Synthetic and immunological studies on clustered modes of mucin-related Tn and TF O-linked antigens: The preparation of a glycopeptide-based vaccine for clinical trials against prostate cancer

Article abstract of DOI:10.1021/ja9825128

The syntheses of two tumor-associated carbohydrate antigens, Tn and TF, have been achieved using glycal assembly and cassette methodologies. These synthetic antigens were subsequently clustered (c) and immunoconjugated to a carrier protein (KLH or BSA) or

Full text of DOI:10.1021/ja9825128

12474
J. Am. Chem. Soc. 1998, 120, 12474-12485
Synthetic and Immunological Studies on Clustered Modes of
Mucin-Related Tn and TF O-Linked Antigens: The Preparation of a
Glycopeptide-Based Vaccine for Clinical Trials against Prostate
Cancer†
Scott D. Kuduk,‡ Jacob B. Schwarz,‡ Xiao-Tao Chen,§ Peter W. Glunz,‡ Dalibor Sames,‡,§
Govindaswami Ragupathi, Philip O. Livingston, and Samuel J. Danishefsky*,‡,§
Contribution from the Laboratory for Bioorganic Chemistry and Laboratory for Tumor Vaccinology,
Sloan-Kettering Institute For Cancer Research, 1275 York AVenue, New York, New York 10021, and
Department of Chemistry, Columbia UniVersity, New York, New York 10027
ReceiVed July 16, 1998
Abstract: The syntheses of two tumor-associated carbohydrate antigens, Tn and TF, have been achieved using
glycal assembly and cassette methodologies. These synthetic antigens were subsequently clustered (c) and
immunoconjugated to a carrier protein (KLH or BSA) or a synthetic lipopeptide (pam) for immunological
study. Three Tn conjugates were used to vaccinate groups of mice, and all preparations proved to be
immunogenic. The Tn(c) covalently linked to KLH (27-KLH) plus the adjuvant QS-21 was the optimal
vaccine, inducing high median IgM and IgG titers against Tn(c) by ELISA. These antibodies were strongly
reactive with the Tn(c) positive human colon cancer cell line LS-C but not the Tn(c) negative colon cancer
cell line LS-B by FACS. The antibodies’ reactivities with natural antigens were inhibited with synthetic Tn(c)
but not with structurally unrelated compounds. On the basis of these results, vaccines containing 27-KLH
and 30-pam plus QS-21 are being tested in patients with prostate cancer.
Introduction
in the search for synthetic antitumor and other vaccines.3 Such
investigations had been greatly aided by preparative powers of
peptide chemistry, and particularly by solid phase synthesis of
peptides and recombinant technology. However, immune
responses to nonpeptidic substances, such as carbohydrates,
although highly abundant on surfaces of viruses, bacteria, and
tumor tissues, remain poorly understood partly due to a lack of
experimentation on telling probe structures. This situation
reflects, in many instances, the complexity in the synthetic
methodology required to reach adequate quantities of informa-
tive goal systems. In our continuing chemical studies, we have
undertaken the development of synthetic methodology of general
applicability for the preparation of carbohydrates, in the form
of glycolipids and glycopeptides, which mimic components of
the accessible cell surface of tumor cells. The disclosure herein
focuses on mucin-related O-linked glycopeptides, in particular
the Tn and TF antigens.
The development of efficient routes for the preparation of
complex oligosaccharide or carbohydrate conjugates has been
our goal for some time.1 Synthetic investigations in this area
can help to provide a detailed knowledge of the structural and
chemical behavior of carbohydrates and their conjugates.
Furthermore, issues related to biological function of glycocon-
jugates can be evaluated, provided suitable quantities of
informative probe structures can be constructed.
With time, we became interested in using chemistry to
examine an exciting possibility. The goal would be that of
recruiting the immune system to respond to malignant lesions.
We were particularly drawn to the concept of inducing “active
immunity” by synthetic vaccines.2 Peptidic molecules, whose
immunobiology has been studied extensively, have been used
† Abbreviations: BSA, bovine serum albumin; DAST, diethylaminosul-
furtrifluoride; DIEA, diisopropylethylamine; ELISA, enzyme-linked im-
munosorbent assay; FMOC, fluorenylmethoxycarbonyl; GalNAc, N-acetyl-
galactosamine; IIDQ, 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihryoquinoline;
HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate; KLH, keyhole limpet hemacyanin; pam, palmitoylcysteine;
MBS, m-maleimimidobenzoyl-N-hydroxysuccinimide ester; NHS, N-hy-
droxysuccinimide; SAMA-OPfp, S-acetylthioglycolic pentafluorophenyl
ester; TFA, trifluoroacetic acid; Tn(c), Tn cluster.
Mucins, which comprise a family of large glycoproteins
expressed on cells of epithelial tissues, carry large glycodomains
in clustered modes.4 Mucin amino acid sequences possess a
very high percentage of serine and threonine residues, often
found in contiguous arrays ranging in number from two to five.
In most cases, the details of the occupancy of such blocks of
serine and threonine subunits is not known in detail.5 Despite
a large variety of mucin glycostructures, the modality wherein
the first residue, an N-acetylgalactosamine moiety, is linked to
‡ Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute For
Cancer Research.
§ Department of Chemistry, Columbia University.
Laboratory for Tumor Vaccinology, Sloan-Kettering Institute For
Cancer Research.
(1) Danishefsky, S. J.; Bilodeau, M. T. Angew. Chem., Int. Ed. Engl.
1996, 35, 1381.
(3) (a) Slingluff, C. L., Jr.; Hunt, D. F.; Engelhard, V. H. Curr. Opin.
Immunol. 1994, 6, 733. (b) Berzofsky, J. A. Ann. N.Y. Acad. Sci. 1993,
609, 256. (c) Hill, A. V. Nature 1992, 360, 434.
(2) Ragupathi, G.; Park, T. K.; Zhang, S.; Kim, I. J.; Graber, L.; Adluri,
S.; Lloyd, K. O.; Danishefsky, S. J.; Livingston, P. O. Angew. Chem., Int.
Ed. Engl. 1997, 36, 125.
(4) Carlstedt, I.; Davies, J. R. Biochem. Soc. Trans. 1997, 25, 214.
(5) Poland, P. A.; Kinlough, C. L.; Rokaw, M. D.; Magarian-Blander,
J.; Finn, O. J.; Hughey, R. P. Glycoconjugate J. 1997, 14, 89.
10.1021/ja9825128 CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/20/1998

Mucin-Related Tn and TF O-Linked Antigens
J. Am. Chem. Soc., Vol. 120, No. 48, 1998 12475
Figure 1.
Figure 2.
a serine or threonine residue Via an R-linkage appears to be
broadly conserved (Figure 1). Interestingly, the significant
changes of the glycopatterns during the malignant transformation
generally result in shorter carbohydrate chains.6 The Tn antigen
represents the simplest member of the family. This antigen, as
well as the related Thomsen-Friedenreich disaccharide (TF)
antigen, is quite common in carcinoma malignancies, particularly
of the colon and prostate.7 Simple carbohydrate antigens have
been synthesized, and their immunogenicity in conjugate
vaccines has been confirmed.8 For example, antibody titers
against STn have been reported to correlate with improved
prognosis in breast cancer patients.9 Comparable studies with
more complex carbohydrates have rarely been described, thus
the clear interest in large clustered forms of antigens.
Given this context, we set for ourselves several goals. The
first was the development of synthetic methodology to gain
access to the clustered antigen motifs. We would go on from
the chemistry phase to evaluate the immunogenicity of the
clustered glycopeptide fragments in mice. The longer range
goal would be the development and evaluation of antitumor
glycopeptide-based vaccines.10 In this paper we report the
realization of these goals up to the point of the clinical trials
which have just begun.
Figure 3.
Synthetic Analysis
suitably conjugated to immunogenic stimulants such as bacterial
lipopeptides or carrier proteins, would then be evaluated in
biological studies for antibody production, and eventual clinical
application.
We have previously demonstrated that even bulky glycosyl
amino acids can be efficiently incorporated into peptide
backbones.11 In light of these results, we opted to construct
the glycosyl amino acids representing the Tn and TF epitopes.
The amino acid domains would be protected in a fashion which
anticipates the requirements of peptide assembly to form the
clustered epitopes as shown in Figure 2. These constructs,
Synthetic Planning
For the synthesis of the Tn and TF antigens, we turned to
our basic glycal assembly1 logic. The key starting point would
be the appropriately protected galactal, which could be used to
produce both mono- and disaccharides. Azidonitration12 of these
glycals according to Lemieux might be used to introduce the
2-azido group and to produce the functionality at the anomeric
carbon required for the fashioning of the prerequisite donor.
Subsequent glycosylation of such a construct with the ap-
propriately protected serine or threonine would pave the way
for reaching our conjugates. An alternative and potentially more
general method would involve building a shorter Tn construct
as a “cassette”, which would have the R-O-linked amino acid
prebuilt into the GalNAc. This cassette might serve as a general
acceptor to be inserted in the late stages of synthesis of virtually
any O-linked glycopeptide goal structures. Such an approach,
captured in Figure 3, might sidestep the serious difficulty of
designing ad hoc methods for achieving high anomeric selectiv-
(6) Schachter, H.; Brockhausen, I. In Glycoconjugates: Composition,
Structure and Function; Allen, H. J., Kisailus, E. C., Eds.; Dekker: New
York, 1992; p 263.
(7) Springer, G. F. Science 1984, 224, 1198.
(8) (a) Springer, G. F. Clin. ReV. Oncogenesis 1995, 6, 57. (b) MacLean,
G. D.; Reddish, M. A.; Bowen-Yacyshyn, B. B.; Poppema, S.; Longenecker,
B. M. Cancer InVest. 1994, 12, 46. (c) Jennings, H. J.; Ashton, F. E.;
Gamian, A.; Michon, F. In Towards Better Carbohydrate Vaccines; Roy,
R., Bell, R., Torrigiani, G., Eds.; London: J. Wiley & Sons, Ltd., 1987; pp
11-17.
(9) Schneerson, R.; Robbinson, J. B.; Szu, S. C.; Yang, Y. Vaccines
composed of polysaccharide-protein conjugates: current status, unanswered
questions, and prospects for the future. In Towards Better Carbohydrate
Vaccines; Bell, R., Torrigiani, G., Eds.; J. Wiley & Sons, Ltd.: London,
1987; pp 307-327.
(10) (a) Koganty, R. R.; Reddish, M. A.; Longenecker, B. M. In
Glycopeptides and Related Compounds. Synthesis, Analysis and Applica-
tions; Large, D. G., Warren, Ch. D., Eds.; Marcel Dekker, Inc.: New York,
1997; p 707. (b) Ragupathi, G.; Koganty, R. R.; Qui, D. X.; Llioyd, K. O.;
Livingston, P. O. Glycoconjugate J. 1998, 15, 217.
(11) Sames, D.; Chen, X. T.; Danishefsky, S. J. Nature 1997, 389, 587.
(12) Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57, 1244.

12476 J. Am. Chem. Soc., Vol. 120, No. 48, 1998
Kuduk et al.
Scheme 1
Scheme 2
°
Scheme 3
ity in glycosylations of the side chain hydroxyls of serine and
threonine for each new construct we hope to build.13
Results and Discussion
The Tn antigen had previously been prepared by a number
of methods.14 We chose a method to prepare the peracetylated
form of the antigen, which is compatible with both solution
and solid phase peptide chemistry. Thus, glycosylation of
known bromide 115 with N-Fmoc-serine or threonine benzyl
ester afforded 2 or 3 as a separable 4:1 R/â mixture of anomers
in 60% yield (Scheme 1). Reductive acetylation using neat
thiolacetic acid produced fully protected Tn antigens 4 and 5.
Careful hydrogenolysis afforded the peracetylated acids 6 and
7 in 75% yield, while Fmoc removal gave 8 and 9, which were
in principle available for glycopeptide assembly.
The mediocre yield of glycosylation and the serious incon-
venience of the need for anomer separation prompted a search
for an alternative method. As matters turned out, another
program in our laboratory, moving forward, had been directed
to a total synthesis of the F1R antigen.16 In screening for
efficient serine or threonine glycosylation reactions, it was found
that compounds 10 and 11 could be prepared as shown in
Scheme 2. In the case of serine-derived acceptor the glycosy-
lation ratio apparently gave only R-product 10. In the synthesis
of the threonine product 11, a small amount of â-product was
noted. In both cases the yields were far superior to those
obtained from 1. The idea then emerged to use 10 and 11 as
general inserts (cassettes) to be installed toward the end of a
complex synthesis. Thus, we need only solve the very difficult
O-linkage problem once for a given “reducing end” and exploit
that capability for building on the desired clustered system.
To implement this strategy, it would be necessary to fashion
from the cassettes a variety of orthogonally protected modules
for further use as glycosyl acceptors. In the case at hand, simple
TBAF-mediated desilylation of 10 or 11 affords position 6
acceptor 12 or 13, respectively, in excellent yield (Scheme 3).
Moreover, removal of both the TIPS and acetonide groups using
I2 in MeOH17 gave rise to the versatile triol 14 or 15, which
could readily be converted to the peracetyl Tn precursors 4 and
5. Alternatively the triols could be transformed, via resilylation
with TBSCl, into diol 3 acceptor 16 or 17,18 or by benzylidene
formation19 to give specific position 3 acceptor 18 or 19. We
have used this methodology to prepare substantial quantities of
the Tn antigen. In addition, we have used these intermediates
for the efficient preparation of several O-linked tumor-associated
antigens such as TF (vide infra) and sialosyl-Tn and sialosyl-T
antigens.20
The amino acid sequence we chose for these clusters was to
have three consecutive serines or threonines. Due to the lack
of reliable information regarding which serine or threonine
residues within a contiguous array constitutes an optimal epitope,
we selected such trimers for the initial evaluation. The synthesis
(13) For reviews on the synthesis of R-O-linked serine or threonine
glycopeptides, see: Kunz, H. Pure Appl. Chem. 1993, 65, 123-1332. Kunz,
H. AdV. Carbohydr. Chem. Biochem. 1994, 50, 277.
(14) For examples of previous Tn syntheses, see: Toyokuni, T.;
Hakomori, S.; Singhal, A. K. Bioorg. Med. Chem. 1994, 11, 1119 and
references therein.
(17) Szarek, W. A.; Zamojski, A.; Tiwari, K. N.; Isoni, E. R. Tetrahedron
Lett. 1986, 27, 3827.
(18) Nakahara, Y.; Nakahara, Y.; Ito, Y.; Ogawa, T. Tetrahedron Lett.
1997, 38, 7211-7214.
(15) (a) Paulsen, H.; Holck, J. P. Carbohydr. Res. 1982, 109, 89-107.
(b) Paulsen, H.; Kolar, C. Chem. Ber. 1981, 114, 306.
(16) Chen, X. T.; Sames, D.; Danishefsky, S. J. J. Am. Chem. Soc. 1998,
120, 7700-7709.
(19) Meinjohanns, E.; Meldal, M.; Schleyer, A.; Paulsen, H.; Bock, K.
J. Chem. Soc., Perkin. Trans. 1996, 1, 985-993.
(20) Schwarz, J. B.; Kuduk, S. D.; Chen, X. T.; Sames, D.; Glunz, P.
W.; Danishefsky, S. J. J. Am. Chem. Soc., submitted.

Mucin-Related Tn and TF O-Linked Antigens
J. Am. Chem. Soc., Vol. 120, No. 48, 1998 12477
Scheme 4
Scheme 5
of the clustered Tn glycopeptides began with IIDQ-mediated
coupling between acid 6 or 7 and amine 8 or 9 to afford the
corresponding dipeptides (see 20 and 21) in 97% yield (Scheme
4). The Fmoc-carbamate was then deprotected with neat
morpholine, and the resulting amine was resubjected to a second
IIDQ coupling. This step was followed by deprotection and
acetyl capping of the N-terminus to afford the tripeptides (see
22 and 23). The glycosylated tripeptide thus obtained was
subjected to hydrogenolysis to afford acids (see 24 and 25),
which could now be further modified and conjugated to either
a synthetic lipopeptide as the immunological activator or to an
immunogenic carrier protein.
As shown in Scheme 5, two pathways were followed for
eventual conjugation. The first involved attachment of a suitable
linker to conjugate with a carrier protein. The mercaptoaceta-
mide unit has proven to be effective for this purpose.21 Acid
24 was coupled with tert-butyl-N-(3-aminopropyl)carbamate Via
the agency of IIDQ. This step was followed by removal of the
BOC cap with TFA. The resulting amine was then coupled
with S-acetylthioglycolic acid pentafluorophenyl ester (SAMA-
OPfp, NovaBiochem) in the presence of Hunig’s base in 81%
two-step yield. The resulting fully protected glycopeptide 26
was then subjected to methanolysis under carefully controlled
conditions (pH ∼9, degassed MeOH) to give 27 in 85% yield.
The latter was now ready to be conjugated to the appropriate
carrier protein.
For the synthesis of a fully synthetic lipopeptide, we followed
Toyokuni’s method attaching tripalmitoyl-S-glycerylcysteinylserine
(Pamcys).22 Pamcys has proven to be a potent macrophage and
B lymphocyte activator, and has been used for purposes similar
to ours by Tokoyuni with one to three epitopes of serine Tn.23
First, careful saponification of 24 or 25 with NaOMe/MeOH
gave the fully deprotected glycopeptide 29 or 30 in 95% yield.
Coupling with amine 28 using either the NHS or HOAt/HATU
method24 afforded glycolipid 31 or 32 in ca. 40% yield.
Fortunately, the starting materials for this reaction can be
recovered.
(22) (a) Toyokuni, T.; Dean, B.; Cai, S.; Boivin, D.; Hakomori, S.;
Singhal, A. K. J. Am. Chem. Soc. 1994, 116, 395. (b) Toyokuni, T.;
Hakomori, S.; Singhal, A. K. Bioorg. Med. Chem. 1994, 11, 1119.
(23) Toyokuni, T.; Singhal, A. K. Chem. Soc. ReV. 1995, 231-242.
(21) Brugghe, H. R. Int. J. Peptide Protein Res. 1994, 43, 166.

12478 J. Am. Chem. Soc., Vol. 120, No. 48, 1998
Kuduk et al.
Scheme 6
42, but gave essentially no selectivity with either AgOTf or
AgClO4, contrary to our previous observations for the 2,6-ST
case.11 Similarly, poor selectivities have been reported for TF
glycosyl bromides and chlorides.30 Likewise, with both R and
â trichloroacetimidates 40, no meaningful selectivity was
observed under a variety of conditions, but yields were generally
good to excellent.31 The glycosyl fluoride 39 also gave no
selectivity and a lower yield. In the past, threonine has in many
cases been observed to give a much higher R-selectivity than
serine. This was not the case for the systems described here.
Also to be noted that the use of BF3 as a promoter, as well as
the use of THF as solvent, afforded no product. Another
observation revealed a curious substrate effect. Thus, with ether
as the solvent, in the case at hand a 3.4:1 â:R ratio was observed.
By contrast in the case of the seemingly similar substrate 43
(Figure 4), R-product was favored 3:1 for this LeY-related
system.32 The only positive feature of this study was that the
R and â glycosylation products could be separated by careful
chromatography.33
Plagued by this poor selectivity, the cassette route was
mobilized for implementation. The idea was to use a glyco-
sylated amino acid with the required R-O-linkage solidly in place
as the glycosyl acceptor. Again, we turned to our glycal
assembly methodology to simplify the construction. Thus, the
epoxide derived from glycal 44 proved to be a powerful donor
in reaction with 19 to afford â-linked disaccharide 45 in 97%
yield (Scheme 7). The disaccharide was readily converted to
42 in high overall yield. The use of such acceptors had been
previously reported by Paulsen for the synthesis of the TF
antigen, using glycosyl bromides34 or trichloroacetimidates.35
The Paulsen method is now complemented by the highly
efficient approach directly from the glycal. It should be noted
that conversion from 6-TIPS to a 6-acetate on the galactal
carbonate was necessary for our glycosylation reaction to
proceed.
Reductive acetylation of 42 was carried out with thiolacetic
acid in 87% yield to afford protected TF antigen 46 (Scheme
8). This step was followed by hydrogenolysis, leading to
protected TF acid 47 in quantitative fashion. At this point, we
decided to attach the protected diamine linker first, and then to
proceed on to the clustering.36 We did so because diminished
yields were encountered for attachment of the linker at a late
stage in the synthesis. This coupling to 48 initially failed when
mediated by IIDQ, but proceeded in 84% yield with HOAt/
HATU and collidine in DMF.37
Synthesis of Clustered TF Antigen. We then turned to the
preparation of the TF disaccharide using the logic of glycal
assembly based upon our experience with 1,2-anhydro sugars
derived from protected galactal. The epoxide derived from
treatment of 33 with DMDO in CH2Cl2 served as an excellent
â-galactosyl donor with 6-TIPS galactal to afford disaccharide
34 in 80% yield25 (Scheme 6). Related approaches for the
synthesis of the TF antigen disaccharide have been reported
using glycosyl bromides and fluorides26 or trichloroacetimidates
as the galactosyl donors.27 At this stage the protecting groups
were exchanged from triisopropylsilyl to acetate to afford 35.
The glycal underwent azidonitration12 efficiently, but with no
selectivity, to furnish 36 as a 1:1 mixture of anomers in 67%
yield. Azidonitration with the TIPS group present at the 6 and
6′ positions gave a substantially lower yield (∼30%) with
significant byproduct formation. The azidonitrate was converted
directly into the labile glycosyl bromide 37 with LiBr in CH3CN
or reduced by PhSH and DIEA in 91% yield. Treatment of 38
with DAST28 in THF afforded glycosyl fluoride 39 in 94% yield
as a 1:1 mixture of R and â isomers, and trichloroacetimidate
formation with Cl3CCN and K2CO3 proceeded smoothly to give
40 as a separable 3:1 mixture of R and â isomers in 92% yield.29
At this stage, the use of 37, 39, and 40 in glycosylation
reactions was investigated. Results are shown in Table 1.
Several interesting observations should be noted. Glycosylation
with N-Fmoc-threonine benzyl ester and bromide 37 afforded
Glycopeptide 48 was then carried through the sequence of
deprotections and coupling with 46 (Scheme 9) similar to that
with Tn except using the HOAt-HATU coupling method37 was
required as IIDQ was extremely sluggish for this system. In
(30) (a) Kunz, H.; Birnbach, S.; Wernig, P. Carbohydr. Res. 1990, 202,
207-223. (b) Paulsen, H.; Peters, S.; Bielfeldt, T.; Meldal, M.; Bock, K.
Carbohydr. Res. 1995, 258, 17-34.
(31) Kinzy, W.; Schmidt, R. R. Carbohydr. Res. 1987, 166, 265-276.
(32) Glunz, P. W.; Hintermann, S.; Danishefsky, S. J. Unpublished
results.
(33) High R-selectivities have been observed for amino acid glycosylation
by Koganty through use of a 4,6-benzylidene acetal on the GalNAc as a
“block donor”, but the yields are slightly reduced: (a) Qui, D.; Gandhi, S.
S.; Koganty, R. R. Tetrahedron Lett. 1996, 37, 595. (b) Yule, J. E.; Wong,
T. C.; Gandhi, S. S.; Qui, D.; Riopel, M. A.; Koganty, R. R. Tetrahedron
Lett. 1995, 38, 6839.
(34) Paulsen, H.; Holck, J. P. Carbohydr. Res. 1982, 109, 89-107.
(35) Meinjohanns, E.; Meldal, M.; Schleyer, A.; Paulsen, H.; Bock, K.
J. Chem. Soc., Perkin. Trans. 1996, 1, 985-993.
(24) (a) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397. (b) Carpino,
L. A.; Ionescu, D.; El-Faham, A. J. Org. Chem. 1996, 61, 2460.
(25) Park, T. K.; Kim, I. J.; Hu, S.; Bilodeau, M. T.; Randolph, J. T.;
Kwon, O.; Danishefsky, S. J. J. Am. Chem. Soc. 1996, 118, 11488.
(26) (a) Kunz, H.; Birnbach, S.; Wernig, P. Carbohydr. Res. 1990, 202,
207-223. (b) Tsuda, T.; Nishimura, S. I. Chem. Commun. 1996, 2779-
2780. (c) Qui, D.; Gandhi, S. S.; Koganty, R. R. Tetrahedron Lett. 1996,
37, 595-598.
(27) Paulsen, H.; Peters, S.; Bielfeldt, T.; Meldal, M.; Bock, K.
Carbohydr. Res. 1995, 268, 17-34.
(28) (a) Rosenbrook, W., Jr.; Riley, D. A.; Larty, P. A. Tetrahedron Lett.
1985, 26, 3. (b) Posner, G. H. and Haines, S. R. Tetrahedron Lett. 1985,
26, 5.
(36) Mono- and diclusters of the TF antigen have been reported: Kunz,
H.; Birnbach, S.; Wernig, P. Carbohydr. Res. 1990, 202, 207-223.
(37) (a) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397. (b) Carpino,
L. A.; Ionescu, D.; El-Faham, A. J. Org. Chem. 1996, 61, 2460.
(29) Schmidt, R. R.; Kinzy, W. AdV. Carbohydr. Biochem. 1994, 50,
84-123.

Mucin-Related Tn and TF O-Linked Antigens
J. Am. Chem. Soc., Vol. 120, No. 48, 1998 12479
a
Table 1.
donor X
37 (Br)
37 (Br)
40 (R-TCA)
40 (â-TCA)
40 (â-TCA)
40 (â-TCA)
39 (â/R-F)
catalyst/solvent/temp
R ) H R:â (yield, %)
R ) CH3 R:â (yield, %)
AgOTf, CH2Cl2, -78 °C to rt
AgClO4, CH2Cl2, -78 °C to rt
TMSOTf (0.5 equiv), CH2Cl2, -30 °C
TMSOTf (0.5 equiv), CH2Cl2, -30 °C
TMSOTf (0.5 equiv), CH2Cl2, -50 °C
TMSOTf (0.5 equiv), Et2O/CH2Cl2 (12:1), -30 °C
Cp2ZrCl2, AgClO4, CH2Cl2, -78 °C to rt
1.2:1 (63)
1.1:1 (73)
2:1 (80)
1.2:1 (72)
1:1 (84)
1:3.4 (75)
1:1 (50)
1:1 (80)
1:1.3 (77)
a Bromide: Donor 37 (1.1 equiv) was added over 30 min to acceptor, catalyst (1.5 equiv), and 4 Å molecular sieves at -78 °C and allowed to
warm to rt overnight. TCA: Donor 40, acceptor (1.5 equiv), and 4 Å molecular sieves were added in the indicated solvent, and TMSOTf was
added at the noted temperatures for 1 h. Fluoride: Donor 37 (1.1 equiv) was added over 30 min to acceptor, catalysts (1.5 equiv of each), and 4
Å molecular sieves at -78 °C and allowed to warm to rt overnight. All yields and ratios are for isolated products.
Scheme 8
Figure 4.
Scheme 9
Scheme 7
Discussion of Immunological Result
The initial experiments were to evaluate the antibody response
to vaccination of mice with either Tn(c) lipopeptide 30 or more
conventional Tn(c)-KLH or -BSA conjugates. The prepara-
tion of these conjugates started with the previously described
27, which was covalently linked with carrier proteins, keyhole
addition, deprotection of the FMOC was facilitated using KF
limpet hemocyanin (KLH), or bovine serum albumin (BSA),
in DMF with catalytic amount of 18-crown-6 as morpholine in
using MBS (m-maleimimidobenzoyl-N-hydroxysuccinamide
DMF showed a proclivity to attack the 3,4-carbonate.38 After
removal of the Boc group of 49 with TFA, coupling with
SAMA-OPfp followed by NaOMe/MeOH deprotection af-
forded 50 in 60% yield for the three steps.
ester), a heterobifunctional reagent which cross-links thiol
groups with amino groups (Scheme 10). For KLH, about 317
clusters per protein were introduced, while BSA showed only
7 clusters per protein.
These conjugates plus the adjuvant QS-21 or Tn(c)-pam (30)
in intralipid and 30 in intralipid plus QS-21 were used to
(38) Jiang, J.; Li, W.-R.; Joullie, M. Synth. Commun. 1994, 24, 187-
195.

12480 J. Am. Chem. Soc., Vol. 120, No. 48, 1998
Kuduk et al.
Table 3. Tn Cluster FACS Analysis: Serum Tested 11 Days Post
Third Vaccination
Scheme 10
IgG (tested 1/9/98)
% positive cells
IgM (tested 1/12/98)
% positive cells
group
30
46.99
12.00
94.72
92.14
39.98
46.41
49.54
51.89
30 + QS-21
27-KLH + QS-21
27-BSA + QS-21
found optimal previously for augmenting the immunogenicity
of ganglioside GD3 and GM2.41
The cell surface reactivity of anti-Tn(c) antibodies was
evaluated using Tn(c) positive LS-C colon cancer cells and Tn(c)
negative LS-B colon cells. Measurements involved flow
cytometry assays and complement dependent cytotoxicity
(CDCX) assays. The median percent positive cells by flow
cytometry with sera from mice 30 or 30 with QS-21-vaccinated
mice was low. However, sera from mice vaccinated with 27-
KLH or 27-BSA with QS-21 showed clear IgM reactivity with
LS-C colon cancer cells by flow cytometry (Table 3). IgG
reactivity was also seen by flow cytometry.
Important characteristics of the antibody response to im-
munization with 27-KLH conjugate plus QS-21 included the
pattern of antibody titers and the specificity of the antibodies.
IgM antibody titers were significantly higher than IgG at most
time points, despite repeated booster immunizations. There was
evidence for neither an IgM to IgG class switch nor a secondary
antibody response, all consistent with the T cell independent
antibody responses characteristic of most carbohydrate antigens.
Specificity analysis of the IgM antibody responses using
inhibition assays demonstrated that the response was polyclonal
with antibody subpopulations recognizing several different
clustered epitopes. Tn(c) expressed on cells was readily
recognized by the induced antibodies, resulting in complement
activation and lysis of Tn(c) positive tumor cells. More detailed
study on the immunology of these vaccines will be described
elsewhere.
Table 2. ELISA Antibody Titers against Tn(c)a
prevaccination
after third vaccination
vaccine
30
30 + QS-21
27-KLH
27-BSA
IgM
IgG
IgM
IgG
0
0
0
0
0
0
0
0
1350
1350
12150
1350
150
50
450
150
a All titers are medians for groups of five mice.
vaccinate groups of five mice. All of these constructs proved
to be immunogenic. There were no detectable anti-Tn(c) IgM
or IgG antibodies present prior to vaccination. The median IgM
and IgG ELISA titers against Tn(c)-pam in sera from the five
groups of mice immunized with 27- or 30-conjugated vaccines
at three weeks are shown in Table 2. Sera of mice immunized
with 10 µg of 30 in conjunction with 10 µg of QS-21 failed to
show strong reaction. In contrast to the case with 30, construct
27 conjugated with KLH or BSA induced high IgM and
moderate IgG titers. The highest titers were elicited by the KLH
vaccine. IgM antibody titers remained higher than IgG titers
at most time points, including the aftermath of two booster
immunizations. In general, titers were no higher after the
booster immunizations than after the initial immunizations.
Immunogenic protein carriers39 have been among the most
well studied approaches to the problem of increasing immuno-
genicity of carbohydrate antigens. Such protein conjugate
vaccines have elicited high IgM and moderate IgG titer
antibodies against the colon ganglioside GM2 in clinical trials.40
The studies here with Tn(c) vaccines were patterned after
previous studies with ganglioside vaccines, and the results were
similar. Of the five carriers and many adjuvants tested, KLH
was found to be the most effective carrier and QS-21, a
homogeneous saponin fraction purified from the bark of Quillaja
saponaria Molina, the most effective adjuvant. KLH conjuga-
tion plus the use of QS-21 as adjuvant was also the approach
Summary
The synthesis and clustering of Tn and Tf antigens has been
achieved. The application of glycal assembly and further
development of the “cassette” methodology has laid the founda-
tion for application to more complex O-linked glycopeptide
synthesis. This report confirms the immunogenicity of such
fully synthetic and characterized complex synthetic carbohydrate
antigens for use in vaccines. It provides a basis for the synthesis
and testing of other complex carbohydrate vaccines such as TF.42
On the basis of these observations we have initiated clinical
trials with the 27-KLH with QS-21 vaccine in patients with
Tn(c) positive cancers. Early indication suggests that the
synthetic vaccines are well tolerated. Immunological and
clinical data will be forthcoming in due course.
Experimental Section
Materials. A cysteine group was introduced to facilitate conjugation
with protein carriers. QS-2143 was obtained from Aquila Biopharma-
ceutical, Inc. (Worcester, MA). Keyhole limpet hemocyanin (KLH)
was obtained from PerImmune Inc. (Rockville, MD). Bovine serum
(41) Livingston, P. O.; Wong, G. Y. C.; Adluri, S.; Tao, Y.; Padavan,
M.; Parente, R.; Hanlon, C.; Calves, M. J.; Helling, F.; Ritter, G.; Oettgen,
H. F.; Old, L. J. J. Clin. Oncol. 1994, 12, 1036.
(42) Longnecker, B. M.; Reddish, M.; Koganty, R. R.; MacLean, G. D.
Ann. N.Y. Acad. Sci. 1993, 690, 276.
(43) Helling, F.; Shang, A.; Calves, M.; Zhang, S.; Ren, S.; Yu, R. K.;
Oettgen, H. F.; Livingston, P. O. Cancer Res. 1994, 54, 197-203, 1994.
(39) Soederstroem, T. Anti-idiotype as surrogate polysaccharide vaccines.
In Towards Better Carbohydrate Vaccines; Bell, R., Torrigiani, G., Eds.;
J. Wiley & Sons, Ltd.: London, 1987; pp 119-138.
(40) Helling, F.; Zhang, S.; Shang, A.; Adluri, S.; Calves, M.; Koganty,
R.; Longenecker, B. M.; Yao, T. J.; Oettgen, H. F.; and Livingston, P. O.
Cancer Res. 1995, 55, 2783.

Mucin-Related Tn and TF O-Linked Antigens
J. Am. Chem. Soc., Vol. 120, No. 48, 1998 12481
albumin (BSA) and sodium cyanoborohydride were obtained from
Sigma Chemical Co. (St. Louis, MO). Monoclonal antibody IE3 (mAb
IE3) was kindly provided by Dr. Singhal. Goat anti-mouse IgG and
IgM conjugated with alkaline phosphatase and goat anti-mouse IgM
fluorescence isothiocyanate (FITC) were obtained from Southern
Biotechnology Associates Inc. (Birmingham, AL). Female CB6F1 mice
were obtained from the Jackson Laboratory (Bar Harbor, ME).
General Procedure for Glycosylation of 1. A flame-dried flask
was charged with silver perchlorate (930 mg, 2 equiv), 4.0 g of 4 Å
molecular sieves, and N-Fmoc-L-threonine benzyl ester (1.21 g, 2.8
mmol, 1.2 equiv) in a drybox. A 30 mL sample of dry CH2Cl2 was
added to the flask, and the mixture was stirred at rt for 10 min. Donor
1 (1.13 g, 2.87 mmol) in 16 mL of CH2Cl2 was added slowly over 30
min via syringe. The reaction was stirred under argon atmosphere at
rt overnight. The mixture was then diluted with CH2Cl2 and washed
twice with water. The solution was dried over Na2SO4 and evaporated,
and the crude material (a 4:1 mixture of R/â isomers) was purified on
a silica gel column (1-1.5-2-2.5% MeOH/CH2Cl2) to provide 3 (1.28
g, 60% yield).
Compound 2: 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J ) 7.5 Hz,
2H), 7.62 (d, J ) 7.4 Hz, 2H), 7.28-7.42 (m), ), 5.94 (d, J ) 8.0 Hz,
1H), 5.39 (d, J ) 2.6 Hz, 1H), 5.21-5.27 (m), 4.86 (d, J ) 3.4, 1H),
4.58-4.62 (m, 1H), 4.40 (d, J ) 7.2 Hz, 2H), 4.24 (t, J ) 7.1, 1H),
4.16 (dd, J ) 3.0, 10.9 Hz, 1H), 3.50-4.08 (m), 3.58 (dd, J ) 3.5,
11.2 Hz, 1H), ), 2.14 (s, 3H), 1.96 (s, 3H). All data are in agreement
with literature reports.14
Compound 3. Compound 15 (0.58 g, 0.94 mmol) was taken up in
5.0 mL of acetic anhydride, and then 1.0 mL of pyridine was added.
The solution was stirred at ambient temperature for 1 h and then
partitioned cautiously between 50 mL of EtOAc and 50 mL of 1 N
HCl(aq). The phases were separated, and the organic phase was washed
with saturated NaHCO3 (2 × 50 mL) and brine (50 mL), dried
(Na2SO4), and concentrated. Flash chromatography of the residue (2:1
hexanes/EtOAc) furnished 0.63 g (90%) of 3 as a colorless foam: 1H
NMR (400 MHz, CDCl3) δ 7.77 (d, J ) 7.6 Hz, 2H), 7.27-7.42 (m),
5.66 (d, J ) 9.5 Hz, 1H), 5.44 (br s, 1H), ), 5.25-5.31 (m), ), 5.37 (d,
J ) 13.2, 1/2AB, 1H), 5.20 (d, J ) 12.1 Hz, 1/2AB, 1H), 4.90 (d, J )
3.6 Hz, 1H), 4.40-4.51 (m), 4.31-4.38 (m), 4.18-4.28 (m), 3.70 (d,
J ) 6.5 Hz, 2H), 3.58 (dd, J ) 3.7, 11.2 Hz, 1H), 2.16 (s, 3H), 2.04
(s, 3H), 1.34 (d, J ) 6.3 Hz, 3H). All data are in agreement with
literature reports.14
68.00, 67.50, 67.33, 62.36, 58.88, 47.62, 47.28, 23.37, 20.87, 20.77,
18.38; IR (neat) 3332, 2978, 1748, 1677 cm-1; HRMS calcd for
C40H44N2O13Na 783.2741, found 783.2756.
General Procedure B for Deprotection of Benzyl Esters. To a
solution of 5 (0.46 g, 0.60 mmol) in 150 mL of methanol and 10 mL
of H2O was added 0.14 g of 5% Pd/C. The system was evacuated and
purged 5× with H2 and then placed under 1 atm of H2 for 90 min. The
suspension was gravity filtered and concentrated. Flash chromatography
of the residue (10 f 15 f 20% MeOH/CH2Cl2) yielded 0.37 g (92%)
of 7 as a colorless crystalline solid.
23
1
Compound 7. [R]D + 90.6° (c 1.55, CHCl3); H NMR (CDCl3)
mixture of rotamers; spectra available in Supporting Information,
confirmed by variable temperature NMR; 13C NMR (CDCl3, mixture
of rotamers) δ 173.78, 173.51, 172.42, 171.52, 171.28, 170.58, 170.27,
170.22, 170.00, 157.73, 156.46, 143.51, 143.47, 143.21, 143.13, 141.24,
141.19, 141.16, 127.77, 127.69, 127.66, 127.12, 127.04, 126.96, 125.10,
124.77, 124.53, 124.46, 119.92, 119.85, 99.48, 98.71, 77.71, 77.20,
75.73, 68.01, 67.53, 67.26, 67.22, 66.96, 66.90, 62.15, 61.96, 58.61,
48.37, 47.77, 47.16, 46.84, 22.82, 22.12, 20.75, 20.62, 20.58, 20.54,
20.51, 18.41, 18.12; IR (neat) 3344, 2938, 1748, 1726 cm-1; HRMS
calcd for C33H38N2O13Na 693.2272, found 693.2298.
General Procedure A for Deprotection of FMOC-carbamates.
Compound 5 (0.18 g, 0.24 mmol) was taken up in 3.0 mL of morpholine
and the solution stirred at ambient temperature for 30 min. Excess
morpholine was then removed by azeotroping with dry toluene under
reduced pressure (3 × 4 mL). Flash chromatography of the residue (4
f 7.5 f 10% MeOH/CH2Cl2) provided 0.11 g (86%) of amine 9 as a
coloroless foam.
Compound 8. To a round-bottom flask were charged 4 (330 mg,
0.44 mmol) and morpholine (3 mL) at 0 °C. The reaction mixture
was stirred at 0 °C to rt for 3 h. After evaporation of the solvent by
a flow of air, the residue was separated by chromatography (4 f 7.5
f 10% MeOH/CH2Cl2) to give the desired amine (230 mg, 99%):
[R]D20 +62.0° (c 1.46, CHCl3); IR (film) 3310, 2960, 1751, 1734, 1654,
1
1542 cm-1; H NMR (CDCl3, 400 MHz) δ 7.31-7.35 (m, 5H), 5.86
(d, J ) 9.8 Hz, 1H), 5.30 (d, J ) 2.3 Hz, 1H), 5.15 (d, J ) 11.5 Hz,
1H), 5.12 (d, J ) 11.5 Hz, 1H), 5.05 (dd, J ) 11.4, 3.0 Hz, 1H), 4.78
(d, J ) 3.6 Hz, 1H), 4.53 (m, 1H), 4.01-4.12 (m, 3H), 3.90 (dd, J )
9.8, 3.6 Hz, 1H), 3.66-3.70 (m, 2H), 2.12 (s, 3H), 2.00 (s, 3H), 1.96
(s, 3H), 1.92 (s, 3H), 1.80 (m, 2H); 13C NMR (CDCl3, 75.5 MHz) δ
173.5, 170.8, 170.3, 170.2, 170.1, 135.2, 128.8, 128.7, 128.3, 98.8,
71.0, 68.4, 67.3, 61.9, 54.8, 47.7, 23.1, 20.7, 20.6; HRMS (FAB) calcd
for C24H33N2O11 [M + H]+ 525.2070, found 525.2090.
Compound 4. To a round-bottom flask were added R-glycoside 2
(1.25 g, 1.71 mmol), pyridine (2 mL), and thiolacetic acid (2 mL) at 0
°C subsequently. The reaction was stirred from 0 °C to room
temperature overnight. After evaporation of the solvent by a flow of
air, the residue was separated by chromatography to give desired product
Compound 9: [R]D23 +55.9° (c 1.16, CHCl3); 1H NMR (CDCl3) δ
7.34 (m, 5H), 6.01 (d, J ) 9.5 Hz, 1H), 5.34 (d, J ) 2.5 Hz, 1Hz),
5.16 (d, J ) 12.0 Hz, 1H), 5.05 (d, J ) 12.1 Hz, 2H), 4.77 (d, J ) 3.7
Hz, 1H), 4.50 (m, 1H), 4.21 (m, 1H), 4.05 (m, 3H), 3.42 (br s, 1H),
2.13 (s, 3H), 2.01 (s, 3H), 1.97 (s, 3H), 1.93 (s, 3H), 1.65 (br s, 2H),
1.33 (d, J ) 6.4 Hz, 3H); 13C NMR (CDCl3) δ 174.16, 170.68, 170.19,
134.75, 128.66, 128.43, 99.75, 78.03, 68.53, 67.28, 67.11, 67.04, 62.01,
59.06, 47.50, 22.99, 20.61, 20.49, 18.09; IR (neat) 3385, 2976, 1745,
1666 cm-1; HRMS calcd for C25H34N2O11Na 561.2060, found 561.2069.
Compound 18. To a solution of 14 (0.24 g, 0.40 mmol) in 5.0 mL
of nitromethane was added benzaldehyde dimethyl acetal (0.11 mL,
0.79 mmol), followed by p-toluenesulfonic acid monohydrate (4.0 mg,
0.02 mmol). The mixture was stirred for 1 h at ambient temperature
at which time an additional 0.11 mL of PhCH(OMe)2 and 4.0 mg of
TsOH were added. Stirring was continued for 2 h, and the mixture
was neutralized with 5 drops of Et3N and concentrated. Flash
chromatography of the residue (2:1 hexanes/EtOAc) afforded 0.26 g
20
(1.13 g, 88%): [R]D +61.0° (c 0.68, CHCl3); IR (film) 3346, 3018,
1
2954, 1748, 1732, 1715, 1668, 1557, 1538 cm-1; H NMR (CDCl3,
400 MHz) δ 7.72 (d, J ) 7.5 Hz, 2H), 7.56 (d, J ) 7.5 Hz, 2H), 7.25-
7.45 (m, 9H), 6.06 (d, J ) 7.8 Hz, 1H), 5.74 (d, J ) 8.8 Hz, 1H), 5.30
(d, J ) 3.0 Hz, 1H), 5.16 (m, 2H), 5.03 (dd, J ) 11.2, 3.0 Hz, 1H),
4.76 (br s, 1H), 4.59 (m, 1H), 4.49 (m, 1H), 4.40 (d, J ) 6.8 Hz, 2H),
4.20 (t, J ) 6.8 Hz, 1H), 3.92-4.15 (m, 5H), 2.12 (s, 3H), 1.96 (s, 3H),
1.92 (s, 3H), 1.89 (s, 3H); 13C NMR (CDCl3, 75.5 MHz) δ 170.7, 170.2,
170.1, 170.0, 168.8, 155.8, 143.6, 141.2, 134.7, 128.7, 128.3, 127.7,
127.0, 124.9, 120.0, 99.1, 69.8, 68.1, 67.6, 67.2, 61.9, 54.6, 47.6, 47.0,
23.1, 20.6, 20.5; HRMS (FAB) calcd for C39H43N2O13 [M + H]+
747.2765, found 747.2766.
Compound 5. Compound 3 (0.40 g, 0.54 mmol) was taken up in
5.0 mL of thiolacetic acid and the solution stirred for 20 h at ambient
temperature. The mixture was concentrated with a N2 flow and the
residue purified by flash chromatography (1:1 hexanes/EtOAc) to give
23
1
(94%) of 18 as a colorless foam: [R]D +79.9° (c 1.35, CHCl3); H
NMR (CDCl3) δ 7.76 (d, J ) 7.5 Hz, 2H), 7.58 (m, 2H), 7.46 (m,
2H), 7.31-7.42 (m, 7H), 5.89 (d, J ) 8.0 Hz, 1H), 5.50 (s, 1H), 5.25
(d, J ) 12.1 Hz, 1H), 5.20 (d, J ) 12.1 Hz, 1H), 4.90 (d, J ) 3.2 Hz,
1H), 4.57 (m, 1H), 4.44 (dd, J ) 10.5, 7.1 Hz, 1H), 4.32 (dd, J )
10.4, 7.3 Hz, 1H), 4.21 (app t, J ) 7.2 Hz, 1H), 4.12-4.18 (m, 3H0,
3.98 (m, 2H), 3.86 (d, J ) 12.7 Hz, 1H), 3.56 (br s, 1H), 3.49 (dd, J
) 10.6, 3.3 Hz, 1H), 2.38 (d, J ) 10.9 Hz, 1H); 13C NMR (CDCl3) δ
169.67, 155.85, 143.72, 143.58, 141.25, 137.17, 134.99, 129.31, 128.62,
128.53, 128.27, 127.74, 127.09, 127.05, 126.11, 125.07, 124.95, 120.02,
101.19, 99.97, 75.18, 69.81, 68.92, 67.77, 67.26, 66.93, 63.21, 60.41,
23
0.31 g (76%) of 5 as a colorless oil: [R]D + 49.6° (c 1.26, CHCl3);
1H NMR (CDCl3, mixture of rotamers) (major rotamer) δ 7.76 (d, J )
7.4 Hz, 2H), 7.62 (d, J ) 7.4 Hz, 2H), 7.33 (m, 9H), 5.78 (m, 1H),
5.60 (m, 1H), 5.36 (br s, 1H), 5.18 (d, J ) 11.8 Hz, 1H), 5.06 (d, J )
11.8 Hz, 1H), 4.78 (d, J ) 3.1 Hz, 1H), 4.52 (m, 1H), 4.44 (m, 3H),
4.21 (m, 3H), 4.05 (m, 3H), 2.15 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H),
1.96 (s, 3H), 1.29 (d, J ) 6.3 Hz, 3H); 13C NMR (CDCl3) δ 171.09,
171.01, 170.53, 170.47, 156.71, 143.84, 141.45, 134.52, 129.08, 129.02,
128.73, 127.95, 127.27, 125.33, 125.21, 120.19, 100.03, 77.14, 68.56,

12482 J. Am. Chem. Soc., Vol. 120, No. 48, 1998
Kuduk et al.
54.58, 46.97; IR (neat) 3423, 2921, 2109, 1721 cm-1; HRMS calcd
for C38H36N4O9Na 715.2380, found 715.2387.
was removed, and the crude was purified by RP chromatography (C18
silica gel, eluted with H2O). The solution was lyophilized to yield
glycopeptide 27 (85%) as a white solid. Further analysis was renounced
to avoid dimerization of the thiol, and the material was stored under
Ar at -78 °C: MS (ES) [M + Na] 1083.4, calcd for C40H68N8O23SNa
1083.4.
Compound 19. 19 was prepared as described for compound 18
and obtained in 73% yield from 15 as a colorless solid: [R]D23 +90.4°
1
(c 1.17, CHCl3); H NMR (CDCl3) δ 7.76 (d, J ) 7.5 Hz, 2H), 7.62
(d, J ) 7.4 Hz, 2H), 7.48 (m, 2H), 7.31-7.39 (m, 7H), 5.79 (d, J )
9.3 Hz, 1H), 5.54 (s, 1H), 5.23 (s, 2H), 4.93 (d, J ) 3.4 Hz, 1H), 4.47
(m, 3H), 4.34 (dd, J ) 10.5, 7.6 Hz, 1H), 4.23 (m, 3H), 4.09 (ddd, J
) 13.3, 10.4, 3.1 Hz, 1H), 4.02 (d, J ) 12.1 Hz, 1H), 3.68 (s, 1H),
3.53 (dd, J ) 10.5, 3.4 Hz, 1H), 2.53 (d, J ) 10.6 Hz, 1H), 1.29 (d, J
) 6.3 Hz, 3H); 13C NMR (CDCl3) δ 170.06, 156.74, 143.86, 143.63,
141.24, 141.21, 137.19, 134.95, 129.35, 128.61, 128.51, 128.29, 127.67,
127.07, 127.05, 126.15, 125.16, 125.10, 119.92, 101.16, 99.20, 76.20,
75.24, 69.02, 67.70, 67.38, 67.28, 63.18, 61.03, 58.71, 47.08, 18.63;
IR (neat) 3427, 2921, 2110, 1725 cm-1; HRMS calcd for C39H38N4O9-
Na 729.2537, found 729.2561.
Compound 29. To a stirred solution of peracetylated cluster 2414
(0.19 g, 0.14 mmol) in 10.0 mL of methanol was added sodium
methoxide (25 wt % solution in MeOH) until the pH of the solution
reached 9 (ca. 10 drops). Stirring was continued for 16 h, at which
time Amberlyst-15 was added to lower the pH of the mixture to ca. 4.
The solution was separated from the resin Via pipet, the resin was rinsed
with methanol (3 × 5 mL), and the combined extracts were concen-
trated. Purification of the residue on LiChroprep RP-18 using 1:1
MeOH/H2O as eluant furnished 0.13 g (95%) of tripeptide 29 as a
colorless crystalline solid. [R]D23 + 160.7° (c 1.30, CHCl3); 1H NMR
(CD3OD) δ 8.32 (J ) 8.5 Hz, 1H), 8.24 (d, J ) 8.8 Hz, 1H), 7.53 (m,
2H), 7.38 (m, 2H), 4.98 (d, J ) 3.2 Hz, 1H), 4.81 (d, J ) 3.5 H, 1H),
4.72 (br s, 1H), 4.66 (br s, 1H), 4.48 (s, 1H), 4.16-4.29 (m, 6H), 3.92
(m, 6H), 3.79 (m, 3H), 3.71 (m, 6H), 2.09 (s, 3H), 2.08 (s, 3H), 2.06
(s, 3H), 2.05 (s, 3H), 1.31 (m, 6H), 1.22 (d, J ) 6.3 Hz, 3H); 13C
NMR (CD3OD) δ 174.33, 174.24, 173.79, 172.69, 171.85, 101.09,
100.79, 78.24, 77.66, 77.37, 72.87, 72.81, 70.81, 70.43, 70.31, 62.76,
62.66, 58.58, 58.31, 57.92, 51.68, 51.59, 51.47, 23.48, 23.43, 23.25,
22.49, 19.32, 19.07, 19.03; IR (neat) 3372, 2933, 1633 cm-1; LRMS
calcd for C38H64N6O23Na 995.3920, found 995.3886.
General Procedure C for Peptide Couplings Using IIDQ. To a
solution of amine 9 (0.11 g, 0.20 mmol) in 3.0 mL of CH2Cl2 at 0 °C
was added quickly a solution of acid 7 (0.15 g, 0.23 mmol) in 3.0 mL
of CH2Cl2. Immediately following the addition, IIDQ (74 mg, 0.25
mmol) in 1.0 mL of CH2Cl2 was added quickly dropwise. The mixture
was allowed to warm slowly to ambient temperature and stirred for 24
h. The solvent was evaporated with an N2 flow, and flash chroma-
tography of the residue (EtOAc) afforded 0.23 g (97%) of dipeptide
21 as colorless crystals.
Compound 25. Benzyl ester 23 (0.21 g, 0.15 mmol) was depro-
tected according to general procedure B. Filtration and concentration
of the crude reaction mixture provided 0.19 g (96%) of pure free acid
25 as a colorless crystalline solid: [R]D23 +103.5° (c 1.76, CHCl3); 1H
NMR (CD3OD) δ 8.40 (d, J ) 9.0 Hz, 1H), 8.31 (d, J ) 9.2 Hz, 1H),
5.40 (m, 3H), 5.15 (m, 4H), 4.99 (d, J ) 3.8 Hz, 1H), 4.91 (J ) 3.7
Hz, 1H), 4.78 (d, J ) 2.1 Hz, 1H), 4.70 (m, 1H), 4.62 (m, 1H), 4.31-
4.46 (m, 10H), 4.11 (m, 6H), 2.14 (s, 3H), 2.13 (s, 3H), 2.13 (s, 3H),
2.12 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 2.01 (s, 3H), 2.00
(s, 3H), 1.99 (s, 3H), 1.95 (s, 3H), 1.93 (s, 3H), 1.93 (s, 3H), 1.38 (d,
J ) 6.2 Hz, 3H), 1.32 (m, 6H); 13C NMR (CD3OD) δ 173.83, 173.51,
173.49, 173.33, 173.16, 172.54, 172.44, 172.09, 172.06, 171.94, 101.25,
100.83, 100.45, 79.62, 77.86, 77.50, 70.45, 70.13, 69.77, 68.92, 68.87,
68.31, 68.25, 63.36, 63.14, 58.22, 58.14, 58.01, 23.41, 23.13, 23.08,
22.50, 20.74, 20.70, 20.66, 20.62, 20.58, 19.54, 19.35, 19.21; IR (neat)
3324, 2981, 1748, 1658 cm-1; HRMS calcd for C56H82N6O32Na
1373.4871, found 1373.4856.
Compound 26. Boc-protected 1,3-diaminopropane (12.1 mg, 0.07
mmol) in 1 mL of CH2Cl2 (anhydrous) was added to a flask charged
with glycopeptide 24 (70 mg, 0.05 mmol). While the solution was
being stirred, IIDQ (21.2 mg, 0.07 mmol) in 0.8 mL of CH2Cl2 was
added at 0 °C. After 5 min the reaction was removed from the bath
and stirred for 48 h. Longer reaction times are required for this
coupling. The reaction mixture was purified directly on a silica column
(7% MeOH/CH2Cl2). The product was dissolved in 0.2 mL of CH2Cl2,
and 0.6 mL of CF3COOH was added. The reaction was complete in
30 min. The solvent was removed with a stream of nitrogen, and the
material was dried in Vacuo. The crude product was dissolved in a
1:1 mixture of tBuOH/AcOH and lyophilized. To a solution of the
crude ammonium salt (27 mg, 0.026 mmol) was added pentafluorophe-
nyl acetylmercaptoacetate (9.1 mg, 1.5 equiv) in 0.7 CH2Cl2 and (iPr)2-
NEt (7.3 µL, 0.04 mmol). The mixture was purified by a silica column
(3-4-5-6-7% MeOH, CH2Cl2) to yield pure product 26 (27 mg,
70% yield): 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J ) 8.3 Hz), 7.72
(d, J ) 7.9 Hz), 7.39-7.59 (m), 7.22 (d, J ) 9.5 Hz), 7.10 (d, J ) 8.9
Hz), 6.70-6.82 (m), 5.32-5.40 (m), 5.08-5.17 (m), 5.03 (d, J ) 2.9
1H), 4.99 (d, J ) 3.0 Hz, 1H), 4.97 (d, J ) 3.3 Hz, 1H), 4.51-4.70
(m), 4.13-3.91 (m), 3.65-3.72 (m), 3.60 (d, J ) 15.0 Hz), 1.95-2.08
(14 acetates), 1.42 (d, J ) 7.14), 1.39 (d, J ) 6.6 Hz); MS (ES) [M +
Na] 1503.6, calcd for C56H84N8O3Na 1503.6.
Compound 31. 31 was prepared as described for 32 in 40% yield:
MS (ES) [M + Na] 1988.5, calcd for C95H172N10O30S 1988.5. Data
are in agreement with literature description.14
Compound 32. To a slurry of tripeptide 30 (52 mg, 0.053 mmol)
and amine acetate 28 (100 mg, 0.090 mmol) in 4.0 mL of dry DMF at
ambient temperature were added HATU (81 mg, 0.21 mmol), HOAt
(14.5 mg, 0.11 mmol), and then diisopropylethylamine (37 µL, 0.21
mmol). The mixture was stirred at ambient temperature for 16 h, at
which time the DMF was removed in Vacuo. The residue was rinsed
with CH2Cl2 (3 × 0.5 mL). The remainder of the residue was purified
by flash chromatography on silica gel (CHCl3/MeOH/H2O, 65:25:4)
to afford 40 mg (38%) of the lipid conjugate as a colorless amorphous
solid. 1H NMR (CDCl3/CD3OD/D2O, 65:25:4) δ 8.37 (d, J ) 9.3 Hz,
1H), 8.33 (d, J ) 8.8 Hz, 1H), 8.03 (d, J ) 7.1 Hz, 3H), 7.92 (m, 1H),
7.72 (app t, J ) 8.2 Hz, 1H), 7.66 (d, J ) 8.8 Hz, 1H), 7.32 (d, J )
9.2 Hz, 1H), 5.19 (m, 1H), 4.87 (d, J ) 3.6 Hz, 1H), 4.83 (d, J ) 3.6
Hz, 1H), 4.80 (d, J ) 3.7 Hz, 1H), 4.67 (m, 2H), 4.57 (m, 2H), 4.13
(m, 6H), 3.89 (br s, 7H), 3.74 (m, 1H), 3.19 (m, 4H), 3.04 (m, 1H),
2.88 (m, 1H), 2.77 (m, 1H), 2.31 (m, 6H), 2.11 (s, 3H), 2.06 (s, 3H),
2.04 (s, 3H), 2.03 (s, 3H), 1.61 (m, 9H), 1.27 (s, 90H), 1.19 (d, J )
6.1 Hz, 3H), 0.88 (t, J ) 6.6 Hz, 9H); LRMS calcd for C98H178N10O30S
2007.2, found 2007.2.
Compound 36. To a solution of protected galactal 35 (1.01 g, 2.01
mmol) in 11 mL of anhydrous CH3CN at -20 °C was added a mixture
of NaN3 (392 mg, 6.03 mmol) and CAN (3.30 g, 6.03 mmol). The
reaction mixture was vigirously stirred at -15 °C for 1 h. Then the
reaction mixture was diluted with diethyl ether, and washed with cold
water and brine subsequently. Finally, the solution was dried over
anhydrous Na2SO4. After evaporation of the solvent, the residue was
purified by chromatography on silica gel (50-75% EtOAc/hexanes).
A mixture of R and â anomers 36 (2.17 g, 67% yield) was obtained.
1
The ratio of R and â anomers was almost 1:1 based on H NMR: IR
(film) 3476, 2963, 2119, 1813, 1747, 1660, 1372, 1227, 1051 cm-1
;
1H NMR (400 MHz, CDCl3) 6.25 (d, J ) 4.2 Hz, 1H, H-1, R anomer),
5.40 (d, J ) 8.6 Hz, 1H, H-1, â anomer); MS (EI) calcd 629.2, found
629.2 (M + Na). Copies of 1H and 13C NMR spectra are available in
the Supporting Information.
Bromide 37. A solution of compound 36 (220 mg, 0.363 mmol) in
1.0 mL of dry acetonitrile was mixed with lithium bromide (140 mg,
1.6 mmol, 5 equiv) and stirred at rt for 3 h in the dark. The
heterogeneous mixture was diluted with dichloromethane, the solution
was washed twice with water and dried over magnesium sulfate, and
the solvent was evaporated without heating. After rapid flash chro-
matography on predried silica gel (EtOAc), R-bromide 37 (200 mg,
88%) was isolated and stored under argon atmosphere at -80 °C to
Compound 27. The protected glycopeptide 26 was dissolved in
anhydrous MeOH (deoxygenated with a stream of nitrogen/argon in
order to prevent oxidative dimerization!). The pH was adjusted to 9-10
with a 25% solution of NaOMe in MeOH, and the mixture was stirred
under argon overnight. Acidic Amberlyst resin was then added until
the solution became neutral or mildly acidic (pH 5-6). The solvent

Mucin-Related Tn and TF O-Linked Antigens
J. Am. Chem. Soc., Vol. 120, No. 48, 1998 12483
prevent hydrolysis unitl use: 1H NMR (400 MHz, CDCl3) δ 5.16 (d,
J ) 3.6 Hz, 1H), 5.63 (d, J ) 2.8 Hz, 1H), 5.19-5.15 (m, 2H), 4.90-
4.81 (m, 2H), 4.41-3.96 (m, 18H); CI (NH3) MS calcd for (M + H)
C21H27N3BrO14 629, found 629.
0.139 mmol), N-Fmoc-L-threonine benzyl ester (70 mg, 0.167 mmol),
and 200 mg of 4 Å molecular sieves in a glovebag. The mixture was
dissolved in 6 mL of dry CH2Cl2. The reaction mixture was cooled to
-30 °C, trimethylsilyl triflate (14 µL, 0.5 equiv) was added, and the
mixture was stirred until completion judged by TLC and then quenched
with TEA. The mixture was then directly separated by flash chroma-
tography on silica gel (50-80% EtOAc/hexane) to yield R-product 42R
(56 mg, 42%) and â-product 42â (57 mg, 42%).
Compound 38. To a solution of a mixture of azidonitrates 36 (390
mg, 6.64 mmol) in 3 mL of anhydrous CH3CN at 0 °C were slowly
added Et(i-Pr)2N (111 µL, 0.64 mmol) and PhSH (198 µL, 1.9 mmol)
subsequently. The reaction mixture was stirred at 0 °C for 1 h, and
then the solvent was blown off by an argon flow. The residue was
purified by chromatography on silica gel (EtOAc) to give a 1:1 mixture
of R and â anomers of 38 (318 mg, 85%) as a white foam: 1H NMR
(400 MHz, CDCl3) 5.35 (d, J ) 3.4 Hz, 1H, H-1 R anomer), 4.86 (d,
J ) 8.4 Hz, 1H, H-1, â anomer); MS (EI) calcd 562.2, found 562.2
(M + H); FAB HRMS calcd for (M + Na) C21H27N3O15Na 584.1340,
General Procedure for Glycosylation with Glycosyl Fluorides.
A flame-dried flask was charged with N-Fmoc-L-threonine benzyl ester
(28 mg, 1.5 equiv), Cp2ZrCl2 (12.9 mg, 0.044 mmol), AgClO4 (18.2
mg, 0.088 mmol), and 200 mg of 4 Å molecular sieves in a glovebag.
The mixture was cooled to -30 °C, and 1 mL of CH2Cl2 was added.
Then, donor 39 (25 mg, 0.044 mmol) in 1 mL of CH2Cl2 was added
dropwise, and the reaction was stopped after judged complete by TLC
(1 h). Purification by silica gel chromatography yielded R-product 42R
(10 mg, 25%) and â-product 42â (10 mg, 25%).
1
found 584.1319. Copies of H and 13C NMR spectra are available in
the Supporting Information.
Fluoride 39. To a solution of 38 (53 mg, 0.081 mmol) in 1 mL of
THF was added 20 mL (0.12 mmol, 1.5 equiv) of DAST at -40 °C,
and the reaction was allowed to warm to rt over 1 h. The reaction was
then quenched with 0.5 mL of MeOH and purified by chromatography
on silica gel (50-80% EtOAc/hexane) to afford 25 mg (47%) of 39R
and 26 mg of 39â (47%). These were used immediately to prevent
degradation of the donor. 39R: 1H NMR (400 MHz, CDCl3) δ 5.68
(dd, J ) 55, 2.5 Hz, 1H), 5.59 (d, J ) 6.4 Hz, 1H), 5.12-5.10 (m,
2H), 4.86 (dd, J ) 8.5, 1.2 Hz, 1H), 4.79 (dd, J ) 8.5 Hz, 1.2 Hz,
1H), 4.39-4.17 (m, 4H), 4.13 (br t, J ) 5.7 Hz, 1H), 4.06 (dd, J )
10.5, 2.9 Hz, 1H), 3.96-3.87 (m, 2H), 2.20 (s, 3 H), 2.15 (s, 3H), 2.11
(s, 3H), 2.03 (s, 3H); 19F NMR (421 MHz, CDCl3) δ 14.2 (dd, J ) 55,
26.5, 1F). 39â: 1H NMR (400 MHz, CDCl3) δ 5.45 (br t, 1H), 5.13-
5.06 (m, 2H), 4.94 (d, J ) 7.6 Hz, 1H), 4.87 (dd, J ) 8.6, 1.6 Hz, 1H),
4.80 (dd, J ) 8.6, 1.6 Hz, 1H), 4.40-4.20 (m, 4H), 4.10 (dt, J ) 6.5,
1.4 Hz, 1H), 3.98 (dd, J ) 11.7, 1.6 Hz, 1H), 3.86-3.79 (m, 2H), 3.56
(dd, J ) 10.3, 3.6 Hz, 1H), 2.21 (s, 3H), 2.18 (s, 3H), 2.11 (s, 3H),
2.07 (s, 3H); 19F NMR (421 MHz, CDCl3) δ 23.5 (dd, J ) 54.8, 14.2,
1F): CI (NH3) MS calcd for (M + H) C21H26FN3O14 564, found 564.
Trichloroacetimidate 40. To a solution of 38 (327 mg, 0.58 mmol)
in 5 mL of CH2Cl2 at 0 °C were added K2CO3 (300 mg) and Cl3CCN
(0.58 mL, 5.8 mmol). The reaction mixture was stirred at 0 °C to
room temperature overnight. The suspension was filtered through a
pad of Celite and washed with CH2Cl2. The filtrate was evaporated,
and the residue was purified by chromatography on silica gel (40-
80% EtOAc/hexane) to give R-trichloroacetimidate 40R (94 mg, 22%)
and â-trichloroacetimidate 40â (270 mg 72%) as white foams. 40â:
[R]20D +36.8° (c 0.28, CHCl3); IR (film) 3318, 2962, 2116, 1750, 1679,
Compound 42r: [R]20 +61.44° (c 0.5, CHCl3); IR (film) 3434,
D
3362, 3065, 2956, 2114, 1815, 1746, 1514, 1371, 1077 cm-1; 1H NMR
(400 MHz, CDCl3) δ 7.83 (d, 2H), 7.67 (d, 2H), 7.47-7.33 (m, 9H),
5.78 (d, J ) 9.4 Hz, 1H), 5.51 (d, J ) 2.0 Hz, 1H), 5.24 (d, J ) 12.3
Hz, 1H), 5.20 (d, J ) 12.3 Hz, 1H), 5.12 (br s, 1H), 4.86-4.76 (m,
3H), 4.49-4.10 (m, 6H), 3.98 (dd, J ) 10.4, 2.6 Hz, 1H), 3.88 (dd, J
) 13.0, 6.9 Hz, 1H), 3.69 (dd, J ) 10.6, 3.5 Hz, 1H), 2.26 (s, 3H),
2.18 (s, 3H), 2.16 (s, 3H), 2.02 (s, 3H), 1.29 (d, J ) 7.0 Hz, 3H); 13
C
NMR 100 MHz, CDCl3) δ 170.2, 170.1, 169.8, 168.4, 156.5, 152.8,
143.7, 143.5, 141.0, 134.7, 132.4, 128.5, 128.4, 128.3, 127.5, 126.9,
124.9, 119.8, 100.2, 98.7, 77.1, 76.4, 72.2, 69.1, 68.1, 68.0, 67.6, 67.5,
67.1, 62.7, 62.1, 59.1, 58.6, 53.3, 46.9, 20.5, 20.4, 18.2. 42â: [R]20
D
+ 17.3° (c 0.64, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.76 (d, 2H),
7.61 (d, 2H), 7.41-7.28 (m, 9H), 5.71 (d, J ) 9.2 Hz, 1H), 5.35 (d, J
) 2.9 Hz, 1H), 5.19 (d, J ) 12.3 Hz, 1H), 5.11 (d, J ) 12.3 Hz, 1H),
5.08 (t, J ) 3.2 Hz, 1H), 5.00 (t, J ) 3.4 Hz, 1H), 4.83-4.79 (m, 3H),
4.50-4.09 (m, 10H), 3.89 (dd, J ) 11.4, 6.9 Hz, 1H), 3.62 (br t, 1H),
3.56 (t, J ) 5.2 Hz, 1H), 3.36 (d, J ) 10.4, 3.3 Hz, 1H), 2.19 (s, 3H),
2.18 (s, 3H), 2.12 (s, 3H), 2.03 (s, 3H), 1.32 (d, J ) 6.3 Hz, 3H); 13C
NMR (100 MHz, CDCl3) δ 170.5, 170.4, 170.0, 169.8, 168.6, 156.8,
143.9, 143.7, 141.2, 135.3, 128.6, 128.4, 128.2, 127.6, 127.0, 125.2,
125.1, 120.0, 100.1, 99.9, 79.1, 74.9, 72.7, 72.4, 71.4, 68.4, 68.2, 67.7,
67.3, 62.5, 62.3, 61.9, 58.4, 47.1, 20.7, 20.6, 16.8; FAB HRMS calcd
for (M + Na) C47H50N4O19Na 997.2967, found 997.2961.
Compound 45. Glycal 44 (65 mg, 0.303 mmol) was dissolved in
1 mL of CH2Cl2 and treated with 6.06 mL of DMDO (0.06 M in
acetone, 0.364 mmol) at 0 °C for 30 min. The solvent was removed
in Vacuo and placed under high vacuum for 1 h. The residue was
dissolved in 2 mL of THF and cooled to -78 °C, and acceptor 19
(177 mg, 0.155 mmol) in 3 mL of THF was added followed by dropwise
addition of 0.155 mL of ZnCl2 (1.0 M in Et2O, 0.155 mL). The reaction
was allowed to warm to room temperature and stirred overnight. The
reaction was quenched with saturated NaHCO3, extracted with Et2O,
dried over MgSO4, filtered, and concentrated. The residue was purified
by chromatography on silica gel (50-75% EtOAc/hexanes) to afford
128 mg (97%) of 45 as a white film: [a]20D +61.84° (c 0.25, CHCl3);
1
1370, 1224, 1071 cm-1; H NMR (400 MHz, CDCl3) δ 8.76 (br s,
1H), 5.58 (d, J ) 8.6 Hz, 1H), 5.47 (d, J ) 3.2 Hz, 1H), 5.10-5.05
(m, 2H), 4.85-4.80 (m, 2H), 4.37 (dd, J ) 10.2, 6.7 Hz, 1H), 4.28
(dd, J ) 10.0, 6.6 Hz, 1H), 3.99-3.89 (m, 7H), 3.62 (dd, J ) 10.3,
3.3 Hz, 1H), 2.21 (s, 3H), 2.11 (s, 3H), 2.07 (s, 3H), 1.98 (s, 3H); 13
C
NMR (100 MHz, CDCl3) δ 171.0, 170.3, 170.2, 169.9, 168.6, 160.6,
152.9, 100.1, 96.4, 90.1, 79.5, 73.0, 72.6, 72.3, 68.3, 67.6, 67.5, 62.1,
61.6, 60.2, 20.8, 20.6, 20.5, 14.0. 40R: 1H NMR (400 MHz, CDCl3)
δ 8.77 (br s, 1H), 6.45 (br s, 1H), 5.61 (br s, 1H), 5.13 (br t, 2H), 4.87
(d, J ) 8.5 Hz, 1H), 4.85 (d, J ) 8.5 Hz, 1H), 4.37-4.08 (m, 16H),
3.86 (dd, J ) 8.2, 4.1 Hz, 1H), 2.17 (s, 3H), 2.12 (s, 3H), 2.07 (s, 3H),
1.99 (s, 3H); FAB HRMS calcd for (M + Na) C23H27N4O15Cl3Na
727.0436, found 727.0446.
General Procedure for Glycosylation with Glycosyl Bromide. A
flame-dried flask was charged with silver perchlorate (185 mg, 0.896
mmol), 300 mg of 4 Å molecular sieves, and N-Fmoc-L-threonine
benzyl ester (283 mg, 0.672 mmol, 1.5 equiv) in a glovebag. Then,
2.5 mL of CH2Cl2 was added to the flask, and the mixture was stirred
at rt for 10 min. Donor 37 (280 mg, 0.448 mmol) in 2.5 mL of CH2-
Cl2 was added slowly over 1 h. The reaction was stirred under argon
atmosphere at rt for 2 h. The mixture was then diluted with CH2Cl2
and filtered through Celite. The precipitate was thoroughly washed
with CH2Cl2, the filtrate was evaporated, and the crude material was
purified on silica gel chromatography (50-80% EtOAc/hexane) to
provide 42R (180 mg, 39%) and 42â (161 mg, 34%).
IR (film) 3425, 3064, 2920, 2112, 1809, 1740, 1513, 1237, 1042 cm-1
;
1H NMR (400 MHz, CDCl3) δ 7.78 (d, J ) 9.0 Hz, 2H), 7.61 (d, J )
8.1 Hz, 2H), 7.55 (d, J ) 8.0 Hz, 2H), 7.43-7.30 (m, 12H), 5.86 (d,
J ) 9.4 Hz, 1H), 5.56 (s, 1H), 5.21 (s, 2H), 4.93 (d, J ) 3.3 Hz, 1H),
4.80 (d, J ) 5.7 Hz, 1H), 4.48-4.44 (m, 2H), 4.25-4.23 (m, 3H),
4.25-4.03 (m, 9H), 3.90 (m, 2H), 3.61 (br s, 1H), 3.01 (br s, 1H),
2.12 (s, 3H), 1.30 (d, J ) 6.2 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ 170.4, 170.1, 156.7, 153.5, 143.8, 143.6, 141.2, 137.4, 134.8, 129.0,
128.6, 128.5, 128.4, 128.1, 127.7, 127.0, 126.9, 126.4, 125.0, 199.9,
101.8, 100.8, 98.8, 76.1, 75.5, 75.4, 73.3, 69.2, 68.9, 68.7, 67.7, 67.2,
63.4, 62.2, 59.2, 58.7, 47.0, 20.6, 18.6; FAB HRMS calcd for (M +
Na) C48H48N4O16Na 959.2963, found 959.2927.
Fully Protected Threonine TF Antigen 46. Compound 45 (120
mg, 0.123 mmol) was treated with thiolacetic acid (5 mL, distilled three
times) for 19 h at rt. The thiolacetic acid was removed with a stream
of nitrogen, followed by toluene evaporation (three times). The crude
product was purified by flash chromatography (80-100% EtOAc/
hexane) to yield 103 mg (87%): [R]20D +90.18° (c 0.055, CHCl3); IR
General Procedure for Glycosylation with Glycosyl Trichloro-
acetimidates. A flame-dried flask was charged with donor 40 (98 mg,

12484 J. Am. Chem. Soc., Vol. 120, No. 48, 1998
Kuduk et al.
(film) 3324, 3064, 2954, 1815, 1747, 1674, 1527, 1372, 1228, 1043
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.78 (d, 2H), 7.65 (d, 2H), 7.43-
7.32 (m, 7H), 5.90 (br d, J ) 9.3 Hz, 1H), 5.76 (br d, 1H), 5.44 (br s,
1H), 5.20 (d, J ) 12.0 Hz, 1H), 5.07 (d, J ) 12.0 Hz, 1H), 4.97 (br d,
J ) 7.7 Hz, 2H), 4.81-4.76 (m, 3H), 4.48-4.41 (m, 4H), 4.18-3.90
(m, 8H), 2.20 (s, 3H), 2.16 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.03 (s,
3H), 1.29 (d, J ) 6.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 170.6,
170.5, 170.3, 170.1, 168.8, 156.5, 152.9, 143.6, 143.5, 141.1, 134.4,
128.7, 128.3, 127.6, 126.9, 124.9, 124.5, 119.9, 99.9, 75.0, 72.9, 72.3,
68.8, 68.2, 67.9, 67.5, 66.9, 62.9, 62.1, 60.2, 58.6, 48.7, 47.0 ,23.2,
20.8, 20.6, 20.5, 18.2, 14.0; FAB HRMS calcd for (M + Na)
C49H54N2O20Na 1013.3158, found 1013.3201.
m, 2H), 1.32 (d, J ) 6.2 Hz, 3H), 1.29 (d, J ) 5.8 Hz, 3H), 1.23 (d,
J ) 5.7 Hz, 3H); CI (NH3) MS calcd for (M + Na) C21H27N3BrO14Na
1611.8, found 1611.8.
Vaccine Preparation. 27-KLH and 27-BSA vaccines were
prepared as described previously for the MUC1-KLH conjugate
vaccine.45 The yield of this procedure was generally around 10%.
Unreacted Tn(c) was removed by a molecular cutoff filter (MW 30 000,
Centriprep, Amicon Inc., Beverly, MA). The samples was filtered
through a 0.22 µm filter under sterile conditions. The protein content
was determined using the BioRad protein assay and the carbohydrate
content by a HPAEC-PAD assay. The epitope ratio of 27-KLH and
-BSA was 317:1 and 7:1, respectively. The resulting more effective
conjugation to KLH versus BSA may reflect uncertainties in the quality
of the reagents employed for the latter. The aliquoted vaccine was
stored at 4 °C.
Glycopeptide 48. Compound 47 (30 mg, 0.03 mmol) and 10 mg
of 10% Pd/C were dissolved in 3 mL of MeOH under a hydrogen
atmosphere (balloon) for 4 h. After consumption of starting material
by TLC, the solution was filtered through Celite and concentrated to
Immunization Protocol. Groups of mice (female CB6F1 mice,
Jackson Laboratory, Bar Harbor, ME) were immunized subcutaneously
five times (weeks 0, 1, 2, 7, and 20) with 10 µg of 30 alone dissolved
in intralipid, 10 µg of 30 alone dissolved in intralipid plus 20 µg of
QS-21, and 27-KLH or 27-BSA (containing 3 µg of synthetic Tn(c)
trisaccharide) plus 20 µg of QS-21. Mice were bled 10 days after the
third, fourth, and fifth vaccinations and, sera were separated and stored
at -30 °C.
afford 27 mg (100%) of 48 as a white film: [R]20 +69.05° (c 0.2,
D
CHCl3); IR (film) 3333, 3064, 2938, 1817, 1746, 1642, 1528, 1372,
1226, 1044 cm-1; 1H NMR (400 MHz, MeOH-d6) δ 7.70 (d, 2H), 7.64
(t, 2H), 7.35 (t, 2H), 7.26-7.30 (m, 2H), 4.88-5.00 (m, 4H), 4.59-
4.65 (m, 2H), 4.15-4.47 (m, 11H), 4.00 (dd, J ) 11.0, 8.1 Hz, 1H),
3.92 (dd, J ) 11.2, 3.0 Hz, 1H), 2.07 s, 3H), 2.05 (s, 3H), 2.03 (s,
3H), 1.98 (s, 3H), 1.95 (s, 3H), 1.18 (d, J ) 6.2 Hz, 3H); 13C NMR
(100 MHz, MeOH-d6) δ 173.6, 173.5, 172.4, 172.3, 172.2, 170.6, 159.1,
159.0, 155.2, 145.4, 145.2, 142.6, 128.9, 128.3, 128.1, 126.2, 126.1,
125.1, 121.1, 101.9, 100.9, 77.6, 76.7, 76.0, 74.9, 71.2, 70.6, 69.1, 67.7,
64.4, 63.5, 60.0, 59.9, 50.0, 48.5, 23.3, 21.0, 20.9, 20.8, 19.2; FAB
HRMS calcd for (M + Na) C42H48N2O20Na 923.2598, found 923.2692.
General Procedure D for Peptide Coupling with HOAt/HATU.
Glycosyl amino acid 14 or 15 (1.0 equiv) and the peptide with a free
amino gruop (1.2 equiv) were dissolved in CH2Cl2 (22 mL, 1 mmol).
The solution was cooled to 0 °C, and IIDQ (1.15-1.3 equiv) was added
(1 mg in ca. 20 mL of CH2Cl2). The reaction was then stirred at rt for
1-2 h. The mixture was then purified directly by chromatography on
silica gel (3-5% MeOH/CH2Cl2) and subsequently deprotected.
Serological Analysis. The serological response was analyzed by
several serological methods.
(1) ELISA. Enzyme-linked immunosorbent assay (ELISA) was used
to determine the titer of antibodies against Tn(c)-pam as described
previously.44 Serially diluted antiserum was added to wells coated with
antigen (0.1 µg) and incubated for 1 h at room temperature. Goat anti-
mouse IgM or IgG conjugated with alkaline phosphatase served as
secondary antibodies. Absorbance was measured at 414 nm. The
antibody titer was defined as the highest serum dilution showing an
absorbance of 0.1 or greater than that of normal mouse sera.
(2) Flow Cytometry. Cell surface reactivity of these antibodies
was assayed by flow cytometry on Tn(c) positive LS-C cells and Tn(c)
negative LS-B cells.45 Single cell suspensions of 2 × 105 cells/tube
were washed in PBS with 3% fetal calf serum and 0.01 M NaN3 and
incubated with 20 µL of 1:20 diluted antisera or mAb IE3 for 30 min
on ice. After the cells were washed twice with 3% FCS in PBS, 20
µL of 1:15 goat anti-mouse IgM labeled with fluorescein-isothiocy-
anate (FITC) was added. The solution was mixed and incubated for
30 min. After the cells were washed, the positive population and mean
fluorescence intensity of stained cells were analyzed by flow cytometry
(EPICS-Profile II, Coulter, Co., Hialeah, FL) as described.46
General Procedure E for FMOC Deprotection with KF.
A
substrate (1 mmol in 36 mL of DMF) was dissolved in anhydrous DMF
followed by addition of KF (10 equiv) and 18-crown-6 (catalytic
amount). The mixture was then stirred for 48-60 h at rt until complete
by TLC. Evaporation of DMF in Vacuo was followed by flash
chromatography on silica gel (7-10% MeOH/CH2Cl2). The resulting
amine was then used directly in peptide coupling or capping with Ac2O.
Glycopeptide 48. [R]20 +52.33° (c 0.7, CHCl3); IR (film) 3249,
D
2942, 2865, 1748, 1684, 1370, 1243, 1034 cm-1; 1H NMR (500 MHz,
CDCl3) δ 7.81 (d, 2H), 7.68 (t, 2H), 7.42-7.30 (m, 4H), 5.43 (d, J )
2.8 Hz, 1H), 4.92-4.83 (m, 3H), 4.57 (dd, J ) 10.8, 6.3 Hz, 1H), 4.48
(dd, J ) 10.8, 6.3 Hz, 1H), 4.39-4.09 (m, 11H), 3.94-3.89 (m, 2H),
3.21-3.15 (m, 2H), 3.02 (br t, 2H), 2.09 (br s, 6H), 2.06 (s, 3H), 2.00
(br s, 6H), 1.58 (t, J ) 6.5 Hz, 2H), 1.45 (s, 9H), 1.20 (d, J ) 6.3 Hz,
3H); 13C NMR (125 MHz, CDCl3) δ 170.9, 170.5, 170.4, 169.9, 168.8,
157.0, 156.9, 152.8, 143.8, 143.7, 141.3, 127.7, 127.1, 125.1, 125.0,
120.0, 100.6, 100.1, 79.7, 77.7, 76.7, 72.6, 72.3, 69.0, 68.3, 68.2, 67.2,
63.0, 62.3, 58.7, 49.3, 47.2, 37.1, 36.0, 30.1, 28.4, 23.4, 20.8, 17.7;
FAB HRMS calcd for (M + Na) C50H64N4O21Na 1079.3961, found
1079.3955.
(3) Complement Dependent Cytotoxicity (CDC). The ability of
these sera to mediate complement lysis was assessed by a chromium
release complement dependent cytotoxicity assay against LS-C cells
(6). Complement dependent cytotoxicity was assayed at a serum
dilution of 1:10 with LS-C cells by a 4 h chromium-release assay as
previously described. All assays were performed in triplicate. Controls
included cells incubated only with culture medium, complement,
antisera, or mAb IE3. Spontaneous release was the europium released
by target cells incubated with complement alone. Percent cytolysis
was calculated according to the formula specific release (%) )
experimental release - spontaneous release/maximum release -
spontaneous release × 100
Glycopeptide 50. Compound 49 (70 mg, 0.03 mmol) was treated
with 1.5 mL of TFA in 0.5 mL of CH2Cl2 for 30 min at rt. The reaction
was concentrated and lyophilized with AcOH/tert-butyl alcohol (1:5).
To the resulting white solid was added 18.3 mg (2.0 equiv, 0.06 mmol)
of SAMA-OPfp in 2.5 mL of CH2Cl2 followed by the dropwise
addition of DIEA (10.6 mL, 0.06 mmol). After 1 h at rt, the reaction
mixture was concentrated and purified by silica gel chromatography
(7-10% MeOH/CH2Cl2) to afford 63 mg of white film. This material
was dissolved in a degassed solution of NaOMe in MeOH (pH ∼10)
under Ar for 2 h. The reaction was treated with Amberlyst-15 resin
until pH ∼5, and then filtered, concentrated, and purified via chroma-
tography on RP-18 silica gel (degassed H2O) to afford 36 mg (60%)
of 50 as a fluffy solid after lyophilization. Further analysis was
renounced to avoid dimerization of the thiol, and the material was stored
under Ar at -78 °C: 1H NMR (400 MHz, D2O) δ 4.89-3.12 (m,
∼60H), 2.13 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H), 1.69 (br
Inhibition Assay. Antisera at 1:1500 dilution or mAb IE3 at 0.1
µg/mL were mixed with various concentrations of structurally related
and unrelated carbohydrate antigens. The mixture was incubated at
room temperature for 30 min, and transferred to an ELISA plate coated
with Tn(c)-pam. ELISAs were performed as described above.
Percentage inhibition was calculated as the difference in absorbance
between the uninhibited and inhibited serum.
(44) Zhang, S.; Helling, F.; Lloyd, K. O.; Livingston, P. O. Cancer
Immunol. Immunother. 1995, 40, 88-94.
(45) Natoli, E. J., Jr.; Livingston, P. O.; Cordon-Cardo, C.; Pukel, C. S.;
Lloyd, K. O.; Wiegandt, H.; Szalay, J.; Oettgen, H. F.; Old, L. J. Cancer
Res. 1986, 46, 4116.
(46) Cahan, L. D.; Irie, R. F.; Singh, R.; Cassidenti, A.; Paulson, J. C.
Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 7629-733.

Mucin-Related Tn and TF O-Linked Antigens
J. Am. Chem. Soc., Vol. 120, No. 48, 1998 12485
ELISAs were also performed with sera that had been inhibited
(absorbed) by incubation with LS-C or LS-B cells. For this assay 5 ×
105 cells were incubated with sera for 1 h and the cells removed by
centrifugation. ELISA was performed as described above.
acknowledge Dr. George Sukenick (NMR Core Facility, Sloan-
Kettering Institute) for NMR and mass spectral analyses.
Supporting Information Available: 1H and 13C spectra for
compounds 5, 7, 9, 14-19, 21, 23, 25, 30, 35-38, 40â, 41R,
Acknowledgment. This research was supported by the
National Institutes of Health (Grant Nos. AI 16943 (S.J.D.) and
CA-61422 (P.O.L.) and Sloan-Kettering Institute Core Grant
(CA-08748)). Postdoctoral fellowship support is gratefully
acknowledged by S.D.K. (US Army Breast Cancer Research
Fellowship DAMD 17-98-1-8154), J.B.S. (Grant NIH-F3218804),
P.G. (American Cancer Society, Grant PF98026), and D.S.
(Irvington Institute for Immunological Research). We gratefully
1
42R, and 45-47, H spectra for compounds 32, 39R,â, 40R,
and 50, and experimental details for compounds 14-17, 20-
23, 35, 41R, 41â, and 49 (60 pages, print/PDF). See any current
masthead page for ordering information and Web access
instructions.
JA9825128