Synthesis of oxime-linked mucin mimics containing the tumor-related T(N) and sialyl T(N) antigens.

Article abstract of DOI:10.1021/ol0166247

[reaction--see text] The synthesis of oxime-linked mucin mimics was accomplished via the incorporation of multiple ketone residues into a peptide followed by reaction with aminooxy sugars corresponding to the tumor-related T(N) and sialyl T(N) (ST(N)) ant

Full text of DOI:10.1021/ol0166247

ORGANIC
LETTERS
2001
Vol. 3, No. 23
3691-3694
Synthesis of Oxime-Linked Mucin
Mimics Containing the Tumor-Related TN
and Sialyl TN Antigens
Lisa A. Marcaurelle, Youngsook Shin, Scarlett Goon, and Carolyn R. Bertozzi*
Center for New Directions in Organic Synthesis,†
Departments of Chemistry and Molecular and Cell Biology, and Howard Hughes
Medical Institute, UniVersity of California, Berkeley, California 94720
bertozzi@cchem.berkeley.edu
Received August 21, 2001
ABSTRACT
The synthesis of oxime-linked mucin mimics was accomplished via the incorporation of multiple ketone residues into a peptide followed by
reaction with aminooxy sugars corresponding to the tumor-related TN and sialyl TN (STN) antigens.
The site-specific attachment of oligosaccharides to proteins
can be accomplished by reaction of nucleophilic sugar
derivatives with aldehydes and ketones.1 Previously, we
reported on the use of ketone-amino acid 1 (Figure 1) for
the synthesis of glycopeptide analogues containing unnatural
sugar-peptide linkages.2 Amino acid 1 can be prepared in
one step via reductive ozonolysis of commercially available
Fmoc-dehydroleucine (2)3 and can be incorporated into
peptides (3) by Fmoc-based solid-phase peptide synthesis
(SPPS) without need for protection of the ketone group.4
The ketone group is chemically orthogonal to all naturally
occurring amino acid side chain functional groups and thus
can be selectively condensed with aminooxy sugars to give
the corresponding oxime-linked products (4). Since glyco-
† The Center for New Directions in Organic Synthesis is supported by
Bristol-Myers Squibb as Sponsoring Member.
(1) Reviewed in: (a) Marcaurelle, L. A.; Bertozzi, C. R. Chem. Eur. J.
1999, 5, 1384. (b) Hang, H.; Bertozzi, C. R. Acc. Chem. Res. 2001, 34,
727.
(2) (a) Marcaurelle, L. A.; Rodriguez, E. C.; Bertozzi, C. R Tetrahedron
Lett. 1998, 39, 8417. (b) Rodriguez, E. C.; Marcaurelle, L. A.; Bertozzi, C.
R. J. Org. Chem. 1998, 63, 9614.
(3) Purchased from BACHEM.
(4) Marcaurelle, L. A.; Bertozzi, C. R. Tetrahedron Lett. 1998, 39,
7279.
Figure 1. Synthesis of oxime-linked glycopeptides.
10.1021/ol0166247 CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/13/2001

proteins often contain more than one site of glycosylation,
we were interested to see if this strategy could be applied to
the synthesis of glycopeptides with clustered oxime-linked
glycans.
We chose fragments of the endothelial mucin GlyCAM-1
(5 and 6, Figure 3) as peptide scaffolds.10 Peptides 5 and 6
Mucins are a class of heavily O-glycosylated proteins that
are abundantly secreted by epthelial cells specialized for
mucus production.5 They are rich in serine and threonine
residues bearing R-linked glycans initiated by N-acetyl-
galactosamine (GalNAc). A variety of carbohydrate ligands
required for cell-surface interactions, including the Lewis
and blood group antigens, can be presented on a mucin
scaffold. In cancer cells, the glycosylation pattern of mucins
is altered, leading to the expression of distinct tumor-related
epitopes.6 Thus, mucin fragments and their cancer-associated
oligosaccharides have attracted much attention as components
of synthetic cancer vaccines.7 In the present study we focused
on the preparation of oxime-linked mucin mimics contain-
ing clusters of the TN and sialyl TN (STN) antigens8 (Figure
2), which are abundantly expressed in many types of
Figure 3. Amino acid sequence of GlyCAM-1 and corresponding
peptide fragments 5 and 6 bearing ketone-amino acid 1 (single letter
code “O”) in place of Ser and Thr.
each replace six Ser or Thr residues within the 12- or 17-
amino acid sequence with amino acid 1 (designated with the
single letter code “O”). The syntheses of 5 and 6 were carried
out on an automated peptide synthesizer using DCC/HOBt-
mediated couplings, on Fmoc-Glu(tBu)-Wang and MBHA
resins, respectively. Amino acid 1 was used without protec-
tion of the ketone group. Following chain assembly the
peptides were cleaved from the resin with 95% aqueous TFA
for 5 h and precipitated from Et2O. Analysis of the crude
peptides by ESI-MS showed the desired ketone-containing
derivatives as the major products.11 Purification of the
peptides by reversed-phase HPLC yielded targets 5 and 6 in
40-46% yield.
Figure 2. Oxime-linked analogues of the TN and STN antigens.
The synthesis of the aminooxy-TN antigen (7) was ac-
complished as previously described2 using a phase transfer
catalyzed (PTC)12 reaction of N-hydroxysuccinimide (NHS)
with glycosyl chloride 813 (Scheme 1). Reductive acetylation
cancer, including tumors of the breast, colon, liver, and
pancreas.9
(5) Reviewed in: (a) Hanisch, F.-G. Biol. Chem. 2001, 143. (b) Strous,
G. J.; Dekker: J. J. Crit. ReV. Biochem. Mol. Biol. 1992, 27, 57. (c)
Carraway, K. L.; Hull, S. R. Glycobiology 1991, 1, 131.
(6) Varki, A. Glycosylation “Changes in Cancer”. In Essentials of
Glycobiology; Varki, A., Cummings, R., Esko, J., Freeze, H., Hart, G.,
Marth, J., Eds.; New York: Cold Spring Harbor Laboratory, 1999; pp 537-
550.
Scheme 1a
(7) Danishefsky, S. J.; Allen, J. R. Angew. Chem., Int. Ed. 2000, 39,
836.
(8) For the synthesis of glycopeptides containing the TN and STN antigens,
see: (a) Liebe, B.; Kunz, H. Angew. Chem., Int. Ed. 1997, 36, 618. (b)
Keil, S.; Claus, C.; Dippold, W.; Kunz, H. Angew. Chem., Int. Ed. 2001,
40, 366. (c) Kuduk, S. D.; Schwarz, J. B.; Chen, X.-T.; Glunz, P. W.; Sames,
D.; Ragupathi, G.; Livingston, P. O.; Danishefsky, S. J. J. Am. Chem. Soc.
1998, 120, 12474. (d) Schwarz, J. B.; Kuduk, S. D.; Chen, X.-T.; Sames,
D.; Glunz, P. W.; Danishefsky, S. J. J. Am. Chem. Soc. 1999, 121, 2662.
(e) Elofsson, M.; Salvador, L. A.; Kilhberg, J. Tetrahedron 1997, 53, 369.
(f) George, S. K.; Holm, B.; Reis, C.; Schwientek, T.; Clausen, H.; Kihlberg,
J. J. Chem. Soc., Perkin Trans. 1 2001, 880.
(9) (a) Brockhausen, I.; Yang, J. M.; Burchell, J.; Whitehouse, C.; Taylor-
Papdimitriou, J. Eur. J. Biochem. 1995, 233, 607. (b) Sasaki, M.; Yamato,
T.; Nakanuma, Y. Pathol. Int. 1999, 49, 325. (c) Itzkowitz, S. H.; Yaun,
M.; Montgomery, C. K.; Kjeldsen, T.; Takahashi, H. K.; Bigbee, W. L.;
Kim, Y. S. Cancer Res. 1989, 49, 197. (d) Itzkowitz, S.; Kjeldsen, T.; Friera,
A.; Hakomori, S.-N.; Yang, U.-S.; Kim, Y. S. Gastroenterology 1991, 100,
1691.
a Reagents: (a) NHS, (nBu)4NHSO4, CH2Cl2, Na2CO3, 57% (R
only); (b) NHS, AgClO4, CH2Cl2, 4 Å MS, 67% (3:1 R/â); (c) H2,
Pd/C, Ac2O, 100%; (d) 10% aq N2H4, 71%.
3692
Org. Lett., Vol. 3, No. 23, 2001

of 9, followed by mild hydrazinolysis, afforded the desired
aminooxy sugar (7). We have recently found that the
intermediate NHS glycoside (9) can also be accessed using
glycosyl bromide 1013 as a donor in a Koenigs-Knorr
glycosylation with NHS. While the stereoselectivity of this
reaction is not as high as that achieved in the PTC reaction,
the preparation of the R-bromide is generally higher yielding
than the â-chloride,13 making this route an attractive alterna-
tive.
Scheme 2a
The synthesis of aminooxy-STN 11 utilized the selectively
protected glycosyl acceptor 12, containing the preinstalled
NHS glycoside, for reaction with known sialyl phosphite 1314
(Figure 4). For the installation of the NHS glycoside we
a Reagents: (a) Me2C(OMe)2, PPTS, DMF, 50 °C, 1 h, 95%;
(b) CAN, NaN3, CH3CN, -20 °C, 15 h, 71%, (c) LiBr, CH3CN,
rt, 5 h, 96%; (d) NHS, AgClO4, CH2Cl2, 4 Å MS, rt, 2 d, 65% (3:1
R/â); (e) TBAF, AcOH, THF, rt, 6 h, 45%; (f) 13, TMSOTf, THF,
4 Å MS, -35 °C, 1 h, 44% (3:1 R/â); (g) p-TsOH, MeOH, rt,
overnight, 60%; (h) Ac2O, pyridine, DMAP, rt, overnight, 54%;
(i) H2, Pd/C, Ac2O, rt, 2 h, 49% after HPLC; (j) (i) NaOMe, MeOH,
rt, 24 h, (ii) LiOH, MeOH, H2O, 4 °C, overnight, (iii) 10% aq
N2H4‚H2O, 70% for 3 steps.
pH 5.5 (Scheme 3).16 The reactions were monitored by
reversed-phase HPLC and judged to be complete after
24 h. The oxime-linked products (19-22) containing the TN
and STN antigens were purified by reversed-phase HPLC
(60-70% yield), and their identity was confirmed by ESI-
MS.17
Figure 4. Retrosynthesis of aminooxy-STN (11).
chose to use a Koenigs-Knorr glycosylation with glycosyl
bromide 14, which was obtained from 6-O-TBDPS-D-galactal
(15).15
These syntheses illustrate that multiple clustered ketone
residues can be incorporated into a peptide and reacted with
aminooxy sugars. Such an approach circumvents the need
to synthesize large quantities of complex glycosyl amino
acids for use in peptide synthesis, a process that can be
extremely labor intensive depending on the complexity of
the pendant glycan. The oxime-based strategy benefits from
convergent assembly of peptides and aminooxy sugars, both
of which are straightforward to prepare. The incorporation
of these glycopeptides into larger, full-length proteins, by
techniques such as native and expressed protein ligation,18
should provide access to homogeneous mucin analogues for
a variety of applications.
As depicted in Scheme 2, glycosyl bromide 14 was
generated in three steps via iosopropylidine formation,
azidonitration12 with CAN and NaN3 and treatment with
LiBr. Reaction of bromide 14 with NHS in the presence of
AgClO4 gave compound 16 in 65% yield as a mixture of
anomers (3:1 R/â). Isolation of the desired R-glycoside (12)
was achieved following removal of the TBDPS group with
TBAF. Glycosylation of 12 with sialyl phosphite 13 using
TMSOTf as the promoter gave disaccharide 17 in 44% yield
as a mixture of anomers (3:1 R/â). Subsequent deprotection
and reductive acetylation of 17 over a series of steps afforded
the target aminooxy-STN 11.
The ligation of 7 and 11 with peptides 5 and 6 was carried
out at 37 °C with an excess of either sugar in NaOAc buffer,
(16) General Procedure for Oxime Formation. To 200 µL of peptide
(50 mM with respect to ketone groups) was added 50 µL of 1 M NaOAc
buffer (pH 5.5) and 250 µL of aminooxy sugar (100 mM). The reaction
mixture was incubated at 37 °C for 24 h, and the oxime-linked product
was isolated by reversed-phase HPLC using a gradient of 0-30% CH3CN
in water (0.1% TFA) over 20 min.
(10) Imai, Y.; Singer, M. S.; Fennie, C.; Lasky, L. A.; Rosen, S. D. J.
Cell. Biol. 1991, 113, 1213.
(11) ESI-MS (neg-ion mode): calcd for 5 1356.1, found 1355.7; calcd
for 6 2021.8, found 2021.7.
(12) Cao, S.; Tropper, F. D.; Roy, R. Tetrahedron 1995, 51, 6679.
(13) Lemieux, R.; Ratcliffe, R. M. Can. J. Chem. 1979, 57, 1244.
(14) (a) Sim, M. M.; Kondo, H.; Wong, C.-H. J. Am. Chem. Soc. 1993,
115, 2260. (b) Bhattacharya, S. K.; Danishefsky, S. J. J. Org. Chem. 2000,
65, 144.
(17) ESI-MS (neg-ion mode): calcd for 19 2665.3, found 2665.6; calcd
for 20 3331.3, found 3330.2; calcd for 21 4412.9, found 4413.2; calcd for
22 5078.9, found 5080.0.
(18) (a) Macmillan: D.; Bertozzi, C. R. Tetrahedron 2000, 56,
9515. (b) Tilbert, T. J.; Wong, C.-H. J. Am. Chem. Soc. 2000, 122, 5421.
(c) Marcaurelle, L. A.; Bertozzi, C. R. Chem Eur. J. 2000, 7, 1129. (d)
Dawson, P. E.; Kent, S. B. H. Annu. ReV. Biochem. 2000, 69, 923. (e)
Muir, T. W.; Sondi, D.; Cole, P. A. Proc. Natl. Acad. Sci. 1998, 95,
6705.
(15) Gervay, J.; Peterson, J. M.; Oriyama, T.; Danishefsky, S. J. J. Org.
Chem. 1993, 58, 5465.
Org. Lett., Vol. 3, No. 23, 2001
3693

Scheme 3. Synthesis of Mucin Mimics 19 and 20, Containing the TN Antigen (A), and Mucin Mimics 21 and 22, Containing the
STN Antigen (B)a
a All ligation reactions were performed by incubating the peptide with an excess of aminooxy sugar at 37 °C in NaOAc buffer, pH 5.516.
Acknowledgment. This research was supported by a
grant from the National Science Foundation (CAREER
Award CHE-9734439) and by the Laboratory Technology
Research Division (SC-32), within the Office of Science,
U.S. Department of Energy under a CRADA (Cooperative
Research and Development Agreement) between Lawrence
Berkeley National Laboratory and CibaVision under U.S.
DOE Contract DE-AC03-76SF00098. L.A.M. thanks the
American Chemical Society Division of Organic Chemistry
for a graduate fellowship.
Supporting Information Available: Full experimental
1
procedures and tabulated H and 13C NMR data for all
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL0166247
3694
Org. Lett., Vol. 3, No. 23, 2001