.6, 13.1 Hz), 3.93-3.75 (6H, m),
3.70 (1H, d, J ) 1.7 Hz), 3.55 (1H, m), 3.46 (1H, m), 2.81 (1H, m),
2.45 (1H, m), 2.30 (1H, m), 1.16 (3H, d, J ) 6.44 Hz), 1.12-1.00
(42H, m); 13C-NMR (100 MHz, CDCl3) δ 155.26, 140.60, 138.66,
138.47, 132.55, 128.75, 128.49, 128.33, 128.24, 128.16, 127.93, 127.85,
127.65, 127.55, 127.48, 127.29, 99.40, 97.82, 83.60, 79.10, 77.49, 76.42,
74.86, 74.07, 72.92, 72.80, 71.48, 71.41, 70.25, 67.82, 67.76, 55.09,
23.51, 17.90, 17.90, 16.69, 14.37, 11.81; HRMS (FAB) calcd for
C66H97O16NS2Si2K 1318.5420 (M + K)+, found 1318.5470.
1092 cm-1; H-NMR (400 MHz, CDCl3) δ 7.68 (2H, br d, J ) 7.2
1
Hz), 7.42-7.14 (63H, m), 5.74-5.66 (1H, m), 5.43 (1H, d, J ) 2.0
Hz), 5.38 (1H, br dd, J ) 15.4 and 8.6 Hz), 5.11 (1H, br d, J ) 2.9
Hz), 4.94-4.89 (3H, m), 4.85-4.79 (3H, m), 4.75 (1H, d, J ) 5.7
Hz), 4.73 (1H, d, J ) 5.1 Hz), 4.70-4.66 (4H, m), 4.64-4.51 (8H,
m), 4.48-4.41 (5H, m), 4.35-4.30 (3H, m), 4.29-4.21 (3H, m), 4.16-
4.08 (3H, m), 4.02-3.90 (8H, m), 3.88-3.83 (2H, m), 3.80-3.73 (6H,
m), 3.68-3.64 (2H, m), 3.56-3.52 (4H, m), 3.47-3.25 (9H, m), 3.08
(1H, br s), 2.10-2.05 (5H, m), 1.83 (3H, s), 1.25 (22H, br s), 1.12-
1.03 (24H, m), 0.97 (21H, br s), 0.88 (3H, t, J ) 7.1 Hz); 13C-NMR
(100 MHz, CDCl3) δ 169.23, 168.56, 153.40, 140.57, 139.25, 139.17,
138.67, 138.55, 138.47, 138.38, 138.34, 138.30, 138.25, 138.15, 138.08,
137.99, 137.91, 132.36, 128.97, 128.52, 128.38, 128.31, 128.28, 128.27,
128.25, 128.22, 128.21, 128.19, 128.13, 128.08, 127.97, 127.93, 127.84,
127.76, 127.63, 127.56, 127.51, 127.49, 127.43, 127.42, 127.40, 127.35,
127.21, 127.12, 127.09, 127.08, 103.32, 102.12, 100.72, 98.95, 98.42,
97.97, 81.53, 80.65, 79.38, 79.24, 78.85, 77.62, 76.25, 75.98, 75.73,
75.42, 75.17, 74.85, 74.80, 74.16, 74.13, 73.51, 73.12, 73.08, 73.01,
72.90, 72.66, 72.45, 72.40, 72.32, 72.27, 70.38, 70.15, 70.11, 69.54,
68.73, 68.25, 68.05, 68.04, 68.03, 67.99, 67.88, 63.78, 61.21, 60.89,
53.58, 32.32, 31.88, 29.64, 29.63, 29.61, 29.59, 29.41, 29.31, 29.13,
29.00, 22.65, 20.85, 20.79, 18.02, 17.97, 17.93, 17.88, 16.64, 14.08,
11.83 (several peaks); HRMS (FAB) calcd for C167H214N4O35SSi2Na
[M + Na]+ 2946.4250, found 2946.4150.
Synthesis of Amide 55. A mixture of azide 54 (50 mg, 0.017
mmol), Lindlar’s catalyst (106 mg), and palmitic anhydride (25.4 mg,
0.051 mmol) in EtOAc (2.2 mL) was stirred at room temperature under
a H2 atmosphere for 18 h. The reaction mixture was filtered through
a pad of silica gel, rinsed with EtOAc (20 mL), and concentrated in
Vacuo. Flash column chromatography of crude material with 20-23%
EtOAc in hexanes provided 55 (32.7 mg, 92%) of amide as a colorless
oil: [R]23D +12.1° (c 0.98, CHCl3); IR (CHCl3 film) 3339, 2924, 2854,
1815, 1749, 1673, 1650, 1454, 1366, 1234, 1094, 734, 697 cm-1; 1H-
NMR (400 MHz, CDCl3) δ 7.67 (2H, d, J ) 7.2 Hz), 7.35-7.13 (63H,
m), 5.65 (1H, d, J ) 9.0 Hz), 5.63-5.57 (1H, m), 5.43 (1H, br s),
5.33-5.27 (1H, m), 5.11 (2H, t-like, J ) 4.0 Hz), 4.94 -4.90 (2H,
m), 4.88-4.79 (4H, m), 4.76-4.73 (2H, m), 4.71-4.64 (5H, m), 4.62-
4.58 (3H, m), 4.56-4.49 (5H, m), 4.44 (1H, dd, J ) 8.6 and 2.2 Hz),
4.40 (1H, d, J ) 7.0 Hz), 4.36-4.32 (2H, m), 4.30-4.11 (9H, m),
4.02-3.88 (7H, m), 3.81-3.65 (8H, m), 3.60-3.50 (4H, m), 3.45-
3.36 (4H, m), 3.34-3.23 (5H, m), 3.09 (1H, br s), 2.10-1.98 (4H, m),
2.04 (3H, s), 1.83 (3H, s), 1.51 (2H, m), 1.30-1.23 (49H, m), 1.10
-0.97 (42H, m), 0.88 (6H, t, J ) 6.9 Hz); 13C-NMR (100 MHz, CDCl3)
δ 172.41, 169.63, 168.58, 153.41, 140.58, 139.34, 139.21, 138.71,
Synthesis of Hexasaccharide 51 and 52. A mixture of acceptor
trisaccharide 32 (92 mg, 0.077 mmol, 1.0 equiv), thiogycoside 50 (198
mg, 2.0 equiv), and freshly activated 4 Å molecular sieves (560 mg)
under N2 at room temperature was suspended in CH2Cl2-Et2O (1:2,
3.9 mL) and stirred for 10 min. The reaction mixture was cooled to 0
°C and then treated with methyl triflate (52.4 µL, 6.0 equiv). The
reaction mixture was stirred for 4.5 h at 0 °C and for 1.5 h while
warming to 15 °C. The reaction was quenched with TEA (1.0 mL),
and the mixture was filtered through a pad of silica and rinsed with
Et2O. The filtrate (70 mL) was washed with saturated NaHCO3 solution
(2 × 50 mL), dried (Na2SO4), and concentrated to dryness. The crude
product was purified by HPLC (MICROSORB Semi-prep Si 80-120-
C5, 17% EtOAc in hexanes, 15 mL/min, 260 nm UV detection) to
give 158 mg (85%) of the desired product 51 and 27.7 mg of
corresponding R-isomer 52 (ca. 55% purity).
51: tR 22 min; [R]23D -13.3° (CHCl3, c 1.4); IR (CHCl3 film) 2940,
1
2865, 1792, 1652, 1454, 1161, 1101, 734 cm-1; H-NMR (400 MHz,
CDCl3) δ 7.8 (2H, m), 7.38-7.06 (58H, m), 6.43 (1H, d, J ) 6.1 Hz),
5.15 (1H, br s), 5.07 (1H, d, J ) 3.6 Hz), 5.03 (1H, d, J ) 3.6 Hz),
4.99 (1H, d, J ) 11.6 Hz), 4.89-4.61 (12H, m), 4.54-4.46 (4H, m),
4.42 (2H, app s), 4.38 (1H, d, J ) 11.9 Hz), 4.34-4.26 (3H, m), 4.21-
4.18 (4H, m), 4.13-4.03 (7H, m), 3.98-3.76 (14H, m), 3.70-3.61
(4H, m), 3.46-3.27 (7H, m), 2.84 (1H, OH), 1.16 (3H, d, J ) 6.4 Hz),
1.13-1.02 (42H, m); 13C-NMR (100 MHz, CDCl3) δ 155.35, 144.55,
140.78, 138.99, 138.75, 138.68, 138.57, 138.54, 138.43, 138.13, 138.03,
137.94, 137.82, 132.31, 128.81, 128.52, 128.51, 128.38, 128.36, 128.27,
128.24, 128.20, 128.16, 128.02, 127.93, 127.72, 127.66, 127.58, 127.48,
127.43, 127.37, 127.20, 103.41, 102.75, 99.79, 99.55, 98.29, 97.76,
80.49, 80.39, 79.03, 78.91, 78.25, 77.68, 77.37, 76.51, 75.88, 75.09,
74.99, 74.91, 74.73, 74.15, 74.02, 73.92, 73.52, 73.19, 73.10, 72.94,
72.67, 72.25, 72.07, 71.76, 71.56, 71.33, 70.33, 69.45, 69.32, 68.48,
68.08, 67.88, 67.86, 67.75, 61.97, 61.60, 56.14, 17.99, 17.96, 17.95,
17.92, 16.75, 11.86; HRMS (FAB) calcd for C138H169NO30SSi2Na (M
+ Na) 2431.0890, found 2431.0970.

Human Breast Tumor (Globo-H) Antigen
J. Am. Chem. Soc., Vol. 118, No. 46, 1996 11499
138.60, 138.56, 138.50, 138.41, 138.39, 138.26, 138.20, 138.12, 138.02,
136.88, 132.36, 128.99, 128.54, 128.39, 128.32, 128.27, 128.23, 128.20,
128.19, 128.15, 128.10, 127.99, 127.94, 127.90, 127.85, 127.77, 127.65,
127.60, 127.58, 127.52, 127.49, 127.43, 127.38, 127.29, 127.23, 127.16,
127.12, 127.09, 103.27, 102.23, 101.11, 99.03, 98.47, 98.17, 79.41,
79.31, 78.88, 77.46, 76.50, 76.37, 76.00, 75.85, 75.22, 74.88, 74.82,
74.32, 74.20, 74.15, 73.58, 73.13, 73.11, 73.03, 72.93, 72.76, 72.47,
72.34, 72.26, 70.38, 70.24, 69.58, 68.75, 68.35, 68.12, 68.04, 67.92,
67.71, 61.25, 60.93, 53.62, 53.38, 51.52, 36.81, 32.27, 31.90, 29.69,
29.52, 29.44, 29.34, 29.27, 25.67, 22.66, 20.91, 20.79, 18.03, 17.98,
17.95, 17.90, 16.66, 14.09, 11.85; HRMS (FAB) calcd for C183H246N2O36-
SSi2Na [M + Na]+ 3158.6640, found 3158.6740.
colorless oil. To a solution of the coupled product (31.1 mg, 0.0109
mmol), DMAP (1.6 mg, 0.013 mmol) and Et3N (3.0 µL, 0.0219 mmol)
in CH2Cl2 (1 mL) and Ac2O (1.2 µL, 0.013 mmol) were added at 0
°C. The reaction mixture was allowed to warm to room temperature
and stirred for 1 h. After the solvent was evaporated, the residue was
diluted with EtOAc (30 mL), washed with dilute NaHCO3 solution
(2x 5 mL) and brine, dried over Na2SO4, and concentrated in Vacuo.
Flash column chromatography (20% EtOAc-hexanes) of the crude
material gave 30.0 mg (95%) of the desired product 57 as a colorless
oil: [R]23D -9.2° (c 0.50, CH2Cl2); IR (CHCl3 film) 3344, 3030, 2924,
2864, 2101, 1789, 1754, 1496, 1453, 1366, 1232 cm-1; 1H-NMR (400
MHz, CDCl3) δ 7.75 (2H, d, J ) 7.2 Hz), 7.46-7.05 (63H, m), 5.75
(1H, dt, J ) 15.2, 6.8 Hz), 5.43 (1H, dd, J ) 15.5, 8.6 Hz), 5.13 (2H,
m), 5.09 (1H, d, J ) 3.6 Hz), 5.05 (1H, d, J ) 11.6 Hz), 5.00 (1H, d,
J ) 11.5 Hz), 4.94-4.86 (5H, m), 4.83-4.65 (14H, m), 4.59 (2H, d,
J ) 11.7 Hz), 4.53-4.43 (4H, m), 4.39-4.31 (4H, m), 4.23 (1H, d, J
) 11.9 Hz), 4.18 (1H, d, J ) 11.9 Hz), 4.15-4.08 (2H, m), 4.05-
3.57 (31H, m), 3.54 (1H, d, J ) 9.1 Hz), 3.49-3.45 (2H, m), 3.38
(1H, m), 3.31-3.23 (3H, m), 2.91 (2H, m), 2.75 (1H, dt, J ) 6.0 Hz),
2.12 (2H, dq, J ) 6.9 Hz), 1.85 (3H, s), 1.20-1.09 (42, m), 0.92 (3H,
t, J ) 6.6 Hz); 13C-NMR (100 MHz, CDCl3) δ 169.1, 165.9, 155.5,
140.9, 139.2, 139.0, 138.8, 138.64, 138.47, 138.43, 138.3, 138.2, 138.10,
138.07, 138.0, 132.1, 129.1, 128.69, 128.65, 128.56, 128.43, 128.36,
128.35, 128.26, 128.17, 128.12, 128.08, 127.97, 127.77, 127.66, 127.64,
127.60, 127.54, 127.49, 127.45, 127.41, 127.3, 126.0, 103.0, 102.7,
100.8, 99.7, 99.2, 98.0, 81.2, 80.6, 79.5, 79.2, 79.0, 78.3, 77.7, 76.8,
76.5, 75.5, 75.1, 75.03, 74.97, 74.91, 74.87, 74.0, 73.2, 73.10, 73.07,
72.98, 72.93, 72.6, 72.3, 72.1, 72.0, 71.32, 71.25, 70.2, 69.4, 69.32,
69.25, 68.1, 67.9, 67.5, 68.3, 62.1, 56.1, 32.4, 31.9, 29.71, 29.68, 29.66,
29.48, 29.2, 29.1, 22.7, 20.7, 18.13, 18.11, 18.01, 17.98, 16.9, 14.2,
11.9; LRMS (FAB) calcd for C165H212O34N4SSi2Na (M + Na)+
2904.4140, found 2904.
Synthesis of 56. A solution of amide 55 (82 mg, 0.026 mmol) in
dry THF (5 mL) was treated with TBAF (1.0 M in THF, 260 µL, 10
molar equiv) under N2 for 19 h at room temperature. Then, MeOH (3
mL) and NaOMe (25 wt % in MeOH, 200 µL) were added. After the
mixture was stirred for 1 h, 200 mg of Dowex 50-X8 was added, and
the reaction mixture was filtered and concentrated in Vacuo. The
residue was dissolved in EtOAc (70 mL), washed with water (3 × 50
mL) and brine (50 mL), dried over Na2SO4, and concentrated to dryness.
Flash column chromatography with 2-5% MeOH in CH2Cl2 gave 67
mg of the product. Na (87 mg) was added to liquid NH3 (ca. 8 mL)
at -78 °C, and the mixture was stirred for 2 min under N2 to form a
blue solution, into which was added the desilylated and decarbonated
substrate (67 mg) in dry THF (2 mL). The resulting mixture was stirred
for 45 min at -78 °C, and then the reaction was quenched with absolute
MeOH (5 mL) at -78 °C. The ammonia was removed by a stream of
N2, and more MeOH was added to a final ca. 10 mL of solution in the
reaction vessel. After neutralization with Dowex 50-X8 (810 mg,
dried), the mixture was then filtered, rinsed with NH3 in methanol (20
mL), and concentrated in Vacuo. The crude product (83 mg) was further
dissolved in DMF-THF (3 mL, 1:1), and DMAP (4 mg), Et3N (1.0
mL), and finally Ac2O (400 µL) were added. The mixture was stirred
for 24 h at room temperature and then diluted to 60 mL with EtOAc.
It was washed with water (2 × 50 mL) and saturated NaHCO3 solution
(2 × 50 mL), dried over Na2SO4, and concentrated in Vacuo.
Chromatography with 60% EtOAc-CH2Cl2 gave 41.2 mg (73%) of
the peracetated product. It was then treated with NaOMe (25 wt %,
102 µL) in MeOH (10 mL) for 15 h at room temperature and neutralized
with Dowex 50-X8. Filtration, concentration, and purification by
reverse phase chromatography (Lichroprep, 6 g, 20-0% H2O in MeOH)
gave 27.2 mg of 56 (68% over three steps) as a white cotton: mp 203
Synthesis of Amide 58. A mixture of azide 57 (66 mg, 0.023
mmol), Lindlar’s catalyst (66 mg), and palmitic anhydride (23 mg, 0.046
mmol) in EtOAc (1 mL) was stirred at room temperature under H2
atmosphere for 24 h. The reaction mixture was filtered through a pad
of silica gel, rinsed with EtOAc (20 mL), and concentrated in Vacuo.
The crude material was purified with HPLC (20% EtOAc in hexanes,
15 mL/min, 260 nm UV detection) to give 58 (64 mg, 90%) as a
colorless oil: [R]23 -17.9° (c 0.65, CH2Cl2); IR (CHCl3 film) 3531,
D
3346, 3063, 3030, 2924, 2854, 1790, 1674, 1496, 1454, 1365, 1236
cm-1; 1H-NMR (400 MHz, CDCl3) δ 7.72 (2H, d, J ) 7.2 Hz), 7.42-
7.02 (63H, m), 5.65 (1H, d, J ) 9.1 Hz), 5.62 (1H, dt, J ) 15.3, 6.6
Hz), 5.31 (1H, dd, J ) 15.3, 8.6 Hz), 5.10 (1H, m), 5.05 (1H, d, J )
3.6 Hz), 5.02 (1H, d, J ) 11.5 Hz), 4.96 (1H, d, J ) 11.4 Hz), 4.90-
4.62 (13H, m), 4.57-4.38 (8H, m), 4.32-4.26 (3H, m), 4.21-4.07
(9H, m), 4.01-3.41 (31H, m), 3.30 (1H, m), 3.23 (3H, m), 2.87 (2H,
m), 2.71 (1H, m), 2.07-1.97 (4H, m), 1.82 (3H, s), 1.52 (2H, m), 1.32-
1.19 (53H, m), 1.15-1.08 (42H, m), 0.88 (6H, t, J ) 6.8 Hz); 13C-
NMR (100 MHz, CDCl3) δ 169.06, 165.86, 155.54, 140.94, 139.23,
139.01, 138.64, 138.47, 138.10, 138.00, 132.00, 128.56, 128.43, 128.40,
128.36, 125.35, 128.26, 128.17, 128.12, 127.66, 127.64, 127.60, 127.54,
127.49, 127.45, 127.41, 126.04, 102.99, 102.71, 100.80, 99.72, 99.15,
97.97, 8.19, 80.60, 79.60, 79.21, 79.02, 78.47, 77.67, 77.40, 76.90,
76.49, 75.03, 74.97, 74.91, 74.87, 74.03, 73.19, 73.10, 73.07, 72.98,
72.80, 72.51, 72.03, 71.83, 71.7, 70.16, 69.20, 68.29, 68.09, 67.86,
67.06, 63.77, 62.11, 62.04, 56.05, 32.39, 31.94, 29.71, 29.68, 29.66,
29.48, 29.38, 29.20, 29.07, 22.71, 20.70, 16.89, 14.16; LRMS (FAB)
calcd for C181H244O35N2SSi2Na (M + Na)+ 3116.6530, found 3116.
Synthesis of 1. A solution of 58 (20 mg, 0.0065 mmol) in THF
(0.5 mL) was treated with TBAF solution (1.0 M in THF, 50 µL) and
stirred for 2 h. Then it was filtered through a pad of silica gel, washed
with EtOAc (30 mL), and concentrated in Vacuo. The crude material
was dissolved in MeOH (1 mL), treated with MeONa (10 mg), and
stirred for 3 h. The reaction was neutralized with Dowex 50-X8 (40
mg), filtered, washed with EtOAc (10 mL), and concentrated in Vacuo.
Flash column chromatography of crude material with 0-5% MeOH
in CH2Cl2 afforded the desired product (16.5 mg, 94%). To liquid
NH3 (ca. 8 mL) under N2 at -78 °C was added Na (12 mg), and the
mixture was stirred for 2 min. To the blue solution was added the
desilylated and decarbonated substrate (16 mg) in THF (1 mL), and
the mixture was stirred for 45 min at -78 °C. The reaction mixture
was quenched by addition of MeOH (4 mL). Ammonia was removed
°C dec; [R]23 +41.7° (c 1.09, DMSO); IR (KBr) 3396 (br), 2926,
D
2853, 1650, 1547, 1377, 1076, 820 cm-1; 1H-NMR (400 MHz, CD3OD)
δ 5.68 (1H, dt, J ) 15.2, 6.9 Hz), 5.44 (1H, dd, J ) 15.2 and 7.7 Hz),
5.19 (1H, d, J ) 3.8 Hz), 5.11 (1H, d, J ) 3.6 Hz), 5.01 (1H, d, J )
3.6 Hz), 4.59-4.58 (1H, m), 4.41-4.36 (2H, m), 4.30 (1H, d, J ) 7.8
Hz), 4.27-4.16 (5H, m), 4.13-4.08 (1H, m), 4.06-4.03 (2H, m), 3.99-
3.88 (5H, m), 3.83-3.80 (3H, m), 3.79-3.64 (11H, m), 3.59-3.51
(6H, m), 3.42-3.40 (1H, m), 3.31-3.25 (3H, m), 2.17 (2H, t, J ) 7.6
Hz), 2.01 (3H, s), 1.57 (2H, br s), 1.34-1.25 (51H), 0.90 (6H, t, J )
6.6 Hz); 13C-NMR (100 MHz, CD3OD) δ 175.99, 173.78, 135.13,
131.41, 105.45, 104.47, 103.90, 102.49, 101.54, 95.55, 80.88, 79.83,
79.49, 77.01, 76.72, 76.50, 76.28, 76.25, 75.29, 74.96, 74.72, 73.54,
73.05, 72.79, 72.59, 71.84, 71.66, 70.57, 70.36, 69.94, 68.98, 68.63,
67.49, 62.93, 62.68, 62.50, 61.77, 61.66, 54.74, 50.75, 49.90, 37.42,
33.51, 33.13, 30.92, 30.87, 30.84, 30.82, 30.80, 30.76, 30.68, 30.53,
30.51 (several peaks), 27.21, 26.70, 23.78, 23.07, 16.89, 14.51; HRMS
(FAB) calcd for C72H130N2O32Na [M + Na]+ 1557.8500, found
1557.8440.
Synthesis of Azide 57. A mixture of hexasaccharide glycal 51 and
freshly activated 4 Å molecular sieves (100 mg) in CH2Cl2 (1 mL)
was treated with dimethyldioxirane (ca. 0.07 M, 1 mL) at 0 °C. After
completion of the reaction, the volatiles were removed by a stream of
N2, and the residue was dried on high vacuum for 20 min. Then, a
solution of azidohydrin 53 in THF (0.8 mL) was added to the epoxide
via cannula and cooled to -40 °C. ZnCl2 (1 M) in Et2O (25 µL) was
added, and the reaction mixture was allowed to warm up to room
temperature and stirred for 12 h. It was then diluted with EtOAc (50
mL), washed with saturated NaHCO3 solution (2 × 20 mL) and brine
(10 mL), dried over Na2SO4, and concentrated to dryness. The crude
material was purified by flash column chromatography (10-22%
EtOAc in hexanes) to give 31.3 mg (53%) of coupled product as a

11500 J. Am. Chem. Soc., Vol. 118, No. 46, 1996
Park et al.
with a stream of N2, and then the solution was diluted with MeOH to
ca. 7 mL. The reaction mixture was neutralized with Dowex 50-X8
(115 mg), filtered, rinsed with NH3 in MeOH (20 mL), and concentrated
in Vacuo. A mixture of the residue and DMAP (3 mg) were dissolved
in DMF (1 mL), THF (1 mL), and Et3N (0.2 mL), then treated with
Ac2O (50 µL), and stirred for 3 h. After the reaction mixture was
concentrated, the residue was purified on 10-40 µm of silica gel using
10% hexanes in EtOAc to give the product (10.7 mg, 80%). A portion
of the peracetate (2.3 mg) dissolved in MeOH (0.5 mL) under N2 was
treated with MeONa in MeOH (25 wt %, 5 µL), and the mixture was
stirred for 4 h. The reaction mixture was neutralized with Dowex 50-
X8 (4.5 mg), filtered, washed with MeOH (5 mL), and concentrated
in Vacuo. The residue was purified with reverse phase column
chromatography (Lichroprep, MeOH) to give 1 (quantitative) as white
4.22-3.95 (13H, m), 3.90-3.77 (3H, m), 2.19-1.92 (51H, m), 1.15
(3H, d, J ) 6.4 Hz); 13C-NMR (100 MHz, CDCl3) δ 172.40, 171.45,
170.84, 170.54, 170.52, 170.48, 170.45, 170.40, 170.39, 170.34, 170.23,
169.99, 169.82, 169.74, 169.36, 169.00, 145.43, 102.01, 101.17, 98.83
(2C), 98.45, 94.24, 75.65, 74.95, 73.98, 73.64, 73.49, 72.32, 71.84,
71.53, 71.44, 70.81, 70.74, 70.66, 70.12, 69.77, 68.97, 68.71, 68.67,
68.02, 67.97, 67.88, 67.60, 67.35, 64.43, 61.88, 61.81, 61.42, 61.29,
61.04, 56.18, 23.06, 21.02, 20.81, 20.76, 20.68, 20.64, 20.62, 20.58,
20.57, 20.55, 20.49, 20.43, 15.88; LRMS (FAB), 1676 [M + Na]+
(100), 1634 (18), 1594 (35), 1404 (10), 849 (95); HRMS (FAB) calcd
for C70H95NO44Na [M + Na]+ 1676.5120, found 1676.5160.
Peracetyl hexasaccharide glycal (52 mg) was divided into two
portions (22 mg and 30 mg). A solution of hexasaccharide glycal (22.0
mg, 13.4 µmol) in dry CH2Cl2 (2 mL) under N2 at 0 °C was treated
with dimethyldioxirane solution (ca. 0.08 M, 500 µL) and stirred for
40 min at 0 °C. The reaction mixture was concentrated to ca. 100 µL
with a stream of dry N2 at 0 °C and then treated with allyl alcohol (5
mL). The mixture was stirred for 15 h at room temperature. Excess
allyl alcohol was removed in Vacuo. The other batch (30 mg) was
treated similarly. The crude products were combined and chromato-
graphed with 85% EtOAc in CH2Cl2 to give 35.8 mg (66%) of less
polar product (â-allyl gluco) and 15.7 mg (29%) of more polar product
(R-allyl manno). A 33.2 mg (19 µmol) portion of the less polar material
under N2 was dissolved in absolute MeOH (14 mL) and treated with
MeONa (25% in MeOH, 165 µL). After 6 h, the reaction mixture was
neutralized with Dowex 50-X8 (200 mg, washed and dried), filtered,
and concentrated to give quantitative yield of the title compound. An
analytical sample was prepared by RP column chromatography (Li-
chroprep, 6.0 g), eluting with water-5% methanolic water, followed
cotton: [R]23 +9.1° (c 0.45, MeOH); IR (KBr) 3356, 2920, 2851,
D
1734, 1652, 1547, 1466, 1372, 1260, 1075, 1042 cm-1; 1H-NMR (400
MHz, DMSO-d6/D2O, 10:1) δ 5.53 (1H, dt, J ) 15.3, 6.7 Hz), 5.35
(1H, dd, J ) 15.2, 7.0 Hz), 4.95 (1H, br s), 4.81 (1H, d, J ) 3.7 Hz),
4.48 (1H, d, J ) 8.4 Hz), 4.44 (1H, d, J ) 8.1 Hz), 4.25 (1H, d, J )
7.6 Hz), 4.16 (1H, d, J ) 7.8 Hz), 4.07 (2H, m), 3.99-3.85 (5H, m),
3.81-3.72 (6H, m), 3.68-3.35 (22H, m), 3.29 (2H, m), 3.03 (1H, t, J
) 7.9 Hz), 2.02 (2H, m), 1.93 (2H, m), 1.82 (3H, s), 1.44 (2H, m),
1.23 (46H, m), 1.08 (3H, d, J ) 6.4 Hz), 0.84 (6H, t, J ) 6.6 Hz);
13C-NMR (125 MHz, CD3OD) δ 176.02, 174.52, 135.21, 131.40,
105.55, 105.44, 104.44, 103.95, 102.86, 101.06, 81.18, 80.49, 80.05,
79.12, 77.97, 76.84, 76.57, 76.44, 76.27, 75.57, 74.96, 74.70, 73.59,
73.06, 72.61, 72.48, 71.56, 70.69, 70.66, 70.37, 69.99, 69.72, 69.73,
68.12, 62.69, 62.64, 61.77, 61.62, 54.89, 54.83, 53.14, 37.43, 33.55,
33.17, 30.96, 30.92, 30.89, 30.87, 30.85, 30.79, 30.73, 30.64, 30.59,
30.54, 30.49, 27.26, 23.83, 23.76, 16.76, 14.46; HRMS (FAB) calcd
for C72H130O32N2Na (M + Na)+ 1557.8500, found 1557.8450.
by lyophilization to obtain white powder: mp 204-206 °C dec; [R]23
D
+5.5° (MeOH, c 0.67); IR (MeOH film) 3356 (br), 2923, 1658, 1374,
1071 cm-1; 1H-NMR (400 MHz, CD3OD) δ 5.99-5.93 (1H, m), 5.35-
5.29 (1H, m), 5.24 (1H, d, J ) 3.8 Hz), 5.18-5.14 (1H, m), 4.93 (1H,
d, J ) 3.9 Hz), 4.56-4.54 (2H, m), 4.42-4.06 (10H, m), 3.99 (1H, s),
3.91-3.47 (26H, m), 3.41-3.37 (1H, m), 3.27 (1H, t, J ) 8.8 Hz),
2.01 (3H, s), 1.24 (3H, d, J ) 6.5 Hz); 13C-NMR (100 MHz, CD3OD,
ref ) δ 49.05) δ 174.55, 135.73, 117.57, 105.48, 105.42, 103.94,
103.26, 102.79, 101.08, 81.21, 80.67, 80.05, 79.20, 78.09, 76.79, 76.56,
76.48, 76.44, 76.41, 75.54, 74.86, 74.68, 73.57, 72.63, 72.50, 71.57,
71.16, 70.64, 70.41, 69.68, 68.16, 62.67, 62.64, 62.57, 61.96, 61.63,
53.11, 23.58, 16.78; LRMS (FAB), 613 (100); HRMS (FAB) calcd
for C41H69NO30Na [M + Na]+ 1078.3800, found 1078.3780.
Synthesis of Allyl Glycoside 64. A solution of hexasaccharide
glycal 51 (85 mg, 0.035 mmol) in THF (6 mL) under N2 at room
temperature was treated with TBAF (1.0 M in THF, 353 µL, 10 equiv).
After 38 h at room temperature, the reaction mixture was concentrated
to ca. 1 mL, then dissolved in EtOAc (60 mL), washed with H2O (2 ×
30 mL), dried (Na2SO4), and concentrated to dryness. Flash column
chromatography of the crude material with 4% MeOH in CH2Cl2 gave
70.0 mg (98%) of the desilyl decarbonated product. To liquid ammonia
(ca. 8 mL) under N2 at -78 °C was added sodium (95 mg). To the
formed blue solution was added a solution of the above desilyl
decarbonate compound (70 mg, 33.8 µmol) in dry THF (2 mL). After
45 min at -78 °C, the reaction was quenched with absolute methanol
(4 mL). Most of the ammonia was removed with a stream of nitrogen
(final volume was ca. 4 mL) and the reaction mixture diluted with
methanol to ca. 10 mL. To the solution was added Dowex 50-X8 (890
mg, washed and dried), and the mixture was stirred for 5 min. The
solution was filtered and rinsed with methanol and finally with NH3 in
methanol (5 mL), and the filtrate was concentrated in Vacuo. The
residue and DMAP (2.4 mg) were placed under N2, suspended in DMF
(1.0 mL), THF (1.0 mL), and Et3N (1.0 mL), and then treated with
Ac2O (0.3 mL). After 20 h (TLC analysis with EtOAc), the reaction
mixture was poured into water (40 mL), extracted with EtOAc (2 ×
40 mL), washed with dilute NaHCO3 solution (30 mL) and water (30
mL), dried over Na2SO4, and concentrated to dryness. Flash column
chromatography of the crude material with 80% EtOAc in CH2Cl2 gave
52.0 mg (93%) of the peracetylhexasaccharide glycal as a white foam.
Acknowledgment. This research was supported by the
National Institutes of Health (Grant No. CA 28824); NIH
National Research Service Award Postdoctoral Fellowships to
J.T.R. (GM15240) and M.T.B. (GM16291) are gratefully
acknowledged, as are Drs. P. O. Livingston, G. Ragupathi, and
S. Zhang (Laboratory for Tumor Vaccinology, SKI) for binding
studies, Dr. George Sukenick (Spectral Core Lab, SKI) for NMR
and mass spectral contributions, and The Synthesis Core Lab
(SKI) for preparative synthesis work. We gratefully acknowl-
edge Maria Colnaghi and Silvana Canevari for providing MAb
MBr1.
Peracetylhexasaccharide glycal: mp 132-134 °C; [R]23 +4.7°
D
(CHCl3, c 1.4); IR (CHCl3 film) 1742, 1652, 1371, 1227, 1069 cm-1
;
Supporting Information Available: Experimental proce-
1
dures and H NMR spectra for 9 f 16 and NMR spectra (1H,
1H-NMR (400 MHz, CDCl3) δ 6.68 (1H, d, J ) 6.8 Hz), 6.42 (1H, d,
J ) 6.0 Hz), 5.58 (1H, d, J ) 3.2 Hz), 5.47 (1H, d, J ) 3.4 Hz),
5.40-5.37 (2H, m), 5.29 (1H, dd, J ) 10.9, 3.1 Hz), 5.25-5.15 (5H,
m), 5.06 (1H, dd, J ) 11.2, 3.3 Hz), 5.02 (1H, d, J ) 3.6 Hz), 4.99-
4.92 (2H, m), 4.84-4.81 (2H, m), 4.67 (1H, d, J ) 7.8 Hz), 4.56-
4.51 (2H, m), 4.45-4.38 (3H, m), 4.29 (1H, dd, J ) 10.6, 3.4 Hz),
13C) for most compounds (1, 22, 23, 24, 28, 32, 35, 37, 38, 46,
47, 49-51, 55, 56, 58, and 64) (50 pages). See any current
masthead page for ordering and Internet access instructions.
JA962048B