A broadly applicable method for the efficient synthesis of α-O-linked glycopeptides and clustered sialic acid residues

Article abstract of DOI:10.1021/ja9833265

The total syntheses of complex sialylated cell-surface antigens have been accomplished. The target systems include 2,3-STF, STn, 2,6-STF, and glycophorin antigens. In addition, an α-O-linked serine glycoside of an entire Lewis blood group (Y) antigen has

Full text of DOI:10.1021/ja9833265

2662
J. Am. Chem. Soc. 1999, 121, 2662-2673
A Broadly Applicable Method for the Efficient Synthesis of
R-O-Linked Glycopeptides and Clustered Sialic Acid Residues
Jacob B. Schwarz, Scott D. Kuduk, Xiao-Tao Chen, Dalibor Sames, Peter W. Glunz, and
Samuel J. Danishefsky*
Contribution from the Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer
Research, 1275 York AVenue, New York, New York 10021, and Department of Chemistry, Columbia
UniVersity, New York, New York 10027
ReceiVed September 17, 1998
Abstract: The total syntheses of complex sialylated cell-surface antigens have been accomplished. The target
systems include 2,3-STF, STn, 2,6-STF, and glycophorin antigens. In addition, an R-O-linked serine glycoside
of an entire Lewis blood group (Y) antigen has been assembled.
Introduction
conduct of the glycosylation in the context of a fully assembled
complex glyco-domain (such as a glycal) is fraught with
considerable uncertainty as to the eventual R/â ratios.2,4 While
success was achieved in a few cases, seemingly small structural
variations in fact profoundly affect the quality of the various
steps in the progression from terminal glycal to O-linked serine
or threonine glycoside in ways that are difficultly interpretable.2
After much study of many cases, we are unable to provide a
generally reliable protocol that would deliver the required serine
or threonine glycoside to mature glycodomains with high
R-selectivity to any substrate of our choosing. Given the
potentially great importance of the synthesis of complex cell-
surface molecular mimics in which serine or threonine residues
are omnipresent in R-linkages, we viewed this uncertain situation
as a serious detriment to progress. Herein we report a remarkably
general solution to reach our goal systems. The method has been
tested under stringent challenges such as are involved in the
stereoselective syntheses of particularly complex and potentially
invaluable cell surface molecular mimics. The assembly logic
we describe is that of a “cassette” modality rather than reliance
on a maximally convergent approach cited above. In the
traditional regime,4 a full glycodomain is assembled. An
R-linkage to serine or threonine is then fashioned at the reducing
end of the fully mature domain. Aside from the unpredictable
glycosylation ratios in any new case, another disadvantage of
this strategy is that difficulties in the yield or stereoselectivity
during installation of the R-O-linked serine or threonine are
borne out by the full glycodomain. Furthermore, in the
traditional approach the protecting groups throughout the domain
must be compatible with installation of the glycosyl donor
functionality, and with the coupling step to the serine or
threonine. Most serious is the nonreliability of the stereochem-
istry of this type of glycosylation in any new case. In our new
approach, a terminal GalNAc residue bearing an in-place serine
or threonine is used as a generalized acceptor to be appended
to a suitable donor linkage.5 Hopefully we would be facing
less problematic glycosylation challenges associated with the
saccharide-saccharide coupling in our assembly process. Ide-
ally, such couplings would be conducted with a suitably
As part of a multidisciplinary program targeted to the
enlistment of the human immune system for recognition of
various tumors and for mounting a clinically helpful response,
we have directed our attention to the mucin class of glycopro-
teins. Mucins represent a family of cell-surface glycoproteins
often associated, in aberrant glycoforms, with tumors of
epithelial tissues.1 In the first phase of our investigations,
reported in an earlier publication, we have demonstrated that
the trimeric clusters of Tn and TF glycoepitopes, conjugated
as such to the Pam3Cys lipophilic immunostimulant2 or to KLH
(keyhole limphet hemocyanin) immuno-protein carrier,3 are
immunogenic as judged by antibody production. Furthermore,
the clustered motifs provoke robust production of antibodies,
with promising cell-surface reactivity for those tumors express-
ing the respective antigen. Seemingly, our fully synthetic vaccine
constructs, with trimeric arrangement of Tn epitopes, are able
to mimic the cell-surface presentation of mucin O-linked
antigens.3 That proof-of-principle study provided particularly
strong incentives to develop generally applicable schemes for
the preparation of even more complex members of this class.
In particular, we focused on the development of synthetic
methodology to reach sialylated members of the TF family of
mucin antigens including the glycophorin family. The state of
sialylation (usually accomplished intracellularly by sialyltrans-
ferases or by de-sialylation within a cell by sialidases) is a
critical determinant in cell-surface recognition of glycoproteins.
In stepping up the complexity level of our goals, we would be
confronting a long-standing problem in oligosaccharide and
glycoconjugate synthesis. The crux of the difficulty has been
the problematic character of synthesizing carbohydrate domains
O-linked to the key amino acids, serine and threonine, with
strong stereochemical control in the formation of the R-glyco-
sidic linkage. In previous publications we have shown that the
(1) Carlstedt, I.; Davies, J. R. Biochem. Soc. Trans. 1997, 25, 214. Finn,
O. J. et al. Immunol. ReV. 1995, 145, 61.
(2) Kuduk, S. D.; Schwarz, J. B.; Chen, X.-T.; Sames, D.; Glunz, P.
W.; Govindaswami, R.; Livingston, P. O.; Danishefsky, S. J. J. Am. Chem.
Soc. 1998, 120, 12474.
(3) 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) Sames, D.; Chen, X.-T.; Danishefsky, S. J. Nature 1997, 389, 587.
10.1021/ja9833265 CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/11/1999

Synthesis of R-O-Linked Glycopeptides
J. Am. Chem. Soc., Vol. 121, No. 12, 1999 2663
Figure 1.
Figure 2.
protected serine or threonine residue (properly protected) in
place at the “reducing end” of the acceptor. Figure 2 contrasts
both approaches drawing particular attention (see bold arrows)
to the bond being established between the cassette and the rest
of the domain.6
Four distinct subgoals had to be achieved to implement the
new program. First we had need to synthesize acceptor systems
(cassettes) with R-O-linked serine or threonine residues, in place,
in the context of GalNAc or latent GalNAc units. Moreover,
these GalNAc units must bear differentiated or readily dif-
ferentiable acceptor sites. Furthermore, the actual glycosylation
with the acceptor cassette bearing the in-place O-linked serine
or threonine had to proceed smoothly. Finally, global depro-
tection of the fully assembled construct must be achievable
without undermining the stability of the potentially fragile
linkage connecting the carbohydrate and amino acid domains.
In the work described herein, we set about to field test our
notions regarding these challenges in the context of synthesizing
sialylated antigens which are R-O-linked to peptides. Clearly,
the inclusion of these sialic acid residues represents a significant
extension in the complexity level of the undertaking. There was
a clear rationale for undertaking such additional chemical
challenges. Thus, the STn antigen 1, which contains a sialic
acid residue at position 6 of galactose is abundantly expressed
in major epithelial tumors of the breast, ovary, colon, and
stomach.7 The presence of sialic acid moieties at either the 6-O
position or the 3′-O position of the TF disaccharide is found in
the 2,6-STF8 and 2,3-STF9 antigens 2 and 3, respectively. The
presence of sialic acid moieties on both the 6-position of the
GalNAc and the 3-position of the galactose is the distinctive
feature of the parent member of the family, glycophorin 4. This
motif is found in the context of a major erythrocyte membrane
(5) In practice since the assembly state of the 2,6-STF antigen (2) worked
reasonably well by convergent glycal assembly, we did not resynthesize
this goal structure through full implementation of the cassette method with
two separate donors.
(6) For other cassette related approaches, see Meinjohanns, E.; Meldal,
M.; Paulsen, H.; Schleyer, A.; Bock, K. J. Chem. Soc., Perkin. Trans. 1
1996, 985. Mathieux, N.; Paulsen, H.; Meldal, M.; Bock, K. J. Chem. Soc.,
Perkin. Trans. 1 1997, 2359. Nakahara, Y.; Iijima, H.; Ogawa, T.
Tetrahedron Lett. 1994, 35, 3321. Liebe, B.; Kunz, H. Tetrahedron Lett.
1994, 35, 8777.
(7) Itzkowitz, S. H.; Yuan, M.; Montgomery, C. K.; Kjeldsen, T.;
Takahashi, H. K.; Bigbee, W. L.; Kim, Y. S. Cancer Res. 1989, 49, 197.
MacLean, G. D.; Reddish, M. A.; Koganty, R. R.; Longenecker, B. M. J.
Immunother. 1996, 19, 59.
(8) (a) Fukuda, M.; Carlsson, S. R.; Klock, J. C.; Dell, A. J. Biol. Chem.
1986, 261, 12796. (b) Saitoh, O.; Gallagher, R. E.; Fukuda, M. Cancer
Res. 1991, 51, 2854.
(9) Lloyd, K. O.; Burchell, J.; Kudryashov, V.; Yin, B. W. T.; Taylor-
Papadimitriou, J. J. Biol. Chem. 1996, 271, 33325.

2664 J. Am. Chem. Soc., Vol. 121, No. 12, 1999
Schwarz et al.
Figure 3.
Scheme 1
glycoprotein.10 Moreover, the presence of the 2,3-STF antigen
3 on breast tumors has been demonstrated,9 and the expression
of the 2,6-STF antigen 2 on cells of myelogenous leukemia has
also been identified.8 Our target structures are identified in
Figure 3. From a chemical standpoint, the effective introduction
of sialic acid residues into glycoconjugates has been a challenge
for many years.11 Careful considerations, as to which sialylation
protocols would prove to be workable with either a glycal
linkage in place or with R-O-linked glycosyl amino acid
acceptors, would be a critical element of the synthetic design.
In the studies described below, the two approaches discussed
above were considered for accommodating the inclusion of sialic
acid residues: (1) assembly of an entire glycodomain in the
form of an advanced glycal, followed by the attachment of the
amino acid; (2) an alternative approach wherein a “cassette”,
comprising a reactive acceptor having the O-linked amino acid
attached to the GalNac residue, is united with a donor already
equipped with a sialic acid. Such an approach would provide a
suitably protected precursor to target structures 1-4 (Figure 2).
Results and Discussion
1. Synthesis of 2,6-STF Glycosyl Serine/Threonine via the
Modular Glycal Approach. To construct the glycosylated
amino acid building blocks corresponding to the 2,6-STF12
antigen for incorporation into a peptide backbone, we turned
to the practice of glycal assembly (Scheme 1).13 In the event,
6-O-TIPS galactal was coupled with compound 5, to provide
disaccharide 6 in 75% yield. After deblocking of the 6-O-TIPS
moiety with buffered TBAF-HOAc, sialylation was attempted
with various sialic acid donors. The diethyl phosphite donor14
8 proved to be most effective and afforded trisaccharide 9 in
84% yield as a separable 4:1 R: â mixture of anomers. Other
donors such as a sialyl chloride15 gave similar yields but with
poorer sialic anomer selectivity.
(12) For previous synthesis of 2,6-STF, see (a) Iijima, H.; Ogawa, T.
Carbohydr. Res. 1989, 186, 95. (b) Qiu, D.; Koganty, R. R. Tetrahedron
Lett. 1996, 37, 595. (c) Qiu, D.; Koganty, R. R. Tetrahedron Lett. 1997,
38, 961.
(13) Danishefsky, S. J.; Bilodeau, M. T. Angew. Chem., Int. Ed. Engl.
1996, 35, 1381.
(14) (a) Martin, T. J.; Schmidt, R. R. Tetrahedron Lett. 1992, 33, 6123.
(b) Martin, T. J.; Brescello, R.; Toepfer, A.; Schmidt, R. R. Glycoconjugate
J. 1993, 10, 16.
(15) Ratcliffe, M. R.; Venot, A. P.; Abbas, Z. S. Eur. Pat. Appl. EP
319, 253. Chem Abstr. 1990, 112, 175281a.
(10) Thomas, D. B.; Winzler, R. J. J. Biol. Chem. 1969, 244, 5943.
(11) Reviews: Okamoto, K.; Goto, T. Tetrahedron 1990, 46, 5835. (a)
Deninno, M. P. Synthesis 1991, 583. (b) Ito. Y.; Gaudino, J. J.; Paulson, J.
C. Pure Appl. Chem. 1993, 65, 753.

Synthesis of R-O-Linked Glycopeptides
J. Am. Chem. Soc., Vol. 121, No. 12, 1999 2665
Table 1.
Scheme 2
We now had to deal with the introduction of the O-linked
amino acid to a donor ultimately derived from glycal 9. The
resultant galactal underwent smooth azidonitration16 resulting
in installation of the latent galactosamine for eventual glyco-
sylation of serine or threonine. Conversion of compound 10 to
a number of candidate donor linkages was achieved. Direct
displacement of the anomeric nitrate to the R-bromide with LiBr/
CH3CN furnished anomeric bromide 11 in 75% yield. Reduction
i
of the nitrate to the hemiacetal (PhSH, Pr2NEt) could be
followed by conversion to the trichloroacetimidate 12 in 80%
yield.17 Alternatively treatment of the hemiacetal with chloro-
diethyl phosphite gave the unstable phosphite donor 13. Both
12 and 13 were obtained as mixtures of anomers in a 1:1 ratio.
The results of attempts to glycosylate N-Fmoc-protected serine
and threonine benzyl ester with donors 11-13 are shown in
Table 1. With these donors, the coupling to protected threonine
acceptors occurred with complete stereoselectivity, exclusively
yielding the desired R-linked product 15. While not proceeding
with equivalent stereocontrol, the coupling of donors 12 and
13 with protected serine acceptors still provided the desired R-O-
linked anomer as the major products in the indicated yields.
The diethyl phosphite donor 13 afforded the highest selectivity.
Noteworthy was the fact that a mixture of 12R and 12â gave a
lower selectivity than pure 12â. We had previously found that
related donors where the 6-O-sialic acid in 11-13 is replaced
by an acetate, the selectivity for both serine and threonine drops
off to 2:1 R: â at best.2 However, the types of difficulties as
exemplified in the glycophorin synthesis (vide infra) in general-
izing serine or threonine glycosylation ratios led us to favor
the cassette approach. At this stage, our initial objective was
achieved: a rapid, efficient glycodomain assembly via glycal
logic, with resident protecting groups suitable for subsequent
peptide coupling steps, and mild basic deprotection to conclude
the synthesis was realized.4 We first describe the construction
of the 2,6-STF antigen motif.
of the benzyl ester, to afford 16 and 17. These compounds served
as suitable building units for the glycopeptide assembly. The
glycopeptide backbone was elaborated from the carboxy to
amino terminus direction (Scheme 2). Trisaccharide 17 was
coupled to Ala-Val-benzyl ester (IIDQ), and iterative peptide
coupling steps between the N-terminus of the peptide and the
protected glycosyl amino acid, gave the desired pentapeptide
18 in high yield (average 85% for each coupling step). Finally,
the carbamate linkage was deprotected and the amine capped
with an acetyl group. After global deprotection (NaOH-H2O,
MeOH), the desired CD43 glycoprotein N-terminus 19 contain-
ing the clustered 2,6-STF epitopes was obtained in 80% yield
and high purity as determined by reverse-phase HPLC (MeOH/
H2O, C18, 215 nm). This cluster is currently being readied for
immunoconjugation and study.
3. Synthesis of STn Glycosyl Serine/Threonine via the
Cassette Approach. In our synthesis of the blood-group
determinant F1R,20 it had been shown that lactosylation of a
building block containing an acceptor hydroxyl at the 6-O-
position could be effected. This strategy provided a fully
protected precursor to free F1R. It was hoped that, in a related
way, we could gain access to the STn antigen with high synthetic
economy using protected glycosyl amino acid derivatives (see
cassettes 20 and 21) and a sialic acid-containing donor as a
counterpart of the lactosylation system.21 A synthesis of the STn
monomer and its incorporation into a peptide was recently
reported by Kunz.22 Indeed, when either of the glycosylated
serine or threonine cassettes 20 or 21, respectively, were exposed
2. Assembly of CD43-Derived Glycopeptides with Clus-
tered 2,6-STF Epitopes. Having accomplished the synthesis
of the 2,6-STF antigen, albeit in a nongeneral fashion, we set
our sights on clustering such a substructure to produce a mimic
portion of the CD43 glycopeptide.18 The azido groups of
trisaccharides 14 and 15 were reduced with thiolacetic acid19
(78% yield) followed by quantitative hydrogenolytic removal
(20) Chen, X.-T.; Sames, D.; Danishefsky, S. J. J. Am. Chem. Soc. 1998,
120, 7700-7709.
(21) For previous synthesis of STn via glycosylation of a disaccharide
see: (a) Iijima, H.; Ogawa, T. Carbohyr. Res. 1988, 172, 183. For a related
example using a similar type of cassette, see (b) Liebe, B.; Kunz, H.
Tetrahedron Lett. 1994, 35, 8777. (c) Elofsson, M.; Salvador, L. A.;
Kihlberg, J. Tetrahedron 1997, 53, 369. (d) Yule, J. E.; Wong, T. C.; Gandhi,
S. S.; Qiu, D.; Riopel, M. A.; Koganty, R. R. Tetrahedron Lett. 1995, 36,
6839.
(16) Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57, 1244.
(17) Schmidt, R. R.; Kinzy, W. AdV. Carbohydr. Biochem. 1994, 50,
84-123.
(18) Carlstedt, I.; Davies, J. R. Biochem. Soc. Trans. 1997, 25, 214.
(19) (a) Bieldfeldt, T.; Peters, S. Meldal, M.; Bock, K.; Paulsen, H.
Angew. Chem., Int. Ed. Engl. 1992, 31, 857. (b) Rosen, T.; Lico, I. M.;
Chu, D. T. W. J. Org. Chem. 1988, 53, 1580.
(22) Liebe, B.; Kunz, H. HelV. Chim. Acta 1997, 80, 1473.

2666 J. Am. Chem. Soc., Vol. 121, No. 12, 1999
Schwarz et al.
Scheme 3
Scheme 4
to a phosphite donor 22 or chloride donor 23, the desired
disaccharides 24 and 25 were obtained (Scheme 3). The
stereochemistry of the addition was determined to be R from
1
the chemical shift pattern at C3 in the H NMR spectrum of
the sialyl residue. In the case of 25, although the overall R: â
selectivity was 4:1, separation of the anomers proved to be
tedious and the yield of pure R-adduct was only 37%. Kunz
has made similar observations in both selectivity and yields,
using sialyl xanthate donors.23 Noteworthy, but not helpful, was
the fact that sialylation using CH2Cl2 as solvent instead of THF
led to the exclusive formation of the undesired â-anomer. In
preparation for the impending peptide coupling, a short synthetic
sequence was necessary. The phase commenced with the
reduction of the azide linkage of 25 (AcSH, pyridine, 87%).
Removal of the acetonide, reprotection as the acetates, and
hydrogenolysis of the benzyl ester provided the compound 26,
necessary for clustering in 84% two-step yield. An iterative
peptide coupling process, as employed above for the 2,6-STF
series, afforded 27. This tripeptide is differentially protected
on each terminus to enable further elaboration. Conjugation of
27 to an immunostimulant or suitable derivative is now
underway.
4. Synthesis of 2,3-STF Glycosyl Threonine via the
Cassette Approach. Given the success of the synthesis and
clustering of the 2,6-STF and STn antigens, we turned our
attention toward the 2,3-STF antigen, which has been associated
with human breast cancer.8 The chemical synthesis of 2,3-STF
by a similar approach has been recently reported24 as has a fully
enzymatic procedure.25 Initially, as implemented in the 2,6-STF
synthesis, it was our strategy that construction of the trisaccha-
ride portion, would be followed by azidonitration and subsequent
attachment of the full carbohydrate donor to a protected serine
or threonine acceptor. Initial efforts at preparing trisaccharide
met with many complications. A serious problem was the
difficult sialylation at the 3′-O-position of a suitably protected
disaccharide glycal (TF core). This approach was also plagued
by extremely poor yields and low selectivities.26
As an alternative approach, we sought to apply the cassette
technology.2,6 The goal would involve coupling of the ap-
propriately protected cassette with a suitable donor derived from
a disaccharide such as 30. Thus, sialylation of compound 29
was carried out using phosphite donor 22 mediated by TM-
SOTf,13 or alternatively with donor chloride 23 mediated by
AgOTf.27 The reaction mixture was then treated with DBU at
0 °C for 1 h to afford compound 30 in 40-50% yield for the
two steps.26,28
The cassette coupling was attempted using the strategy
previously employed by us for the TF antigen.2 Thus, treatment
of 30 with DMDO was followed by exposure to acceptor 32 in
the presence of ZnCl2. However, no coupling was observed. It
was thought that the TIPS group in 30 was again too demanding
as a steric presence. However, even with an acetate at the
corresponding position (see 31), no coupling was observed.
In previous studies, we and others have shown that thioethyl
glycosides, suitably protected at C2 (pivalate, benzoate), can
serve as excellent â-glycosidation donors in a variety of
glycosylation reactions.29 We were intrigued with the possibility
of a convergent coupling of an inactivated thio donor such as
compound 33 with acceptor 32. In the event, treatment of 30
(23) Liebe, B.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1997, 36, 618.
(24) Nakahara, Y.; Nakahara, Y.; Ito, Y.; Ogawa, T. Tetrahedron Lett.
1997, 38, 7211.
(25) Synthesis: Kren, V.; Thiem, J. Angew. Chem., Int. Ed. Engl. 1995,
34, 893.
(26) Woo, J. W.; Kuduk, S. D.; Danishefsky, S. J. Unpublished results.
(27) Gervay, J.; Peterson, J. M.; Oriyama, T.; Danishefsky, S. J. J. Org.
Chem. 1993, 58, 5465.
(28) Ercegovic, T.; Magnusson, G. J. Org. Chem. 1995, 60, 3378.

Synthesis of R-O-Linked Glycopeptides
J. Am. Chem. Soc., Vol. 121, No. 12, 1999 2667
Scheme 5
Scheme 8
Scheme 6
Scheme 7
coupling products were obtained, the reaction proved quite
inefficient and gave generally poor yields (ca. 25%). Another
consequence of using methyl triflate as the promoter, was the
formation of the compound 36 at the C-5 amide of the sialic
acid. Cleavage of the methyl group required an additional
hydrolytic step.
After surveying a variety of alternative coupling conditions,
it was found that recourse to the Fraser-Reid N-iodosuccinim-
ide-triflic acid promoter system30 afforded compound 37 in
ca. 60% yield with good reproducibility. Of course, in the
absence of a methylating potential in the promoter, no imidate
byproduct was observed.
A somewhat related approach had been previously employed
by Ogawa using trichloroacetimidate 38.31 It should be noted
here that thiodonor 35 was highly unactivated due the presence
of the acetates, while donor 38 was an electron rich perbenzy-
lated system. Thus, although we have exploited a “disarmed”32
glycosyl donor in the case of thiodonor 35, the use of the NIS/
TfOH30 promoter system was successful toward 2,3-STF. We
have also extended this approach to other “disarmed” glycosyl
donors (vide infra). Global deprotection starting with 37 to
expose the free antigen was accomplished in a straightforward
manner (Scheme 8). Hence, removal of the 6-O-TBS group on
the GalNAc residue was achieved with 1 N HCl. Acetylation
of the free hydroxyl groups provided trisaccharide 39 with all
resident hydroxyl groups protected as esters in 85% yield. At
this stage the stereochemistry of the NIS coupling product was
determined to be â by COSY and HETCOR experiments.
Reduction of the azide with thiolacetic acid proceeded in 56%
yield. Deprotection of the amino acid portion was effected by
with DMDO, followed by epoxide opening with ethanethiol in
the presence of catalytic trifluoroacetic acid, provided 33 in 61%
yield, to serve as the thiodonor. Benzoylation at C2 afforded
34, and subsequent conversion of the 6-O-TIPS group to acetate
furnished the projected donor 35 in 71% yield with only a single
purification step. The critical coupling step was at hand.
Glycosylation of 34 with acceptor 32 was first investigated
using our previously developed conditions (MeOTf, DTBP).29a
No coupling was observed. Our first response to this setback
was to replace the 6-O-TIPS group with an acetoxy function as
in donor 35, which would hopefully be less encumbered based
upon our previous observations with TF.2 Although some
(30) Konradsson, P.; Udodong, U. E.; Fraser-Reid, B. Tetrahedron Lett.
1990, 31, 4313.
(31) Nakahara, Y.; Nakahara, Y.; Ito, Y.; Ogawa, T. Tetrahedron Lett.
1997, 38, 7211.
(29) (a) Seeberger, P. H.; Eckhardt, M.; Gutteridge, C. E.; Danishefsky,
S. J. J. Am. Chem. Soc. 1997, 119, 10064. (b) Garegg, P. J.; Hentrichson,
C.; Norberg, T. Carbohydr. Res. 1983, 116, 162. Reviews on thioglyco-
sides: (c) Fujed, P.; Garegg, P. J.; Lonn, H.; Norberg, T. Glyconconjugate
J. 1987, 4, 97. (d) Toshima, K.; Tatsuta, K. Chem. ReV. 1993, 93, 1503.
(32) (a) Mootoo, D. R.; Konradsson, P.; Udodong, U. E.; Fraser-Reid,
B. J. Am. Chem. Soc. 1988, 110, 5583. (b) Halcomb, R. L.; Danishefsky,
S. J. J. Am. Chem. Soc. 1989, 111, 6661. (c) Veeneman, G. H.; van Boom,
J. H. Tetrahedron Lett. 1990, 31, 275. (d) Veeneman, G. H.; van Leeuwen,
S. H.; van Boom, J. H. Tetrahedron Lett. 1990, 31, 1331.

2668 J. Am. Chem. Soc., Vol. 121, No. 12, 1999
Schwarz et al.
Scheme 9
carbamate removal (morpholine), and capping of the free amine
was achieved by acetylation. Subsequent hydrogenolysis of the
benzyl ester, hydrolysis of the acetates with sodium hydroxide
in aqueous methanol, and removal of the 2′-O-benzoate
(NaOMe, MeOH, 65 °C) provided N-acetyl-2,3-STF 40. While
the yield of the global deprotection sequence was only 44%
overall, some of the loss could be accounted for via â-elimina-
tion of the amino acid under the harsh basic conditions.33
Clustering of the antigen and immunological evaluation can be
realized in an analogous fashion as for the 2,6-STF and STn
glycopeptides and will be reported in due course.
even more ambitious “disarmed” glycosylation30 process related
to an ongoing Ley project in our laboratory.37 In this regard,
we were interested in producing R-O-linked Ley structures. One
such construct was hexasaccharide 45, which could be obtained
in a [5 + 1] cassette coupling with a pentasaccharide donor. In
the event, thio-donor 43 and cassette 44 were coupled via NIS/
TfOH in an exemplary 79% yield. It will not go unnoticed that
45 constitutes the basic substructure by which a Lewis Y blood
group determinant can be presented in the context of an
adVanced cell-surface mimic. We are currently exploiting this
remarkable cassette coupling method.
5. Synthesis of a Glycophorin Glycosyl Threonine Precur-
sor via a Modified [2 + 2] Cassette Approach. Having
successfully accessed the other members of the ST antigen
family, we directed our attentions toward an even more
complicated venture. The glycophorin antigen is encountered
on a major glycoprotein found on the human erythrocyte
membranes.34 To date, only Ogawa has reported a total synthesis
of an O-linked glycophorin. However, the Ogawa routes were
plagued by poor selectivity in the amino acid glycosylations,35
or by recourse to a series of mono-glycosylations, which
required significant manipulating of protecting groups.36 For
application of our methodology, a properly O-linked disaccha-
ride acceptor would be required. For this purpose we required
an undifferentiated acceptor such as 41. In this undertaking, in
an overall sense (see arrows) we would be using cassette 20
with two different donors in a properly timed way. Accordingly,
sialylation of 20 with sialic acid donor 23 afforded 24, followed
by acetonide cleavage using AcOH to yield compound 41 in
48% yield for the two steps. We hoped that the glycosylation
(i.e. sialylgalactosylation) would occur at C3 of the galactose
segment of 41 (see arrow) which is equatorial. In the event,
coupling of 41, prepared as shown, with 35 using the Fraser-
Reid conditions30,32 afforded the protected O-linked tetrasac-
charide 42 in 53% yield.
Summary
In conclusion, we have successfully prepared the 2,3-STF,
STn, 2,6-STF, and glycophorin antigens utilizing both the
classical and cassette approaches. Clustered forms of these
antigens are now being studied for their immunological profiles.
Also, the synthesis of an R-O-linked serine glycoside of an entire
Lewis blood group (Y) antigen has been accomplished via the
cassette methodology. While complexities will undoubtedly be
encountered on a case-to-case basis, we believe that the results
shown here constitute validation and broad demonstration that
the required chemistry can be achieved in the general case.
Increasingly sophisticated and, we hope, increasingly realistic
cell-surface molecular mimics can be now assembled and
evaluated both as regards to spectroscopy and immune recogni-
tion.
Experimental Section
Glycal 6. Galactal 5 (1.96 g, 9.89 mmol) was dissolved in 100 mL
of anhydrous CH2Cl2 and cooled to 0 °C. DMDO (0.06 M solution in
acetone, 200 mL, 12 mmol) was added via cannula to the reaction flask.
After 1 h the starting material was consumed as judged by TLC. The
solvent was removed with a stream of N2, and the crude epoxide was
dried in vacuo for 1 h at ambient temperature. The crude residue was
dissolved up in 33 mL of THF, and 6-O-TIPS galactal (2.50 g, 8.24
mmol) in 20 mL THF was added. The resulting mixture was cooled to
-78 °C, and ZnCl2 (1.0 M solution in ether, 9.80 mL, 9.80 mmol)
was added dropwise. The reaction was allowed to warm slowly to
ambient temperature and stirred 12 h. The mixture was diluted with
EtOAc and washed with NaHCO3 and brine, dried (MgSO4), and
concentrated. The residue was purified by flash chromatography
5. Synthesis of a Ley Hexasaccharide Glycosyl Serine
Precursor via a Modified [5 + 1] Cassette Approach. As a
further extension of this new methodology, we examined an
(33) Meldal, M.; Bielfeldt, T.; Peters, S.; Jensen, K. J.; H. Paulsen,; Bock,
K. Int. J. Pept. Protein Res. 1994, 43, 529.
(34) Thomas, D. B.; Winzler, R. J. J. Biol. Chem. 1969, 244, 5943.
(35) Iijima, H.; Ogawa, T. Carbohydr. Res. 1989, 186, 107.
(36) (a) Nakahara, Y.; Iijima, H.; Ogawa, T. Tetrahedron Lett. 1994,
35, 3321. (b) Nakahara, Y.; Iijima, H.; Ogawa, T. Carbohydr. Lett. 1994,
1, 99.
(37) Glunz, P. W.; Hintermann, S.; Lloyd, K. O.; Danishefsky, S. J.
Unpublished results.

Synthesis of R-O-Linked Glycopeptides
J. Am. Chem. Soc., Vol. 121, No. 12, 1999 2669
Scheme 10
(40 f 45 f 50 f 60% EtOAc in hexanes) to yield 3.36 g of product
which was immediately acetylated. The material was dissolved in 50
mL of dry CH2Cl2, and triethylamine (19.2 mL) and DMAP (20 mg)
were added. The solution was cooled to 0 °C at which time acetic
anhydride (9.90 mL) was added dropwise. The reaction was stirred at
ambient temperature 12 h. The solvent was removed in vacuo and the
crude material purified by flash chromatography (50% EtOAc in
hexanes) to give 3.30 g (75%) of glycal 6: 1H NMR (CDCl3) δ 6.42
(d, J ) 6.3 Hz, 1H, H-1, glycal), 4.35 (1/2 AB, dd, J ) 11.5, 6.8 Hz,
1H, H-6′a), 4.28 (1/2 AB, dd, J ) 11.5, 6.1 Hz, 1H, H-6′b). HRMS
(EI) calcd for C28H44O13SiK (M + K): 655.2188; found: 655.2154.
Glycal 7. Glycal 6 (1.50 g, 2.43 mmol) was dissolved in 24 mL of
THF and cooled to 0 °C. A mixture of TBAF (5.80 mL, 5.83 mmol)
and acetic acid (0.34 mL, 5.83 mmol) was added to the substrate at 0
°C. The reaction was stirred at 30 °C for 5 h. The solution was
partitioned between EtOAc and saturated NaHCO3, and the phases
separated. The organic phase was washed with saturated NaHCO3, brine,
dried (MgSO4), and concentrated. The residue was purified by flash
chromatography (80 f 85 f 90% EtOAc/ hexane) to yield 0.9 g (80%)
of glycal 7: 1H NMR (CDCl3) δ 6.38 (dd, J ) 6.3, 1.8 Hz, 1H, H-1,
glycal), 5.39 (m, 1H, H-4), 2.22 (s, 3H), 2.16 (s, 3H), 2.13 (s, 3H).
HRMS (EI) calcd for C19H24O13K (M + K): 499.0854; found:
499.0885.
stirred at -15 °C for 12 h. The heterogeneous mixture was diluted
with ethyl acetate, washed twice with ice cold water, dried (Na2SO4),
and concentrated. The residue was purified by flash chromatography
to provide 0.25 g (60%) of R: â-anomeric mixture 10: 1H NMR
(CDCl3) δ 6.35 (d, J ) 4.2 Hz, 1H, H-1, R-nitrate), 3.79 (s, 3H, methyl
ester), 3.41 (dd, J ) 11.0, 4.7 Hz, 1H, H-2), 2.54 (dd, J ) 4.6, 12.8,
H-2eq NeuNAc); 13C NMR (CDCl3, selected characteristic peaks)
170.99, 170.82, 170.30, 170.22, 169.81, 168.58 (acetates), 100.36,
98.34, 97.33, 94.76 (anomeric centers), 72.23, 71.77, 69.29, 68.91,
68.46, 67.70, 67.22, 62.53, 62.50, 57.53, 52.95, 21.06, 20.93, 20.83,
20.78; FTIR (neat): 2117, 1734 cm-1. MS (EI) calcd: 1037.8; found
1038.4 (M + H). The azidonitrate mixture was of limited stability which
precluded HRMS analysis.
R-Bromide 11. To a solution of the azidonitrates 10 (0.15 g, 0.15
mmol) in 0.6 mL of dry acetonitrile was added lithium bromide (62.7
mg, 0.73 mmol) and stirred at ambient temperature for 3 h in the dark.
The heterogeneous mixture was diluted with dichloromethane, and the
solution was washed twice with water, dried (MgSO4), and concentrated
(without heating). The residue was purified by flash chromatography
(5% MeOH in CH2Cl2) to afford 0.12 g (75%) of R-bromide 12 which
was stored under an argon atmosphere at -80 °C: 1H NMR (CDCl3)
δ 6.54 (d, J ) 3.7 Hz, 1H, H-1), 3.40 (dd, J ) 10.8, 4.5 Hz, 1H, H-2),
2.57 (dd, J ) 12.9, 4.5 Hz, 1H, H-2eq NeuNAc), 2.20 (s, 3H), 2.15 (s,
3H), 2.14 (s, 3H), 2.12 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.87 (s, 3H,
CH3CONH); MS (EI) calcd for C39H51N4O25Br: 1055.7; found: 1057.4
(M + H).
Trichloroacetimidates 12. Azidonitrate 10 (0.60 g, 0.58 mmol) was
dissolved in 3.6 mL of acetonitrile, and the resultant solution was treated
with thiophenol (0.18 mL) and diisopropylethylamine (0.10 mL). After
10 min, the solvent was removed with a stream of nitrogen. The crude
material was purified by flash chromatography (2 f 2.5 f 3 f 3.5%
MeOH in CH2Cl2) to provide 0.47 g (82%) of intermediate hemiacetal
as a 1:1 mixture of R: â-anomers: 1H NMR (CDCl3, selected peaks) δ
5.57 (d, J ) 2.9 Hz, 1H, 1R), 5.30-5.35 (m, 4H), 5.21 (d, J ) 8.6 Hz,
1H, 1â), 5.11 (m, 4H), 5.04 (dd, J ) 3.7, 0.5 Hz, 1H), 4.82-4.91 (m,
4H), 4.79 (dd, J ) 8.4, 3.2 Hz, 2H), 4.56 (dd, J ) 7.6, 5.9 Hz, 1H),
3.79 (s, 3H), 3.47 (dd, J ) 10.4, 4.2 Hz, 1H), 3.34 (dd, J ) 10.7, 7.2
Hz, 1H); 13C NMR (CDCl3, selected peaks) δ 100.45, 100.31, 98.98,
98.30, 96.62, 92.19, 23.17, 21.00, 20.85, 20.71; FTIR (neat): 2112,
1747 cm-1. HRMS (EI) calcd for C39H52N4O26 (M + K): 1031.2507;
found: 1031.2539. To this hemiacetal (60 mg, 0.06 mmol) in 2.0 mL
of CH2Cl2 were added trichloroacetonitrile (60 µL) and potassium
carbonate (60 mg). After 6 h the mixture was diluted with CH2Cl2,
filtered of excess K2CO3, and concentrated. The residue was purified
by flash chromatography (5% MeOH in CH2Cl2) to provide 53.2 mg
(64%, two steps) of 12 as a 1:1 mixture of R: â-anomers: 1H NMR
(CDCl3) δ 8.76 (d, J ) 5.1 Hz, 1H), 6.50 (d, J ) 3.1 Hz, 1H,
R-anomer), 5.65 (d, J ) 8.4 Hz, 1H, â-anomer). LRMS (ES) calcd for
C41H52N5O26Cl3Na (M + Na): 1158.1; found: 1158.2. The low stability
Glycal 9. A flame-dried flask was charged with sialyl phosphite
donor 8 (69 mg, 0.11 mmol) and acceptor 7 (40 mg, 0.085 mmol) in
the drybox (argon atmosphere). The mixture was dissolved in 0.6 mL
of dry THF. Dry toluene (0.6 mL) was added, and the solution was
slowly cooled to -60 °C to avoid precipitation. Trimethylsilyl triflate
(2.4 µL) was added, and the mixture was stirred at -45 °C for 2 h.
The reaction was quenched at -45 °C with 2 mL of saturated NaHCO3,
warmed until the aqueous phase melted, and poured into EtOAc. The
organic phase was washed with sat. NaHCO3, dried (Na2SO4), and
1
concentrated. H NMR analysis of the crude material revealed a 4:1
ratio of R: â isomers. The mixture was separated by flash chromatog-
raphy on silica gel (2 f 2.5 f 3 f 3.5 f 4% MeOH in CH2Cl2) to
yield 50 mg (63%) of glycal 9: 1H NMR (CDCl3) δ 6.42 (d, J ) 6.2
Hz, 1H), 5.37 (m, 1H), 5.32-5.29 (m, 4H), 5.26-5.24 (m, 1H), 5.12-
5.10 (m, 2H), 4.98 (d, J ) 3.5 Hz, 1H), 4.92-4.85 (m, 1H), 4.83-
4.80 (m, 3H), 4.54 (m, 1H), 4.45 (dd, J ) 3.0, 13.5 Hz, 1H), 4.33-
4.20 (m, 3H), 4.22-4.02 (m, 7H), 3.96 (dd, J ) 10.9, 7.6 Hz, 1H,
H-2), 2.59 (dd, J ) 12.9, 4.6 Hz, 1H, H-2eq NeuNAc), 2.30 (dd, J )
12.9, 7.2 Hz, 1H, H-2ax NeuNAc), 2.16 (s, 3H), 2.14 (s, 3H), 2.13 (s,
3H), 2.12 (s, 3H), 2.06 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.88 (s, 3H,
CH3CONH); FTIR (neat) 2959, 1816, 1745, 1684, 1662 cm-1. HRMS
(EI) calcd for C39H51NO25K (M + K): 972.2386; found: 972.2407.
Azidonitration Product 10. Glycal 9 (0.37 g, 0.40 mmol) was
dissolved in 2.2 mL of dry acetonitrile, and the solution was cooled to
-20 °C. Sodium azide (38.6 mg, 0.59 mmol) and ammonium cerium
nitrate (0.65 g, 1.20 mmol) were added, and the mixture was vigorously

2670 J. Am. Chem. Soc., Vol. 121, No. 12, 1999
Schwarz et al.
of 12 precluded further characterization. It is recommended to use 12
immediately in the following step.
Protected 2,6-STF Serine Acid 16. The compound 16 was prepared
in 80% yield from 14 following the same procedure for the preparation
1
of 17: [R]23 +40.0 (c 1.75, CDCl3); H NMR (CDCl3) mixture of
Compound 14. A flame-dried flask was charged with donor 11 (50
mg, 0.04 mmol), 80 mg of 4 Å mol sieves, and N-Fmoc-L-serine benzyl
ester (27.5 mg, 0.07 mmol) in the drybox. THF (0.6 mL) was added to
the flask, and the mixture was cooled to -30 °C. Boron trifluoride
diethyl etherate (2.8 µL, 0.022 mmol) was added, and the reaction was
allowed to warm to -10 °C over 3 h. The mixture was diluted with
EtOAc and washed with sat. NaHCO3 while still cold. The organic
phase was dried (Na2SO4) and concentrated, and the residue was purified
by flash chromatography (2 f 2.5 f 3% MeOH in CH2Cl2) to provide
40 mg (66%) of 14 as a 4:1 mixture of R: â-isomers. The pure R-anomer
was separated by flash chromatography (80 f 85 f 90 f 100%
EtOAc:hexanes). 14R: [R]23D +34.3 (c 0.44, CDCl3); 1H NMR (CDCl3)
δ 7.77 (d, J ) 7.5 Hz, 2H), 7.62 (br d, 2H), 7.30-7.48 (m, 9H), 6.09
(br d, J ) 7.9 Hz, 1H), 5.70 (d, J ) 8.8 Hz, 1H), 5.34-5.39 (m, 2H),
5.30 (m, 1H), 5.20-5.27 (m, 1H), 5.17 (br t, J ) 9.9 Hz, 2H), 4.79-
4.95 (m, 5H), 4.80 (dd, J ) 3.1 Hz, 1H), 4.60 (br s, 1H), 4.38-4.48
(m, 4H), 4.22-4.31 (m, 3H), 3.99-4.11 (m, 5H), 3.82-3.95 (m, 3H),
3.75 (s, 3H), 3.33 (dd, J ) 10.2, 4.4 Hz, 1H), 2.55 (dd, J ) 12.8, 4.5
Hz, 1H, H2eq of NeuNAc), 2.11 (s, 3H), 2.10 (s, 3H), 2.08 (s, 6H),
2.07 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H), 1.99 (s, 3H), 1.93
(app t, J ) 12.5 Hz, 1H, H2ax of NeuNAc); 13C NMR (CDCl3) δ 170.92,
170.73, 170.60, 170.22, 170.19, 170.11, 169.81, 168.96, 167.81, 152.68,
143.79, 141.34, 134.90, 128.82, 128.70, 128.41, 127.85, 127.15, 125.12,
120.06, 100.43, 99.08, 98.66, 75.80, 73.04, 72.69, 72.25, 69.23, 69.09,
68.73, 68.49, 67.59, 67.40, 67.24, 63.78, 62.48, 62.24, 54.71, 52.91,
49.35, 48.87, 47.18, 37.64, 23.30, 23.22, 21.00, 20.84, 20.79, 20.76;
IR (neat): 1746, 1673 cm-1. HRMS: Calcd for C66H77N3O31K:
1446.4178; found: 1446.4140.
D
rotamers, selected characteristic peaks: δ 3.36 (br s), 3.28 (br s), 2.52
(br d), 2.48 (br d); 13C NMR (CDCl3) selected peaks: δ 98.90, 98.60,
77.04, 76.79, 72.61, 69.10, 68.56, 68.43, 67.56, 62.68, 62.42, 53.09,
49.33, 47.16, 23.18, 21.02, 20.84, 20.75, 20.66, 20.22; FTIR (KBr
pellet): 3362, 1750 cm-1. HRMS (EI) calcd for C59H71N3O31K (M +
K): 1356.3708; found: 1356.3820.
General Procedure for Peptide Coupling. Glycosyl amino acid
16 or 17 (1 equiv) and the peptide with a free amino group (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. 0.02 mL
of CH2Cl2). The reaction was then stirred at ambient temperature for 8
h. The mixture was directly loaded onto a silica gel column for
purification by flash chromatography.
General Procedure for N-Fmoc Deprotection. 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 ether (catalytic amount).
The mixture was then stirred for 48 h at ambient temperature.
Evaporation of DMF in vacuo was followed by purification by flash
chromatography on silica gel.
1
Protected Glycopeptide 18. H NMR (CDCl3) δ 3.45-3.30 (m, 3
× 1H, H-2), 3.74 (s, 3H, methyl ester), 2.58-2.49 (m, 3 × 1H, H-2eq
NeuNAc); FTIR (neat) 2962, 1819, 1747, 1664; MS (EI) calcd: 3760;
found: 1903.8/doubly charged ) 3806 (M + 2Na).
Deprotected Glycopeptide 19. The benzyl ester was hydrogenolyzed
according to the procedure set forth for compound 17. The acid was
dissolved in methanol to yield a 5 µM solution. 1 M NaOH (aq) was
added dropwise until the pH reached 10-10.5, and the mixture was
stirred for 12 h at ambient temperature. Amberlyst-18 was added to
bring the pH to ca. 4. The resin was filtered off and the mixture
concentrated. The crude product was purified by reverse phase flash
chromatography (LiChroprep RP-18, H2O eluant) to provide fully
deprotected 19 in 80% yield (two steps): 1H NMR (D2O) δ 4.73 (m,
2H, 2 × H-1), 4.70 (d, 1H, H-1), 4.64 (m, 3H, 3 × H-1′), 4.26-4.20
(m, 5H), 4.12-4.00 (m, 7H), 3.95-3.82 (m, 7H), 3.77-3.27 (m, 51H),
2.55-2.51 (m, 3H, 3 × H-2eq NeuNAc), 1.84-1.82 (m, 21H, CH3-
CONH), 1.52-1.45 (m, 3H, H-2ax NeuNAc), 1.20 (d, J ) 7.2 Hz,
3H), 1.18 (d, J ) 6.6 Hz, 3H), 1.12 (d, J ) 6.2 Hz, 3H), 0.71 (d, J )
6.6 Hz, 6H, Val); 13C NMR (D2O) anomeric carbons: δ 105.06, 105.01,
100.60, 100.57, 100.53, 100.11, 99.52, 98.70; MS (FAB) C96H157N11O64
2489 (M + H); MS(MALDI) 2497.
Compound 15. A flame-dried flask was charged with silver
perchlorate (27.3 mg, 0.14 mmol), 0.12 g of 4 Å mol sieves, and
N-Fmoc-L-threonine benzyl ester (37.3 mg, 0.086 mmol) in the drybox.
Dry CH2Cl2 (0.72 mL) was added to the flask, and the mixture was
stirred at ambient temperature for 10 min. Bromide 11 (76 mg, 0.072
mmol) in 0.46 mL of CH2Cl2 was added slowly over 40 min. The
reaction was stirred under argon atmosphere at ambient temperature
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 by flash chroma-
tography (1 f 1.5 f 2 f 2.5% MeOH in CH2Cl2) to provide 74 mg
1
(74%) of 15. The undesired â-anomer was not detected by H NMR
and HPLC analysis of the crude material. 15: [R]23 +26.8 (c 0.45,
D
1
CH2Cl2); H NMR (CDCl3) δ 7.77 (d, J ) 7.5 Hz, 2H), 7.63 (d, J )
7.2 Hz, 2H), 7.40-7.25 (m, 8H), 5.72 (d, J ) 9.2 Hz, 1H), 5.46 (s,
1H), 5.33 (m, 1H), 5.29 (d, J ) 8.2 Hz, 1H), 5.23 (s, 2H), 5.11-5.04
(m, 3H), 4.87-4.71 (m, 4H), 4.4-4.39 (m, 3H), 4.33-4.25 (m, 4H),
4.09-3.97 (m, 6H), 3.79 (s, 3H), 3.66 (dd, J ) 10.6, 3.7 Hz, 1H, H-3),
3.38 (dd, J ) 10.7, 3.0 Hz, 1H, H-2), 2.52 (dd, J ) 12.7, 4.3 Hz, 1H,
H-2eq NeuNAc), 2.20 (s, 3H), 2.13 (s, 3H), 2.11 (s, 3H), 2.10 (s, 3H),
2.04 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.87 (s, 3H, CH3CONH), 1.35
(d, J ) 6.2 Hz, Thr-CH3); FTIR (neat) 2110, 1749; HRMS (EI) calcd
for C65H75N5O30K (M + K): 1444.4130; found: 1444.4155.
Protected 2,6-STF Threonine Acid 17. Azide 15 (47 mg, 33.4
µmol) was treated with thiolacetic acid (3 mL, distilled three times)
for 27 h at ambient temperature. Excess thiolacetic acid was removed
with a stream of nitrogen, followed by toluene azeotrope (4 × 5 mL).
The crude product was purified by flash chromatography (1.5 f 2 f
2.5 f 3 f 3.5% MeOH in CH2Cl2) to yield 37 mg (78%) of reduced
product which was immediately dissolved in 7.6 mL of methanol and
0.5 mL of water. After purging the system with argon, 6.5 mg of
palladium catalyst (10% Pd-C) was added and the system placed under
1 atm of H2. After 8 h the H2 was removed by an argon flow, the
catalyst was removed by filtration, and the mixture was concentrated.
Compound 25. To a suspension of 6-acceptor 21 (R ) Me, 0.41 g,
0.62 mmol), sialyl phosphite 22 (X ) OP(OBn)2, 0.42 g, 0.57 mmol),
and 0.2 g 4 Å mol sieves in 15 mL of dry THF at -45 °C was added
trimethylsilyl trifluoromethanesulfonate (23 µL, 0.11 mmol) dropwise
over 2 min. The mixture was stirred at -45 °C for 12 h at which time
an additional 23 µL of TMSOTf was added dropwise. The mixture
was stirred 6 h at -45 °C, and then filtered through a pad of Celite
and concentrated. Flash chromatography of the residue (1:1 hexanes:
EtOAc f EtOAc) yielded a mixture of R- and â-anomers, which was
subjected to further chromatography (4:1 EtOAc:hexanes) to afford 0.26
g (37%) of pure R anomer 25 (R ) Me) as a colorless foam. [R]23
D
+32.6 (c 1.18, CHCl3); 1H NMR (CDCl3) δ 7.73 (d, J ) 7.5 Hz, 2H),
7.58 (d, J ) 7.5 Hz, 2H), 7.20-7.37 (m, 10H), 5.65 (d, J ) 9.5 Hz,
1H), 5.41 (d, J ) 9.3 Hz, 1H), 5.36 (m, 1H), 5.30 (d, J ) 7.5 Hz, 1H),
5.18 (dd, J ) 12.1, 5.8 Hz, 2H), 4.85 (m, 1H), 4.76 (d, J ) 3.6 Hz,
1H), 4.41 (m, 3H), 4.32 (dd, J ) 12.4, 2.6 Hz, 1H), 4.27 (m, 2H), 4.20
(app t, J ) 7.3 Hz, 1H), 4.04-4.12 (m, 5H), 3.87 (m, 1H), 3.74 (s,
3H), 3.63 (m, 1H), 3.29 (dd, J ) 7.6, 3.6 Hz, 1H), 2.56 (dd, J ) 12.9,
4.7 Hz, 1H), 2.09 (s, 3H), 2.08 (s, 3H), 2.02 (m, 1H), 1.98 (s, 6H),
1.84 (s, 3H), 1.45 (s, 3H), 1.32 (d, J ) 5.5 Hz, 3H), 1.31 (s, 3H); 13C
NMR (CDCl3) δ 170.81, 170.44, 170.14, 170.00, 169.94, 169.90,
156.63, 143.84, 143.57, 141.18, 141.15, 134.93, 128.56, 128.51, 128.47,
127.62, 127.59, 127.01, 126.97, 125.14, 125.06, 119.85, 109.82, 98.84,
98.59, 77.20, 72.97, 72.70, 72.37, 69.07, 68.98, 67.55, 67.48, 67.24,
67.17, 63.50, 62.26, 60.75, 58.86, 52.65, 49.22, 47.03, 37.35, 27.87,
25.97, 23.04, 20.95, 20.72, 20.68, 20.63, 18.56; IR (neat): 2986, 2109,
Flash chromatography of the residue (10% MeOH in CH2Cl2) provided
1
36 mg (78%) of acid 17: [R]23 +34.7 (c 1.75, CDCl3); H NMR
D
(CDCl3) mixture of rotamers, selected characteristic peaks: δ 3.80 (s,
3H, methyl ester), 3.41 (m, 1H, H-2), 2.53 (m, 1H, H-2e NeuNAc)),
1.45 (d, J ) 5.1 Hz, Thr-CH3), 1.35 (d, J ) 5.8 Hz, Thr-CH3); FTIR
(neat) 1818, 1747; HRMS (EI) calcd for C60H73N3O31K (M + K):
1370.3870; found: 1370.3911.
1745, 1668, 1666 cm-1
.

Synthesis of R-O-Linked Glycopeptides
J. Am. Chem. Soc., Vol. 121, No. 12, 1999 2671
1
Protected STn Acid 26. [R]23 +36.7 (c 1.04, CHCl3); H NMR
to ambient temperature and stirred for 16 h. The solution was partitioned
between EtOAc (60 mL) and H2O (60 mL), and the phases were
separated. The organic phase was washed with sat. NaHCO3 (60 mL)
and brine (60 mL), dried (Na2SO4), and concentrated. To the crude
alcohol was added triethylamine (0.72 mmol, 5.20 mmol), acetic
anhydride (0.49 mL, 5.20 mmol), and DMAP (10 mg, 0.08 mmol),
and the solution was stirred for 6 h at ambient temperature. The mixture
was partitioned between EtOAc (50 mL) and sat. NaHCO3 (50 mL),
and the phases were separated. The organic phase was washed with
brine (50 mL), dried (Na2SO4), and concentrated. Flash chromatography
of the residue (4:1, EtOAc:hexanes) provided 0.15 g (71% from 33)
of thioethyl glycoside 35 as a pale yellow solid. [R]23D +1.56 (c 1.58,
CHCl3); 1H NMR (CDCl3) δ 8.00 (d, J ) 7.2 Hz, 2H), 7.58 (t, J ) 7.4
Hz, 1H), 7.44 (t, J ) 7.6 Hz, 2H), 5.50 (ddd, J ) 16.1, 11.0, 5.4 Hz,
1H), 5.40 (m, 1H), 5.24 (m, 3H), 5.08 (d, J ) 3.6 Hz, 1H), 4.63 (dd,
J ) 12.0, 3.1 Hz, 1H), 4.55 (d, J ) 10.0 Hz, 1H), 4.34 (m, 3H), 4.10
(ABq, J ) 10.4 Hz, 1H), 3.94 (m, 2H), 3.65 (d, J ) 10.5 Hz, 1H),
2.68 (m, 2H), 2.48 (dd, J ) 13.8, 5.4 Hz, 1H), 2.10 (s, 3H), 2.09 (s,
3H), 2.07 (s, 3H), 2.00 (s, 3H), 1.97 (s, 3H), 1.85 (s, 3H), 1.77 (dd, J
) 13.7, 11.7 Hz, 1H), 1.22 (t, J ) 7.4 Hz, 3H); 13C NMR (CDCl3) δ
170.82, 170.65, 170.61, 170.39, 170.30, 169.90, 165.16, 163.14, 133.39,
129.77, 129.14, 128.39, 95.09, 82.87, 74.59, 72.71, 72.62, 69.23, 68.87,
68.42, 66.93, 62.79, 62.18, 48.99, 38.04, 24.07, 23.03, 20.77, 20.70,
20.66, 20.55, 14.84; IR (neat): 2966, 1746, 1676 cm-1. HRMS: Calcd
for C36H45NO18SNa: 834.2255; found: 834.2269.
D
(CDCl3) δ 7.83 (d, J ) 7.5 Hz, 2H), 7.70 (app t, J ) 6.7 Hz, 2H), 7.41
(m, 2H), 7.32 (m, 2H), 5.41 (m, 2H), 5.33 (dd, J ) 9.2, 2.0 Hz, 1H),
5.03 (dd, J ) 11.7, 3.2 Hz, 1H), 4.93 (d, J ) 3.8 Hz, 1H), 4.81 (m,
1H), 4.64 (dd, J ) 10.8, 6.3 Hz, 1H), 4.46 (dd, J ) 10.7, 5.9 Hz, 1H),
4.37 (m, 2H), 4.27 (m, 3H), 4.14 (m, 2H), 4.08 (dd, J ) 12.4, 5.1 Hz,
1H), 3.97 (app t, J ) 10.5 Hz, 1H), 3.87 (m, 1H), 3.82 (s, 3H), 3.32
(m, 1H), 2.60 (dd, J ) 12.7, 4.6 Hz, 1H), 2.16 (s, 3H), 2.11 (s, 3H),
2.09 (s, 3H), 2.03 (m, 1H), 2.00 (s, 3H), 1.98 (s, 3H), 1.94 (s, 3H),
1.93 (s, 3H), 1.83 (s, 3H), 1.23 (d, J ) 6.4 Hz, 3H); 13C NMR (CDCl3)
δ 175.61, 175.47, 175.34, 174.19, 174.08, 173.83, 173.63, 173.44,
173.30, 171.07, 160.91, 147.30, 147.00, 144.61, 144.58, 130.70, 130.65,
130.11, 130.05, 128.04, 127.87, 122.90, 122.83, 102.71, 101.75, 79.98,
75.16, 72.49, 71.90, 71.12, 70.89, 70.38, 69.38, 66.45, 65.33, 61.94,
55.29, 51.89, 40.74, 24.75, 24.56, 23.08, 22.78, 22.62, 22.57, 21.20;
IR (neat): 3361, 2956, 1746, 1659 cm-1. HRMS: Calcd for C51H63N3O24-
Na: 1124.3699; found: 1124.3748.
Protected STn Cluster 27. [R]23D +42.0 (c 1.67, CHCl3); 1H NMR
(CD3OD) δ 7.83 (d, J ) 7.5 Hz, 2H), 7.72 (app t, J ) 6.5 Hz, 2H),
7.41 (app t, J ) 7.5 Hz, 2H), 7.34 (app t, J ) 7.4 Hz, 2H), 5.34 (m,
8H), 5.17 (m, 2H), 5.08 (m, 2H), 4.98 (d, J ) 3.6 Hz, 1H), 4.90 (d, J
) 2.7 Hz, 1H), 4.78 (m, 2H), 4.69 (br s, 1H), 4.58 (dd, J ) 10.7, 6.6
Hz, 1H), 4.49 (dd, J ) 10.6, 6.2 Hz, 1H), 4.42 (m, 3H), 4.27-4.35
(m, 8H), 4.18 (app t, J ) 5.9 Hz, 1H), 4.09 (m, 6H), 3.96 (m, 2H),
3.88 (m, 2H), 3.81 (s, 3H), 3.71 (s, 3H), 3.08 (m, 3H), 2.59 (dd, J )
12.7, 4.6 Hz, 2H), 2.54 (dd, J ) 12.8, 4.6 Hz, 1H), 2.17 (s, 3H), 2.16
(s, 3H), 2.11 (s, 9H), 2.09 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.00 (s,
3H), 2.00 (s, 3H), 1.99 (s, 3H), 1.98 (s, 6H), 1.97 (s, 3H), 1.97 (s, 3H),
1.94 (s, 3H), 1.93 (s, 3H), 1.92 (s, 3H), 1.83 (s, 3H), 1.82 (s, 6H), 1.45
(s, 9H), 1.37 (m, 6H), 1.32 (d, J ) 6.2 Hz, 3H); 13C NMR (CD3OD)
δ 173.78, 173.53, 173.25, 172.70, 172.40, 172.36, 172.31, 172.23,
172.06, 171.82, 171.79, 171.77, 171.54, 171.50, 169.24, 169.19, 169.14,
159.12, 158.56, 145.49, 145.22, 142.76, 128.91, 128.29, 126.32, 126.20,
121.11, 121.05, 101.24, 100.63, 100.01, 99.98, 99.95, 80.06, 79.41,
78.35, 73.34, 71.02, 70.71, 70.40, 69.24, 68.55, 67.79, 64.76, 63.44,
60.32, 58.53, 58.24, 53.48, 50.06, 38.95, 38.08, 30.91, 28.94, 23.55,
23.44, 23.39, 22.74, 21.30, 21.04, 20.87, 20.79, 19.87, 19.62; IR
Compound 37. A mixture of thiodonor 35 (0.10 g, 0.12 mmol) and
3-acceptor 32 (0.18 g, 0.25 mmol) was azeotroped with dry benzene
(4 × 5 mL), and the flask was backfilled with nitrogen and placed
under high vacuum for 1 h. Molecular sieves (4 Å, 0.5 g), CH2Cl2 (5.0
mL), and NIS (69 mg, 0.31 mmol) were added, and the mixture was
cooled to 0 °C. Triflic acid (freshly prepared 1% solution in CH2Cl2,
1.84 mL, 0.12 mmol) was added dropwise over 5 min. The mixture
was warmed to ambient temperature immediately following addition,
allowed to stir for 10 min, and then partitioned between EtOAc (50
mL) and sat. NaHCO3 (50 mL). The phases were separated, and the
aqueous phase was back-extracted with EtOAc (20 mL). The combined
organic phases were washed with sat. Na2S2O3 (50 mL) and brine (50
mL), dried (Na2SO4), and concentrated. Flash chromatography of the
(neat): 2934, 2470, 1746, 1654 cm-1
.
residue (4:1 EtOAc:hexanes) furnished 0.11 g (62%) of trisaccharide
Thioglycoside Donor 33. To a solution of lactone 30 (0.19 g, 0.26
mmol) in 8.5 mL of CH2Cl2 at 0 °C was added freshly prepared DMDO
(0.06 M solution in acetone, 8.5 mL, 0.51 mmol). The solution was
stirred for 1 h, and the solvent was evaporated with an N2 flow. To the
residue was added 3.0 mL of CH2Cl2, and the solution was cooled to
-78 °C. Ethanethiol (0.38 mL, 5.11 mmol) was added, followed by
trifluoroacetic anhydride (2 µL, 0.01 mmol). The mixture was allowed
to warm slowly to ambient temperature over 6 h, and concentrated
with an N2 flow. Flash chromatography of the residue (4:1, EtOAc:
hexanes) yielded 0.13 g (61%) of thioglycoside 33 as colorless
crystals: [R]23D -22.8 (c 4.2, CHCl3); 1H NMR (CDCl3) δ 5.50 (ddd,
J ) 7.6, 2.0 Hz, 1H), 5.41 (d, J ) 10.2 Hz, 1H), 5.23 (dd, J ) 8.2, 1.6
Hz, 1H), 5.18 (ddd, J ) 6.2, 2.2 Hz, 1H), 4.83 (d, J ) 4.0 Hz, 1H),
4.36-4.31 (m, 2H), 4.23-4.08 (m, 3H), 4.00-3.88 (m, 2H), 3.72 (d,
J ) 7.1 Hz, 1H), 3.65 (dd, J ) 10.3, 1.4 Hz, 1H), 3.51 (t, J ) 9.4 Hz,
1H), 2.81-2.57 (m, 3H), 2.46 (dd, J ) 13.5, 5.4 Hz, 1H), 2.14 (s,
3H), 2.04 (s, 3H), 2.03 (s, 3H), 2.01 (m, 1H), 2.01 (s, 3H), 2.00 (s,
3H), 1.87 (s, 3H), 1.30 (t, J ) 7.3 Hz, 3H), 1.13 (bs, 21H); 13C NMR
(CDCl3) δ 171.0, 170.6, 170.5, 170.4, 170.0, 169.8, 164.0, 95.0, 85.1,
78.6, 77.2, 74.5, 73.2, 69.7, 69.5, 68.7, 66.9, 62.7, 62.2, 49.1, 38.5,
24.3, 23.0, 20.8, 20.6, 20.5, 17.8, 15.1, 11.8; IR (film) 3364, 2942,
1749, 1667 cm-1; HRMS: Calcd for C36H59NO16SSiNa: 844.3222;
found: 844.3227.
Benzoate Thioglycoside Donor 35. To a solution of thio-donor 33
(0.21 g, 0.26 mmol) in 4.0 mL of CH2Cl2 were added triethylamine
(0.18 mL, 1.28 mmol), benzoyl chloride (0.15 mL, 1.28 mmol), and
DMAP (0.31 g, 2.55 mmol), and the solution was stirred at ambient
temperature for 16 h. The mixture was partitioned between EtOAc (50
mL) and sat. NaHCO3 (50 mL), and the phases were separated. The
organic phase was washed with brine (50 mL), dried (Na2SO4), and
concentrated. To the crude benzoate 34 in 10 mL of THF at 0 °C were
added acetic acid (0.89 mL, 15.6 mmol) and TBAF (1.0 M solution in
THF, 3.90 mL, 3.90 mmol). The solution was allowed to warm slowly
1
37 as a colorless crystalline solid. [R]23 +29.6 (c 1.65, CHCl3); H
D
NMR (CDCl3) δ 8.02 (d, J ) 7.3 Hz, 2H), 7.77 (d, J ) 7.7 Hz, 2H),
7.56 (m, 2H), 7.26-7.50 (m, 12H), 5.59 (d, J ) 9.5 Hz, 1H), 5.51
(ddd, J ) 15.9, 11.2, 5.5 Hz, 1H), 5.59 (d, J ) 9.5 Hz, 1H), 5.21 (br
s, 4H), 5.07 (m, 3H), 4.85 (d, J ) 8.0 Hz, 1H), 4.66 (m, 2H), 4.19-
4.48 (m, 10H), 4.13 (br s, 1H), 4.66 (m, 2H), 4.19-4.48 (m, 10H),
4.13 (br s, 1H), 4.09 (d, J ) 10.4 Hz, 1H), 4.04 (m, 1H), 3.94 (m, 3H),
3.78 (m, 4H), 3.64 (d, J ) 10.4 Hz, 1H), 3.45 (dd, J ) 10.5, 3.9 Hz,
1H), 2.11 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 2.00 (s,3H), 1.99 (s, 3H),
1.86 (s, 3H), 1.78 (m, 1H), 1.29 (d, J ) 6.3 Hz, 3H), 0.86 (s, 9H) 0.03
(s, 6H); 13C NMR (CDCl3) δ 170.95, 170.66, 170.39, 169.95, 165.30,
163.02, 156.70, 143.92, 143.63, 141.24, 134.81, 133.41, 129.74, 129.11,
128.58, 128.54, 128.49, 128.36, 128.01, 127.71, 127.09, 127.02, 125.17,
125.11, 119.96, 100.80, 99.49, 95.16, 78.46, 76.17, 72.78, 72.14, 71.75,
71.54, 71.25, 70.92, 70.05, 69.18, 68.57, 68.33, 67.61, 67.33, 67.07,
63.05, 62.25, 62.21, 58.79, 58.70, 49.23, 47.11, 37.97, 25.83, 23.10,
20.82, 20.73, 20.71, 20.63, 20.55, 18.78, 18.28, 18.00, 17.88, 17.84,
11.89, -5.35, -5.50; IR (neat): 2953, 2931, 2111, 1744, 1689 cm-1
.
HRMS: Calcd for C72H87N5O27SiNa: 1504.5255; found: 1504.5202.
Peracetylated 2,3-STF Trisaccharide 39. To trisaccharide 37 (70
mg, 0.05 mmol) in 1.0 mL of THF was added 1.0 mL of 1 N HCl. The
solution was stirred for 30 min and then partitioned between EtOAc
(30 mL) and sat NaHCO3 (30 mL). The phases were separated, and
the organic phase was washed with brine (30 mL), dried (Na2SO4),
and concentrated. To the residue were added CH2Cl2 (2.0 mL), acetic
anhydride (0.1 mL), triethylamine (0.1 mL), and DMAP (2 mg), and
the solution was stirred at ambient temperature for 12 h. The mixture
was partitioned between EtOAc (30 mL) and sat. NaHCO3 (30 mL).
The phases were separated, and the organic phase was washed with
brine (30 mL), dried (Na2SO4), and concentrated. The residue was
purified by flash chromatography (4:1 hexanes:EtOAc) to afford 58
mg (85%) of peracetylated trisaccharide 39 as a colorless crystalline

2672 J. Am. Chem. Soc., Vol. 121, No. 12, 1999
Schwarz et al.
solid. [R]23D +40.0 (c 0.90, CHCl3); 1H NMR (CDCl3) δ 8.02 (d, J )
7.4 Hz, 2H), 7.78 (d, J ) 7.4 Hz, 2H), 7.59 (d, J ) 7.4 Hz, 2H), 7.37-
7.53 (m, 6H), 7.29 (m, 6H), 5.61 (d, J ) 9.4 Hz, 1H), 5.51 (m, 2H),
5.19 (m, 4H), 5.08 (d, J ) 4.3 Hz, 1H), 5.00 (d, J ) 3.5 Hz, 1H), 4.81
(d, J ) 7.9 Hz, 1H), 4.75 (d, J ) 3.8 Hz, 1H), 4.58 (dd, J ) 11.9, 3.3
Hz, 1H), 4.47 (dd, J ) 10.3, 7.1 Hz, 1H), 4.27-4.42 (m, 5H), 4.17-
4.25 (m, 2H), 4.10 (m, 2H), 3.90-4.02 (m, 3H), 3.86 (dd, J ) 11.8,
8.2 Hz, 1H), 3.63 (d, J ) 10.5 Hz, 1H), 3.46 (dd, J ) 10.6, 3.7 Hz,
1H), 2.49 (dd, J ) 13.8, 5.5 Hz, 1H), 2.11 (s, 3H), 2.10 (s, 3H), 2.09
(s, 3H), 2.09 (s, 3H), 2.00 (s, 6H), 1.98 (s, 3H), 1.85 (s, 3H), 1.75 (app
t, J ) 13.7 Hz, 1H), 1.26 (d, J ) 6.3 Hz, 3H); 13C NMR (CDCl3) δ
170.89, 170.65, 170.36, 169.99, 169.82, 169.50, 165.25, 162.92, 156.70,
143.83, 143.61, 141.27, 134.78, 133.40, 129.72, 129.19, 128.63, 128.59,
128.45, 128.37, 127.76, 127.09, 127.04, 125.11, 125.06, 120.01, 100.95,
99.22, 95.08, 75.29, 72.51, 72.26, 71.74, 71.19, 70.20, 69.54, 69.20,
68.21, 68.05, 67.67, 67.36, 66.78, 63.18, 62.38, 62.22, 59.34, 58.72,
49.28, 47.10, 38.03, 23.11, 20.82, 20.75, 20.71, 20.65, 20.56, 18.38;
IR (neat): 3356, 2961, 2111, 1744 cm-1. HRMS: Calcd for C70H77N5O29-
Na: 1474.4602; found: 1474.4595.
(m, 4H), 3.51 (dd, J ) 9.6, 8.0 Hz, 1H), 2.75 (dd, J ) 12.4, 4.6 Hz,
1H), 2.12 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.78 (t, J ) 12.2 Hz, 1H),
1.24 (d, J ) 6.4 Hz, 3H); 13C NMR (D2O, external CS2 reference) δ
176.14, 174.97, 174.61, 174.10, 173.98, 104.57, 99.63, 99.00, 77.23,
76.74, 75.62, 74.78, 72.78, 71.91, 70.95, 69.00, 68.80, 68.45, 68.12,
67.37, 62.53, 61.28, 61.02, 59.26, 51.66, 48.57, 39.77, 22.39, 22.05,
21.97, 18.26; HRMS: Calcd for C31H49N3O22Na3: 884.2501; found:
884.2551.
Disaccharide Acceptor 41. 6-Acceptor 20 (190 mg, 0.29 mmol),
CaSO4 (400 mg), and AgOTf (125 mg, 0.58 mmol) were combined in
a flask under argon, at which time 5.0 mL of THF and DTBP (0.15
mL, 0.58 mmol) were added. After stirring at ambient temperature for
30 min, the reaction was cooled to -78 °C, and chloride donor 23 (X
) Cl, 0.30 g, 0.58 mmol) in 5.0 mL of THF was added dropwise over
1 h. The reaction was allowed to warm to -10 °C and held at this
temperature 4 h and then filtered through Celite and concentrated. The
crude product 24 was treated with 80% HOAc for 16 h and
concentrated. The resultant oil was purified by silica gel chromatography
(2 f 3 f 4% MeOH in CH2Cl2) to provide 0.15 g of 41 (48% from
20) as a white foam: [R]23D +61.8 (c ) 0.25, CHCl3); 1H NMR (CDCl3)
δ 1.87 (s, 3H), 1.90 (s, 3H), 2.02 (s, 3H), 2.03 (s, 3H), 2.11 (s, 3H),
2.15 (s, 3H), 2.27 (dt, J ) 13.0, 1.7 Hz, 1H), 2.53 (dd, J ) 12.9, 4.6
Hz, 1H), 2.77 (d, J ) 8.9 Hz, 1H), 3.29 (d, J ) 4.4 Hz, 1H), 3.69-
4.57 (m, 12H), 4.84-4.87 (m, 2H), 5.16 (d, J ) 9.2 Hz, 1H), 5.02-
5.34 (m, 6H), 5.39 (dt, J ) 11.0, 5.4 Hz, 1H), 5.50 (d, J ) 9.6 Hz,
1H), 5.88 (d, J ) 8.2 Hz, 1H), 7.22-7.47 (m, 9H), 7.60 (d, 2H), 7.75
(d, 2H); 13C NMR (CDCl3) δ 171.1, 170.8, 170.5, 170.3, 170.1, 170.0,
169.6, 169.0, 168.0, 155.8, 143.7, 143.6, 141.1, 135.0, 128.5, 128.4,
127.6, 127.0, 125.1, 125.0, 119.0, 99.2, 98.6, 94.8, 72.7, 69.2, 69.0,
68.8, 68.1, 67.5, 63.3, 62.7, 62.5, 60.1, 54.4, 53.2, 53.0, 49.1, 46.9,
36.8, 36.0, 22.9, 20.9, 20.8, 20.7, 20.6; IR (film) 3357, 3067, 2956,
2110, 1745, 1664, cm-1. FAB HRMS m/e calcd for (M + Na)
C51H59N5O21Na 1100.3600, found 1100.3589.
Tetrasaccharide 42. Sialyated acceptor 41 (58 mg, 0.054 mmol)
and thioglycoside 35 (22 mg, 0.027 mmol) were azeotroped with
benzene (3 × 5 mL). NIS (15.2 mg, 0.068 mmol), 0.1 g of 4 Å mol
sieves, and 2.0 mL of CH2Cl2 were then added. A freshly prepared
solution of triflic acid (1% solution in CH2Cl2, 0.24 mL) was then added
dropwise. After 5 min, the reaction was judged complete by TLC and
quenched with triethylamine. Flash chromatography (3 f 3.5 f 4 f
4.5 f 5% MeOH in CH2Cl2) afforded 26 mg (53%) of 42 as a white
film: [R]23D +20.8 (c ) 1.25, CHCl3); 1H NMR (CDCl3) δ 8.02 (d, J
) 6.7 Hz, 2H), 7.77 (d, J ) 6.7 Hz, 2H), 7.60 (t, J ) 6.8 Hz, 2H),
7.53 (t, J ) 7.2 Hz, 1H), 7.04-7.44 (m, 11H), 5.84 (d, J ) 8.3 Hz,
1H), 5.51 (dt, J ) 10.7, 5.4 Hz, 1H), 5.16-5.38 (m, 10H), 5.06 (bs,
1H), 4.85 (bm, 1H), 4.77 (d, J ) 7.9 Hz, 1H), 4.75 (bs, 1H), 4.61 (bd,
J ) 8.3 Hz, 2H), 3.75-4.48 (m, 22H), 3.65 (d, J ) 10.5 Hz, 1H), 3.55
(dd, J ) 9.7, 5.8 Hz, 1H), 3.48 (dd, J ) 10.4, 3.4 Hz, 1H), 2.61 (bs,
1H), 2.56 (dd, J ) 12.8, 4.6 Hz, 1H), 2.51 (dd, J ) 13.9, 5.5 Hz, 1H),
2.12 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 2.00 (s, 3H), 1.99
(s, 3H), 1.87 (s, 3H), 1.86 (s, 3H); 13C NMR (CDCl3) δ 171.0, 170.9,
170.7, 170.6, 170.4, 170.3, 170.2, 170.0, 169.9, 169.8, 168.0, 165.3,
163.0, 155.8, 143.8, 143.7, 141.2, 135.0, 133.4, 129.7, 129.1, 128.6,
128.5, 128.4, 128.3, 127.8, 127.1, 125.2, 120.0, 100.8, 99.0, 98.7, 95.1,
72.8, 72.7, 72.2, 71.2, 69.4, 69.2, 69.0, 68.9, 68.8, 68.0, 67.7, 67.6,
67.2, 67.0, 66.3, 62.5, 62.0, 58.3, 54.4, 53.4, 52.8, 49.3, 47.1, 38.0,
37.5, 29.7, 23.1, 23.0, 21.0, 20.8, 20.7, 20.6, 20.5; IR (film) 3366, 3065,
2959, 2111, 1744, 1687, 1533, 1369, 1225 cm-1. FAB HRMS m/e calcd
for (M + Na) C85H98N6O39Na 1849.5767, found 1849.5766.
Azide Reduction of 39. Trisaccharide 39 (77 mg, 0.053 mmol) was
deprotected according to the procedure set forth for azide reduction of
17 with AcSH. Purification by flash chromatography (1:1 hexanes:
EtOAc f EtOAc) afforded 44 mg (56%) of a colorless oil. [R]23D +43.2
1
(c 1.65, CHCl3); H NMR (CDCl3) δ 8.00 (d, J ) 7.5 Hz, 2H), 7.79
(d, J ) 7.5 Hz, 2H), 7.62 (m, 2H), 7.25-7.53 (m, 12 H), 5.65 (d, J )
9.4 Hz, 1H), 5.45 (m, 1H), 5.41 (br s, 2H), 5.27 (d, J ) 10.3 Hz, 1H),
5.15 (m, 2H), 5.01 (d, J ) 3.3 Hz, 1H), 4.90 (d, J ) 11.9 Hz, 1H),
4.76 (d, J ) 3.4 Hz, 1H), 4.72 (d, J ) 7.7 Hz, 1H), 4.63 (m, 1H), 4.52
(m, 2H), 4.25-4.43 (m, 6H), 4.18 (m, 2H), 4.08 (m, 2H), 3.93 (m,
2H), 3.84 (dd, J ) 11.5, 8.2 Hz, 1H), 3.64 (d, J ) 10.5 Hz, 1H), 2.50
(dd, J ) 13.5, 5.0 Hz, 1H), 2.11 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H),
2.01 (s, 3H), 1.99 (s, 6H), 1.95 (s, 3H), 1.81 (s, 3H), 1.73 (app t, J )
12.7 Hz, 1H), 1.23 (s, 3H), 1.22 (d, J ) 5.8 Hz, 3H); 13C NMR (CDCl3)
δ 171.13, 170.02, 170.66, 170.41, 170.37, 169.96, 169.77, 164.89,
162.95, 156.58, 143.72, 141.37, 141.32, 134.41, 133.62, 129.99, 129.81,
129.15, 128.83, 128.76, 128.55, 128.49, 127.80, 127.13, 127.08, 124.93,
124.87, 120.08, 99.85, 99.61, 95.11, 74.15, 72.53, 72.33, 71.57, 71.40,
70.25, 69.15, 68.74, 68.25, 67.95, 67.60, 66.94, 66.78, 63.25, 62.67,
62.21, 58.64, 49.28, 48.71, 47.20, 37.97, 29.65, 23.08, 22.64, 20.80,
20.74, 20.72, 20.65, 20.56, 18.25; IR (neat): 3333, 2927, 1745, 1670
cm-1. HRMS: Calcd for C72H81N3O30Na: 1490.4802; found: 1490.4814.
To this oil (29 mg, 0.020 mmol) was added morpholine (2 mL), and
the mixture was stirred at ambient temperature 1 h. Excess morpholine
was removed by azeotroping with toluene (3 × 5 mL). To the crude
free amine in 2.0 mL of CH2Cl2 were added acetic anhydride (0.1 mL)
and diisopropylethylamine (0.1 mL), and the solution was stirred at
ambient temperature 1 h. The mixture was partitioned between EtOAc
(20 mL) and saturated NaHCO3 (20 mL). The phases were separated,
and the organic phase was dried (Na2SO4) and concentrated. Flash
chromatography of the residue (3 f 4 f 5 f 6 f 7 f 8% MeOH in
CH2Cl2) provided 22.8 mg of a colorless solid. The product contained
ca. 10% of a byproduct, presumably owing to some lactone opening
by morpholine (supported by mass spectral analysis). The mixture was
used in the next step without purification. Hydrogenolysis of the benzyl
ester according to the procedure set forth for compound 17 was effected
and the product used without purification. To the crude acid was added
methanol (0.5 mL), 0.1 N aq NaOH (0.5 mL), and the mixture was
stirred at ambient temperature 24 h. DOWEX-50H was added to lower
the pH to ca. 4, the mixture was filtered through a cotton plug to remove
the resin, and the solution was concentrated. To the residue was added
methanol (2.0 mL), and then sodium methoxide (25 wt % in MeOH)
dropwise until the pH reached 10-10.5 (ca. 5 drops). The solution
was heated to reflux 16 h and cooled, and DOWEX-50H was added to
lower the pH of the solution to ca. 4. The mixture was filtered through
a cotton plug to remove the resin and concentrated. Purification of the
residue by reverse-phase column chromatography (LiChroprep RP-18,
H2O eluant) provided 7.1 mg (44%) of N-acetyl-2,3-STF 40 as a
Hexasaccharide 45. A mixture of thioglycoside 43 (70.8 mg, 0.05
mmol) and 3-acceptor 44 (68.3 mg, 0.1 mmol) was azeotroped with
toluene (3 × 5 mL). To the mixture was added powdered 4 Å mol
sieves (0.2 g), and 3.0 mL of CH2Cl2, and the mixture was stirred 30
min at ambient temperature. The mixture was cooled to 0 °C, at which
time NIS (26.7 mg, 0.12 mmol) and freshly prepared triflic acid solution
(1% solution in CH2Cl2, 0.42 mL, 0.05 mmol) were added. The red
mixture was stirred at 0 °C for 15 min and then diluted with EtOAc.
The organic phase was washed with sat. NaHCO3, 10% Na2S2O3 and
brine, dried (MgSO4), and concentrated. The residue was purified by
colorless crystalline solid. [R]23 +56.1 (c 0.27, MeOH); 1H NMR
D
(D2O) δ 4.95 (d, J ) 3.8 Hz, 1H), 4.53 (d, J ) 7.8 Hz, 1H), 4.36 (dd,
J ) 6.6, 2.6 Hz, 1H), 4.26 (m, 2H), 4.20 (br d, J ) 2.6 Hz, 1H), 4.05
(m, 3H), 3.92 (d, J ) 3.2 Hz, 1H), 3.86 (m, 4H), 3.72 (m, 4H), 3.62
flash chromatography (1:1 f 2:1 EtOAc:CH2Cl2) to afford 80.1 mg
1
(79%) of 45 as a colorless solid. [R]23 -26.4 (c 1.00, CHCl3); H
D

Synthesis of R-O-Linked Glycopeptides
J. Am. Chem. Soc., Vol. 121, No. 12, 1999 2673
NMR (CDCl3) δ 8.10 (d, J ) 7.4 Hz, 2H), 7.79 (d, J ) 7.5 Hz, 2H),
7.59 (d, J ) 7.0 Hz, 2H), 7.54 (t, J ) 7.2 Hz, 1H), 7.43-7.24 (m,
12H), 5.86 (d, J ) 8.5 Hz, 1H), 5.52-5.47 (m, 2H), 5.35-5.32 (m,
4H), 5.18-5.05 (m, 5H), 5.04-4.98 (m, 3H), 4.95-4.88 (m, 3H), 4.80
(d, J ) 7.9 Hz, 1H), 4.72 (d, J ) 3.3 Hz, 1H), 4.59-4.56 (m, 2H),
4.51 (dd, J ) 11.7, 5.7 Hz, 1H), 4.43-4.37 (m, 2H), 4.33-4.23 (m,
2H), 4.21-4.07 (m, 6H), 4.03-3.84 (m, 5H), 3.80-3.73 (m, 4H), 3.44
(d, J ) 10.3 Hz, 1H), 3.43 (d, J ) 10.5 Hz, 1H), 3.21-3.13 (m, 1H),
2.83 (s, 1H), 2.21 (s, 3H), 2.18 (s, 3H), 2.16 (s, 3H), 2.14 (s, 3H), 2.12
(s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 1.99 (s,
6H), 1.27 (s, 3H), 1.14 (d, J ) 5.6 Hz, 6H), 0.86 (s, 9H), 0.04 (s, 6H);
13CNMR (CDCl3) δ 171.37, 171.23, 171.10, 170.96, 170.91, 170.87,
170.85, 170.74, 170.54, 170.39, 170.17, 169.96, 169.92, 165.79, 156.31,
144.18, 141.69, 135.43, 134.09, 130.24, 129.51, 129.05, 129.01, 128.92,
128.84, 128.17, 127.50, 125.58, 125.54, 120.43, 102.39, 100.83, 100.69,
99.87, 96.62, 96.09, 78.11, 77.30, 74.25, 73.76, 73.52, 73.30, 72.96,
72.04, 71.81, 71.33, 71.26, 71.10, 71.03, 69.81, 69.38, 68.71, 68.61,
68.23, 68.10, 67.99, 67.95, 67.67, 67.29, 65.45, 64.36, 62.95, 62.20,
60.95, 58.84, 58.76, 54.87, 47.51, 26.25, 22.97, 21.47, 21.30, 21.26,
21.14, 21.08, 21.05, 20.99, 18.69, 16.28, 15.99, -4.98, -5.07; IR
(neat): 2935, 2110, 1746 cm-1. FAB HRMS m/e calcd for (M + Na)
Acknowledgment. This research was supported by the
National Institutes of Health (Grant numbers: AI 16943 (S.J.D.),
Sloan-Kettering Institute Core Grant (CA-08748)). Postdoctoral
Fellowship Support is gratefully acknowledged by J.B.S. (NIH-
F3218804), S.D.K. (US Army Breast Cancer Research Fellow-
ship DAMD 17-98-1-8154), P.W.G. (American Cancer Society,
PF98026), and D.S. (Irvington Institute for Immunological
Research). We gratefully acknowledge Dr. George Sukenick
(NMR Core Facility, Sloan-Kettering Institute) for NMR and
mass spectral analyses.
Supporting Information Available: 1H and 13C NMR
spectra for compounds 9, 15, 16, 17, 18, 19, 25, 26, 27, 33, 35,
1
37, 39, 39b, 40, 41, 42, 45; H NMR spectra for compounds
11, 12, 14; full chemical names for new compounds 25, 27,
33, 39, 40, 41, 42, 45 (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
C100H127N5O45SiNa: 2168.7470; found 2168.7545.
JA9833265