Synthesis of selected LeY and KH-1 analogues: A medicinal chemistry approach to vaccine optimization

Article abstract of DOI:10.1021/jo048016l

(Chemical Equation Presented) As part of our ongoing anticancer vaccine program, we recently found that antibodies generated in response to the KH-1-KLH construct recognized not only KH-1 antigen but also the Lewis Y (Le ^y) antigen as well, with antibody titer levels much higher than those observed after immunization with individual Le^y-KLH vaccine constructs. In an attempt to explore the structure-antigenic relationship of these carbohydrate epitopes, several analogues of both KH-1 and Le^y were synthesized. A convergent synthetic approach to the analogues was designed on the basis of well-established glycal methodology, employing a minimum number of building blocks to generate competent antigens with high stereoselectivity and reasonable yield.

Full text of DOI:10.1021/jo048016l

Synthesis of Selected LeY and KH-1 Analogues: A Medicinal
Chemistry Approach to Vaccine Optimization
Maria K. Spassova,† William G. Bornmann,‡ Govindaswami Ragupathi,§ George Sukenick,|
Philip O. Livingston,§ and Samuel J. Danishefsky*,
Organic Synthesis Core Facility, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center,
New York, New York 10021, Department of Experimental Diagnostic Imaging,
M.D. Anderson Cancer Center, Houston, Texas 77030, Laboratory of Tumor Vaccinology,
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021,
NMR Core Facility, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center,
New York, New York, and The Laboratory for Bioorganic Chemistry,
Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York 10021
s-danishefsky@mskcc.org
Received November 8, 2004
As part of our ongoing anticancer vaccine program, we recently found that antibodies generated in
response to the KH-1-KLH construct recognized not only KH-1 antigen but also the Lewis Y (Ley)
antigen as well, with antibody titer levels much higher than those observed after immunization
with individual Ley-KLH vaccine constructs. In an attempt to explore the structure-antigenic
relationship of these carbohydrate epitopes, several analogues of both KH-1 and Ley were
synthesized. A convergent synthetic approach to the analogues was designed on the basis of well-
established glycal methodology, employing a minimum number of building blocks to generate
competent antigens with high stereoselectivity and reasonable yield.
Introduction
active and passive cancer immunotherapies still await
full development. The challenge is that the cancer host
immune system, unlike the case in other invasive dis-
eases, is unable to develop effective endogenous im-
munity. Tumor cells are seemingly able to evade immune
surveillance. Recent advances in the cellular and molec-
ular immunology of the complex interactions between the
immune system and tumor cells lead to a better under-
standing of these phenomena. Several mechanisms have
been advanced to explain these differences, including the
development of tumor variants lacking certain tumor
antigens,4 loss of MHC expression,5 downregulation of the
Lewis Y (Ley) and KH-1 are carbohydrate tumor-
associated antigens that are often overexpressed on the
surfaces of breast and prostate cancer cells as membrane
glycolipids.1 Like many other carbohydrate epitopes,
these antigens are of potential interest in the search for
an effective anticancer vaccine. Although research in the
field of tumor immunology was initiated about 100 years
ago through the pioneering work of Coley2 and Ehrlich,3
† Organic Synthesis Core Facility, Sloan-Kettering Institute.
‡ Department of Experimental Diagnostic Imaging, M.D. Anderson
Cancer Center.
§ Department of Medicine, Memorial Sloan-Kettering Cancer Center.
| NMR Core Facility, Sloan-Kettering Institute.
(3) Ehrlich, P. In The Collected Papers of Paul Ehrlich; Himmelweir,
Ed.; Pergamon Press: Elmsford, NY, 1956; Vol. II, p 148.
The Laboratory for Bioorganic Chemistry, Memorial Sloan-Ket-
tering Cancer Center.
(4) (a) Espinoza-Delgaro, I. Oncologist 2002, 7, 20. (b) Urban, J. L.;
Burton, R. C.; Holland, J. M. et al. J. Exp. Med. 1982, 155, 557. (c)
Uyttenhove, C.; Marianski, J.; Boon, T. J. Exp. Med. 1983, 157, 1040.
(d) Wortzel, R. D.; Filipps, C.; Schreiber, H. Nature 1983, 304, 165.
(5) (a) Hui, K.; Grosveld, F.; Festenstein, H. Nature 1984, 311, 750.
(b) Wallich, R.; Bulbuc N.; Hammerling, G. J. et al. Nature 1985, 315,
301. (c) Haywood, G. R.; McKhann, C. F. J. Exp. Med. 1971, 133, 1171.
(1) (a) Lloyd, K. O. Am. J. Clin. Pathol. 1987, 87, 129. (b) Lloyd, K.
O. Cancer Biol. 1991, 2, 421. (c) Nudelman, E.; Levery, S. B.; Kaizu,
T.; Hakomori, S. J. Biol. Chem. 1986, 261, 11247. (d) Hakomori, S.
Adv. Cancer Res. 1989, 52, 257. (e) Hakomori, S.; Zhang, Y. Chem.
Biol. 1997, 4, 97.
(2) Coley, W. Johns Hopkins Hosp. Bull. 1896, 7, 157.
10.1021/jo048016l CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/05/2005
J. Org. Chem. 2005, 70, 3383-3395
3383

Spassova et al.
FIGURE 1. Structures of Ley, KH-1, and proposed analogues.
antigen-processing mechanism,6 and expression of inhibi-
tory molecules, such as TGF-â, which may promote
escape from immune surveillance.7 However, despite
increasing amounts of information developed from recent
studies, the problem is still far from being fully under-
stood. For instance, the observation, in rare cases, of
spontaneous regression of human tumors remains un-
explained. A particularly problematic observation is that
in many cancer types there are no chemically defined
“tumor-specific” structures8 that cleanly differentiate
tumor immunity from immunity in other diseases, espe-
cially in the case of carbohydrate antigens. Extensive
studies with monoclonal antibodies have detected many
carbohydrate tumor-associated antigens.8 However, most
of them are self-antigens, i.e., antigens present in normal
cells or tissue, but overexpressed at the tumor cell
surface. In some instances, the antigens, although present
on the tumor cell surface and absent in progenitor cells,
are expressed in other normal tissues (e.g., di- or trimeric
LeX, and sialyl Lea in gastrointestinal cancer). Due to
altered glycosylation patterns during the oncogenic trans-
formation, the antigens are accumulated as precursors
with incomplete or truncated structures, neoglycolipids
(di- or trimeric Lex, trifucosyl Ley, sialyl Lea), or branched
structures.9
didates for anticancer vaccines. Most of the carbohydrate-
based antigens are, as a practical matter, not available
by isolation. Their presence is inferred from degradative
studies. Total syntheses of these epitopes had to be
developed to overcome this problem.10-14
Recent preclinical15 and clinical trials16 with mixtures
of individual KLH-conjugates and multivalent KLH-
conjugate vaccines with the same antigens revealed the
strong immunogenic properties of KH-1. Antibodies
generated in response to immunization with KH-1-KLH
constructs recognized not only KH-1 antigen but Ley as
well, and their antibody titers were found to be much
higher than those of Ley antibodies generated after
immunization with individual Ley-KLH vaccine con-
structs. The superiority of KH-1 relative to Ley as an
antigen is not surprising. Thus, compounds that are
endogenously expressed at high levels, such as Ley, are
typically less effective as antigens than are those which
are naturally present in only low levels, such as KH-1.
Having established the potency of KH-1 as an antigen
to induce Ley antibodies, we hoped to design and execute
the syntheses of a series of truncated analogues, each
containing the crucial Ley tetrasaccharide fragment in
the context of a more elaborate construct, which is not
normally encountered. In designing routes to fully syn-
Ley pentasaccharide (1) and KH-1 (2) are membrane
glycolipids that are expressed as ceramides. Both anti-
gens contain (Fuc1-2)Gal1-4(Fuc1-3)GlcNAc, a core
motif known as Ley tetrasaccharide (Figure 1, in blue).
In Ley pentasaccharide (1), the core structure is extended
with a Gal unit through a 1-3 glycosidic bond. In KH-1
(2), the tetrasaccharide core structure is attached to
3Gal1-4(Fuc1-3)GlcNAc1-3Gal1-4Glc, known as Lex
pentasaccharide. Ley, KH-1, and other carbohydrate
tumor associated antigens, such as Globo H, GM2 and
MUC1, have been extensively studied as potential can-
(10) Danishefsky, S. J.; Bilodeau M. T. Angew. Chem., Int. Ed Engl.
1996, 35, 1380.
(11) Danishefsky, S. J.; Behar, V.; Randolf, J. T.; Lloyd, K. O. J.
Am. Chem. Soc. 1995, 117, 5701.
(12) Deshpande, P. P.; Kim, H. M.; Zatorski, A.; Park, T. P.;
Ragupathi, G.; Livingston, P.; Live, D.; Danishefsky, S. J. J. Am. Chem.
Soc. 1998, 120, 1600.
(13) Allen, J. R.; Danishefsky, S. J. J. Am. Chem. Soc. 1999, 121,
10875.
(14) Boss, F.; Marcaurelle, L. A.; Seeberger, P. H. J. Org. Chem.
2002, 6659.
(15) Kudryashov, V.; Kim, H. A.; Ragupathi, G.; Danishefsky, S.;
Livingston, P. O.; Lloyd, K. O. Cancer Immunol. Immunother. 1997,
45, 281.
(16) (a) Sabbatini, P.; Kudryashov, V.; Ragupathi, G.; Danishefsky,
S. J.; Livingston, P. O.; Bornmann, W. G.; Spassova, M.; Spriggs, D.;
Aghajanian, C.; Soignet, S.; Peyton, M.; O’Flaherty, C.; Curtin, J.;
Lloyd, K. Int. J. Cancer 2000, 87, 79. (b) Wang Z.-G.; Wiliams, L. J.;
Zhang, X.-F.; Zatorski, A.; Kudryashov, V.; Ragupathi, G.; Spassova,
M.; Bornmann, W.; Slovin, S. F.; Scher, H. I.; Livingston, P. O.; Lloyd,
K. O.; Danishefsky, S. J. Proc. Natl Acad. Sci. U.S.A. 2000, 97, 2719.
(c) Ragupathi, G.; Cappello, S.; Yi, S. S.; Spassova, M.; Bornmann, W.;
Danishefsky, S. J.; Livingston, P. Vaccine 2002, 20, 1030. (d) Ragu-
pathi, G.; Koide, F.; Kagan, E.; Spassova, M.; Bornmann, W.; Gregor,
P.; Reis, C.; Clausen, H.; Danishefsky, S. J.; Livingston, P. Cancer
Immunol. Immunother. 2003, 52, 608.
(6) Restifo, N. P.; Esquivel, F.; Asher, A. L. et al. J. Immunol. 1991,
147, 1453.
(7) (a) Torre-Amione, G.; Beauchamp, R. D.; Koeppen, H. et al. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 1486. (b) Hahne, M.; Rimoldi, D.;
Schroter, M. et al. Science 1996, 274, 1363.
(8) Hakomori, S. In Gangliosides and Cancer; Oettgen, H. F., Ed.;
VCH: Weinheim, Germany, 1989; p 59.
(9) Hart, G., Marth, J., Eds. Varki, A. Glycosylation Changes in
Cancer. In Essentials of Glycobiology; Varki, A., Cummings, R. D., Esco,
J., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor,
NY, 1999; p 537.
3384 J. Org. Chem., Vol. 70, No. 9, 2005

Synthesis of Selected LeY and KH-1 Analogues
FIGURE 2. Proposed building blocks.
SCHEME 1. Synthesis of Ley Core Donors 6 and 7a
a Key: (a) 30% HBr/HOAc; (b) Zn/HOAc, two steps, 78%; (c) 4 M NH3/MeOH, 98%; (d) TBDPSCl, imidazole, -10 °C, DMF, 84%;
(e) CDI, imidazole, THF, 58%; (f) 10, Sn(OTf)2, 4 Å mol. sieves, 2,6-di-tert-butyl-4-methylpyridine, toluene, THF 60%; (g) PHSO2NH2,
I(sym-coll)2ClO4, 4 Å mol. sieves, CH2Cl2, 83%; (h) 1 M TBAF/THF, 89%; (i) BnBr, NaH, TBAI, DMF, 88%; (j) PHSO2NH2,
I(sym-coll)2ClO4, 4 Å mol. sieves, CH2Cl2, 87%.
thetic carbohydrate-based antigens, we are in a unique
position of being able to apply hard won capabilities in
oligosaccharide assembly, to bring to bear the mindset
of medicinal chemistry in optimizing antigen design.
Since KH-1 contains within it the Ley tetrasaccharide
motif (Figure 1, shown in blue), this substructure was
considered “conserved”. As the “variable” part of the
molecules (shown in red) we chose different oligosaccha-
ride linkers, attached to the core tetrasaccharide struc-
ture through a â-1,3-glycosidic bond: lactose (A-1, 3); a
tetrasaccharide consisting of acetyl lactosamine attached
to lactose (A-2, 4); and R-fucosylated lactose (B-1, 5). In
addition, we planned to synthesize KH-1 (2). The pres-
ence of both fucose and acetylaminosugars in oligosac-
charides are known to impart structural rigidity to the
molecular framework.8
Our synthetic strategy was to prepare a number of
common synthetic “building blocks” that could be com-
bined, as needed, to fashion the core target structures.
Following examination of the most complex target struc-
ture, KH-1 (2), three disconnection points present them-
selves. First, the conserved Ley tetrasaccharide could
serve as a donor in an azaglycosylation reaction (discon-
nection a). Second, the R-fucosyl fragment (shown in
green), required for the KH-1 and B-1 syntheses, could
be introduced through formation of an R-(1-3) glycosidic
bond at a differentiated hydroxyl group (disconnection
b). Finally, the tetrasaccharide, required for the KH-1
and A-2 syntheses, could be assembled by a second
Results and Discussion
As a part of this program, we developed an approach
(vide infra) to the synthesis of Ley and KH-1 analogues,
which might clarify the structure-antigenic relationship
of key carbohydrate epitopes. The concise preparation of
complex oligosaccharides in the laboratory still remains
a significant challenge for synthetic organic chemists. The
demands of regio- and stereoselectivity in glycosylation
reactions often lead to lengthy synthetic schemes and
extensive protecting group manipulations. Here we have
tried to take advantage of the existing synthetic methods
for the preparation of Ley pentasaccharide (1) and KH-1
nonasaccharide (2), and to design a convergent synthetic
approach to analogues based on well-established glycal
methodology,10 using a minimum number of building
blocks with high stereoselectivity and reasonable glyco-
sylation reaction yields. The main consideration in the
selection of analogues (Figure 1) was that of including
compounds with more rigid conformations, of the type
likely to enhance immunogenicity.8
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Spassova et al.
FIGURE 3. Screening of acceptors.
SCHEME 2. Preparation of the Acceptor
Fragmentsa
preferable to the known donor 6. With these consider-
ations in mind, we identified five common building blocks
which, when suitably combined, should allow for efficient
access to our four target molecules (Figure 2).
The synthesis of 6 from 11 has previously been
described (Scheme 1).10,12 Perbenzylated core donor 7 was
prepared from intermediate 12 in three steps through
simultaneous silyl and carbonate deprotection, followed
by benzylation of the resulting tetraol and, finally,
iodosulfonamidation.
With the goal of achieving maximum convergency in
the synthesis of the tetrasaccharide-containing analogues
(A-2 and KH-1), we explored the possibility of coupling
the Ley core donors, 6 and 7, with tetrasaccharide
acceptors. Unfortunately, when subjected to azaglycos-
ylation conditions, a sampling of preassembled tetrasac-
charides containing a variety of permutations of resident
protecting groups (Figure 3, 15 to 17) failed to efficiently
couple with either 6 or 7 (yields ∼10%). The coupling of
disaccharide 18 was similarly low-yielding. However, the
coupling of disaccharides 19 and 20 with the Ley core
donors did proceed in somewhat better yields (28% to
46%) and with high stereoselectivity. Hence, the idea of
coupling preassembled tetrasaccharide moieties with 6
and 7 in the construction of A-2 and KH-1 was abandoned
a Key: (a) (Bu3Sn)2O, C6H6.
azaglycosylation reaction with a disaccharide acceptor
(disconnection c). In this manner, the synthesis of each
analogue might be achieved in a convergent fashion
through the assembly of several readily synthesizable,
suitably protected building blocks.
One factor influencing the selection of protecting
groups was the need to install the R-fucosyl fragment in
the course of the KH-1 and B-1 syntheses. The disaccha-
ride component used in these cases would have to
incorporate a differentially protected alcohol that would
be unmasked prior to fucosylation. We planned to employ
the commonly used TES protecting group (cf. 9) to
achieve this end. In addition, since fucosylation would
occur at a late stage, the Ley core donor must be
appropriately protected so as to be stable to TES depro-
tection conditions. In this case, Ley donor 7 might be
SCHEME 3. Synthesis of A-1a
a Key: (a) AgBF4, THF, 4 Å mol. sieves, 28%; (b) 1 M TBAF/THF; (c) Na/NH3, THF; (d) Ac2O, TEA, DMAP, THF/DMF, 44% for three
steps; (e) DMDO, CH2Cl2, then allyl alcohol, 88% for two steps; (f) MeONa/MeOH, 90%.
3386 J. Org. Chem., Vol. 70, No. 9, 2005

Synthesis of Selected LeY and KH-1 Analogues
SCHEME 4. Alternate Synthesis of 22 (en Route to A-1)a
a Key: (a) AgBF4, THF, 4 Å mol. sieves, 42%; (b) Na/NH3, THF; (c) Ac2O, TEA, DMAP, THF/DMF, 81% for two steps.
SCHEME 5. Synthesis of A-2a
a Key: (a) Ac2O, TEA, DMAP, CH2Cl2, 77%; (b) AgBF4, THF, 4 Å mol. sieves, 65%; (c) Na/NH3; (d) Ac2O, TEA, DMAP, THF/DMF, 80%;
(e) DMDO, CH2Cl2; (f) allyl alcohol, 90% for two steps; (g) MeONa/MeOH, 89%.
SCHEME 6. Alternate Synthesis of 29 (en Route to A-2)a
a Key: (a) Ac2O, TEA, DMAP, CH2Cl2; (b) C6H5SO2NH2, I(sym-coll)2ClO4, CH2Cl2, 56% for two steps; (c) AgBF4, THF, 4 Å mol. sieves,
52%; (d) 1 M TBAF/THF; (e) Na/NH3, then Ac2O, TEA, DMAP, THF/DMF, 60% for two steps.
J. Org. Chem, Vol. 70, No. 9, 2005 3387

Spassova et al.
SCHEME 7. Synthesis of B-1a
a Key: (a) AgBF4, THF, 4 Å mol. sieves, 46%; (b) Ac2O, TEA, DMAP, CH2Cl2, 80%; (c) 1 M TBAF/THF: CH3COOH (1:1), 97%; (d)
di-tert-butyl pyridine, Sn(OTf)2, THF, 48%; (e) Na/NH3, then Ac2O, TEA, DMAP, THF/DMF, 67%; (f) DMDO, CH2Cl2; (g) allyl alcohol,
92% for two steps; (h) MeONa/MeOH, 90%.
SCHEME 8. KH-1 Synthesisa
a Key: (a) C6H5SO2NH2, I(sym-coll)2ClO4, CH2Cl2, (b) AgBF4, THF, 4 Å mol. sieves, 32% for two steps.
in favor of stepwise addition of disaccharide lactal
acceptors to the core structures.
The disaccharide acceptor fragments (8 and 9) were
prepared through the conversion of the known disaccha-
rides 19 and 20 to their corresponding tributylstannyl
ethers (Scheme 2).
With the donor and acceptor fragments in hand, we
set out to assemble our target molecules. We well
recognized that the azaglycosylation steps would be
critical to the syntheses, and an optimized protocol was
used in all cases. The coupling of the stannylated
acceptors and the donors was carried out in an acceptor-
to-donor ratio of 3 to 3.5:1 in the presence of silver
tetrafluoroborate at -70 °C. Following slow warming to
room temperature, the reaction was allowed to proceed
for 3 days at rt. As discussed below, we found the yield
of the coupling step to be highly dependent on the nature
of the protecting groups on the Ley core donor fragment.
In the event, the synthesis of A-1 commenced with the
coupling of donor 6 and acceptor 8. This reaction pro-
ceeded in only 28% yield to afford 21, which was
subsequently converted to hexasaccharide 24 through a
series of straightforward manipulations (Scheme 3). By
contrast, the coupling of the same acceptor with the
perbenzylated donor (7) gave rise to hexasaccharide 25
in 42% yield (Scheme 4). The latter was readily converted
to the acetylated hexasaccharide 22.
Interestingly, the impact of the nature of the seemingly
remote Ley tetrasaccharide protecting group pattern was
also pertinent to the success of the coupling reactions,
as evidenced in the elaboration of the A-1 framework to
produce A-2. Thus, hexasaccharide 25, which incorpo-
3388 J. Org. Chem., Vol. 70, No. 9, 2005

Synthesis of Selected LeY and KH-1 Analogues
rated the perbenzylated Ley core, was converted to 27.
The latter was subjected to azaglycosylation with accep-
tor 8 to afford 28 in 65% yield. The latter was readily
converted to 31 (Scheme 5). By comparison, the analogous
coupling of intermediate 32 with the same acceptor gave
rise to 33, albeit in only 52% yield (Scheme 6). The latter
was transformed to the acetylated intermediate, 29.
Although far from the glycosylation donor site, it is
evident that the presence of the deactivating silyl and
carbonate groups of the Ley tetrasaccharide decreases the
efficiency of the glycosylation reaction.
It is also of note that the yields of the second set of
glycosylations (65% for 27 f 28 and 52% for 32 f 33)
were markedly improved over the first (42% for 7 f 25
and 28% for 6 f 21). This difference can perhaps be
attributed to steric effects, with the reaction efficiency
increasing when the anomeric center is further removed
from the hindered tetrasaccharide core structure.
The synthesis of the B-1 analogue commenced with the
coupling of 7 and 9 to afford 34. The latter was advanced
to provide 36 in two steps (Scheme 7). The latter was
treated with â-tribenzylfucosyl fluoride (10) in the pres-
ence of di-tert-butyl pyridine to generate 37 through the
formation of an R-(1-3) glycosidic bond at the free
hydroxyl group. This reaction proceeded in 50% yield and
with apparently complete R-selectivity. The remaining
steps to 40 were accomplished through a series of well-
established protocols.
Most of the reactions with air- or moisture-sensitive re-
agents and reaction products were performed under argon or
nitrogen atmosphere. Tetrahydrofuran (THF), diethyl ether
(Et2O), methylene chloride (CH2Cl2), and toluene were obtained
from a dry solvent system (activated alumina columns, positive
pressure of argon). All other dry solvents were purchased in
Sure/Seal bottles (Aldrich). Triethylamine was distilled from
CaH2 prior to use. All chemicals were high-quality reagents
and used without further purification.
Synthesis of LeYTetrasaccharide Glycal 14. LeY tetra-
saccharide glycal 12 (4 g, 2.44 mmol) was dissolved in THF
(20 mL). TBAF/THF (1 M, 30 mL) was added, and the reaction
mixture was stirred at room temperature for 24 h to complete
disappearance of the starting material. The reaction mixture
was concentrated and the residue chromatographed (EtOAc/
Hexanes 2:1) to give 2.45 g (89%) of the corresponding tetraole
13 as a white solid. Deprotected glycal tetraol and a few
crystals of TBAI were taken up in DMF (25 mL) and cooled to
0 °C. NaH (1.2 g 60% dispersion in mineral oil, 30.05 mmol,
14 equiv) was added in portions, followed by BnBr (3.57 mL,
30.05 mmol, 14 equiv). The reaction mixture was allowed to
reach room temperature and stirred to disappearance of the
starting tetraole. The reaction mixture was poured into ice-
water (250 mL) and extracted with EtOAc (3 × 250 mL). The
combined organics were washed with water (2 × 300 mL) and
brine (1 × 200 mL), dried over MgSO4, and concentrated. The
residue was chromatographed (FC, 30% EtOAc/hexanes) to
give 2.82 g (88%) of 14 as a white solid: [R]23 ) -61.6 (c 1,
D
CHCl3); IR (thin film) 3425, 3060, 3025, 2919, 2860, 1954,
1878, 1813, 1725, 1649, 1606, 1580, 1496, 1455, 1361, 1261,
1208, 1072, 1026, 912, 836, 735, 697; 1H NMR (500 MHz,
CDCl3) δ 7.35-7.02 (m, 50 H), 6.33 (d, J ) 6.2 Hz, 1 H), 5.69
(d, J ) 3.7 Hz, 1H), 4.91-3.26 (m, 42 H), 1.09 (d, J ) 6.4 Hz,
3 H), 1.02 (d, J ) 6.4 Hz, 3 H); 13C NMR (500 MHz, CDCl3) δ
144.96, 139.58, 139.53, 139.31, 139.21, 139.18, 139.09, 139.01,
138.89, 138.68, 138.42, 138.16, 129.05, 128.96, 128.94, 128.85,
128.78, 128.71, 128.70, 128.65, 128.58, 128.50, 128.44, 128.40,
128, 36, 128.34, 128.31, 128.24, 128.17, 128.04, 127.97, 127.94,
127.93, 127.89, 127.86, 127.81, 127.78, 127.76, 127.70, 127.64,
127.57, 127.45, 126.60, 126.50, anomeric carbons (100.91,
99.52, 97.84, 95.06), 84.65, 80.16, 79.93, 79.60, 78.74, 78.28,
78.13, 77.74, 77.49, 77.41, 77.24, 76.33, 75.98, 75.49, 75.30,
75.14, 73.88, 73.62, 73.45, 73.23, 73.12, 73.02, 72.90, 72.83,
72.76, 72.68, 72.20, 71.89, 71.52, 71.32, 69.53, 68.34, 68.30,
66.89, 66.86, 17.01, 16.73; MS (ESI) 1523 [M + Na+]; HRMS
(FAB) calcd for C94H100O17Na 1523.6857, found 1523.6876.
The synthesis of KH-1 was completed by converting
intermediate 37 from the B-1 synthetic route to the
corresponding â-2-iodo-R-1-sulfonamide 41. The latter
was coupled with acceptor 8 to afford nonasaccharide 42
in 32% overall yield from 37 (Scheme 8).
Conclusion
In summary, KH-1 and three analogues have been
prepared with high stereoselectivity and in reasonable
yields using five common building blocks. Conjugation
of these analogues to carrier protein by previously applied
sequences has been accomplished and immunological
tests are currently being completed. Clearly, the advent
of the general notion of glycal assembly,10 and the
development of a menu of reactions directed to glycal
linkages, enables the assembly of large oligosaccharide
arrays on a scale and in a time frame that would not have
been anticipated earlier. Possibilities for developing fully
synthetic vaccines for the selective targeting of certain
tumors are now being pursued.
Synthesis of Iodo Sulfonamide 7. A mixture of tetrasac-
charide glycal 14 (939 mg, 0.625 mmol) and benzenesulfona-
mide (295 mg, 1.875 mmol) was azeotroped with benzene (3
× 20 mL) and then dried under high vacuum for 3 h. Freshly
activated 4 Å powdered molecular sieves (900 mg) were added,
and the mixture was dissolved in CH2Cl2 (12 mL). The
resulting solution was cooled to 0 °C, and iodonium di-sym-
collidine perchlorate was added in one portion. The reaction
was stirred for 40 min at 0 °C, quenched with saturated
aqueous sodium thiosulfate (6 mL), diluted with ethyl acetate,
and filtered through a pad of Celite. The filtrate was washed
with saturated aqueous sodium thiosulfate, saturated aqueous
copper sulfate, and saturated brine. The organics were dried
over MgSO4, filtered, and concentrated. Purification of the
crude product by FC (12-25% EtOAc/hexanes) afforded 975
mg (87%) of 7 as a white foam: [R] ) -73.9 (c 1, CHCl3); IR
(thin film) 3260, 3031, 2868, 1951, 1878, 1811, 1729, 1603,
1490, 1449, 1360, 1220, 107, 915, 768, 692; 1H NMR (500 MHz,
CDCl3) δ 7.84 (d, J ) 7.67 Hz, 2H), 7.33-7.09 (m, 55 H), 5.67
(d, J ) 3.70, 1 H), 5.51 (bs, 1 H), 4.91 (s, 1 H), 4.89 (s, 1 H),
4.73-3.24 (m, 43 H), 1.07 (d, J ) 6.32, 3 H), 0.99 (d, J ) 6.28,
3 H); 13C NMR (500 MHz, CDCl3) δ 141.16, 139.39, 139.30,
139.19, 139.11, 139.03, 138.83, 138.67, 138.34, 138.31, 133.13,
129.232, 129.01, 128.80, 128.76, 128.67, 128.59, 128.55, 128.51,
128.42, 128.36, 128.20, 128.15, 128.10, 128.02, 127.96, 127.87,
127.81, 127.75, 127.66, 127.50, 127.42, 126.62, 126.50, ano-
Experimental Procedures
General Methods. NMR (1H, 13C) spectra were recorded
on an AMX-400 MHz or a DRX-500 MHz spectrometer.
Chemical shifts are reported in ppm, referenced to TMS (1H
NMR and 13C NMR 0.00), CDCl3 (13C NMR, 77.0), unless
otherwise stated. Coupling constants (J) (H, H) are given in
Hz. Signal splitting patterns are designated as singlet (s),
doublet (d), triplet (t), quadruplet (q), multiplet and overlap-
ping signals (m), and broad signals (br). IR spectra were
recorded on an FTIR spectrometer and absorption bands
presented in cm-1. Optical rotations were measured with a
digital polarimeter using a 10 and 5 cm path length cell at rt
(23 °C). Low-resolution mass spectral analyses were performed
using electrospray ionization with a single quad analyzer.
High-resolution mass spectra were acquired using electrospray
ionization with a time-of-flight analyzer.
J. Org. Chem, Vol. 70, No. 9, 2005 3389

Spassova et al.
meric carbons (100.54, 98.42, 98.08, 96.40), 84.42, 79.96, 79.89,
79.06, 78.34, 78.10, 76.46, 75.83, 75.35, 75.22, 75.14, 74.01,
73.78, 73.72, 73.43, 73.18, 73.04, 72.95, 72.84, 72.76, 72.67,
71.67, 67.72, 67.55, 66.74, 17.32, 17.14, 16.97, 16.77, 16.65;
MS (ESI) 1807.6 [M + Na+].
6.53 Hz, 3H); LRMS (ESI) 1618.6 [M + Na+]; HRMS (FAB)
calcd for C68H93NO42Na 1618.5069, found 1618.5073.
Alternative Synthesis of Peracetate LeY Hexasaccha-
ride Glycal 22: (a) Synthesis of Perbenzylated LeY Hexa-
saccharide Glycal 25. To a solution of compound 19 (378
mg, 0.0654 mmol) in dry benzene (150 mL) was added bis-
(tributyltin) oxide (0.359 mmol, 0.183 mL), and the solution
was refluxed overnight with removal of water with a Dean-
Stark trap. The resulting tin ether 8 was concentrated in
vacuo, diluted with THF (5 mL), and added to a mixture of
azeotropically dried (3 × 10 mL of benzene) iodosulfonamide
7 (388 mg, 0.218 mmol) and freshly activated 4 Å molecular
sieves (500 mg). The suspension was cooled to -70 °C, and a
solution of AgBF4 (138 mg) in THF (2.5 mL) was added to it
via cannula. The reaction mixture was stirred for 2 days with
exclusion of light while slowly being allowed to reach room
temperature. The reaction was quenched with saturated
aqueous solution of NaHCO3 (4 mL), diluted with EtOAc (50
mL), and filtered through a pad of Celite. The filtrate was
washed with saturated aqueous solution of NaHCO3 (3 × 30
mL) and brine (1 × 30 mL), dried over MgSO4, and filtered.
The filtrate was concentrated and the crude product purified
by FC (20-30% EtOAc/hexanes) to give 204 mg (42%) of 25
as white foam: [R] ) -60.2 (c 1, CHCl3); IR (thin film) 3496,
3280, 3075, 3020, 2870, 1957, 1871, 1808, 1723, 1643, 1580,
1501, 1455, 1358, 1313, 1250, 1210, 1153, 1096, 1022, 914, 732;
1H NMR (CDCl3) δ 7.65-7.15 (m, 65H), 6.39 (d, J ) 6.07 Hz,
1H), 5.81 (d, J ) 6.58 Hz, 1H), 5.68 (d, J ) 3.74 Hz, 1H), 5.03-
4.52 (m, 24H), 4.40-4.34 (m, 8H), 4.32-4.20 (m, 6H), 4.14-
4.09 (q, 4H), 4.03-3.26 (m, 26H), 2.82 (bs, 1H), 0.95-0.90 (m,
6H); 13C NMR (500 MHz, CDCl3) δ 144.59, 140.86, 138.97,
138.83, 138.76, 138.65, 138.51, 13840, 138.22, 138.12, 137.91,
137.80, 137.63, 137.56, 137.44, 132. 43, 129.22, 129.12, 128.92,
128.88, 128.64, 128.56, 128.51, 128.44, 128.42, 128.40, 128.34,
128.30, 128.24, 128.16, 128.12, 128.07, 128.03, 127.97, 127.87,
127.75, 127.71, 127.63, 127.61, 127.59, 127.51, 127.47, 127.40,
127.37, 127.33, 127.24, 127.20, 126.93, 126.85, 126.26, 126.13,
125.63, 124.06, anomeric carbons (102.89, 101.63, 100.47,
100.34, 98.59, 97.55), 84.11, 84.05, 80.61, 80.11, 80.01, 79.85,
79.72, 79.39, 78.05, 77.78, 77.65, 77.27, 77.02, 76.76, 76.61,
76.22, 75.69, 75.60, 75.39, 75.11, 74.91, 74.83, 74.79, 74.36,
74.09, 73.73, 73.58, 73.46, 73.42, 73.36, 73.27, 73.24, 72.99,
72.80, 72.64, 72.58, 72.39, 72.24, 71.75, 71.26, 71.07, 70.76,
70.61, 70.42, 69.42, 69.04, 68.21, 67.89, 67.84, 67.15, 66.96,
66.88, 66.78, 60.39, 27.84, 26.85, 22.91, 21.06, 17.52, 16.68,
16.60, 16.39, 16.25, 14.20, 13.61, 8.25, 4.14; LRMS (ESI)
2256.9; HRMS (FAB) calcd for C133H143O28NSNa 2256.9414,
found 2256.9421 [M + Na+]. (b) Ley Hexasaccharide Glycal
Peracetate 22 . Ley glycal 25 (120 mg, 0.0537 mmol) was
dissolved in THF (3 mL) and added via cannula to a solution
of sodium (150 mg, 6.52 mmol) in liquid ammonia (10 mL)
under Ar at -78 °C and stirred for 40 min. The reaction
mixture was quenched with MeOH (3 mL), stirred for 15 min
and concentrated with a stream of dry Ar. MeOH (5 mL) was
added, followed by NH4Cl (349 mg, 6.52 mmol), and the
solution stirred for 15-20 min and then concentrated again
to dryness. The crude product was suspended in a mixture of
THF (1 mL), DMF (0.5 mL), and TEA (1 mL). To this were
added Ac2O (0.82 mL, 8.91 mmol) and a catalytic amount of
DMAP, and the mixture was stirred at room temperature for
10 h, poured into ice-water (40 mL), and extracted with EtOAc
(3 × 70 mL). The organics were washed with saturated
aqueous NaHCO3 and H2O, dried over MgSO4, filtered, and
concentrated. The purification of the crude product by FC (65%
EtOAc/CH2Cl2) afforded 69 mg (81%) of 22.
LeY Hexasaccharide Glycal 21. To a solution of compound
19 (552 mg, 0.954 mmol) in dry benzene (200 mL) was added
bis(tributyltin) oxide (0.55 equiv, 0.524 mmol, 0.267 mL), and
the resulting solution was refluxed overnight with removal of
water with a Dean-Stark trap. The resulting solution of tin
ether 8 was concentrated in vacuo, and the residue was diluted
with THF (9 mL) and added to a mixture of azeotropically
dried (3 × 10 mL of benzene) iodosulfonamide 6 (612 mg,
0.3179 mmol) and freshly activated 4 Å molecular sieves (600
mg). The resulting suspension was cooled to -70 °C, and a
solution of AgBF4 (134 mg) in THF (3 mL) was added to it via
cannula. The reaction mixture was stirred for 2 days with
exclusion of light while slowly being allowed to reach room
temperature. The reaction was quenched with saturated
aqueous solution of NaHCO3 (5 mL), diluted with EtOAc (60
mL), and filtered through a pad of Celite. The filtrate was
washed with a saturated aqueous solution of NaHCO3 (3 ×
30 mL) and brine (1 × 30 mL), dried over MgSO4, and filtered.
The filtrate was concentrated and the crude product purified
by FC (20-30% EtOAc/hexane) to give 224 mg (28%) of 21 as
a white foam: [R] ) -43.2 (c 1, CHCl3); IR (thin film) 3498,
3242, 3063, 2935, 2845, 1814, 1642, 1456, 1347, 1162, 1104,
1
1050, 820, 743, 698; H NMR (CDCl3) δ 7.60-7.00 (m, 70H),
6.63 (d, J ) 3.0 Hz, 1H), 6.42 (d, J ) 6.1 Hz, 1H), 5.14 (d, J )
3.5 Hz, 1H), 4.98-3.42 (m, 54), 3.01 (bs, 1H), 2.74 (bs, 1H),
1.04-0.90 (m, 21H), 0.83(d, J ) 6.4, 3H); 13C (500 MHz, CDCl3)
δ 153.42, 144.49, 141.91, 138.55, 138.52, 135.74, 135.56,
135.45, 135.16, 132.29, 130.00, 128.63, 128.59, 128.51, 128.45,
128.36, 128.34, 128.29, 128.21, 128.17, 127.91, 127.86, 127.83,
127.77, 127.72, 1276.67, 127.59, 127.57, 127.42, 127.42, 126.75,
anomeric carbons (102.71, 101.81, 100.42, 100.29, 98.32, 97.72),
79.03, 78.57, 78.06, 77.55, 76.29, 74.90, 74.73, 74.64, 74.50,
74.45, 74.23, 73.68, 73.58, 73.48, 72.74, 72.07, 71.81, 70.80,
70.73, 69.20, 68.61, 68.09, 67.51, 61.55, 61.14, 27.94, 27.85,
27.77, 27.11, 26.86, 26.81, 26.64, 19.37, 19.20, 18.87, 17.53,
16.69, 16.44, 16.26, 16.20, 13.62; LRMS (ESI) calcd for C138
-
H153NO29SSi2Na 2398.9684, found 2398.9 [M + Na+].
Peracetate LeY Hexasaccharide Glycal 22. To a solution
of hexasaccharide 21 (260 mg, 0.11 mmol) in THF (4 mL) was
added 1 M TBAF/THF (0.95 mL, 0.95 mmol), and the resulting
solution was stirred for 10 h at room temperature. The reaction
mixture was concentrated and purified by FC (5%MeOH/
CHCl3). The resulted white solid was dissolved in THF (3 mL)
and added via cannula to a solution of sodium (300 mg, 13.04
mmol) in liquid ammonia (18 mL) under Ar at -78 °C and
stirred for 40 min. The reaction mixture was quenched with
MeOH (6 mL), stirred for 15 min, and concentrated with a
stream of dry Ar. MeOH (10 mL) was added, followed by NH4-
Cl (698 mg, 13.04 mmol), and the solution stirred for 15-20
min and then concentrated again to dryness. The crude
product was suspended in a mixture of THF (5 mL), DMF (2
mL), and TEA (1.5 mL). To this were added Ac2O (1.64 mL,
17.82 mmol) and a catalytic amount of DMAP, the mixture
was stirred at room temperature for 10 h and then poured into
ice-water (40 mL) and extracted with EtOAc (3 × 70 mL).
The organics were washed with saturated aqueous NaHCO3
and H2O, dried over MgSO4, filtered, and concentrated.
Purification of the crude product by FC (65% EtOAc/CH2Cl2)
afforded 76 mg (44% overall from the three steps) of 22 as a
white solid: [R] ) -55 (c 1, CHCl3); IR (thin film) 3394, 2974,
2963, 2904, 2834, 1752, 1723, 1685, 1461, 1374, 1263, 1129,
1076, 1030, 960, 907; 1H NMR (CDCl3) δ 6.40 (D, J ) 6.1 Hz,
1H), 5.45 (d, J ) 7.11, 1H), 5.38-5.29 (m, 7H), 5.28 (d, J )
3.9, 1H), 5.09 (dd, J ) 6.9, J ) 3.1, 1H), 5.07-4.95 (m, 8H),
4.85 (dd, J ) 6.11, J ) 3.3, 1H), 4.62 (m, 2H), 4.60-4.45 (m,
4H), 4.25-3.76 (m, 12H), 3.42 (d, J ) 9.9 Hz, 1H), 2.18-1.96
(m, 45H), 1.91 (s, 3H), 1.21 (d, J ) 6.54 Hz, 3H), 1.18 (d, J )
Allyl Glycoside of Ley Hexasaccharide Peracetate 23.
A mixture of Ley hexasaccharide glycal 22 (32 mg, 0.019 mmol),
dried azeotropically with benzene (3 × 5 mL) and under high
vacuum for 2 h, and freshly activated 4 Å powdered molecular
sieves (50 mg) was suspended in CH2Cl2 (1 mL) and cooled to
0 °C, and DMDO (0.70 mL 0.1 M solution in Me2CO) was
added. The reaction mixture was stirred at 0 °C for 1 h, the
3390 J. Org. Chem., Vol. 70, No. 9, 2005

Synthesis of Selected LeY and KH-1 Analogues
solvent was evaporated, and the residue was taken up in allyl
alcohol (6 mL). This mixture was stirred at room temperature
for 48 h, the solvent was evaporated, and the product was
purified by FC (60% EtOAc/CH2Cl2) affording 30 mg (88%) of
23 as a white solid: [R] ) -51 (c 1, CHCl3); 1H NMR (CDCl3)
δ 5.84 (d, 1H, J ) 6.9), 5.28-4.89 (m, 19 H), 4.55 (d, J ) 7.9
Hz, 1H), 4.37-3.34 (m, 23), 2.81 (bs, 1H), 2.10-1.85 (m, 48H),
1.15 (d, J ) 6.5 Hz, 3H), 1.11 (d, J ) 6.4 Hz, 3H); 13C NMR
(CDCl3) δ 170.35, 169.89, 169.74, 169.62, 169.52, 169.42,
169.37, 169.33, 169.25, 169.18, 169.00, 168.79, 168.52, 168.50,
132.38, 117.37, anomeric protons (100.40, 99.66, 99.40, 97.96,
95.24, 94.66), 75.33, 74.58, 73.53, 72.89, 72.29, 71.96, 71.78,
71.57, 71.54, 70.38, 70.13, 69.89, 69.77, 69.44, 68.40, 67.64,
66.84, 66.69, 66.46, 65.82, 64.10, 62.95, 61.10, 60.46, 59.42,
58.96, 58.66, 28.67, 28.30, 28.20, 22.49, 21.67, 20.09, 19.99,
19.93, 19.90, 19.81, 19.76, 19.71, 19.68, 19.65, 19.60, 14.89,
14.52,; LRMS (ESI) 1692 [M + Na +]; HRMS calcd for C71H99-
NO44Na 1692.5437, found 1692.5440.
Allyl Glycoside of Ley Hexasaccharide 24. A solution
of hexasaccharide 23 (26 mg, 0.0154 mmol) in MeOH (1 mL)
was treated with NaOMe/MeOH (5%, 0.283 mL). After 12 h,
the mixture was neutralized with Dowex 50-X8, filtered, and
concentrated. Purification of the residue with RP-18 reversed-
phase silica gel (5% MeOH/H2O) gave 14 mg (90%) of 24 as a
white solid: [R] ) -41.1 (c 0.9, MeOH); IR (thin film) 3359,
2974, 2893, 1723, 1665, 1630, 1578, 1467, 1368, 1315, 1070,
1024; lH NMR (D2O) δ 5.99 (m, 1H), 5.39 (d, J ) 17.3 Hz, 1H,
CHdCH2), 5.30-5.28 (m, 2H), 5.12 (d, J ) 3.9, 1H), 4.89-
4.78 (multiple protons), 4.72 (d, J ) 8.2, 1H), 4.54-4.43 (m,
5H), 4.26-4.14 (m, 4H), 4.01-3.56 (m, 35H), 3.46 (bs, 1H),
3.35-3.32 (m, 1H), 2.03 (s, 3H), 1.24 (d, J ) 6.6, 3H), 1.18 (d,
J ) 6.7, 3H); 13C NMR (D2O) δ 188.49, 135.92, 121.44, 105.58,
105.15, 103.68, 102.87, 102.09, 101.26, 84.66, 80.99, 79.04,
77.51, 77.42, 77.07, 75.45, 74.60, 74.36, 73.34, 72.66, 72.39,
71.83, 71.41, 70.94, 70.36, 69.59, 64.13, 63.57, 62.74, 62.43,
60.00, 33.78, 24.94, 18.12, 14.43; LRMS (ESI) 1062.4 [M +
Na+]; HRMS (FAB) calcd for C41H69NO29Na 1062.3851, found
1062.3854.
°C for 40 min, quenched with saturated aqueous Na2S2O3
(3 mL), diluted with EtOAc (60 mL), and then filtered through
Celite. The filtrate was washed with saturated aqueous
Na2S2O3 (2 × 30 mL), saturated aqueous CuSO4 (2 × 30 mL),
and brine (1 × 30 mL), and the organics were dried over
MgSO4, filtered, and concentrated. The residue was taken up
in 30% EtOAc/hexanes and passed through a short SiO2
column to give 108 mg 27 (75%). This was azeotroped with
benzene (3 × 3 mL), kept under high vacuum for 3 h, and used
without further purification. Freshly activated 4 Å molecular
sieves (110 mg) were added to it under Ar, followed by a
solution of tin ether of 8 (prepared from 83 mg of 19 and 0.042
mL of (Bu3Sn)2O as described for 21). The resulting suspension
was cooled to -70 °C and AgBF4 (28 mg, 0.143 mmol) in THF
(1 mL) added via cannula. The reaction mixture was stirred
with exclusion of light, allowed to warm slowly over about 5 h
to room temperature, and stirred for 3 days. EtOAc was added,
and the mixture was filtered through Celite. The filtrates were
diluted additionally with EtOAc and washed with saturated
aqueous solution of NaHCO3 (3 × 30 mL) and brine (1 × 30
mL). The organics were dried over MgSO4, filtered, concen-
trated, and chromatographed (FC 30-40% EtOAc/hexanes) to
give 82 mg of 28 (65% for this step and 50% overall for the
two steps): [R] ) -18.5 (c 0.87, CHCl3); IR (thin film) 3483,
3319, 3048, 2872, 1742, 1702, 1642, 1578, 1454, 1366, 1308,
1237, 1166, 1096, 1061, 1020, 908, 802, 732, 700; 1H NMR (500
MHz, CDCl3) δ 8.10(d, J ) 8.1 Hz, 2H), 7.80 (d, J ) 8.1 Hz,
2H), 7.53 (d, J ) 6.1 Hz, 2H), 7.46-7.09 (m, 80 H), 7.02 (d,
7.4 Hz, 2H), 6.92 (d, J ) 7.5 Hz, 2H), 6.39 (d, J ) 6.1 Hz, 1H),
5.67 (d, J ) 3.6 Hz, 1H), 5.41-5.37 (m, 2H), 5.13-5.03 (m,
2H), 4.82-3.21 (m, 82H), 2.00 (s, 6H), 1.89 (s, 3H), 1.35 (d,
J ) 6.1 Hz, 3H), 1.14 (d, J ) 6.0 Hz, 3H); 13C NMR (500 MHz,
CDCl3) δ 170.36, 170.08, 167.78, 144.66, 140.56, 139.78, 138.94,
138.81, 138.73, 138.71, 138.67, 138.60, 138.50, 138.25, 138.13,
138.07, 137.82, 133.88, 132.51, 130.45, 129.07, 128.90, 128.83,
128.76, 128.65, 128.61, 128.56, 128.46, 128.40, 128.35, 128.32,
128.16, 128.10, 128.05, 128.01, 127.98, 127.94, 127.91, 127.84,
127.82, 127.79, 127.76, 127.71, 127.64, 127.60, 127.48, 127.43,
127.38, 127.34, 127.31, 127.13, 127.10, 127.00, 126.92, 126.85,
126.05, anomeric protons (103.02, 102.38, 101.98, 100.56,
100.52, 99.72, 98.06, 96.60), 84.01, 82.17, 80.12, 79.89, 79.12,
79.08, 78.84, 78.33, 78.25, 77.23, 76.78, 75.64, 75.61, 75.39,
74.98, 74.69, 74.59, 74.34, 74.14, 73.98, 73.88, 73.73, 73.60,
73.55, 73.43, 73.40, 72.78, 72.62, 72.45, 72.35, 72.29, 71.58,
70.99, 70.95, 70.28, 69.35, 68.99, 68.48, 68.31, 68.18, 68.03,
67.70, 67.11, 66.98, 66.87, 57.95, 55.13, 38.76, 31.95, 30.39,
29.72, 29.39, 28.95, 24.63, 23.77, 23.01, 22.72, 21.07, 20.80,
16.62, 16.29, 14.15, 14.08, 10.99; LRMS calcd for C178H192O42-
N2S2Na 3116.2287, found 3116 [M + Na+].
LeY Octasaccharide Glycal Peracetate 29. A solution
of LeY octasaccharide glycal 28 (72 mg, 0.018 mmol) in THF
(1 mL) was added via cannula to a solution of sodium (280
mg, 12.17 mmol) in liquid ammonia (10 mL) under Ar at -78
°C and stirred for 40 min. The reaction mixture was quenched
with MeOH (5 mL), stirred for 15 min, and concentrated with
a stream of dry Ar. MeOH (10 mL) was added, followed by
NH4Cl (651 mg, 12.18 mmol), and the solution stirred for 15-
20 min and then concentrated again to dryness. The crude
product was suspended in a mixture of THF (1.5 mL), DMF
(1 mL), and TEA (0.4 mL). To this were added Ac2O (0.4 mL,
1.48 mmol) and a catalytic amount of DMAP, and the reaction
mixture was stirred at room temperature for 10 h. The reaction
mixture was poured into ice-water (40 mL) and extracted with
EtOAc (3 × 70 mL). The organics were washed with saturated
aqueous NaHCO3 and H2O, dried over MgSO4, filtered, and
concentrated. The purification of the crude product by FC (85%
EtOAc/CH2Cl2) afforded 32 mg (80%) of 29 as a white solid:
[R] ) -46.2 (c 1, CHCl3); IR (thin film) 3372, 2971, 1746, 1673,
1534, 1434, 1373, 1223, 1156, 1123, 1067, 1034, 945; 1H NMR
(500 MHz, CDCl3) δ 6.40 (d, J ) 6.1 Hz, 1H), 5.58 (d, J ) 7.0
Hz, 1H), 5.43 (d, 1H), 5.36-5.31 (m, 7H), 5.25-5.12 (m, 3H),
5.10-4.95 (m, 9H), 4.83-4.82 (m, 2H), 4.74 (d, J ) 6.9 Hz,
Acetylated Hexasaccharide 26. To a solution of LeY
hexasaccharide 25 (160 mg, 0.0716 mmol), a catalytic amount
of DMAP, and TEA (0.132 mL, 1 mmol) in CH2Cl2 (3 mL) was
added Ac2O (0.095 mL, 1.0 mmol). The reaction mixture was
stirred for 24 h. EtOAc (100 mL) was added and the solution
washed with saturated aqueous NaHCO3 (3 × 60 mL), dried
over MgSO4, filtered, concentrated, and purified by FC (25%
EtOAc/hexane) to afford 131 mg (77%) of 26 as a white solid:
[R] ) -35.70 (c 1, CHCl3); IR (thin film) 3315, 2870, 1748, 1716,
1
1600, 1450, 1350, 1220, 1120, 1102, 909, 728; H NMR (500
MHz, CDCl3) δ 7.44-7.12 (m, 70H), 6.38 (d, J ) 6.1 Hz, 1H),
5.67 (d, J ) 3.2 Hz, 1H), 5.40m, 2H), 5.17 (m, 1H), 4.92(d, 2H),
4.82, 4.66 (m, 9H), 4.53-4.28 (m, 22H0, 4.15-3.94 (m, 8H),
3.84-3.61 (m, 13H), 3.53-3.21 (m, 5H), 2.00 (s, 3H), 1.96 (s,
6H), 1.35 (d, J ) 6.0 Hz, 3H), 1.18 (d, J ) 6.1 Hz, 3H); 13C
NMR (CDCl3) δ 172.71, 170.34, 170.30, 144.50, 139.72, 138.79,
138.71, 138.68, 138.04, 137.85, 137.83, 137.76, 137.14, 133.84,
130.38, 129.33, 129.22, 128.63, 128.51, 128.46, 128.43, 128.40,
128.36, 128.33, 128.21, 128.15, 128.07, 128.02, 127.94, 127.88,
127.76, 127.64, 127.58, 127.38, 127.05, 126.94, 126.87, 126.00,
anomeric protons (101.72, 100.16, 99.63, 99.44, 98.00, 96.65),
83.98, 79.86, 78.98, 78.29, 75.73, 75.55, 74.96, 74.34, 74.11,
73.85, 73.81, 73.70, 73.70, 73.67, 73.51, 73.37, 73.31, 72.65,
72.57, 72.38, 72.20, 72.12, 70.12, 69.42, 67.97, 67.05, 66.97,
66.81, 24.59, 22.64, 21.00, 20.93, 16.26, 14.12; LRMS (ESI)
calcd for C139H149O31NSNa 2382.9731, found 2381.6 [M + Na+].
LeY Octasaccharide Glycal 28. A mixture of LeY hexa-
saccharide 26 (129 mg, 0.055 mmol) and benzenesulfonamide
(26 mg, 0.16 mmol) was azeotropically dried with benzene
(3 × 4 mL) under high vacuum for 2 h. To the dried mixture
was added freshly activated 4 Å molecular sieves (130 mg)
under Ar, followed by CH2Cl2 (4 mL). The resulting suspension
was cooled to 0 °C, and I (sym-coll)2ClO4 (90 mg, 0.191 mmol)
was added under Ar. The reaction mixture was stirred at 0
J. Org. Chem, Vol. 70, No. 9, 2005 3391

Spassova et al.
1H), 4.62 (d, J ) 7.8 Hz, 1H), 4.50 (d, J ) 7.6 Hz, 1H), 4.45 (q,
2H), 4.43-4.35 (m, 3H), 4.29-4.25 (m, 2H), 4.21-3.75 (m,
17H), 3.51-3.41 (m, 2H), 2.1 (s, 3H), 2.15-2.14 (m, 12H),
2.13-2.11 (m, 15H), 2.10-2.04 (m, 18H), 2.01-1.98 (m, 12 H),
1.91 (s, 3H), 1.89 (s, 3H), 1.21 (d, 6.5 Hz, 3H), 1.18 (d, 6.5 Hz,
3H); 13C NMR (500 MHz, CDCl3) δ 171.52, 171.30, 170.93,
170.75, 170.68, 170.64, 170.61, 170.54, 170.49, 170.44, 170.34,
170.25, 170.23, 170.21, 170.11, 169.97, 169.84, 169.81, 169.46,
169.22, 145.34, anomeric carbons (100.90, 100.70, 100.41,
100.27, 98.98 (two carbons), 96.22, 95.68), 75.26, 74.26, 74.16,
73.88, 73.32, 73.01, 72.69, 72.46, 71.39, 71.20, 71.09, 71.02,
70.87, 70.80, 68.77, 68.72, 67.85, 67.69, 67.50, 66.85, 65.09,
63.99, 61.93, 61.65, 61.32, 29.70, 29.66, 29.36, 27.16, 23.50,
23.19, 23.09, 22.69, 21.09, 20.96, 20.94, 20.91, 20.87, 20.82,
20.78, 20.74, 20.69, 20.67, 20.62, 20.60, 15.91, 15.53, 14.20,
14.13, 1.029; LRMS (ESI) 2194.8; HRMS (FAB) calcd for
C92H126O57N2Na 2193.6919, found 2193.6989 [M + Na+].
128.85, 128.82, 128.68, 128.53, 128.50, 128.48, 128.44, 128.41,
128.35, 128.24, 128.20, 128.13, 128.06, 128.01, 127.95, 127.91,
127.82, 127.71, 127.60, 127.56, 127.51, 127.49, 127.46, 127.40,
127.36, 127.31, 127.22, 126.99, 126.85, anomeric carbons
(103.65, 100.12, 99.73, 99.17, 98.15, 98.10, 96.88, 95.14), 78.65,
78.29, 78.17, 77.95, 77.66, 77.54, 76.26, 76.22, 75.28, 75.15,
75.05, 74.88, 74.70, 74.60, 74.09, 74.04, 73.82, 73.74, 73.71,
73.65, 73.61, 73.54, 73.28, 72.60, 72.26, 72.11, 71.80, 70.66,
70.38, 70.02, 69.84, 69.39, 68.12, 68.01, 67.19, 66.23, 65.74,
64.09, 60.87, 57.13, 78.65, 78.29, 78.17, 77.95, 77.66, 77.54,
76.26, 76.22, 75.28, 75.15, 75.05, 74.88, 74.70, 74.60, 74.09,
74.04, 73.82, 73.74, 73.71, 73.65, 73.61, 73.54, 73.28, 72.60,
72.26, 72.11, 71.80, 70.66, 70.38, 70.02, 69.84, 69.39, 68.12,
68.01, 67.19, 66.23, 65.74, 64.09, 60.87, 57.13, 29.71, 26.86,
26.77, 26.75, 26.66, 26.05, 20.64, 20.62, 19.34, 19.13, 16.61,
16.50, 16.42, 1.03; LRMS (ESI) calcd for C183H202N2O43S2Si2-
Na2 3281.2455, found 3281.4 [M + 2Na2+]. (b) LeY Octasac-
charide Glycal Peracetate 29. To a solution of LeY octasac-
charide glycal 33 (62 mg, 0.019 mmol) dissolved in THF (3
mL) was added 1 M TBAF/THF (0.230 mL, 0.230 mmol). The
reaction mixture was stirred for 24 h at room temperature and
then concentrated and chromatographed (FC, 5%MeOH/
CHCl3). The white solid residue was taken up in THF (3 mL)
and added via cannula to a solution of sodium (61 mg, 2.65
mmol) in liquid ammonia (8 mL) under Ar at -78 °C. The
reaction mixture was stirred for 40 min, quenched with MeOH
(3 mL), stirred for 15 min, and concentrated with a stream of
dry Ar. MeOH (5 mL) was added, followed by NH4Cl (141 mg,
2.65 mmol), and the mixture stirred for 15-20 min and then
concentrated again to dryness. The crude product was sus-
pended in a mixture of THF (1.0 mL), DMF (0.3 mL), and TEA
(0.6 mL). To this were added Ac2O (0.395 mL, 4.18 mmol) and
a catalytic amount of DMAP. The mixture was stirred at room
temperature for 10 h and then poured into ice-water (15 mL)
and extracted with EtOAc (3 × 60 mL). The organics were
washed with saturated aqueous NaHCO3 and H2O, dried over
MgSO4, filtered, and concentrated. Purification of the crude
product by FC (65% EtOAc/CH2Cl2) afforded 25 mg (60%
overall yield) of 29.
Allyl Glycoside of Ley Octasaccharide Peracetate 30.
A mixture of Ley octasaccharide glycal 29 (32 mg, 0.0147 mmol,
azeotropically dried with benzene (3 × 5 mL) and under high
vacuum for 2 h) and freshly activated 4 Å powdered molecular
sieves (50 mg) was suspended in CH2Cl2 (1 mL) under Ar and
cooled to 0 °C. DMDO (220 mL 0.12 M solution in Me2CO)
was then added. The reaction mixture was stirred at 0 °C for
1 h, the solvent was evaporated, and the residue was taken
up in allyl alcohol (5 mL). The reaction mixture was stirred
at room temperature for 48 h, the solvent was evaporated, and
the product was purified by FC (40-70% EtOAc/CH2Cl2) to
afford 30 mg (90%) of 30 as a white solid: [R] ) -17.3 (c 1,
CH2Cl2); IR (thin film) 3486, 3377, 3087, 2961, 2873, 1748,
1683, 1539, 1430, 1372, 1228, 1066, 976, 944; 1H NMR (CDCl3)
δ 5.90 (m, 1H), 5.53 (d, J ) 7.08 Hz, 1H), 5.45 (d, J ) 8.3 Hz,
1H), 5.36-4.96 (m, 20 H), 4.84 (d, J ) 2.00 Hz, 1H), 4.81 (m,
1H), 4.69 (d, J ) 7.8 Hz, 1H), 4.62 (d, J ) 7.9 Hz, 1H), 4.50-
4.49 (m, 2H), 4.45-4.34 (m, 5H), 4.28 (q, 2H), 4.08-3.75 (m,
15H), 3.73 (dd, J ) 9.8 Hz, J ) 3.6 Hz, 2H), 3.69-3.41 (m,
6H), 2.84 (bs, 1H), 2.15-1.89 (m, 66H), 1.21 (d, J ) 6.5 Hz,
3H), 1.18 (d, J ) 6.5 Hz, 3H); 13C NMR (CDCl3) δ 171.39,
171.21, 170.96, 170.78, 170.73, 170.66, 170.62, 170.54, 170.48,
170.37, 170.28, 170.24, 170.15, 169.88, 169.84, 169.77, 169.61,
169.51, 169.00, 133.41, 133.29, 128.33, 118.43, 118.13, ano-
meric carbons (101.46, 100.90, 100.43, 100.24, 99.04, 98.39,
96.23, 95.70), 79.06, 75.25, 73.89, 73.35, 73.01, 72.83, 72.72,
72.61, 72.48, 71.42, 71.19, 71.07, 70.91, 70.82, 70.52, 69.42,
68.70, 67.87, 67.72, 67.53, 66.88, 65.11, 64.02, 61.64, 61.36,
60.48, 60.43, 55.07, 41.84, 31.94, 30.07, 29.72, 29.38, 27.21,
23.91, 23.51, 23.22, 23.10, 22.71, 21.11, 21.08, 21.01, 20.96,
20.94, 20.88, 20.84, 20.80, 20.75, 20.73, 20.69, 20.64, 19.84,
15.93, 15.55, 14, 22, 14.15; LRMS (ESI) calcd for C95H132N2-
O59Na 2267.7287, found 2266.6 [M + Na+].
Alternative synthesis of LeY Octasaccharide Glycal
Peracetate 29: (a) Synthesis of LeY Octasaccharide Gly-
cal 33. To a solution of LeY hexasaccharide 21 (156 mg, 0.066
mmol), a catalytic amount of DMAP, and TEA (0.090 mL, 0.722
mmol) in CH2Cl2 (2 mL) was added Ac2O (0.070 mL, 0.722
mmol), and the mixture was stirred for 24 h. EtOAc (60 mL)
was added and the solution washed with cold saturated
aqueous NaHCO3 (3 × 40 mL), dried over MgSO4, filtered,
concentrated, and passed through a short column of silica gel
(25% EtOAc/hexanes) to give 118 mg (72%) of acetylated 21:
LRMS (ESI) calcd for C144H159NO32SSi2Na 2525.0001, found
2525.9 [M + Na+]. A mixture of the product and benzene-
sulfonamide (20 mg, 0.126 mmol) was dried azeotropically with
benzene (3 × 4 mL) under high vacuum for 2 h. Freshly
activated 4 Å molecular sieves (100 mg) were added to the
mixture under Ar, followed by CH2Cl2 (2 mL). The resulting
suspension was cooled to 0 °C and I(sym-coll)2ClO4 (69 mg,
0.0.146 mmol) added under Ar. The reaction mixture was
stirred at 0 °C for 40 min and quenched with saturated
aqueous Na2S2O3 (2 mL), diluted with EtOAc (60 mL), and
then filtered through Celite. The filtrate was washed with
saturated aqueous Na2S2O3 (2 × 20 mL), saturated aqueous
CuSO4 (2 × 20 mL), and brine (1 × 20 mL). The organics were
dried over MgSO4, filtered, and concentrated. The residue was
taken up in 30%EtOAc/hexanes and passed through a short
SiO2 column. The collected iodosulfonamide 32 (101 mg, 77%)
was dried azeotropically with benzene (3 × 3 mL) and under
high vacuum for 3 h and used without further purification.
Freshly activated 4 Å molecular sieves (80 mg) were added to
it under Ar, followed by a solution of 8 in THF (prepared from
70 mg of 19 and 0.035 mL of (Bu3Sn)2O as described for 21).
The resulting suspension was cooled to -70 °C and AgBF4 (22
mg, 0.112 mmol) in THF (1 mL) added via cannula. The
reaction mixture was stirred with exclusion of light, allowed
to warm slowly to room temperature over about 5 h, and then
stirred for 3 days. EtOAc was added, and the mixture was
filtered through Celite. The filtrates were diluted additionally
with EtOAc and washed with a saturated aqueous solution of
NaHCO3 (3 × 30 mL) and brine (1 × 30 mL). The organics
were dried over MgSO4, filtered, and concentrated, and the
residue was chromatographed (FC 30-40% EtOAc/hexanes)
to give 62 mg (52%, 30% overall for the three steps) of 33 as
a white foam: [R] ) -45.6 (c 1, CHCl3); IR (thin film) 3485,
3305, 3029, 2929, 2858, 1835, 1821, 1754, 1694, 1495, 1453,
1
1360, 1214, 1159, 1103, 1050, 1028, 739, 698; H NMR (500
MHz, CDCl3) δ 8.10 (d, J ) 7.5 Hz, 2H), 7.81 (d, J ) 7.2 Hz,
2H), 7.54-6.90 (m, 86 H), 6.39 (d, J ) 6.0 Hz, 1H), 5.52 (d,
J ) 4.1 Hz, 1H), 5.38 (s, 1H), 5.19 (m, 1H), 5.00-3.22 (m, 80H),
2.12 (s, 3H), 1.94 (s, 3H), 1.76 (s, 3H), 1.19 (d, J ) 6.3 Hz),
1.12 (s, 9H), 1.00 (s, 9H), 0.90 (d, J ) 6.4 Hz); 13C NMR (500
MHz, CDCl3) δ 170.98, 168.97, 168.75, 153.36, 144.52, 140.28,
139.70, 138.92, 138.80, 138.71, 138.60, 138.54, 138.47, 138.44,
138.02, 137.90, 137.72, 137.57, 137.54, 136.09, 135.98, 135.46,
135.39, 135.33, 135.28, 133.97, 133.70, 132.47, 132.30, 132.25,
131.91, 130.20, 130.12, 130.06, 130.00, 129.88, 129.80, 129.20,
3392 J. Org. Chem., Vol. 70, No. 9, 2005

Synthesis of Selected LeY and KH-1 Analogues
Allyl Glycoside of Ley Octasaccharide 31. To a solution
of octasaccharide 30 (28 mg, 0.0124 mmol) in MeOH (0.5 mL)
was added NaOMe/MeOH (5%, 0.294 mL). After 12 h, the
mixture was neutralized with Dowex 50-X8, filtered, and
concentrated. Purification of the residue with RP-18 reversed-
phase silica gel (5% MeOH/H2O) gave 15.8 mg (89%) of 31 as
a white solid: [R] ) -34.32 (c 0.88, MeOH); IR (thin film) 3369,
2933, 2848, 1643, 1558, 1473, 1377, 1318, 1249, 1074; lH NMR
(D2O) δ 5.98 (m, 1H), 5.39 (d, J ) 17.3 Hz, 1H, CHdCH2), 5.37
(s, 1H), 5.28 (d, J ) 2.7 Hz, 1H), 5.12 (d, J ) 3.9, 1H), 4.92
(bs, 1H), 4.88 (d, J ) 7.8 Hz, 1H), 4.84-4.82 (m, 2H), 4.71
(J ) 8.1, 2H), 4.53-4.38 (m, 5H), 4.23 (m, 2H), 4.15 (bs, 2H),
4.10-3.31 (m, 59H), 2.04 (s, 3H-Ac), 2.03 (s, 3H-Ac), 1.70
(bs, 1H), 1.28 (d, J ) 6.7, 3H), 1.24 (d, J ) 6.6, 3H); 13C NMR
(D2O) δ 175.24, 175.07, 161.14, 133.61, 133.55, 119.10, 118.83,
anomeric carbons (103.30, 103.23, 103.10, 102.85, 101.38,
100.57, 99.79, 98.96), 82.38, 78.66, 78.48, 76.73, 75.71, 75.22,
75.12, 74.98, 74.77, 73.91, 73.39, 73.15, 72.53, 72.30, 72.06,
71.03, 70.36, 70.09, 69.53, 69.11, 68.64, 68.08, 67.28, 67.15,
62.31, 61.83, 61.29, 60.41, 60.28, 60.12, 56.46, 55.51, 49.21,
48.91, 48.73, 48.56, 48.39, 48.21, 48.04, 47.87, 22.62, 22.52,
15.81; LRMS (ESI) 1421.1 [M + Na+] HRMS (FAB) calcd for
C55H92N2O39Na 1427.5174, found 1427.5181 [M + Na+].
1452, 1367, 1237, 1222, 1166, 1097, 1068, 990, 913, 843, 804,
1
739; H NMR (500 MHz, CDCl3) δ 8.11 (d, J ) 8.2 Hz, 2H),
7.56-6.94 (m, 63H), 6.30 (d, J ) 6.1 Hz, 1H), 5.67 (d, J ) 3.5
Hz, 1H), 5.44 (d, J ) 2.8 Hz, 1H), 5.39 (d, J ) 8.0 Hz, 1H),
5.14 (t, J ) 8.4 Hz, 1H), 4.92 (d, J ) 11.2 Hz, 1H), 4.76-4.70
(m, 8H), 4.65 (d, J ) 6.3 Hz, 1H), 4.60 (s, 1H), 4.57-4.30 (m,
19H), 4.18-4.11 (m, 3H), 4.07 (dd, J ) 10.2, J ) 3.7 Hz, 1H),
4.03-3.61 (m, 17H), 3.54 (m, 1H), 3.47 (m, 2H), 3.37 (q, J )
8.9 Hz, J ) 4.7 Hz, 1H), 3.22 (s, 1H), 1.97 (m, 9H), 1.36 (d,
J ) 6.3 Hz, 3H, CH3), 1.18 (d, J ) 6.2 Hz, 3H, CH3), 0.91 (t,
J ) 7.9 Hz, 9H, -Si-CH2CH3), 0.55 (q, J ) 7.8 Hz, 6H, -Si-
CH2CH3); 13C NMR (500 MHz, CDCl3) δ 172.73, 170.31, 170.25,
143.11, 139.75, 139.08, 138.81, 138.69, 138.62, 138.54, 138.08,
138.04, 137.92, 137.87, 137.78, 133.81, 130.38, 128.61, 128.54,
128.45, 128.40, 128.37, 128.34, 128.17, 128.09, 128.06, 128.03,
127.96, 127.92, 127.85, 127.75, 127.70, 127.62, 127.51, 127.43,
127.40, 127.33, 127.10, 127.06, 126.98, 126.92, 126.01, ano-
meric carbons (102.11 101.68, 100.05, 99.65, 98.03, 96.68),
83.99, 79.88, 79.18, 78.98, 78.29, 75.57, 75.35, 75.15, 74.98,
74.37, 74.13, 73.86, 73.75, 73.44, 73.38, 73.29, 72.61, 72.39,
72.21, 71.57, 69.77, 69.21, 67.97, 67.11, 66.99, 64.65, 24.58,
20.99, 20.90, 16.58, 16.29, 6.86, 4.85; LRMS (ESI) 2407 [M +
Na+] HRMS (FAB) calcd for C138H157O31NSSiNa, 2407.0126,
found 2407.0127 [M + Na+].
LeY Hexasaccharide Glycal 34. To a solution of compound
20 (419 mg, 0.69 mmol) in dry benzene (200 mL) was added
bis(tributyltin) oxide (0.55 equiv, 0.359 mmol, 0.183 mL). The
solution was refluxed overnight with removal of water with a
Dean-Stark trap. The resulting tin ether 9 was concentrated
in vacuo, diluted with THF (5 mL), and added to a mixture of
azeotropically dried (3 × 10 mL of benzene) iodosulfonamide
7 (235 mg, 0.132 mmol) and freshly activated 4 Å molecular
sieves (300 mg). The resulting suspension was cooled to -70
°C, and a solution of AgBF4 (85 mg) in THF (0.5 mL) was added
to it via cannula. The reaction mixture was stirred for 48 h
with exclusion of light while slowly being allowed to reach
room temperature and then quenched with saturated aqueous
NaHCO3 (1 mL), diluted with EtOAc (50 mL), and filtered
through a pad of Celite. The filtrate was washed with
saturated aqueous NaHCO3 (3 × 25 mL) and brine (1 × 25
mL), dried over MgSO4, and filtered. The filtrate was concen-
trated and the residue purified by FC (20-30% EtOAc/
hexanes) to give 136 mg (46%) of 34 as a white foam: IR (thin
film) 3488, 3282, 3067, 3029, 2952, 2868, 2247, 1951, 1874,
1809, 1729, 1648, 1606, 1495, 1449, 1361, 1315; 1H NMR
(CDCl3) δ 7.64 (d, J ) 8.2 Hz, 2H), 7.37-7.05 (m, 63H), 6.35
(d, J ) 6.2 Hz, 1H), 5.87 (d, J ) 6.6 Hz, 1H), 5.68 (d, J ) 3.6
Hz, 1H), 5.08 (d, J ) 2.2 Hz, 1H), 4.92 (d, J ) 11.4 Hz, 1H),
4.82 (d, J ) 6.3 Hz, 2H), 4.79-4.55 (m, 12H), 4.52 (d, J ) 4.6
Hz, 2H), 4.46 (s, 2H), 4.42-4.19 (m, 11H), 4.12 (q, J ) 8.2 Hz,
1H), 4.05 (dd, J ) 6.3 Hz, J ) 3.7 Hz, 1H), 4.01-3.35 (m, 26H),
3.26 (bs, 1H), 3.19 (bs, 1H), 2.93 (bs, 1H), 1.24 (d, J ) 6.5 Hz,
3H, -CH3), 0.90 (m, 12H), 0.58 (q, J ) 8.0 Hz, 6H); 13C NMR
(500 MHz, CDCl3) δ 143.26, 140.86, 138.83, 138.77, 138.19,
138.13, 138.03, 137.81, 137.68, 128.51, 128.45, 128.42, 128.39,
128.37, 128.33, 128.29, 128.16, 128.12, 128.09, 128.03, 127.84,
127.72, 127.63, 127.49, 127.45, 127.36, 127.22, 126.28, ano-
meric carbons (102.70, 102.35, 101.66, 100.44, 98.58, 97.50),
84.10, 79.86, 79.72, 78.07, 77.77, 76.13, 76.01, 75.62, 75.08,
74.92, 74.82, 73.66, 73.50, 73.41, 73.28, 73.24, 73.01, 72.96,
72.64, 72.57, 72.45, 71.01, 68.67, 68.13, 67.76, 65.88, 60.39,
27.84, 26.84, 17.52, 16.66, 16.38, 14.20, 13.60, 6.88, 4.90; LRMS
(ESI) 2281[M + Na+] HRMS (FAB) calcd for C132H151NO28-
SSiNa, 2280.9809 found 2280.9812 [M + Na+].
LeY Hexasaccharide Glycal 36. To a solution of 35 (208
mg, 0.0871 mmol) in THF (5 mL) was added a solution of 1 M
TBAF/THF and CH3COOH (1:1, 0.348 mmol, 4 equiv). The
resulting solution was stirred at room temperature for 15 h,
diluted with EtOAc (60 mL), and washed with saturated
aqueous NaHCO3 (2 × 30 mL). The organics were dried over
MgSO4, filtered, and concentrated. The crude product was
purified by FC (1:1 EtOAc/hexanes) to give 192 mg (97%) of
36 as a white foam: [R] ) -21.8 (c 1, CHCl3); IR (thin film)
3377, 2931, 2860, 1742, 1466, 1396, 1366, 1266, 1049, 1008,
1
949, 861, 793, 723; H NMR (500 MHz, CDCl3) δ 8.08 (d, J )
7.7 Hz, 2H), 7.50-7.03 (multiple protons, Bn, Ph), 6.34 (d,
J ) 6.0 Hz, 1H), 5.68 (d, J ) 2.7 Hz, 1H), 5.41 (d, J ) 8.0 Hz,
1H), 5.31 (d, J ) 3.5 Hz, 1H), 5.21 (d, m, 1H), 4.92 (d, J ) 9.6
Hz, 1H), 4.81-4.64 (m, 11 H), 4.62 (d, J ) 11.3 Hz, 2H), 4.55-
4.28 (m, 20 H), 4.15-3.98 (m, 4H), 3.94 (d, J ) 1.6 Hz, 1H),
3.90-3.70 (m, 12 H), 3.64 (dd, J ) 6.1 Hz, J ) 3.3 Hz, 1H),
3.55 (dd, J ) 10.2 Hz, J ) 2.8 Hz, 1H), 3.44 (dd, J ) 9.6 Hz,
J ) 2.5 Hz, 1H), 3.40-3.22 (m, 5H), 2.02 (s, 3H), 1.95 (s, 6H),
1.35 (d, J ) 6.6 Hz, 3H), 1.18 (d, J ) 6.4 Hz, 3H); 13C NMR
(500 MHz, CDCl3) δ 172.66, 170.64, 170.38, 143.76, 139.70,
138.77, 138.68, 138.39, 138.23, 137.73, 137.28, 130.31, 128.63,
128.52, 128.47, 128.42, 128.39, 128.37, 128.33, 128.16, 128.08,
128.06, 128.03, 128.01, 127.95, 127.89, 127.61, 127.58, 127. 33,
127.31, 126.94, 125.99, anomeric carbons (103.18, 101.87,
101.66, 99.69, 97.97, 96.69), 83.98, 80.64, 79.87, 78.98, 78.25,
76.76, 76.02, 75.55, 74.87, 74.39, 74.35, 74.17, 73.74, 73.65,
73.64, 73.58, 73.38, 73.22, 72.52, 72.40, 72.35, 72.16, 71.56,
68.34, 66.92, 66.84, 53.26, 29.70, 24.51, 21.02, 20.85, 16.56,
16.26, 14.22, 14.03; LRMS (ESI) 2292.9[M + Na+] HRMS
(FAB) calcd for
C132H143O31NSNa 2292.9261818, found
2292.9232[M + Na+].
LeY Heptasaccharide Glycal 37. A mixture of LeY hexa-
saccharide glycal 36 (192 mg, 0.0845 mmol) and â-fluoroucose
10 (148 mg, 0.338 mmol, 4 equiv) was dried azeotropically with
benzene (3 × 3 mL) and under high vacuum for 2 h. Freshly
activated 4 Å molecular sieves (300 mg) were added to the
mixture under Ar, followed by toluene (5 mL). The suspension
was cooled to 0 °C, and 2,6-di-tert-butyl pyridine (0.190 mL,
0.85 mmol, 10 equiv) was added, followed by a solution of Sn-
(OTf)2 (71 mg, 0.169 mmol, 2 equiv) in THF (0.55 mL). The
suspension was allowed to warm slowly to room temperature,
stirred for 48 h, and then quenched with Et3N (1 mL), diluted
with EtOAc (60 mL), and washed with saturated aqueous
NaHCO3 and brine. The organics were dried over MgSO4,
filtered, and concentrated. The crude product was purified by
FC (15-25% EtOAc/hexane to afford 109 mg (48%) of 37 as
white foam: [R] ) -78 (c 0.7, CHCl3); IR (thin film) 3360, 3027,
Acetylated Hexasaccharide 35. To a solution of LeY
hexasaccharide 34 (256 mg, 0.113 mmol), a catalytic amount
of DMAP, and TEA (0.108 mL) in CH2Cl2 (4 mL) was added
Ac2O (0.107 mL, 1.13 mmol), and the mixture was stirred for
24 h. EtOAc (60 mL) was added and the solution washed with
saturated aqueous NaHCO3 (3 × 30 mL), dried over MgSO4,
filtered, concentrated, and purified by FC (25% EtOAc/hex-
anes) to afford 217 mg (80%) of 35 as a white solid: [R] ) -68.8
(c 1, CHCl3); IR (thin film) 2948, 2868, 1746, 1706, 1646, 1497,
J. Org. Chem, Vol. 70, No. 9, 2005 3393

Spassova et al.
2871, 1957, 1879, 1816, 1749, 1702, 1650, 1577, 1452, 1362,
3.5 Hz, 1H), 5.07-4.95 (m, 8H), 4.67 (d, J ) 11.0 Hz, 1H), 4.62
(d, J ) 7.9 Hz, 1H), 4.57 (d, J ) 8.1 Hz, 1H), 4.51 (q, 2H), 4.42
(d, J ) 7.6 Hz, 1H), 4.40 (d, J ) 8.2 Hz, 1H), 4.35-4.30 (m,
3H), 4.26 (d, J ) 7.8 Hz, 1H), 4.10 (q, J ) 11.5 Hz, J ) 3.8 Hz,
1H), 4.08-3.69 (m, 11H), 3.46 (m, 3H), 2.92(bs, 1H), 2.19-
1.89 (m, 54H), 1.22 (d, J ) 6.6 Hz, 6H), 1.18 (d, J ) 6.3 Hz,
3H); 13C NMR (CDCl3) δ 171.56, 171.34, 171.27, 171.16, 171.05,
170.90, 170.83, 170.79, 170.76, 170.65, 170.59, 170.38, 170.20,
170.16, 169.81, 133.75, 119.06, anomeric carbons (101.74,
101.01, 100.85, 99.39, 96.69, 96.38, 96.08), 78.11, 76.76, 75.69,
74.35, 73.90, 73.76, 73.38, 73.05, 72.01, 71.82, 71.68, 71.37,
71.22, 71.11, 70.98, 69.01, 68.61, 68.55, 68.32, 68.12, 67.88,
67.21, 65.54, 64.38, 61.27, 60.87, 30.09, 23.86, 21.52, 21.38,
21.35, 21.29, 21.26, 21.18, 21.14, 21.08, 21.0116.32, 15.98;
LRMS (ESI) 1922.8 [M + Na+]; HRMS calcd for C81H113NO50-
Na 1922.6226, found 1922.6227.
Allyl Glycoside of Ley Heptasaccharide 40. To a solution
of hexasaccharide 39 (23 mg, 0.0121 mmol) in MeOH (0.5 mL)
was added NaOMe (5%, 0.235 mL). After 12 h, the mixture
was neutralized with Dowex 50-x8, filtered, and concentrated.
Purification of the residue with RP-18 reversed-phase silica
gel (5% MeOH/H2O) gave 12.9 mg (90%) of 40 as a white
solid: [R] ) -31 (c 0.23, MeOH); IR (thin film) 3411, 2975,
2891, 1585, 1416, 1355, 1077, 1022, 768; lH NMR (D2O) δ 5.86
(m, 1H, CHdCH2), 5.34 (d, J ) 3.9 Hz, 1H), 5.24 (dd, J ) 17.2
Hz, J ) 1.7, 1H), 5.07 (d, J ) 3.3, 2H), 5.06 (d, J ) 1.7 Hz,
1H), 4.7 (d, J ) 3.9 Hz, 1H), 4.59 (d, 8.3 Hz, 1H), 4.42 (m,
1H), 4.32 (d, J ) 7.8 Hz, 1H), 4.22 (d, J ) 7.9 Hz, 1H), 4.11-
3.25 (multiple protons), 1.9 (s, 3H), 1.15 (m, 6H), 1.06 (d, J )
6.6 Hz, 3H); LRMS (ESI) 1208.4 [M + Na+]; HRMS (FAB) calcd
for C47H79NO33Na 1208.4431, found 1208.4433.
1
1301, 1223, 1156, 1098, 1020, 739, 690; H NMR (500 MHz,
CDCl3) δ 8.09 (d, J ) 7.5 Hz, 2H), 7.53-7.10 (multiple protons,
Bn, Ph), 7.01 (m, 2H), 6.94 (d, J ) 6.8 Hz, 2H), 6.41 (d, J )
6.1 Hz, 1H), 5.67 (d, J ) 4.6 Hz, 1H), 5.45-5.37 (m, 2H), 5,
19-4.90 (m, 3H), 4.87 (d, J ) 4.4 Hz, 1H), 4.82-4.44 (m, 26H),
4.41-3.44 (m, 35H), 3.40 (d, J ) 2.8 Hz, 1H), 3.36 (d, J ) 10.5
Hz, 1H), 3.26-3.21 (m, 2H), 2.02 -1.83 (m, 9H), 1.35 (d, J )
6.1 Hz, 3H), 1.18 (d, J ) 6.1, 1.05 (d, J ) 6.4 Hz, 3H); 13C
NMR (500 MHz, CDCl3) δ 172.73, 170.22, 144.86, 139.75,
138.79, 138.69, 138.62, 138.06, 137.78, 130.40, 128.63, 128.54,
128.40, 128.25, 128.19, 128.15, 128.10, 128.05, 127.96, 127.91,
127.73, 127.61, 127.54, 127.48, 127.43, 127.39, 127.07, 126.97,
126.87, 126.02, anomeric carbons (101.77, 101.67, 99.90, 99.61,
98.04, 96.67, 94.14), 83.99, 79.87, 78.29, 75.58, 75.37, 74.99,
74.47, 74.37, 74.22, 74.13, 73.79, 73.59, 73.40, 73.15, 73.08,
72.56, 72.38, 72.23, 71.58, 70.90, 67.81, 67.74, 66.99, 66.85,
29.72, 24.60, 20.98, 20.87, 16.66, 16.58, 16.30; LRMS (ESI)
calcd for C159H171O35NSNa 2709.1249, found 2708.6 [M + Na+].
LeY Heptasaccharide Glycal Peracetate 38. A solution
of heptasaccharide 37 (51 mg, 0.0188 mmol) in THF (1.5 mL)
was added via cannula to a solution of sodium (72 mg, 3.13
mmol) in liquid ammonia (10 mL) under Ar at -78 °C. The
reaction mixture was stirred for 40 min, quenched with MeOH
(2 mL), stirred for 15 min, and concentrated with a stream of
dry Ar. MeOH (5 mL) was added to the residue, followed by
NH4Cl (168 mg, 3.13 mmol), and the resulting solution was
stirred for 80 min and then concentrated to dryness. The crude
product was suspended in a mixture of THF (1.5 mL), DMF
(0.5 mL), and TEA (0.6 mL). To this were added Ac2O (0.32
mL, 3.4 mmol) and a catalytic amount of DMAP, and the
reaction mixture was stirred at room temperature for 10 h and
then poured into ice-water (10 mL) and extracted with EtOAc
(3 × 50 mL). The organics were washed with saturated
aqueous NaHCO3 and H2O, dried over MgSO4, filtered, and
concentrated. Purification of the crude product by FC (65%
EtOAc/CH2Cl2) afforded 23 mg (67%) of 38 as a white solid:
[R] ) -27.3 (c 0.7, CH2Cl2); IR (thin film) 3365, 2954, 1742,
1678, 1537, 1443, 1372, 1231, 1137, 1072, 967, 912, 737, 696;
1H NMR (CDCl3) δ 6.30 (d, J ) 6.2 Hz, 1H), 5.38-5.26 (m,
6H), 5.21 (d, J ) 4.1 Hz, 1H), 5.18 (d, J ) 3.4 Hz, 1H), 5.15
(m, 1H), 5.10(dd, J ) 11 Hz, J ) 3.1 Hz, 1H), 5.06-4.87 (m,
8H), 4.68 (q, J ) 6.2 Hz, J ) 3.3 Hz, 1H), 4.61 (d, J ) 7.9 Hz,
1H)), 4.55-4.41 (m, 4H), 4.36 (d, J ) 6.6 Hz, 1H), 4.30 (t, J )
9 Hz, 1H), 4.25-3.97 (m, 9H), 3.88-3.69 (m, 6H), 3.37 (d, J )
9.8 Hz, 1H), 2.86 (bs, 1H), 2.12-1.83 (m, 54 H), 1.14 (d, J )
6.5 Hz, 3H), 1.11 (d, J ) 6. Hz, 6H); 13C NMR (500 MHz,
CDCl3) δ 170.16, 169.92, 169.73, 169.68, 169.64, 169.56, 169.42,
169.25, 169.17, 168.98, 168.85, 168.78, 168.67, 143.77, ano-
meric carbons (99.42, 99.00, 98.11, 96.78, 95.25, 94.65, 91.49),
75.15, 73.64, 72.97, 72.31, 71.97, 71.55, 71.30, 70.49, 70.39,
70.26, 69.91, 69.81, 69.52, 67.67, 67.11, 67.07, 66.87, 66.70,
66.49, 65.86, 64.09, 63.54, 62.97, 60.44, 60.20, 59.48, 28.68,
22.44, 20.12, 19.96, 19.91, 19.77, 19.75, 19.72, 19.69, 19.65,
19.60, 14.90, 14.54; LRMS (ESI) 1848.5 [M + Na+]; HRMS
(FAB) calcd for C78H107NO48Na 1848.5859, found 1848.5867.
LeY Nonasaccharide Glycal 42. A mixture of LeY hep-
tasaccharide 37 (59 mg, 0.022 mmol) and benzenesulfonamide
(10 mg, 0.065 mmol) was dried azeotropically with benzene
(3 × 3 mL) and under high vacuum for 2 h. Freshly activated
4 Å molecular sieves (60 mg) were added to the mixture under
Ar, followed by CH2Cl2 (1 mL). The resulting suspension was
cooled to 0 °C, and I (sym-coll)2ClO4 (36 mg, 0.077 mmol) was
added under Ar. The reaction mixture was stirred at 0 °C for
40 min, quenched with saturated aqueous Na2S2O3 (1 mL),
diluted with EtOAc (20 mL), and then filtered through Celite.
The filtrate was washed with saturated aqueous Na2S2O3
(2 × 10 mL), saturated aqueous CuSO4 (2 × 10 mL), and brine
(1 × 10 mL). The organics were dried over MgSO4, filtered,
and concentrated. The residue was taken up in 30% EtOAc/
hexanes and passed through a short SiO2 column. The collected
iodosulfonamide 41 was dried (azeotropically with benzene and
under high vacuum for 3 h) and used without further purifica-
tion. Freshly activated 4 Å molecular sieves (40 mg) were
added to 41 under Ar, followed by solution of tin ether of 8
(prepared from 35 mg of 19 and 0.018 mL of (Bu3Sn)2O (0.018
mL) as described for 21). The suspension was cooled to -70
°C and AgBF4 (10 mg) in THF (1 mL) added via cannula. The
reaction mixture was stirred with exclusion of light, allowed
to warm slowly during about 5 h, and then stirred at room
temperature for 3 days. EtOAc was added, and the mixture
was filtered through Celite. The filtrates were diluted ad-
ditionally with EtOAc and washed with saturated aqueous
solution of NaHCO3 (3 × 30 mL) and brine (1 × 30 mL). The
organics were dried over MgSO4, filtered, concentrated, and
chromatographed (FC 27-50% EtOAc/hexanes) to give 34 mg
of 42 (32% overall yield for the two steps): [R] ) -25.7 (c 1,
CH2Cl2); IR (thin film) 3477, 3335, 3050, 2931, 2860, 1741,
Allyl Glycoside of Ley Heptasaccharide Peracetate 39.
A mixture of Ley heptasaccharide glycal 38 (23 mg, 0.0125
mmol, dried azeotropically with benzene (3 × 5 mL) and under
high vacuum for 2 h) and freshly activated 4 Å powdered
molecular sieves (30 mg) was suspended in CH2Cl2 (1 mL) and
cooled to 0 °C. DMDO (0.6 mL, 0.11 M solution in Me2CO)
was then added. The reaction mixture was stirred at 0 °C for
1 h, the solvent was evaporated, and the residue was taken
up in allyl alcohol (5 mL). This mixture was stirred at room
temperature for 48 h, the solvent was evaporated, and the
product was purified by FC (60% EtOAc/CH2Cl2) to afford 23
mg (92%) 39 as a white solid: [R] ) -30.5 (c 1, CH2Cl2); IR
(thin film) 3375, 2999, 1749, 1664, 1531, 1428, 1367, 1222,
1071, 1035, 974, 944, 732; 1H NMR (CDCl3) δ 5.90 (m, 1H,
CHdCH2), 5.63 (d, J ) 3.7 Hz, 1H), 5.41 (d, J ) 3.5 Hz, 2H),
5.35-5.22 (m, 9H), 5.16 (dd, J ) 10.6 Hz, 1H),5.12 (ds, J )
1
1706, 1445, 1362, 1225, 1166, 1101, 1053, 733; H NMR (500
MHz, CDCl3) δ 8.10(d, J ) 7.7 Hz, 2H), 7.80 (d, J ) 7.5 Hz,
1H), 7.55-6.92 (m, 98H), 6.40 (d, J ) 6.0 Hz, 1H), 5.67 (d,
J ) 3.7 Hz, 1H), 5.42-5.39 (m, 2H), 5.21-5.15 (m, 1H), 4.94-
3.21 (m, 90H), 2.03 (s, 3H), 2.01 (s, 3H), 1.98 (s, 3H), 1.35 (d,
J ) 6.4 Hz, 3H), 1.18 (d, J ) 6.1 Hz, 3H), 0.90 (s, J ) 6.4 Hz,
3H); 13C NMR (500 MHz, CDCl3) δ 172.74, 171.01, 170.45,
144.58 (anomeric olefinic), 139.71, 138.84, 138.69, 138.54,
138.43, 137.82, 137.76, 133.94, 132.40, 130.38, 128.88, 128.63,
3394 J. Org. Chem., Vol. 70, No. 9, 2005

Synthesis of Selected LeY and KH-1 Analogues
128.55, 128.48, 128.45, 128.40, 128.36, 128.32, 128.26, 128.22,
128.11, 128.07, 127.97, 127.92, 127.87, 127.75, 127.69, 127.65,
127.61, 127.57, 127.52, 127.43, 127.36, 127.28, 127.14, 127.00,
126.97, 126.88, 126.44, 126.02, 109.20, anomeric carbons
(103.29, 102.84, 101.69, 100.50, 100.30, 99.61, 98.03, 96.69,
96.13), 83.93, 79.87, 78.97, 78.33, 75.60, 75.41, 74.98, 74.67,
74.40, 74.15, 74.11, 73.82, 73.49, 73.39, 72.65, 72.43, 72.28,
71.63, 70.90, 70.74, 69.06, 67.69, 67.11, 66.86, 55.34, 53.74,
48.11, 46.47, 44.53, 29.71, 24.57, 21.07, 20.95, 16.57, 16.28,
14.15, 11.47; LRMS calcd for C198H214O46N2S2‚2Na 3465.3703,
found 3465.4 [M + 2Na2+].
Acknowledgment. We thank Prof. W. F. Berkowitz
for discussions and editorial help, Mrs. R. Lambert for
assistance in the preparation of the manuscript, and Dr.
Sylvi Rusli, M.D. and Dr. Anna Dudkina for MS and
HPLC/MS.
Supporting Information Available: Experimental de-
tails and 1H and 13C NMR spectra. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO048016L
J. Org. Chem, Vol. 70, No. 9, 2005 3395