Chemoselective elaboration of O-linked glycopeptide mimetics by alkylation of 3-thioGalNAc

Article abstract of DOI:10.1021/ja003713q

A critical branch point in mucin-type oligosaccharides is the β1 → 3 glycosidic linkage to the core α-N-acetylgalactosamine (GalNAc) residue. We report here a strategy for the synthesis of O-linked glycopeptide analogues that replaces this linkage with a

Full text of DOI:10.1021/ja003713q

J. Am. Chem. Soc. 2001, 123, 1587-1595
1587
Chemoselective Elaboration of O-Linked Glycopeptide Mimetics by
Alkylation of 3-ThioGalNAc
,§
Lisa A. Marcaurelle‡ and Carolyn R. Bertozzi*,‡,
Contribution from the Center for New Directions in Organic Synthesis, Departments of Chemistry and
Molecular and Cellular Biology, and Howard Hughes Medical Institute, UniVersity of California,
Berkeley, California 94720
ReceiVed October 18, 2000
Abstract: A critical branch point in mucin-type oligosaccharides is the â1 f 3 glycosidic linkage to the core
R-N-acetylgalactosamine (GalNAc) residue. We report here a strategy for the synthesis of O-linked glycopeptide
analogues that replaces this linkage with a thioether amenable to construction by chemoselective ligation. The
key building block was a 2-azido-3-thiogalactose-Thr analogue that was incorporated into a peptide by
fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis. Higher order oligosaccharides were
readily generated by alkylation of the corresponding 3-thioGalNAc with N-bromoacetamido sugars. The rapid
assembly of “core 1”and “core 3” O-linked glycopeptide mimetics was accomplished in this fashion.
Introduction
Mucin-type glycosylation, in which oligosaccharides are
attached to proteins via the â-hydroxyl group of either serine
or threonine, is a prevalent form of O-linked glycosylation found
in mammals and other eukaryotes.1 Mucin-type oligosaccharides
may occur singly or in clusters, and may contain up to 20
monosaccharides per glycan. The initial step in the biosynthesis
of this class of O-linked glycans is the addition of a single
N-acetylgalactosamine (GalNAc) residue to the acceptor protein
by one of several polypeptide GalNAc transferases. The resulting
GalNAc(R1-O)Ser/Thr structure, commonly referred to as the
TN antigen (Figure 1), forms the foundation for the biosynthesis
of some common “core” structures arising from branching at
C-3 and/or C-6.2 Of the eight core structures that have been
identified, cores 1 and 2 are the most abundant and are widely
distributed in both mucins and nonmucin glycoproteins. Other
core structures, such as 3 and 4, occur less frequently and tend
to be confined to mucins. Once in place, the core O-linked
glycans may be elongated by the addition of sialic acid, fucose,
and/or repeating units of galactose and N-acetylglucosamine
(GlcNAc) to give poly-N-acetyllactosamine (LacNAc) chains.
Together with further modifications, such as sulfation, these
elaborations give rise to highly complex structures, often
containing important recognition elements such as the Lewis
and blood group antigens.3
Figure 1.
corresponding sugar-nucleotide donorssit is not surprising that
mucin-type glycoproteins exist as a heterogeneous mixture of
glycoforms (i.e., proteins that differ only with respect to the
positions and structures of the pendant glycans). As a result of
this heterogeneity, it is difficult to isolate or generate (through
traditional genetic methods) quantities of well-defined glyco-
proteins for structural and functional studies. Access to chemi-
cally defined material is especially desirable in the case of
glycoprotein-based pharmaceuticals.4 In light of this, much effort
has been devoted to the synthesis of this important class of
biomolecules.5 Impressive mucin-related oligosaccharides have
been synthesized by employing chemical and/or enzymatic
methods, which allows their use in structural and functional
studies.6 Advances in the field of protein chemistry have also
Given the complex nature of their biosynthesissrelying on
the availability of a variety of glycosyltransfersases and the
* To whom correspondence should be addressed. Phone: (510) 643-1682.
Fax: (510) 643-2628.
‡ Department of Chemistry.
Department of Molecular and Cellular Biology.
§ Howard Hughes Medical Institute.
(1) (a) Carraway, K. L.; Hull, S. R. Glycobiology 1991, 1, 131. (b) Strous,
G. J.; Dekker: J. J. Crit. ReV. Biochem. Mol. Biol. 1992, 27, 57. (c) Hilkens,
J.; Ligtenberg, M. J. L.; Vos, H. L.; Litvinov, S. V. Trends Biochem. Sci.
1992, 17, 359. (d) Shimizu, Y.; Shaw, S. Nature 1993, 366, 630.
(2) For a review on the biosynthesis of O-linked glycans, see: (a) Bill,
R. M.; Revers, L.; Wilson, I. B. H. Protein Glycosylation; Kluwer
Academic: Boston, 1998; Chapter 6. (b) Brockhausen, I. In Glycoproteins;
Montreuil, J., Vliegenthart, J. F. G., Schachter, H., Eds.; Elvesier:
Amsterdam, 1995; pp 201-259.
(3) Varki, A.; Cummings, R.; Esko, J.; Freeze, H.; Hart, G.; Marth, J.
Essentials of Glycobiology; Cold Spring Harbor: New York, 1999; Chapter
16.
(4) Cumming, D. A. Glycobiology 1991, 1, 115.
(5) For a recent review, see: Herzner, H.; Reipen, T.; Schultz, M.; Kunz,
H. Chem. ReV. 2000, 100, 4495.
10.1021/ja003713q CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/03/2001

1588 J. Am. Chem. Soc., Vol. 123, No. 8, 2001
Marcaurelle and Bertozzi
Figure 2.
made it possible to produce full-length glycoproteins using the
technique of native chemical ligation.7 Although great progress
has been made in this area of research, the synthesis of mucin-
type glycoproteins is still an arduous task, requiring a great deal
of synthetic expertise or access to the necessary glycosyltrans-
ferases and sugar-nucleotide donors.
replacement of glycosidic linkages to the core R-O-GalNAc with
alternate, more facile linkages that are amenable to convergent
fragment condensation. To this end, we chose to employ the
principle of chemoselective ligation for the attachment of
oligosaccharides to glycopeptides bearing the R-O-GalNAc
epitope.
To address this need for homogeneous glycoproteins, we have
been working to develop new methods for the synthesis of
glycopeptide mimetics.8,9 Ideally, the designed analogues should
be more easily generated than the corresponding native struc-
tures yet possess the desired biological activity. Mindful of the
importance of the core GalNAc(R1-O)Ser/Thr for establishing
proper organization of the glycopeptide backbone,10 we sought
to preserve this structural element. Because it is often the
peripheral sugars of O-linked glycans that contain the informa-
tion required for molecular recognition,3 we desired a means
for presenting these “ligands” in the context of the native core
structure. We reasoned that a viable route might involve
Analogous to enzymatic reactions, chemoselective ligation
reactions are mild and selective and, thus, can be performed in
the presence of richly functionalized peptides and carbohy-
drates.11 The technique is based on the introduction of two
mutually and uniquely reactive functional groups onto unpro-
tected fragments and the convergent coupling of these fragments
in an aqueous environment. One reaction that we have employed
for the construction of glycopeptide mimetics is the coupling
of ketones or aldehydes with aminooxy sugars.8 Using this
reaction, we synthesized glycopeptide analogues in which the
â1 f 6 glycosidic linkage to R-O-GalNAc was replaced by an
oxime ether.8c As shown in Figure 2A, a glycopeptide bearing
the core R-O-GalNAc structure was subjected to oxidation by
galactose oxidase to furnish the C-6 aldehyde. This uniquely
functionalized peptide was then reacted with a variety of
aminooxy sugars to give the corresponding oxime-linked
products. By this method we were able to generate an analogue
of the anti-microbial glycopeptide drosocin that retained its
biological activity.
Because elongation of the core GalNAc residue of O-linked
glycoproteins also occurs at C-3, we sought to introduce an
orthogonal chemical handle at this position for coupling to
appropriately functionalized sugars. The reaction of cysteine
containing proteins with N-haloacetamido sugars has proven
useful for the site-selective introduction of carbohydrates into
a variety of neoglycoproteins.12 This procedure has generally
been exploited for the production of glycoproteins that mimic
N-linked structures. Because thioether formation is compatible
with and chemically orthogonal to oxime formation, we reasoned
that the introduction of a thiol group at the 3-position of a
peptide-bound GalNAc residue would allow for elaboration at
both branch points. As an initial step toward this goal, we
achieved the synthesis of O-linked glycopeptides bearing C-3
thioethers via alkylation of 3-thioGalNAc (Figure 2B) with
N-bromoacetamido sugars. The syntheses of these analogues
are described herein.
(6) (a) Schwarz, J. B.; Kuduk, S. D.; Chen, X.-T.; Sames, D.; Glunz, P.
W.; Danishefsky, S. J. J. Am. Chem. Soc. 1999, 121, 2662. (b) Glunz, P.
W.; Hintermann, S.; Schwarz, J. B.; Kuduk, S. D.; Chen, X.-T.; Williams,
L. J.; Sames, D.; Danishefsky, S. J.; Kudryashov, V.; Lloyd, K. O. J. Am.
Chem. Soc. 1999, 121, 10636. (c) Glunz, P. W.; Hintermann, S.; Williams,
L. J.; Schwarz, J. B.; Kuduk, S. D.; Kudryashov, V.; Lloyd, K. O.;
Danishefsky, S. J. J. Am. Chem. Soc. 2000, 122, 7273. (d) Mathieux, N.;
Paulsen, H.; Meldal, M.; Bock, K. J. Chem. Soc., Perkin Trans. 1 1997,
2359. (e) Wang, Z.-G.; Zhang, X.-F.; Ito, Y.; Nakahara, Y.; Ogawa, T.
Carbohydr. Res. 1996, 295, 25; (f) Singh, L.; Nakahara, Y.; Ito, Y.;
Nakahara, Y. Tetrahedron Lett. 1999, 40, 3769. (g) Xia, J.; Alderfer, J. L.;
Piskorz, C. F.; Matta, K. L. Chem. Eur. J. 2000, 6, 3442. (h) Leppanen,
A.; Mehta, P.; Ouyang. Y.-B.; Ju, T.; Helin, J.; Moore, K. L.; van Die, I.;
Canfield, W. M.; McEver, R. P.; Cummings, R. D. J. Biol. Chem. 1999,
274, 24838. (i) Koeller, K. M.; Smith, M. E.; Huang, R.-F.; Wong, C.-H.
J. Am. Chem. Soc. 2000, 122, 4244. (j) Koeller, K. M.; Wong, C.-H. Chem.
Eur. J. 2000, 6, 1243.
(7) (a) Marcaurelle, L. A.; Mizoue, L. S.; Wilken, J.; Oldham, L.; Kent,
S. B. H.; Handel, T. M.; Bertozzi, C. R. Chem. Eur. J. in press. (b) Shin,
Y.; Backes, B. J.; Kent, S. B. H.; Ellman, J. A.; Bertozzi, C. R. J. Am.
Chem. Soc. 1999, 121, 11684. (c) MacMillan, D.; Bertozzi, C. R.
Tetrahedron 2000, 56, 9515.
(8) (a) Marcaurelle, L. A.; Bertozzi, C. R. Chem. Eur. J. 1999, 5, 1384.
(b) Marcaurelle, L. A.; Rodriguez, E. C.; Bertozzi, C. R. Tetrahedron Lett.
1998, 39, 8417. (c) Rodriguez, E. C.; Winans, K. A.; King, D. S.; Bertozzi,
C. R. J. Am. Chem. Soc. 1997, 119, 9905. (d) Rodriguez, E. C.; Marcaurelle,
L. A.; Bertozzi, C. R. J. Org. Chem. 1998, 63, 7134.
(9) See also: (a) Davis, B. G.; Jones, J. B. Synlett 1999, 9, 1495. (b)
Davis, B. G. J. Chem. Soc., Perkin Trans. 1 1999, 22, 3215.
(10) (a) Live, D. H.; Williams, L. J.; Kuduk, S. D.; Schwarz, J. B.; Glunz,
P. W.; Chen, X.-T.; Sames, D.; Kumar, R. A.; Danishefsky, S. J. Proc.
Natl. Acad. Sci. 1999, 96, 3489. (b) Huang, X.; Barchi, J. J. Jr.; Lung,
F.-D. T.; Roller, P. P.; Nara, P. L.; Muschik, J.; Garrity, R. R. Biochemistry
1997, 36, 10846. (c) Gerken, T.; Butenhof, K. J.; Shogren, R. Biochemistry
1989, 28, 8, 5536. (d) Liang, R.; Andreotti, A. H.; Kahne, D. J. Am. Chem.
Soc. 1995, 117, 10395.
(11) For a review, see: Lemieux, G. A.; Bertozzi, C. R. Trends Biotech.
1998, 16, 506.
(12) (a) Wong, S. Y. C.; Guile, G. R.; Dwek, R. A.; Arsequell, G.
Biochem. J. 1994, 300, 843. (b) Davis, N. J.; Flitsch, S. L. Tetrahedron
Lett. 1991, 32, 6793.

O-Linked Glycopeptide Mimetics
J. Am. Chem. Soc., Vol. 123, No. 8, 2001 1589
Figure 4.
obtained during displacement reactions at C-3.17 Interestingly,
the formation of neither 8 nor 9 was observed. Because an azide
(or another nonparticipating amine equivalent) at C-2 was
required to achieve R-selectivity in the intended glycosylation
with threonine 2, we decided to seek an alternate method for
introducing the 3-thiol.
Figure 3.
Scheme 1a
As depicted in Figure 4, we envisioned that the glycosyl donor
1 could be obtained from a 1,6-anhydrosugar (10) via an
episulfide intermediate (11). The rigid, bicyclic framework of
1,6-anhydrosugars renders them useful for the generation of
modified carbohydrates. The constrained internal acetal serves
to protect the 1- and 6-hydroxyl groups and allows for
modification at positions 2, 3, and 4 in a regio- and stereose-
lective manner.18 On the basis of analogous reactions with 2,3-
epoxysugars,19 we anticipated that opening of the 2,3-episulfide
with azide ion would yield the desired trans diaxial product (10).
The precursor to episulfide 11 (the 1,6-anhydro pseudo-glycal,
1220) has been prepared by a number of methods, including acid-
catalyzed intramolecular Ferrier-rearrangements of D-galactal21
and reduction of isolevoglucosenone (13, Scheme 2).22 Although
our attempts to employ Ferrier-type reactions were of limited
success, reduction of isolevoglucosenone (13) proved to be a
viable route. Isolevoglucosenone (13) was most easily prepared
from tri-O-acetyl-D-glucal (15) as described by Honda et al,23
with the exception that tetrapropylammoniumperruthenate (TPAP)/
N-methylmorpholine N-oxide (NMO)24 was used in place of
MnO2 for the oxidation of allylic alcohol 16.20 Owing to the
instability of enone 13, TPAP was found to be a superior
oxidant, allowing for the direct conversion of 16 to 12 by a
a Reagents: (a) (i) Tf2O, pyr, CH3Cl2, -15 °C; (ii) TBANO2,
CH3CN, rt, overnight, 70%; (b) (i) KSAc, DMF, 60 °C, 5 h, 68% 7,
8% 6.
Results and Discussion
1. Synthesis of 2-Azido-3-ThioGal-Thr. To gain access to
glycopeptides bearing the 3-thioGalNAc epitope, we required
a suitably protected glycosyl amino acid for use in peptide
synthesis. A key intermediate would be a glycosyl donor
containing the desired thiol functionality at C-3 and an azide at
C-2 (1, Figure 3). Glycosylation of this donor with an ap-
propriately protected serine or threonine, such as the known
threonine derivative 213 (Figure 3) would yield the desired amino
acid building block (3).
The first approach that we explored for the synthesis of the
target glycosyl donor involved a double inversion at C-3 of the
known benzylidene derivative 414 (Scheme 1). Treatment of 4
with trifluoromethanesulfonic anhydride (Tf2O) in the presence
of pyridine, followed by reaction with tetrabutylammonium
nitrite (TBANO2),15 afforded 2-azido gulose 5 in 70% yield.
Subsequent 3-O-triflate formation and treatment with potassium
thioacetate (KSAc) in DMF at 60 °C yielded the desired
3-thiogalactose derivative 6 but in very poor yield (∼8%).
Instead, the major product of this reaction was the 2,3-
unsaturated 2-acetamido compound 7, which was obtained in
68% yield. Given that thioacetic acid is often used for the
reductive acetylation of azides, this result was not entirely
unexpected.6a,16 Furthermore, elimination products are often
(15) (a) Lattrell, R.; Lohaus, G. Liebigs Ann. Chem. 1974, 901. (b) Eisle,
T.; Schmidt, R. R. Liebigs Ann. 1997, 865.
(16) (a) Rosen, T.; Lico, I. M.; Chu, D. T. W. J. Org. Chem. 1988, 53,
1580. (b) Bieldfeldt, T.; Peters, S.; Meldal, M.; Bock, K.; Paulsen, H. Angew.
Chem., Int. Ed. Engl. 1992, 31, 857.
(17) (a) Aguilera, B.; Ferna´ndez-Mayoralas, A.; Jaramillo, C. Tetrahedron
1997, 53, 5863. (b) Aguilera, B.; Ferna´ndez-Mayoralas, A. Chem. Commun.
1996, 127.
(18) Cerny, M.; Stanek, J. AdV. Carbohydr. Chem. Biochem. 1977, 34,
23.
(19) (a) Paulsen, H.; Kola´r, C.; Stenzel, W. Chem. Ber. 1978, 111, 2358.
(b) Boons, G.-J.; Heskamp, B. In Carbohydrate Chemistry; Boons, G.-J.,
Ed.; Thomson Science: New York, 1998; pp 50-53.
(20) Ranganayakulu, K.; Singh, U. P.; Murray, T. P.; Brown, R. K. Can.
J. Chem. 1974, 52, 988.
(21) (a) Oberdorfer, F.; Haeckel, R.; Lauer, G. Synthesis 1998, 201. (b)
Haekel, R.; Lauer, G.; Oberdorfer, F. Synlett 1996, 21. (c) Sharma, G. V.
M.; Ramanaiah, K. C. V.; Krishnudu, K. Tetrahedron: Asymmetry 1994,
5, 1905. (d) Mereyala, H. B.; Venkataramanaiah, K. C.; Dalvoy, V. S.
Carbohydr. Res. 1992, 225, 151.
(22) (a) Ko¨ll, P.; Schultek, T.; Rennecke, R.-W. Chem. Ber. 1976, 109,
7. (b) Horton, D.; Roski, J. P.; Norris, P. J. Org. Chem. 1996, 61, 3783. (c)
Taniguchi, T.; Nakamura, K.; Ogasawara, K. Synlett, 1996, 971.
(23) Honda, I.; Shibagaki, M.; Kimino, H.; Takahashi, K.; Matsushita,
H. (Nippon Tobacco Sangyo, Japan). Preparation of Isolevoglucosenone.
Jpn. Kokai Tokyo Koho JP 05125080, 1993.
(13) (a) Kunz, H. In PreparatiVe Carbohydrate Chemistry; Hanessian,
S., Ed.; Marcel Dekker: New York, 1997; pp 274-275. (b) Paulsen, H.;
Adermann, K. Liebigs Ann. Chem. 1989, 751.
(24) For a pertinent review, see: Ley, S. V.; Norman, J.; Griffith, W.
P.; Marsden, S. P. Synthesis 1994, 639.
(14) Grundler, G.; Schmidt, R. R. Liebigs Ann. Chem. 1984, 1826-1847.

1590 J. Am. Chem. Soc., Vol. 123, No. 8, 2001
Marcaurelle and Bertozzi
Scheme 2a
Scheme 3a
a Reagents: (a) PhOH, PhCl, reflux, 4 h; (b) NaOH, H2O, reflux, 3
h, 76%; (c) TPAP, NMO, CH2Cl2, 0 °C to rt, 30 min; (d) NaBH4, CeCl3,
CH2Cl2, -78 °C, 1 h, 70% (2 steps).
a Reagents: (a) NaH, PMBCl, TBAI, DMF, 62%; (b) OsO4, NMO,
acetone/H2O, 0 °C, 90%; (c) (i) SOCl2, Et3N, CH2Cl2; (ii) RuCl3‚H2O,
NaIO4, CCl4/CH3CN/H2O, 66%; (d) (i) KSAc, DMF, 60 °C, 2 h; (ii)
NaOMe, MeOH, rt, 30 min, 91%; (e) TBAN3, Hg(OAc)2, DMF, 0 °C
to rt overnight, 68% 10, 17% 22; (f) dinitrofluorobenzene, DIEA,
CH2Cl2, rt, 12 h, 65% 23, 10% 24.
one-pot oxidation/reduction sequence. Thus, without isolation,
enone 13 was immediately reduced under Luche conditions
(NaBH4/CeCl3, -78 °C)25 to give the 1,6-anhydro compound
(12) in 70% yield. It should be noted that attempts to generate
12 directly from tri-O-acetyl-D-galactal (17) via intermediate
18 were not successful, necessitating this oxidation/reduction
sequence.
Scheme 4a
With ample quantities of 12 in hand, we turned our attention
to the synthesis of episulfide 11. The most common method
for the preparation of episulfides involves the opening of
epoxides with thiourea or thiocyanate ions.26 Unfortunately, such
reactions are not feasible for strained cyclic substrates,26g such
as 2,3-epoxides derived from 1,6-anhydro sugars. Thus, to gain
access to episulfide 11, we chose to use an alternate strategy
based on the double inversion of cyclic sulfates.27 Analogous
to epoxides, cyclic sulfates can be opened by nucleophilic attack
at either carbon center. Opening of the cyclic sulfate generates
a sulfate monoester, which allows for further transformations,
such as intramolecular attack by an internal nucleophile.28 In
this manner the one-pot synthesis of episulfides can be achieved
via reaction with KSAc or KSCN, followed by treatment with
NaOMe.27 By this approach, episulfide 11 was generated from
cyclic sulfate 19 as depicted in Scheme 3. First, allylic alcohol
12 was protected as the p-methoxybenzyl (PMB) ether (20) and
subsequently treated with OsO4/NMO at 0 °C to give diol 21
in 90% yield. Reaction of 21 with SOCl2 and Et3N produced a
diastereomeric mixture of cyclic sulfites, which was directly
converted to cyclic sulfate 19 by oxidation with NaIO4-RuCl3‚
3H2O.29 Finally, opening of the cyclic sulfate (19) was achieved
by reaction with KSAc at 60 °C, followed by treatment with
NaOMe to give the desired 2,3-episulfide (11) in high yield
(91%).
a Reagents: (a) 9:1 Ac2O/TFA, 60 °C, 18 h, 5:1 R/â, 100%; (b)
TiBr4, CH2Cl2, rt, 14 h, 92%; (c) 2, AgClO4, collidine, 4 Å MS, CH2Cl2,
rt, 4 h, 55%; (d) 95% aq TFA, 100%.
yielding only unreacted starting material. In the end, we found
it necessary to activate the episulfide with a thiophilic promoter.
Thus, treatment of 11 with TBAN3 in the presence of Hg(OAc)2
afforded the desired trans diaxial product 10 in 68% yield. While
a small amount of the undesired diequatorial product (22) was
also obtained in the reaction (17%), the diaxial compound could
be isolated in pure form after conversion to the corresponding
2,4-dinitrophenyl (DNP)-derivative 23. Formation of the DNP
thioether served not only to aid in the purification of the desired
isomer, which crystallized from CH2Cl2, but also to protect the
3-thiol during subsequent reactions, such as glycosylation and
peptide synthesis (vide infra).
Having installed the desired thiol functionality at C-3, we
then completed the synthesis of the glycosyl donor. Opening
of the 1,6-anhydro ring was achieved by dissolving 23 in Ac2O/
trifluoroacetic acid (TFA) and stirring overnight at 60 °C
(Scheme 4). In the event, acetolysis of the PMB ether also
occurred, yielding the peracetylated compound 25 in excellent
yield. Based on previous positive experience30 with glycosyl
bromides for constructing R-O-linked GalNAc-Thr/Ser building
blocks, we chose to use bromide 1 as the glycosyl donor.
Although attempts to generate the R-bromide with HBr/AcOH
lead to a mixture of products, 1 could be obtained in high yield
when TiBr4 was employed (91%). Glycosylation of 1 with
To our surprise, nucleophilic ring opening of episulfide 11
with azide ion proved to be somewhat difficult. Attempts to
open the episulfide with LiN3, TMS-N3 or TBAN3 under a
variety of conditions did not produce the desired product,
(25) Luche, J.-L. J. Am. Chem. Soc. 1978, 110, 2226.
(26) (a) Dittmer, D. C. In ComprehensiVe Heterocyclic Chemistry;
Katritzky, A. R., Reese, C. W., Lowski, W., Eds.; Pergamon Press: New
York, 1984; Vol. 7, p 171. (b) van Tamelen, E. E. J. Am. Chem. Soc. 1951,
73, 3444. (c) Ettinger, M. G. J. Am. Chem. Soc. 1950, 72, 4792. (d)
Culvenor, C. C. J.; Davies, W.; Savige, W. E. J. Chem. Soc. 1952, 4480.
(e) Bordwell, F. G.; Andersen, H. M. J. Am. Chem. Soc. 1953, 75, 4959.
(f) Gao, Y.; Sharpless, K. B.; J. Org. Chem. 1988, 53, 4114. (g) Fokin, A.
V.; Kolomiets, A. F. Russ. Chem. ReV. 1975, 44, 138 and references therein.
(27) Calvo-Flores, F. G.; Garc´ıa-Mendoza, P.; Herna´ndez-Mateo, F.; Isac
Garc´ıa, J.; Santoyo-Gonza´lez, F. J. Org. Chem. 1997, 62, 3944.
(28) For recent reviews on cyclic sulfates, see: (a) Byun, H.-S.; He, L.;
Bittman, R. Tetrahedron 2000, 56, 7051. (b) Kolb, H. C.; VanNieuwenhze,
M. S.; Sharpless, K. B. Chem. ReV. 1994, 94, 2483. (c) Lohray, B. B.
Synthesis, 1992, 1035.
(30) Winans, K. A.; King, D. S.; Rao, V. R.; Bertozzi, C. R. Biochemistry
1999, 38, 11701.
(29) Gao, Y.; Sharpless, K. B. J. Am. Chem. Soc. 1988, 110, 7538.

O-Linked Glycopeptide Mimetics
J. Am. Chem. Soc., Vol. 123, No. 8, 2001 1591
Scheme 5a
Figure 5.
N-fluorenylmethoxycarbonyl (Fmoc) threonine tert-butyl ester
(2)13 using AgClO4 as the promoter furnished the desired
R-linked glycosyl amino acid (26) in 55% yield. The only side
product of this reaction was the hydrolyzed donor, which could
be recovered and acetylated to give 25. The DNP thioether
proved to be stable in the glycosylation reaction, and formation
of the undesired â-anomer was not observed. Finally, cleavage
of the tert-butyl ester was accomplished by treatment with TFA
to yield building block 3, primed for use in peptide synthesis.
2. Glycopeptide Synthesis. To evaluate the stability of 3
during Fmoc-based peptide synthesis, we chose to synthesize a
17-amino acid fragment of P-selectin glycoprotein ligand-1
(PSGL-1), a dimeric membrane-bound mucin found on leuko-
cytes that binds to the selectins and initiates the inflammation-
adhesion cascade.31 The target glycopeptide (27, Figure 5)
corresponds to the N-terminal region of the glycoprotein, which
contains a core 2 O-linked glycan at Thr12. The presence of
this O-linked glycan, along with tyrosine sulfation, is essential
for high-affinity binding to P-selectin.6h,31 The target thioether
analogues (28 and 29) correspond to core 1 and core 3 structures.
The synthesis of glycopeptide 27 was carried out on MBHA
Rink amide resin on a 0.1 mmol scale using NR-Fmoc-amino
acids. Chain assembly to produce glycopeptide 30 (Scheme 5)
was accomplished on an automated peptide synthesizer using
N,N′-dicyclohexylcarbodiimide (DCC)/N-hydroxybenzotriazole
(HOBt)-mediated couplings, except for the coupling of 3, which
was performed manually using 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU)/HOBt. Fol-
lowing deprotection and acetylation of the N-terminus, reduction
of the C-2 azide in 30 was performed on resin by reaction with
dithiothreitol (DTT)/DBU in DMF as described by Meldal and
co-workers.32 As anticipated, DNP removal was also ac-
complished in this step. Following acetylation of the resulting
amine and thiol groups with Ac2O/pyridine, cleavage from the
resin was achieved by using treatment with TFA to yield
acetylated glycopeptide 31 as the major product (Figure 6). After
purification by RP-HPLC, 31 was treated with 10% aqueous
hydrazine in the presence of DTT to afford the deacetylated
target glycopeptide 27.
a Reagents: (a) (i) 20% piperidine in DMF, rt, 20 min; (ii) Ac2O/
DMF, rt, 20 min; (b) (i) DTT, DBU, DMF, rt, 30 min; (ii) Ac2O/
pyridine, rt, 20 min; (c) 10% aq N2H4, excess DTT, rt, 30 min.
our attention to the N-bromoacetamido sugars (32-33, Figure
7) required to generate analogues 28 and 30. The synthesis of
N-bromoacetamido sugars has been described previously by
several groups.12,33 Accordingly, 32 and 33 were generated as
reported by Thomas33a via reaction of the commercially available
glycosylamines of galactose and GlcNAc, respectively, with 1
equiv of bromoacetic anhydride in DMF. The resulting N-
bromoacetamido sugars, 32 and 33, were easily purified by
crystallization from MeOH/Et2O. Although we chose to use only
simple monosaccharides for our initial thiol alkylation experi-
ments, it should be noted that N-haloacetamido sugars bearing
up to 10 monosaccharides have been prepared from the
corresponding free sugars via their glycosylamines.33b,c
4. Alkylation of 3-ThioGalNAc. Formation of the target
thioether-linked glycopeptides was achieved by treatment of 27
with an excess of the N-bromoacetamido sugar (32 or 33) in
sodium phosphate buffer (pH 7.0) at 37 °C (Figure 8). The
reactions were monitored by RP-HPLC and judged to be
complete after 6 h. In each case, the only product obtained was
the desired thioether-linked glycopeptide. No formation of the
disulfide-bound homodimer was observed. Purification of 28
and 29 was accomplished by RP-HPLC, and their identities were
confirmed by ESI-MS.
One concern we had with the general approach was the
potential for competitive alkylation of free cysteine residues.
Indeed, in model reactions with cysteine-containing peptides,
alkylation with compound 33 was found to occur at a compa-
rable rate as that observed with 3-thioGalNAc peptide 27 (not
shown). Thus, to achieve site-selectivity in glycan elongation,
free cysteine residues should be avoided. Their participation in
disulfide bonds or orthogonal protection would circumvent this
problem.
Summary
The synthesis of thioether-linked glycopeptides was ac-
complished via the incorporation of a novel glycosyl amino acid
(3) bearing a thiol group at C-3 into solid-phase peptide
3. Synthesis of N-Bromoacetamido Sugars. Having suc-
cessfully achieved the synthesis of glycopeptide 27, we turned
(31) (a) McEver, R. P.; Cummings, R. D. J. Clin. InVest. 1997, 100,
485. (b) Epperson, T. K.; Patel, K. D.; McEver, R. P.; Cumming, R. D. J.
Biol. Chem. 2000, 275, 7839. (c) Li, F.; Erickson, H. P.; James, J. A.; Moore,
K. L.; Cummings, R. D.; McEver, R. P. J. Biol. Chem. 1996, 271, 6342.
(32) St. Hilaire, P. M.; Cipolla, L.; Franco, A.; Tedebark, U.; Tilly, D.
A.; Meldal, M. J. Chem. Soc., Perkin Trans. 1 1999, 3559.
(33) (a) Thomas, E. W. Methods Enzymol. 1997, 46, 362. (b) Manger, I.
D.; Rademacher, T. W.; Dwek, R. A. Biochemistry 1992, 31, 10742. (c)
Wong, S. Y. C.; Manger, I. D.; Guile, G. R.; Rademacher, T. W.; Dwek,
R. A. Biochem. J. 1993, 296, 817. (d) Black, T. S.; Kiss, L.; Tull, D.;
Withers, S. G. Carbohydr. Res. 1993, 250, 195.

1592 J. Am. Chem. Soc., Vol. 123, No. 8, 2001
Marcaurelle and Bertozzi
Figure 6. (a) Analytical RP-HPLC of crude glycopeptide 31 after cleavage from resin (10 f 60% CH3CN in H2O, 0.1% TFA, 50 min); (b)
analytical RP-HPLC of purified, deacetylated glycopeptide 27 (same conditions as above); (c) electrospray mass spectrum of glycopeptide 27.
with Bruker AMX 300 and DRX 500 spectrometers. Chemical shifts
are reported in parts per million (δ) relative to CHCl3 (7.26 ppm) for
spectra run in CDCl3. 13C NMR spectra were obtained at 100 MHz on
a Bruker DRX 500 and are reported in δ relative to CDCl3 (77.00 ppm)
as an internal reference. High-resolution FAB mass spectra were
recorded at the Mass Spectrometry Facility at the University of
California at Berkeley. Electrospray ionization mass spectrometry (ESI-
MS) was performed on a Hewlett-Packard 1100 mass spectrometer.
Figure 7.
synthesis. Alkylation of the 3-thiol with derivatives 32 and
33 proceeded smoothly to yield the target glycopeptides (29
and 30) as the only products. Building block 3 was generated
in 12 steps from readily available tri-O-acetyl-D-glucal and
FmocThrOtBu 2.13 A key feature of this method is that it
circumvents the requirement of higher order glycosylated amino
acid building blocks, the synthetic complexity of which should
not be underestimated. Rather, a modest synthetic effort
produces the 3-thioGalNAc building block (3), enabling the
convergent assembly of numerous complex structures from a
single precursor. It should be noted that the coupling partners,
N-haloacetamido sugars, can be generated in two steps from
unprotected oligosaccharides. Moreover, these derivatives have
been prepared from large glycans (i.e., decasaccharides) that
have been isolated in small quantities from natural sources.33b,c
Having demonstrated that amino acid 3 is compatible with
Fmoc-based SPPS, we are now in position to gain access to
biantennary O-linked glycopeptides (elaborated at both C-6 and
C-3) via oxime/thioether formation. The synthesis and evaluation
of these analogues will be reported in due course.
Tert-butyldimethylsilyl 2-azido-2-deoxy-4,6-O-benzylidene-â-D-
gulose (5). A solution of compound 414 (217 mg, 0.53 mmol) in dry
CH2Cl2 (5 mL) was cooled to -15 °C and treated with pyridine (77
µL, 0.95 mmol) and Tf2O (143 µL, 0.85 mmol). After stirring for ∼45
min at -15 °C, the reaction was diluted with CH2Cl2 (20 mL) and
washed with saturated (satd) aq NaHCO3 (2 × 10 mL). The organic
layer was dried (MgSO4), filtered, and concentrated in vacuo. The crude
residue was redissolved in CH3CN (5 mL) and treated with tetra-n-
butylammonium nitrite (442 mg, 1.6 mmol). After stirring for 24 h at
room temperature the solvent was evaporated in vacuo, and the resultant
residue was dissolved in EtOAc (40 mL), washed with water (3 × 10
mL), and dried (MgSO4). Removal of the solvent and purification of
the residue by flash chromatography (hexanes:EtOAc, 6:1) gave 5 (152
mg, 70%) as a white solid: IR (film) 3469, 2928, 2856, 2110, 1724,
1
1393 cm-1; H NMR (300 MHz, CDCl3) δ 7.50 (m, 2H), 7.38 (m,
3H), 5.54 (s, 1H), 5.06 (d, 8.0 Hz), 4.25 (m, 1H), 4.05 (m, 2H), 3.99
(dd, 1H, J ) 3.0, 1.3 Hz), 3.75 (d, 1H, J ) 1.4 Hz), 3.69 (dd, 1H, J )
8.0, 3.0 Hz), 2.41 (s, 1H), 0.96 (s, 9H), 0.21 (s, 3H), 0.19 (s, 3H); 13
C
NMR (100 MHz, CDCl3) δ 138.0, 129.4, 128.6, 126.5, 126.3, 101.4,
94.9, 75.1, 70.1, 69.6, 65.9, 63.9, 25.9, 18.2, -3.8, -4.6; HRMS (FAB)
[M + Li]+ calcd for C19H29N3O5SiLi, 414.2037; found, 414.2040.
Compounds 6 and 7. A solution of compound 5 (55 mg, 0.14 mmol)
in dry CH2Cl2 (1 mL) and pyridine (25 µL) was treated at 0 °C with
Tf2O (45 µL, 0.28 mmol) and stirred for 30 min at 0 °C and for 1 h at
room temperature. The reaction mixture was diluted with CH2Cl2 (10
mL) and washed with satd. aq NaHCO3 solution (5 mL). The organic
layer was dried (MgSO4), filtered, and concentrated in vacuo. The
residue was redissolved in anhydrous DMF (1 mL) and treated for 5 h
with KSAc (46 mg, 0.40 mmol) at 60 °C. The reaction mixture was
concentrated in vacuo, redissolved in EtOAc (10 mL), washed with
water (3 × 5 mL), and dried (MgSO4). After removal of the solvent in
vacuo, the residue was purified by flash chromatography (hexanes:
EtOAc, 6:1) to yield 6 (43 mg, 68%) and 7 (5 mg, 8%). (6): IR (film)
2926, 2111, 1694, 1362, 1254, 1164, 1084 cm-1; 1H NMR (500 MHz,
CDCl3) δ 7.50 (dd, 2H, J ) 7.5, 1.5 Hz), 7.39 (m, 3H), 5.52 (s, 1H),
4.71 (d, 1H, J ) 7.5 Hz), 4.26 (dd, 1H, J ) 12.5, 1.5 Hz), 4.03 (dd,
1H, J ) 12.5, 2.0 Hz), 3.99 (d, 1H, J ) 2.0 Hz), 3.74 (dd, 1H, J )
12.0, 3.5 Hz), 3.57 (m, 2H), 2.41 (s, 3H), 0.94 (s, 9H), 0.20 (s, 3H),
0.19 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 137.5, 129.0, 128.2, 126.1,
101.1, 98.9, 74.9, 69.1, 63.1, 46.5, 30.5, 25.6, 18.0, -4.1, -5.0; HRMS
(FAB) [M + Li]+ calcd for C21H31N3O5SSiLi, 472.1914; found,
472.1922. (7): IR (film) 3373, 2928, 2855, 2357, 1666, 1516, 1400,
Experimental Section
General Methods NR-Fmoc-amino acids, Rink amide MBHA resin,
HBTU, HOBt, and DCC were purchased from Novabiochem. All other
chemical reagents were obtained from commercial suppliers and used
without further purification. The following solvents were distilled under
a nitrogen atmosphere prior to use: THF was dried and deoxygenated
over Na and benzophenone, CH2Cl2 and CH3CN were dried over CaH2,
and methanol was dried over Mg and I2. Unless otherwise noted, all
air and moisture sensitive reactions were performed under an argon or
nitrogen atmosphere. Analytical thin-layer chromatography (TLC) was
conducted on Analtech Uniplate silica gel plates with detection by ceric
ammonium molybdate (CAM), PPh3/ninhydrin or UV light. For flash
chromatography, 60-Å silical gel (Bodman) was employed. Reversed-
phase high-pressure liquid chromatography (RP-HPLC) was performed
on a Rainin Dynamax SD-200 HPLC system using Microsorb and
Dynamax C18 reversed-phase columns (analytical: 4.6 × 250 mm, 1
mL/min; preparative: 25 × 250 mm, 20 mL/min) and UV detection
(230 nm) was performed with a Rainin Dynamax UV-1 detector.
Infared spectra were recorded on a Perkin-Elmer FT-IR 1600 series
spectrometer. 1H NMR spectra were obtained at either 300 or 500 MHz

O-Linked Glycopeptide Mimetics
J. Am. Chem. Soc., Vol. 123, No. 8, 2001 1593
Figure 8. (a) Alkylation of 27 with 32 or 33 to give analogs 28 and 29; (b) RP-HPLC analysis of ligation reaction between 27 and 32 at various
time points (10 f 60% CH3CN in H2O, 0.1% TFA, 50 min). Similar results were obtained for the alkylation of 27 with 33 to give 29.
1
1367, 1254, 1158, 1093 cm-1; H NMR (500 MHz, CDCl3) δ 7.49
(dd, 2H, J ) 9.5, 2.0 Hz), 7.34 (m, 3H), 7.07 (s, 1H), 6.60 (d, 1H, J
) 5.5 Hz), 5.56 (s, 1H), 5.35 (s, 1H), 4.39 (app dt, 1H, J ) 5.5, 2.0
Hz), 4.32 (d, 1H, J ) 12.5 Hz), 4.17 (dd, 1H, J ) 12.5, 2.5 Hz), 3.53
(s, 1H), 2.06 (s, 3H), 0.95 (s, 9H), 0.25 (s, 3H), 0.24 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 138.1, 128.9, 128.2, 126.4, 125.8, 107.5, 100.9,
92.8, 69.9, 69.0, 66.9, 25.9, 25.6, 24.5, 18.0, -3.7, -4.2; HRMS (FAB)
[M + Li]+ calcd for C21H31NO5SiLi, 412.2132; found, 412.2140.
water (50 mL) was added, and the mixture was concentrated in vacuo.
Purification of the residue by flash chromatography (hexanes:EtOAc,
5:1) yielded compound 20 (4.2 g, 62%) as a colorless oil: IR (film)
3041, 2970, 2902, 2862, 2836, 1730, 1612, 1513, 1465, 1383, 1249,
1174, 1122, 1087, 1032 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.25 (d,
2H, J ) 8.7 Hz), 6.89 (d, 2H, J ) 8.7 Hz), 5.86 (ddd, 1H, J ) 9.8,
3.0, 1.3 Hz), 5.77 (ddt, 1H, J ) 9.8, 1.9, 0.7 Hz), 5.48 (d, 1H, J ) 3.1
Hz), 4.59 (d, 1H, J ) 11.6 Hz), 4.51 (m, 2H), 4.48 (d, 1H, J ) 11.6
Hz), 4.23 (m, 1H), 3.84 (m, 1H), 3.81 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 159.4, 130.0, 129.3, 128.2, 127.5, 113.9, 96.0, 74.6, 73.7,
71.0, 62.8, 55.3; HRMS (FAB) [M+] calcd for C14H16O4, 248.1049;
found, 248.1049.
Modified Procedure23 for the Synthesis of 1,6-Anhydro-2,3-
dideoxy-â-threo-hex-2-enopyranose (12).20 Tri-O-acetyl-D-glucal (15
g, 55 mmol) and phenol (40 g) were dissolved in chlorobenzene (300
mL) and heated at reflux for 4 h. The solvent and excess phenol were
removed under high vacuum, and the resulting residue was dissolved
in CH2Cl2, washed with 1 M aq NaOH and brine, and dried (MgSO4)
to give 1423 as a mixture of anomers. Without purification, 14 was
combined with 10 g of NaOH and heated at reflux for 3 h in H2O (200
mL). After cooling to room temperature, the mixture was extracted
several times with EtOAc, dried (MgSO4), and concentrated. Purifica-
tion of the resulting residue by flash chromatography (hexanes:EtOAc,
2:1 f 1:2) gave 1220 (5.2 g, 76%) as a colorless oil. Compound 12
(4.7 g, 36.7 mmol) was dissolved in dry CH2Cl2 (75 mL) in the presence
of 4-Å molecular sieves (9 g) and NMO (6.45 g, 55.1 mmol). After
cooling to 0 °C, TPAP (645 mg, 1.80 mmol) was slowly added. The
mixture was stirred at room temperature for 1 h and then filtered through
a short plug of silica gel (CH2Cl2). Fractions containing the desired
enone (13)22 were combined, concentrated to a total volume of 80 mL,
and cooled to -78 °C. A solution of CeCl3 in MeOH (96 mL, 0.4 M)
was added, and the mixture was stirred at -78 °C for 30 min. NaBH4
(1.4 g, 37.0 mmol) was added portionwise over a period of 10 min
and stirring was continued until TLC (hexanes:EtOAc, 1:1) showed
complete consumption of 13. The reaction was quenched with acetone
and concentrated in vacuo. The residue was purified by flash chroma-
tography (hexanes:EtOAc, 1:1) to give 12 (3.3 g, 70%) as a colorless
oil: 1H NMR (300 MHz, CDCl3) δ 5.85 (ddd, 1H, J ) 9.7, 3.0, 1.7
Hz), 5.67 (ddt, 1H, J ) 9.7, 1.9, 1.4 Hz), 5.47 (d, 1H, J ) 3.0 Hz),
4.74 (m, 1H), 4.46 (m, 1H), 4.15 (dd, 1H, J ) 8.1, 2.0 Hz), 3.87 (app
t, 1H, J ) 6.8 Hz), 2.04 (br s, 1H). All data are in agreement with
literature reports.19
1,6-Anhydro-4-O-p-methoxybenzyl-â-D-gulopyranose (21). To 4.2
g (20 mmol) of 20 in acetone (80 mL) was added 3.96 g of NMO
(33.8 mmol) followed by water (10 mL). The mixture was cooled to 0
°C, and a solution of OsO4 in t-BuOH (7 mL, 2.5wt %/v) was added.
After stirring for 3 h, saturated Na2SO3 (50 mL) was added. The reaction
mixture was then extracted with Et2O (3 × 100 mL), washed with brine
(100 mL), dried (MgSO4), and concentrated in vacuo. Purification of
the residue by flash chromatography (hexanes:EtOAc, 1:1) yielded diol
21 (4.3 g, 90%) as a white solid: TLC (EtOAc:hexanes, 2:1) Rf )
0.39; IR (film) 3424, 2895, 1611, 1514, 1302, 1249, 1181, 1130, 1089,
1
1025 cm-1; H NMR (300 MHz, CDCl3) δ 7.26 (d, 2H, J ) 8.5 Hz),
6.88 (d, 2H, J ) 8.5 Hz), 5.41 (d, 1H, J ) 2.0 Hz), 4.67 (d, 1H, J )
11.6 Hz), 4.59 (d, 1H, J ) 11.6 Hz), 4.39 (app t, 1H, J ) 4.4 Hz),
4.00 (d, 1H, J ) 7.6 Hz), 3.77 (m, 2H), 3.80 (s, 3H), 3.60 (m, 2H),
2.41 (br s, 2H); 13C NMR (100 MHz, CDCl3) δ 159.5, 130.0, 129.4,
114.0, 101.0, 77.6, 72.5, 72.4, 70.7, 69.5, 63.9, 55.3; HRMS (FAB)
[M + Li]+ calcd for C14H18O6Li, 289.1263; found, 289.1260.
1,6-Anhydro-4-O-p-methoxybenzyl-â-D-gulopyranose 2,3-cyclic
sulfate (19). An ice-cooled and magnetically stirred solution of diol
21 (4.28 g, 15.1 mmol) and Et3N (8.4 mL, 60 mmol) in dry CH2Cl2
(60 mL) was added to a solution of SOCl2 (1.66 mL, 22.7 mmol) in
CH2Cl2 (40 mL) over a period of 10 min. Stirring was continued at 0
°C until TLC (hexanes:EtOAc, 1:1) indicated complete disappearance
of the starting material. The mixture was diluted with CH2Cl2 (200
mL) and washed with water (2 × 100 mL) and brine (100 mL). The
organic solution was dried (Na2SO4) and concentrated in vacuo to afford
a mixture of the cyclic sulfites, which were purified by a short column
of silica gel (hexanes:EtOAc, 4:1) to ensure removal of any residual
Et3N. To a solution of the cyclic sulfites in a 1:1 mixture of CH3CN/
CCl4 (100 mL) was added NaIO4 (6.46 g, 30.2 mmol), followed by a
catalytic amount of RuCl3•3H2O (150 mg) and water (50 mL). The
1,6-Anhydro-4-O-p-methoxybenzyl-2,3-dideoxy-â-threo-hex-2-
enopyranose (20). To a solution of 12 in anhydrous DMF (150 mL)
was added NaH (1.23 g, 51.2 mmol) and TBAI (473 mg, 1.28 mmol)
at 0 °C. After stirring for 10 min, p-methoxylbenzyl chloride (7.0 mL,
51.2 mmol) was added. After stirring at room temperature for 12 h,

1594 J. Am. Chem. Soc., Vol. 123, No. 8, 2001
Marcaurelle and Bertozzi
reaction mixture was stirred vigorously for 15 min at room temperature
and then diluted with ether (800 mL). The organic layer was washed
with water (2 × 200 mL) and brine (200 mL), dried (Na2SO4), and
concentrated in vacuo. Purification of the crude product by column
chromatography (hexanes:EtOAc, 4:1) gave cyclic sulfate 19 (3.6 g,
66%) as a white solid: TLC (hexanes:EtOAc, 2:1) Rf ) 0.62; IR (film)
129.6, 129.3, 114.0, 103.2, 78.0, 73.2, 72.8, 68.3, 65.3, 55.3, 46.4;
HRMS (FAB) [M + Li]+ calcd for C14H17N3O4SLi, 330.1099; found,
330.1098.
1,6-Anhydro-2-azido-2-deoxy-3-S-(2,4-dinitrophenyl)-4-O-p-meth-
oxybenzyl-3-thio-â-D-galactopyranose (23) and 1,6-Anhydro-3-
azido-3-deoxy-2-S-(2,4-dinitrophenyl)-4-O-p-methoxybenzyl-2-thio-
â-D-idopyranose (24). A mixture of compounds 10 and 22 (1.24 g,
3.80 mmol) was dissolved in dry CH2Cl2 (40 mL) and treated with
2,4-dinitrofluorobenzene (784 mg, 4.20 mmol) in the presence of DIEA
(1.0 mL, 5.8 mmol) at room temperature. After stirring for 18 h, the
solution was concentrated in vacuo. Compound 23 (1.2 g, 65%) was
isolated as a bright yellow solid by trituration with ice-cold CH2Cl2.
The undesired diequatorial product (24) was obtained by evaporation
of the filtrate and purification of the resulting residue by flash
chromatography (hexanes:EtOAc, 6:1) (180 mg, 10%). (23): IR (film)
2962, 2358, 2103, 1592, 1514, 1340, 1250, 1133 cm-1; 1H NMR (500
MHz, CDCl3) δ 8.93 (d, 1H, J ) 2.5 Hz), 8.22 (dd, 1H, J ) 9.0, 2.6
Hz), 7.55 (d, 1H, J ) 9.2 Hz), 7.14 (d, 1H, J ) 8.7 Hz), 6.82 (d, 1H,
J ) 8.7 Hz), 5.52 (s, 1H), 4.62 (d, 1H, J ) 8.1 Hz), 4.53 (m, 3H), 4.29
(dd, 1H, J ) 6.9, 4.0 Hz), 3.95 (d, 1H, J ) 6.9 Hz), 3.80 (s 3H), 3.73
(dd, 1H, J ) 7.5, 4.0 Hz), 3.68 (s, 1H); 13C NMR (100 MHz, CDCl3)
δ 159.8, 146.4, 145.7, 144.2, 129.7, 128.6, 128.4, 126.6, 121.2, 114.0,
100.1, 73.1, 72.2, 70.9, 64.5, 63.7, 55.2, 47.6; LRMS (FAB) [M +
Li]+ C20H19N5O8SLi, 496.2. (24): 1H NMR (300 MHz, CDCl3) δ 9.01
(s, 1H, J ) 2.5 Hz), 8.35 (dd, 1H, J ) 9.0, 2.5 Hz), 7.79 (d, 1H, J )
9.1 Hz), 7.29 (d, 1H, J ) 8.7 Hz), 6.91 (d, 1H, J ) 8.7 Hz), 5.45 (s,
1H), 4.69 (d, 1H, J ) 11.4 Hz), 4.61 (d, 1H, J ) 11.5 Hz), 4.48 (m,
1H), 4.18 (d, 1H, J ) 8.0 Hz), 3.83 (s, 3H), 3.76 (m, 3H), 3.28 (d, 1H,
J ) 9.5 Hz); LRMS (FAB) [M + Li]+ C20H19N5O8SLi, 496.2.
1,4,6-Tri-O-acetyl-2-azido-2-deoxy-3-S-(2-4-dinitrophenyl)-3-
thio-r/â-D-galactopyranose (25). Compound 23 (938 mg, 1.92 mmol)
was dissolved in 100 mL of Ac2O/TFA (9:1) and stirred at 60 °C for
22 h. The reaction mixture was coevaporated with toluene (3 × 20
mL), and the resulting residue was purified by flash chromatography
(hexanes:EtOAc, 5:1 f 3:1) to give 25r (822 mg, 83%) and 25â (164
mg, 17%). (25r): IR (film) 3098, 2111, 1747, 1588, 1521, 1362, 1343,
1210, 1137, 1044, 1011 cm-1; 1H NMR (500 MHz, CDCl3) δ 9.00 (d,
1H, J ) 2.5 Hz), 8.44 (dd, 1H, J ) 8.9, 2.5 Hz), 7.79 (d, 1H, J ) 8.9
Hz), 6.44 (d, 1H, J ) 3.3 Hz), 5.44 (app s, 1H), 4.35 (app t, 1H, J )
6.5 Hz), 4.08 (dd, 1H, J ) 11.5, 6.3 Hz), 4.00 (m, 2H), 3.95 (dd, 1H,
J ) 11.9, 3.2 Hz), 2.24 (s, 3H), 2.16 (s, 3H), 2.02 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 170.4, 169.6, 168.4, 147.3, 145.1, 141.9, 129.0,
127.1, 121.6, 90.0, 69.3, 66.7, 61.7, 58.1, 47.5, 21.0, 20.6, 20.3; HRMS
(FAB) [M + Li]+ calcd for C18H19N5O11SLi, 520.0962; found,
520.0966. (25â): 1H NMR (500 MHz, CDCl3) δ 9.00 (d, 1H, J ) 2.5
Hz), 8.43 (dd, 1H, J ) 8.9, 2.5 Hz), 7.80 (d, 1H, J ) 8.9 Hz), 5.72 (d,
1H, J ) 8.0 Hz), 5.40 (d, 1H, J ) 2.5 Hz), 4.11 (m, 3H), 3.91 (dd, 1H,
J ) 12.0, 8.5 Hz), 3.57 (dd, 1H, J ) 11.5, 3.0 Hz), 2.26 (s, 3H), 2.22
(s, 3H), 2.07 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 170.4, 169.6,
168.6, 147.0, 145.1, 142.1, 129.3, 127.1, 121.5, 94.4, 66.6, 61.5, 60.8,
50.9, 20.9, 20.6, 20.3; HRMS (FAB) [M + Li]+ calcd for C18H19N5O11-
SLi, 520.0962; found, 520.0966.
2909, 2839, 1612, 1514, 1390, 1251, 1212, 1136, 1101, 1040 cm-1
;
1H NMR (300 MHz, CDCl3) δ 7.26 (d, 2H, J ) 8.7 Hz), 6.90 (d, 2H,
J ) 8.7 Hz), 5.67 (d, 1H, J ) 1.5 Hz), 4.92 (app t, 1H, J ) 6.3 Hz),
4.84 (dd, 1H, J ) 6.4, 1.6 Hz), 4.72 (d, 1H, J ) 11.4 Hz), 4.54 (d, 1H,
J ) 11.3 Hz), 4.52 (app t, 1H, J ) 4.6 Hz), 4.18 (app t, 1H, J ) 6.1
Hz), 4.04 (dd, 1H, J ) 8.2, 0.8 Hz), 3.82 (s, 3H), 3.69 (dd, 1H, J )
8.2, 5.3 Hz); 13C NMR (100 Hz, CDCl3) δ 159.8, 129.8, 128.5, 114.0,
96.6, 83.2, 77.5, 74.5, 72.6, 72.5, 63.8, 55.3; HRMS (FAB) [M+] calcd
for C14H16O8S, 344.0566; found, 344.0563.
1,6:2,3-Dianhydro-2,3-epithio-4-O-p-methoxybenzyl-â-D-talopy-
ranose (11). To a solution of cyclic sulfate 19 (3.57 g, 9.96 mmol) in
anhydrous DMF (50 mL) was added 1.7 g (15 mmol) of KSAc. The
resultant mixture was stirred at 60 °C until TLC analysis (hexanes:
EtOAc, 2:1) showed complete conversion of the cyclic sulfate into
baseline material (2 h). The mixture was concentrated and dissolved
in anhydrous MeOH (180 mL). A solution of NaOMe (30 mL, 1 M in
MeOH) was added, and the mixture was stirred at room temperature
until TLC (hexanes:EtOAc, 2:1) showed complete disappearance of
the starting material (30 min). A saturated solution of NH4Cl (100 mL)
was added, and the MeOH was removed in vacuo. Water (100 mL)
was added, and the resulting solution was extracted with EtOAc (3 ×
300 mL). The organic layers were collected, dried (MgSO4), and
evaporated. Purifcation of the crude product by flash chromatogaphy
(hexanes:EtOAc, 5:1) yielded episulfide 11 (2.5 g, 91%) as a yellow
syrup: TLC (hexanes:EtOAc, 2:1) Rf ) 0.59; IR (film) 2959, 2899,
2840, 2356, 1608, 1508, 1462, 1302, 1243, 1137, 1110, 1091 cm-1;1H
NMR (300 MHz, CDCl3) δ 7.31 (d, 2H, J ) 8.3 Hz), 6.90 (d, 2H, J )
7.8 Hz), 5.87 (d, 1H, J ) 3.9 Hz), 4.80 (d, 1H, J ) 11.3 Hz), 4.56 (d,
1H, J ) 11.3 Hz), 4.33 (m, 2H), 4.12 (d, 1H, J ) 7.4 Hz), 3.81 (s,
3H), 3.58 (dd, 1H, J ) 6.7, 4.0 Hz), 3.45 (app t, 1H, J ) 6.7 Hz), 3.09
(app t, 1H, J ) 6.2 Hz); 13C NMR (100 MHz, CDCl3) δ 159.5, 129.6,
129.5, 113.9, 97.9, 72.0, 69.8, 69.9, 62.8, 55.3, 43.1, 30.0; HRMS (FAB)
[M+] calcd for C14H16O4S, 280.0769; found, 280.0764.
1,6-Anhydro-2-azido-2-deoxy-4-O-p-methoxybenzyl-3-thio-â-D-
galactopyranose (10) and 1,6-Anhydro-3-azido-3-deoxy-4-O-p-meth-
oxybenzyl-2-thio-â-D-idopyranose (22). Episulfide 11 (2.53 g, 9.0
mmol) was dissolved in anhydrous DMF (45 mL) and cooled to 0 °C.
TBAN3 (14.4 g, 45.0 mmol) was added, followed by Hg(OAc)2 (2.4 g,
9.0 mmol). The reaction mixure was allowed to warm slowly to room
temperature. After stirring fo 24 h, the reaction was quenched with an
excess of â-mercaptoethanol (2.0 mL) and concentrated in vacuo. The
residue was dissolved in EtOAc (400 mL) and washed with water (2
× 100 mL) and brine (100 mL). The organic layer was dried (Na2-
SO4), filtered, and concentrated under reduced pressure. Purification
of the crude product by flash chromatography (hexanes:EtOAc, 7:1)
gave 10 (1.96 g, 68%) and 22 (0.49 g, 17%) as colorless oils. (In
practice, compounds 10 and 22 can be used as a mixture in the next
reaction. The DNP derivative of the diaxial isomer (23) can be isolated
in pure form by precipitation from CH2Cl2, while the undesired isomer
(24) remains in solution.) (10): TLC (hexanes:EtOAc, 3:1) Rf ) 0.59;
IR (film) 2957, 2901, 2869, 2836, 2568, 2102, 1611, 1514, 1250, 1130,
4,6-Di-O-acetyl-2-azido-2-deoxy-3-S-(2,4-dinitrophenyl)-3-thio-r-
D-galactopyranosyl bromide (1). A solution of 25 (768 mg, 1.50
mmol) in anhydrous CH2Cl2 (20 mL) was stirred for 12 h in the presence
of TiBr4 (1.37 g, 3.7 mmol), then diluted with CH2Cl2 (300 mL), washed
with ice water (2 × 100 mL), dried (Na2SO4), and concentrated. The
residue was promptly purified by flash chromatography (hexanes:
EtOAc, 3:1) to give the bromide 1 (734 mg, 92%) as a yellow foam,
which was dried under high vacuum and stored at -20 °C until use:
1H NMR (300 MHz, CDCl3) δ 9.00 (d, 1H, J ) 2.5 Hz), 8.45 (dd, 1H,
J ) 8.9, 2.5 Hz), 7.87 (d, 1H, J ) 9.0 Hz), 6.67 (d, 1H, J ) 3.0 Hz),
5.50 (s, 1H), 4.50 (app t, 1H, J ) 6.5 Hz), 4.18 (dd, 1H, J ) 11.7, 5.6
Hz), 4.06 (m, 2H), 4.08 (dd, 1H, J ) 11.0, 2.4 Hz), 2.19 (s, 3H), 2.05
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 70.3, 169.3, 146.8, 145.1, 142.1,
129.0, 127.4, 121.5, 90.5, 72.2, 67.2, 61.2, 60.7, 48.2, 20.6, 20.3.
N-(9-Fluorenylmethoxycarbonyl)-O-(4,6-di-O-acetyl-2-azido-2-
deoxy-3-S-(2,4-dinitrophenyl)-3-thio-r-D-galactopyranosyl)-L-thre-
onine tert-butyl ester (26). To a flame-dried flask containing 4 Å
molecular sieves (1.0 g) was added dry CH2Cl2 (10 mL), AgClO4 (440
mg, 2.1 mmol), 2,4,6-collidine (290 µL, 2.1 mmol) and N-Fmoc-
1
1033 cm-1; H NMR (300 MHz, CDCl3) δ 7.30 (d, 2H, J ) 8.7 Hz),
6.90 (d, 2H, J ) 8.7 Hz), 5.42 (app s, 1H), 4.64 (d, 1H, J ) 11.2 Hz),
4.63 (d, 1H, J ) 7.8 Hz), 4.46 (d, 1H, J ) 11.2 Hz), 4.43 (app t, 1H,
J ) 4.6 Hz), 3.96 (dd, 1H, J ) 6.4, 4.5 Hz), 3.81 (s, 3H), 3.68 (app s,
1H), 3.58 (m, 2H), 2.24 (d, 1H, J ) 7.0 Hz); 13C NMR (100 MHz,
CDCl3) δ 159.6, 129.6, 129.1, 114.0, 100.9, 73.8, 71.6, 70.5, 66.0, 64.1,
55.3, 38.9; HRMS (FAB) [M + Li]+ calcd for C14H17N3O4SLi,
330.1099; found, 330.1098. (22): Rf ) 0.50; 1H NMR (300 MHz,
CDCl3) δ 7.27 (d, 2H, J ) 8.7 Hz), 6.90 (d, 2H, J ) 8.7 Hz), 5.33 (d,
1H, J ) 1.3 Hz), 4.67 (d, 1H, J ) 11.3 Hz), 4.55 (d, 1H, J ) 11.3
Hz), 4.40 (app t, 1H, J ) 4.5 Hz), 4.07 (d, 1H, J ) 7.8 Hz), 3.81 (s,
3H), 3.69 (dd, 1H, J ) 7.2, 5.2 Hz), 3.54 (ddd, 1H, J ) 8.7, 4.0, 1.0
Hz), 3.41 (dd 1H, J ) 9.8, 8.8 Hz), 2.57 (app t, 1H, J ) 10.4 Hz),
1.76 (d, 1H, J ) 11.0 Hz); 13C NMR (100 MHz, CDCl3) δ 159.6,

O-Linked Glycopeptide Mimetics
J. Am. Chem. Soc., Vol. 123, No. 8, 2001 1595
threonine-OtBu (2)13 (495 mg, 1.25 mmol). The mixture was stirred at
room temperature for 10 min. A solution of bromide 1 (734 mg, 1.37
mmol) in dry CH2Cl2 (10 mL) was added slowly over 30 min via
syringe. After stirring for 5 h at room temperature, the reaction was
diluted with CH2Cl2 and filtered through Celite. The precipitate was
thoroughly washed with CH2Cl2, and the filtrate was evaporated.
Purification of the resulting residue by flash chromatography (hexanes:
EtOAc, 4:1 f 3:1) afforded 25 (534 mg, 55%) as a yellow foam: IR
(film) 2979, 2939, 2358, 2108, 1745, 1595, 1522, 1450, 1370, 1342,
1218, 1153, 1128, 1049 cm-1; 1H NMR (500 MHz, CDCl3, mixture of
rotamers) (major rotamer) δ 8.98 (d, 1H, J ) 2.0 Hz), 8.43 (dd, 1H, J
) 9.0, 2.0 Hz), 7.81 (d, 1H, J ) 9.0 Hz), 7.75 (d, 2H, J ) 7.5 Hz),
7.59 (m, 2H), 7.39 (m, 2H), 7.28 (m, 2H), 5.62 (d, 1H, J ) 9.5 Hz),
5.39 (app s, 1H), 5.17 (d, 1H, J ) 3.5 Hz), 4.50 (dd, 1H, J ) 10.5, 7.0
Hz), 4.34 (m, 3H), 4.23 (m, 1H), 4.11 (m, 4H), 3.70 (dd, 1H, J )
12.0, 3.0 Hz), 2.14 (s, 3H), 2.03 (s, 3H), 1.52 (s, 9H), 1.40 (d, 3H, J
) 6.5 Hz); 13C NMR (100 MHz, CDCl3, mixture of rotamers) δ 171.1,
170.3, 169.7, 169.1, 156.7, 146.9, 144.9, 143.7, 143.5, 142.2, 141.3,
129.0, 128.9, 127.8, 127.3, 127.0, 126.9, 125.0, 124.9, 121.6, 120.0,
98.5, 98.3, 83.2, 68.1, 67.5, 67.4, 67.3, 67.2, 62.2, 62.1, 60.4, 59.2,
58.8, 58.5, 55.7, 47.1, 47.0, 46.5, 28.0, 27.3, 25.5, 21.0, 20.7, 20.6,
20.3, 18.7, 14.2; HRMS (FAB) [M + Li]+ calcd for C39H42N6O14SLi,
857.2639; found, 857.2637.
Deacetylation of Glycopeptide 27. Glycopeptide 31 was treated
with 10% aq hydrazine hydrate in the presence of excess DTT at room
temperature for 20 min. The resulting glycopeptide (27) was directly
purified by preparative RP-HPLC with a gradient of 10-60% CH3CN
in water (0.1% TFA) over 50 min, and its identity confirmed by ESI-
MS (calcd, 2409.6; found, 2409.3).
Synthesis of N-bromoacetamido Sugars 32 and 33.33a The corre-
sponding glycosylamine (100 mg) (Sigma) was dissolved in anhydrous
DMF (1 mL), and bromoacetic anhydride was added (1.2 equivs). After
stirring for 4-6 h, the product was precipitated from ice-cold Et2O
and crystallized (MeOH/Et2O) to give 32 and 33 as white solids (70-
1
85%). (32): H NMR (500 MHz, D2O) δ 4.90 (d, 1H, J ) 9.0 Hz),
3.95 (m, 2H), 3.77 (app t, 1H, J ) 6.5 Hz), 3.69 (m, 3H), 3.63 (app t,
1
1H, J ) 9.5 Hz). (33): H NMR (500 MHz, D2O) δ 5.04 (d, 1H, J )
9.5 Hz), 3.81 (m, 4H), 3.72, (1H, dd, J ) 12.5, 5.0 Hz), 3.59 (app t,
1H, J ) 9 Hz), 3.48 (m, 2H), 1.98 (s, 3H).
Thiol Alkylation: To peptide 27 (2 mg) in 250 µL of sodium
phosphate buffer (0.1 M, pH 7.0) was added 32 or 33 (0.5 mg). The
reaction mixture was incubated at 37 °C for 4 h, and the alkylated
peptide (28 or 29) was isolated by RP-HPLC using a gradient of 10-
60% CH3CN in water (0.1% TFA) over 50 min. The product was
lyophilized and analyzed by ESI-MS (pos). (28): calcd, 2628.8; found,
2629; (29): calcd, 2669.8; found, 2670.
N-(9-Fluorenylmethoxycarbonyl)-O-(4,6-di-O-acetyl-2-azido-2-
deoxy-3-S-(2,4-dinitrophenyl)-3-thio-r-D-galactopyranosyl)-L-threo-
nine (3). A solution of 26 (268 mg, 0.310 mmol) in 95% aq TFA (10
mL) was stirred at room temperature for 1 h. The solution was then
coevaporated with toluene several times to give compound 3 (246 mg,
100%) as a yellow solid: IR (film) 2932, 2104, 1740, 1588, 1522,
1449, 1343, 1217, 1045 cm-1; 1H NMR (500 MHz, CDCl3, mixture of
rotamers) (major rotamer) δ 8.94 (d, 1H, J ) 2.0 Hz), 8.40 (dd, 1H, J
) 9.0, 2.0 Hz), 7.76 (m, 3H), 7.57 (m, 2H), 7.38 (m, 2H), 7.28 (m,
2H), 5.67 (d, 1H, J ) 9.0 Hz), 5.37 (app s, 1H), 5.16 (d, 1H, J ) 3.0
Hz), 4.48 (m, 2H), 4.31 (m, 1H), 4.21 (m, 2H), 4.08 (m, 4H), 3.77
(dd, 1H, J ) 12.0, 3.5 Hz), 2.15 (s, 3H), 2.00 (s, 3H), 1.35 (d, 1H, J
) 5.0 Hz); 13C NMR (100 MHz, CDCl3, mixture of rotamers) δ 173.7,
170.6, 169.8, 169.7, 156.6, 146.8, 144.9, 143.6, 143.4, 142.5, 142.1,
141.2, 129.1, 129.0, 128.9, 128.2, 127.8, 127.2, 127.1, 127.0, 124.9,
121.6, 121.5, 120.0, 98.5, 98.3, 68.1, 67.5, 67.4, 67.3, 62.3, 62.2, 59.2,
58.5, 58.4, 55.7, 47.1, 47.0, 46.8, 20.7, 20.6, 20.3, 18.0; HRMS (FAB)
[M - H + 2 Li]+ calcd for C35H33N6O14SLi2, 807.2096; found,
807.2122.
Synthesis of Glycopeptide 31. Glycopeptide 31 was synthesized
on Rink amide MBHA resin (0.1 mmol) using NR-Fmoc-protected
amino acids and DCC-mediated HOBt ester activation in NMP (Perkin-
Elmer ABI 431A synthesizer, user-devised cycles). Glycosyl amino
acid 3 was manually coupled, using 3.0 equivs of amino acid and
activation with HBTU (3.0 equivs) in the presence of HOBt and DIEA
(3.0 equivs each). After removal of the N-terminal Fmoc group, the
resin-bound peptide was treated with 20% Ac2O in DMF (v/v) for 20
min and then was washed successively with DMF (3×), CH2Cl2 (3 x)
and DMF (3×). Azide reduction/DNP cleavage was achieved by
treatment with DTT (3.0 equivs) and DBU (3.0 equivs) in DMF for 1
h. Following acetylation with Ac2O:pyridine (1:2), the resin was washed
thoroughly with DMF (3×) and CH2Cl2 (5×). Peptide cleavage/
deprotection was accomplished by treatment with 95% aq TFA at room
temperature for 4 h. The crude peptide was precipitated with tert-butyl
methyl ether, dissolved in 50% aq CH3CN, and lyophilized. The
acetylated glycopeptide (31) was purified by preparative RP-HPLC with
a gradient of 10-60% CH3CN in water (0.1% TFA) over 50 min, and
its identity confirmed by ESI-MS (calcd, 2535.7; found, 2535.5).
Synthesis of Cysteine-Containing Peptide (34). A peptide with the
sequence AcNH-EYELDYDYFLPECEPPE-CONH2 (34) was synthe-
sized on Rink amide MBHA resin (0.1 mmol) using NR-Fmoc-protected
amino acids and DCC-mediated HOBt ester activation in NMP (Perkin-
Elmer ABI 431A synthesizer, user-devised cycles). After removal of
the N-terminal Fmoc, the resin-bound peptide was treated with 20%
Ac2O in DMF for 20 min and then washed successively with DMF
(3×), CH2Cl2 (3×), and DMF (3×). Peptide cleavage/deprotection was
accomplished by treatment with 94.5% TFA, 2.5% H2O, 2.5%
ethanedithiol, and 1% triethylsilane at room temperature for 4 h. The
crude peptide was precipitated with tert-butyl methyl ether, purified
by preparative RP-HPLC (10-60% CH3CN in water with 0.1% TFA
over 50 min), and analyzed by ESI-MS (calcd, 2192.3; found, 2192).
Selective Alkylation Experiment. Peptide 27 (2 mg) and 34 (2 mg)
were dissolved in 300 µL of 0.1 M sodium phosphate buffer at pH
6.2, 6.6, and 7.0. Derivative 33 (1.1 equivs) was added, and the reaction
mixtures were kept at room temperature or incubated at 37 °C. The
reactions were monitored by RP-HPLC (10-60% CH3CN in water with
0.1% TFA over 50 min). In all cases, no selective alkylation was
observed. At pH 6.2, the disulfide-linked dimers were obtained in
addition to the alkylated products.
Acknowledgment. This research was supported by a grant
to C.R.B. from the National Science Foundation (CAREER
Award CHE-9734439) and a graduate fellowship to L.A.M.
from Roche Bioscience. The Center for New Directions in
Organic Synthesis is supported by Bristol-Myers Squibb as a
sponsoring member.
Supporting Information Available: 1H and 13C NMR
spectra for compounds 1, 3, 5-7, 10-11, 19-23, 25R, 25â,
and 26. 1H NMR spectra for compounds 24, 32, and 33 (PDF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
JA003713Q