Highly efficient stereospecific preparation of Tn and TF building blocks using thioglycosyl donors and the Ph2SO/Tf2O promotor system

Article abstract of DOI:10.1081/CAR-200067091

The activation of 2-azido-2-deoxy Tn and TF thioglycosyl donors by the powerful thiophilic promoter system Ph₂SO/Tf₂O has been investigated. Glycosylation of an Fmoc-protected threonine derivative gave 1,2-cis glycosides in high yiel

Full text of DOI:10.1081/CAR-200067091

This article was downloaded by: [Case Western Reserve University]
On: 22 October 2014, At: 10:47
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Carbohydrate Chemistry
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lcar20
Highly Efficient Stereospecific
Preparation of Tn and TF Building Blocks
Using Thioglycosyl Donors and the
Ph2SO/Tf2O Promotor System
David Cato a , Therese Buskas a & Geert‐Jan Boons a
a Complex Carbohydrate Research Center , University of Georgia ,
Athens, Georgia, USA
Published online: 16 Aug 2006.
To cite this article: David Cato , Therese Buskas & Geert‐Jan Boons (2005) Highly Efficient
Stereospecific Preparation of Tn and TF Building Blocks Using Thioglycosyl Donors and the Ph2SO/Tf2O
Promotor System , Journal of Carbohydrate Chemistry, 24:4-6, 503-516
To link to this article: http://dx.doi.org/10.1081/CAR-200067091
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Carbohydrate Chemistry, 24:503–516, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1081/CAR-200067091
Highly Efficient
Stereospecific Preparation
of Tn and TF Building Blocks
Using Thioglycosyl Donors
and the Ph2SO/Tf2O
Promotor System
David Cato, Therese Buskas, and Geert-Jan Boons
Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA
The activation of 2-azido-2-deoxy Tn and TF thioglycosyl donors by the powerful
thiophilic promoter system Ph2SO/Tf2O has been investigated. Glycosylation of an
Fmoc-protected threonine derivative gave 1,2-cis glycosides in high yields and excellent
stereoselectivities. The galactosylation of phenyl 2-azido-4,6-O-benzylidene-2-deoxy-1-
thio-b-D-galactopyranoside was achieved in high yield and without orthoester formation
using
a trichloroacetimidate donor carrying a 2-O-(2,5-difluorobenzoyl) group.
The anomeric thiophenyl group of the constructed TF disaccharide could directly be
activated by the van Boom promotor for the glycosylation of a threonine derivative.
Keywords Glycosylation, Thioglycoside, Tn antigen, TF antigen, Glycopeptide
INTRODUCTION
Mucins are a family of highly glycosylated proteins that are expressed on most
epithelial cells.[1] The polypeptide backbone of mucins consists of highly
conserved tandem repeats that are heavily O-glycosylated through clustered
serine and threonine residues. The large and complex mucin-type carbohydrate
structures serve as important recognition motifs for interactions with proteins.
Received February 22, 2005; accepted April 4, 2005.
Dedicated to the memory of Professor Jacques H. van Boom.
Address correspondence to Geert-Jan Boons, Complex Carbohydrate Research Center,
University of Georgia, 315 Riverbend Rd., Athens, GA 30603, USA. E-mail: gjboons@
ccrc.uga.edu
503

D. Cato, T. Buskas, and G.-J. Boons
504
Although the glycoforms of mucins display extreme heterogeneity in regards to
saccharide composition, length, and linkages, the biosynthesis of mucins is
initiated by the addition of N-acetylgalactosamine to serine and threonine
residues. Further elongation of this structure leads to the large family of
O-glycans, classified into eight groups depending on their core stucture. This
elongation takes place at the 3-O and/or 6-O-position by addition of galactose
and/or polylactosamine residues followed by chain terminations with sialic
acids, fucoses, or sulfation.
In malignant cells, altered expression levels of glycosyl transferases such
as the downregulation of glucosaminyltransferases and concomitant up-regu-
lation of sialyltransferases lead to simpler truncated forms of the glycans.
This aberrant glycosylation has been correlated with specific disease states.
For example, it is known that the presence of the Tn antigen and the related
structure Galpb(1-3)GalpNaca-Ser/Thr, also known as the TF antigen (Fig. 1),
is common on human epithelial tumor cells,[2] such as colon and prostate
cancers.[3] The presence of these antigens has spurred intense studies aimed
at the development of immunotherapy for cancer.[4,5] However, the enormous
structural diversity that is introduced by glycosylation renders the isolation of
well-defined glycopeptides from natural sources an almost impossible task,
thus presenting a major obstacle to the study of the structure-activity relation-
ship of these compounds. It is thus not surprising that homogeneous synthetic
glycopeptides would be most valuable tools for unraveling the specific roles of
glycopeptides derived from mucins in biological processes.[6]
Our goal is to develop synthetic carbohydrate-based anticancer vaccines
and to construct complex glycosulfopeptides derived from mucins. With this
goal in mind, we needed a facile route to substantial quantities of Tn and TF
building blocks that could be used for Fmoc solid phase synthesis. To this
end, the formation of the a-glycosidic linkage between N-acetylgalactosamine
and serine or threonine is a key step. This particular glycosylation has
garnered much attention and has been extensively reviewed in the liter-
ature.[7–9] Here we report the use of the van Boom/van der Marel promotor
Figure 1: Tn and TF.

Stereospecific Preparation of Tn and TF Building Blocks
505
system for the activation of thioglycosides in the synthesis of Tn and TF deriva-
tives useful for solid phase glycopeptide synthesis.
RESULTS AND DISCUSSION
Despite recent advances, the chemical synthesis of glycopeptides remains a
difficult task.[10,11] A crucial step in any glycopeptide synthesis is the incor-
poration of the saccharide part to the peptide backbone. Currently, the most
general synthetic methodology employs preformed glycosylated amino acids
for the stepwise solid-phase synthesis of peptides. The protecting groups for
these glycosylated amino acids must be carefully chosen and are rather
limited, as the O-glycosidic bond is acid labile and the O-linked glycopeptide
can undergo b-elimination upon treatment with strong bases. Presently, the
use of acetyl esters as hydroxyl protection for the oligosaccharide part and
Na-Fmoc-protected amino acids is a standard technique in solid-phase
glycopeptide synthesis. The formation of the a-glycosidic linkage between
N-acetylgalactosamine and serine or threonine is a key step in the preparation
of suitable glycosylated amino acid derivatives. For the installment of the
1,2-cis linkage, the nonparticipating 2-azido-2-deoxy group is commonly
employed to mask the amino function. Recent reports have shown that
2-acetamido-2-deoxy galactose derivatives carrying a 4,6-benzylidene give
a-selectivity in the preparation of Tn,[12] TF,[13] and sialyl-TF[14] building
blocks. Anomeric halides[4,15–18] and trichloroacetimidates[19,20] are the
most commonly used glycosyl donors to prepare the GalpNAca-Ser/Thr
linkage.[8,9] A typical example of this glycosylation is the activation of fully
acetylated 2-azido-2-deoxy bromides by AgClO4/Ag2CO3 in the presence of
serine or threonine derivatives (Sch. 1).[21] The reported yields and stereoselec-
tivities are highly dependent on the protecting group patterns of both the
saccharide donor and amino acid acceptor. Anomeric fluorides have also been
utilized for the preparation of Tn derivatives with Cp2ZrCl2/AgClO4 as promo-
ters.[4,20,22] Although high yielding in glycosylations of both serine and threo-
nine derivatives, the observed stereoselectivity was markedly decreased for
the Na-Fmoc-Ser-OBn derivative.[20]
The resulting poor stereoselectivities from the use of various TF disacchar-
ide donors[4,23–25] for the glycosylation of serine and threonine derivatives led
Scheme 1: (i): AgClO4/Ag2CO3, DCM/toluene, 48 hr, 64%.

D. Cato, T. Buskas, and G.-J. Boons
506
Danishefsky and coworkers to implement the “cassette method.”[4] This
method involves the use of a properly protected Tn derivative as a general
acceptor in the synthesis of practically any O-linked glycopeptide.
Most reported methods depend on conventional labile donors that must be
prepared just prior to glycosylation, thus clearly diminishing synthetic flexi-
bility. More stable donors such as the n-pentenyl-[26] and seleno-glycosides[27]
are capable of withstanding protecting group manipulations and may be
directly activated for glycosidations, but have not found widespread use for
the synthesis of Tn and TF building blocks.[28–32]
A new generation of thioglycosyl promoters has recently been introduced
by Crich and coworkers.[33,34] In the most successful promoter system,
1-benzenesulfinylpiperidine (BSP) was reacted with triflic anhydride (Tf2O)
to form a sulfonium species, which could convert disarmed thioglycosides
into reactive triflates at very low temperatures.[34] However, it was found
that the BSP/Tf2O promoter pair was unable to successfully activate the
disarmed phenylthioglycosides of 2-azido-2-deoxy-mannose and 2-azido-2-
deoxy-glucose. Thus, building upon the foundation of the Crich discovery,
this promoter system was further refined by van Boom and van der
Marel.[35,36] Reacting diphenylsulfoxide (Ph2SO) with Tf2O led to a highly elec-
trophilic species capable of activating highly unreactive donors. This potent
promoter resulted in high yields and excellent stereoselectivities. By exploiting
differences in reactivity, it was shown to be useful in chemoselective glycosyla-
tion sequences with the BSP/Tf2O promoter system. In light of this discovery,
we wished to explore the use of thioglycosides as donors in the formation of the
a-glycosidic linkage between 2-azido-2-deoxy derivatives of the Tn and TF
saccharides.
To streamline the synthesis of Tn and TF derivatives, we wanted to explore
the use of the Ph2SO/Tf2O promoter system for the direct activation of 2-azido-
2-deoxy thiogalactoside donors in glycosylations with threonine derivatives. In
an attempt to accomplish this, bromide 5[37] was converted into the corres-
ponding thiophenyl derivative by reaction with sodium thiophenolate in a
mixture of dichloromethane and ethanol (Sch. 2).[38] Activation of thiophenyl
glycoside 8[39] with the Ph2SO/Tf2O promoter system for the glycosylation of
Na-Fmoc-Thr(OH)-OBn (6) proceeded with high efficiency and produced the
glycosyl amino acid 7 in an excellent yield of 85%. The reaction proceeded
Scheme 2: (i): NaS Ph, EtOH/DCM, 3 hr, %; (ii): 6, Ph2SO/Tf2O, DCM, 2 608C, 1 hr, 85%.

Stereospecific Preparation of Tn and TF Building Blocks
507
with complete stereochemical control as the a-anomer was formed exclusively.
Compared to similar glycosylations using MeOTf,[28] DMTST,[28,29] or NBS/
TBAOTf,[30–32] the Ph2SO/Tf2O promoter provides far superior yield and
stereoselectivity. Encouraged by this result, we directed our attention to the
preparation of the TF antigen. By deacetylation and the introduction of a
4,6-benzylidene, thioglycoside 8 was easily converted into the known
acceptor 10.[39] For the galactosylation, several glycosyl donors were investi-
gated. The results are summarized in Table 1. The commonly used donor
per-O-acetylated galactosyl bromide 11 activated by AgOTf[40] (entry 1) gave
in our experiments unreliable results. Disaccharide 12 was often accompanied
by a formation of substantial amounts of the corresponding orthoester.
Changing the promoter system or anomeric leaving group did not improve
the outcome (entries 2–4).
The fact that the Ph2SO/Tf2O promoter system uses nearly stoichiometric
quantities of activator and the thioglycoside is converted into the correspond-
ing triflate before the glycosyl acceptor is added to the reaction mixture,[35]
prompted us to explore the possibility of using the readily available thiogal-
actoside 14. Activation of 14 by Ph2SO/Tf2O in the presence of 2,5-di-tbutyl-
4-methylpyridine (DTBMP) and subsequent reaction with acceptor 10 (entry
5) gave mainly the corresponding orthoester. We were encouraged to find
only trace amounts of the disaccharide corresponding to the activation and
self-coupling of acceptor 10. In an attempt to avoid the orthoester formation,
a similar reaction was performed with the omission of the base. Unfortunately,
this reaction gave the same disappointing result (entry 6).
Replacing a 2-O-acetyl by a 2-O-benzoyl is a common way to avoid orthoe-
ster formation. Indeed, using fully benzoylated bromide 15 in an AgOTf-
activated glycosylation of acceptor 10 gave disaccharide 16 in 76% yield
(entry 7). However, due to the more severe basic conditions required for the
removal of O-benzoates and in particular a 2-O-benzoate of galactose, this
derivative would not be suitable for use in glycopeptide synthesis. Instead,
we turned our attention to the 2,5-di-fluorobenzoyl group (dFBz) that was
recently introduced for glycopeptide synthesis by Kihlberg and coworkers.[41]
An advantage of this protecting group is that the difluorobenzoyl possesses a
combination of the positive qualities associated with 2-O-benzoyl groups in gly-
cosylations and the ease of removal of acetyl esters. Using the easily accessible
fully difluorobenzoylated galactosyl bromide 17[41] in an AgOTf-mediated
reaction, the desired disaccharide 18 was obtained, but only in 51% yield
(entry 8). The donor was found to be very unreactive and the reaction
sluggish. An attempt to improve the reaction by gentle heating was unsuccess-
ful. A positive feature of this donor was that no orthoester was isolated. The
mediocre yield prompted us to prepare glycosyl donors 21 and 23 (Sch. 3).
Carrying a C-2 dFBz and acetyl esters at the C-3, C-4, and C-6 position, it
was believed that these donors would exhibit a higher reactivity. To accomplish

Table 1.
Entry
Donor
R1
R2
Ac
R3
Ac
Promotor/solvent/temp.
Product
1
2
11
11
13
14
14
14
15
17
21
23
a-Br
a-Br
AgOTf/DCM/–408C-RT
Orthoester/12
Orthoester/12
Orthoester/12
Orthoester/12
Orthoester
Orthoester
16 (76%)
Ac
Ac
Ac
Ac
Ac
Bz
Ac
Ac
Ac
Ac
Ac
Bz
HgO/HgCl2/DCM/508C
TMSOT f/DCM/–208C-RT
NIS/TMSOTf/DCM/–208C-RT
Ph2SO/Tf2O/DTBMP/DCM/–608C-RT
Ph2SO/Tf2O/DCM/–608C-RT
AgOTf/DCM/–408C-RT
3
a/b-OC(NH)CCl3
4
b-SEt
5
b-SEt
6
b-SEt
7
a-Br
8
9
10
a-Br
a-Br
dFBz
dFBz
dFBz
dFBz
Ac
Ac
AgOTf/DCM/–408C-RT
AgOTf/DCM/–408C-RT
TMSOTf/DCM/–208C-RT
18 (51%)
25 (63%)
25 (74%)
a/b-OC(NH)CCl3

Stereospecific Preparation of Tn and TF Building Blocks
509
Scheme 3: (i): dF BzCl, DMAP, pyridine, 18 hr, 96%; (ii): 30% HBr/HOAc, Ac2O, 508C, 89%;
(iii): NH2NH2-HOAc, DMF, 608C, 3 hr, 92%; (iv): Trichloroacetonitrile, DBU, DCM, 0oC, 96%.
this goal, alcohol 19[42] was acylated with difluorobenzoyl chloride in the
presence of 4-dimethylaminopyridine (DMAP), which afforded C-2 dFBz
derivative 20 in 96% yield. Conversion of 20 into glycosyl donor 21 was
achieved by treatment with 30% hydrogen bromide in acetic acid at 508C. Selec-
tive cleavage of the anomeric acetate of 20 gave hemiacetal 22, which was
transformed into trichloroacetimidate 23 using standard conditions.[43]
AgOTf activation of bromide 21 at –408C in the presence of glycosyl acceptor
10 provided disaccharide 25 in 63% yield (entry 9). The superior reactivity of
bromide 21 as compared to that of fully difluorobenzoylated bromide 17 was
reflected by a slightly increased yield. The condensation of trichloroacetimidate
23 activated by TMSOTf at –208C and alcohol 10 furnished disaccharide 25 in
a further improved yield of 74%.
Having established a reliable and efficient route to the thiophenyl TF
disaccharide, we chose to evaluate this glycosyl donor with construction of
the a-O-linkage to threonine in mind. As depicted in Scheme 4, thioglycoside
25 was activated by Ph2SO/Tf2O in the presence of DTBMP at 2608C for the
glycosylation of threonine derivative 6. Remarkably, the reaction yielded exclu-
sively the a-anomer product 26 in an excellent yield of 82%. It should be noted
that these results are not only the best results reported for the TF disaccharide
thioglycosides, but also the selectivity of this reaction is more superior than
what is observed in most procedures using halides and trichloroacetimidates
as glycosyl donors. To illustrate this feature, thiophenyl 25 was converted
into the corresponding bromide 27 by treatment with molecular bromine.
Subsequently, activation of the bromide with AgClO4 in a glycosidation with
threonine acceptor 6 gave 26 in an acceptable overall yield, but as expected,
the a/b-selectivity was lowered and isolation of 26 required careful
chromatography.

D. Cato, T. Buskas, and G.-J. Boons
510
Scheme 4: (i): 6, Ph2SO/Tf2O, DCM, 2608C, 1 hr, 82%; (ii): Br2, DCM, 08C; (iii): 6, AgClO4, DCM,
rt, 48 hr, 68% yield over two steps.
In conclusion, we have described an efficient route for Tn and TF antigen
building blocks that are useful in the solid-phase synthesis of glycopeptides
derived from mucins. Using the promotor system introduced by van Boom
and van der Marel for the activation of disarmed thioglycosides, we found
that the activation of Tn and TF thioglycoside donors proceeded smoothly
and provided the a-O-glycosidic bond to Na-Fmoc-Thr benzyl ester in high
yields and with exclusive formation of the a-product. Additionally, the TF
derivative may serve as an intermediate for further extension in the synthesis
of other mucin-derived glycopeptides.
EXPERIMENTAL
General
NIS was purchased from Fluka and recrystallized from dioxane/CCl4. All
other chemicals were purchased from Aldrich, Acros, and Fluka and used
without further purification. Molecular sieves were activated at 1458C for
10 hr. All solvents employed were of reagent grade and dried by refluxing
over appropriate drying agents. TLC was performed using Kieselgel 60 F254
(Merck) plates, with detection by UV light (254 nm) and/or by charring with
8% sulfuric acid in ethanol. Column chromatography was performed on silica
gel (Merck, mesh 70–230). Extracts were concentrated under reduced
pressure at ꢀ 408C (water bath). 1H NMR and 13C NMR spectra were
recorded on a Varian Inova300 spectrometer and a Varian Inova500

Stereospecific Preparation of Tn and TF Building Blocks
511
1
spectrometer equipped with Sun workstations. H spectra recorded in CDCl3
were referenced to residue CHCl3 at 7.26 ppm or TMS, and 13C spectra to the
central peak of CDCl3 at 77.0 ppm. Assignments were made using standard
1D and gCOSY, gHSQC, and TOCSY 2D experiments. Positive ion matrix-
assisted laser desorption ionization time of flight (MALDI-TOF) mass spectra
were recorded using an HP-MALDI instrument using gentisic acid as a matrix.
N-(9-Fluorenylmethyloxycarbonyl)-O-(3,4,6-tri-O-acetyl-2-azido-2-
deoxy-a-D-galactopyranosyl)-L-threonine benzylester (7). To a solution
of compound 8 (43 mg, 101 mmol) and Ph2SO (58 mg, 284 mmol) in dry CH2Cl2
(5 mL) was added, at 2608C, trifluoromethanesulfonic anhydride (24 mL,
141 mmol). The reaction mixture was stirred for 10 min, after which a
solution of acceptor 6 (87 mg, 202 mmol) in CH2Cl2 (1 mL) was added. The
mixture was stirred at 2608C for 1 hr after which it was slowly warmed to rt
and quenched by the addition of saturated aqueous NaHCO3 (3 mL). The
organic phase was washed with brine, dried (MgSO4), and concentrated.
Purification of the residue by silica gel chromatography (hexane/EtOAc 3 : 1)
yielded 7 (64 mg, 86.0 mmol, 85%); TLC (hexane/EtOAc 2 : 1) Rf ¼ 0.39; NMR
data was in agreement with reported data. HR MALDI-TOF MS: m/z: Calc
for C38H40N4O12: 744.2643; found 767.2541 [M þ Na]þ.
1,3,4,6-Tetra-O-acetyl-2-O-(2,5-difluorobenzoyl)-a-D-galactopyranose
(20). To a solution of 1,3,4,6-tetra-O-acetyl-a-D-galactopyranose (700 mg,
2.01 mmol) in dry pyridine (8 mL) was added 4-(dimethylamino)pyridine
(49 mg, 0.402 mmol) and the solution was stirred at rt for 30 min. 2,5-Difluoro-
benzoyl chloride (0.5 mL, 4.02 mmol) was added dropwise over a 10-min period
and the stirring was continued for 18 hr. The reaction was quenched by
addition of methanol (4 mL) and after stirring for 1 hr, the solution was
diluted with CH2Cl2 (120 mL) and washed with water (150 mL). The aqueous
phase was extracted with CH2Cl2 (50 mL) and the combined organic phases
were dried (MgSO4) and evaporated to dryness. After purification by silica
gel chromatography (hexane/EtOAc 8 : 1), 20 (942 mg, 1.93 mmol, 96%) was
afforded as a white solid; TLC (hexane/EtOAc 1 : 1), Rf ¼ 0.68; [a]D þ 20.0
1
(c 2 mg/mL, CHCl3); NMR data (CDCl3): H, d 7.55–7.05 (m, 3H, dFBz), 6.53
(d, 1H, J1,2 3.3 Hz, H-1), 5.60–5.41 (m, 3H, H-2, H-3, H-4), 4.39 (t, 1H, J5,6
6.6 Hz, H-5), 4.16–4.07 (m, 2H, H-6), 2.18, 2.14, 2.04, 2.03 (s, 12H,
4 ꢁ CH3CO); 13C, d 20.7, 20.8, 20.9, 21.0 (4 ꢁ CH3CO), 61.4 (C-6), 67.6 (C-5),
67.7 (C-3), 68.0 (C-4), 69.1 (C-2), 89.7 (C-1), 118.3–122.2 (aromatic C), 170.6,
170.3, 170.2, 170.1 (4 ꢁ CH3CO); HR MALDI-TOF MS: m/z: Calc for
C21H22F2O11: 488.1130; found 511.1029 [M þ Na]þ.
3,4,6-Tri-O-acetyl-2-O-(2,5-difluorobenzoyl)-a-D-galactopyranosyl
bromide (21). Compound 20 (300 mg, 614 mmol) was dissolved in a mixture

D. Cato, T. Buskas, and G.-J. Boons
512
of acetic acid (3 mL) and acetic anhydride (2 mL). Thirty-three percent
hydrogen bromide in acetic acid (4 mL) was added and the mixture was
stirred at 508C for 6 hr. The solution was allowed to cool to rt, diluted with
CH2Cl2 (100 mL), and washed with water (125 mL) and saturated aqueous
NaHCO3 (125 mL). The organic phase was dried (MgSO4) and concentrated.
Purification of the residue by silica gel chromatography (hexane/EtOAc 3 : 1)
furnished 21 (278 mg, 545 mmol, 89%); TLC (hexane/EtOAc 2 : 1), Rf ¼ 0.51;
[a]D þ 31.4 (c 2.0 mg/mL, CHCl3); NMR data (CDCl3): 1H, d 7.61–7.08
(m, 3H, dFBz), 6.80 (d, 1H, J1,2 3.8 Hz, H-1), 5.60–5.55 (m, 2H, H-3, H-4),
5.30 (dd, 1H, J1,2 2.2 Hz, J2,3 9.6 Hz, H-2), 4.54 (t, 1H, J5,6 6.6 Hz, H-5), 4.25–
4.09 (m, 2H, H-6), 2.17, 2.06, 1.98 (s, 9H, 3 ꢁ CH3CO); 13C, d 20.7, 20.8, 20.9,
(3 ꢁ CH3CO), 61.0 (C-6), 67.3 (C-5), 68.2 (C-2), 69.0 (C-4), 71.5 (C-3), 87.8
(C-1), 118.4–122.7 (aromatic C), 170.6, 170.1, 169.9 (3 ꢁ CH3CO); HR
MALDI-TOF MS: m/z: Calc for C19H19BrF2O9: 508.0181; found 531.0079
[M þ Na]þ.
3,4,6-Tri-O-acetyl-2-O-(2,5-difluorobenzoyl)-D-galactopyranose (22).
Hydrazine acetate (192 mg, 2.08 mmol) was added to a solution of 20 (925 mg,
1.89 mmol) in DMF (9 mL) heated at 608C. The mixture was kept at 608C for
3 hr, allowed to return to rt, diluted with EtOAc (75 mL), and washed with
20% aqueous NaCl (75 mL). The aqueous phase was extracted with EtOAc
(40 mL) and the combined organic layers were dried (MgSO4) and concentrated
under reduced pressure. Silica gel chromatography purification (hexane/
EtOAc 2 : 1) gave 22 (776 mg, 1.74 mmol, 92%); TLC (hexane/EtOAc 2 : 1),
1
Rf ¼ 0.22; [a]D þ 7.4 (c 2.0 mg/mL, CHCl3); NMR data (CDCl3): H, d 7.60–
1
7.10 (m, 3H, dFBz), 5.68 (d, 1H, J1,2 3.6 Hz, H-1), 5.59 (dd, H, J2,3 10.5 Hz,
J3,4 3.0 Hz, H-3), 5.52 (d, 1H, J4,5 2.5 Hz, H-4), 5.36 (dd, 1H, J1,2 10.7 Hz, J2,3
3.6 Hz, H-2), 4.52 (t, 1H, J5,6 6.6 Hz, H-5), 4.20–4.10 (m, 2H, H-6), 3.10 (bs,
1H, OH), 2.18, 2.17, 2.06 (s, 9H, 3 ꢁ CH3CO); 13C, d 20.8, 20.9, 21.0
(3 ꢁ CH3CO), 62.0 (C-6), 66.7 (C-5), 68.5 (C-3), 69.9 (C-4), 71.5 (C-2), 90.8
(C-1), 118.2–122.2 (aromatic C), 170.2, 170.4, 170.7 (3 ꢁ CH3CO); HR
MALDI-TOF MS: m/z: Calc for C19H20F2O10: 446.1025; found 469.0920
[M þ Na]þ.
3,4,6-Tri-O-acetyl-2-O-(2,5-difluorobenzoyl)-a-D-galactopyranosyl
trichloroacetimidate (23). Compound 22 (250 mg, 560 mmol) was dissolved
in dry CH2Cl2 (5 mL) at 08C, and trichloroacetonitrile (1.2 mL, 8.40 mmol) was
added followed by 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU, 8 mL, 53.5 mmol).
The mixture was stirred for 2 hr at 08C, concentrated to dryness, and
purified by silica gel chromatography (hexane/EtOAc/TEA 5 : 1 : 0.01) to
yield 23 (317 mg, 538 mmol, 96%); TLC (hexane/EtOAc 2 : 1), Rf ¼ 0.39;
[a]D þ 26.25 (c 2.0 mg/mL, CHCl3); NMR data (CDCl3): 1H, d 7.57–7.06
(m, 3H, dFBz), 6.74 (d, 1H, J1,2 2.2 Hz, H-1), 5.55–5.70 (m, 3H, H-2, H-3,

Stereospecific Preparation of Tn and TF Building Blocks
513
H-4) 4.51 (t, 1H, J5,6 6.6 Hz, H-5), 4.24–4.08 (m, 2H, H-6), 2.20, 2.03, 1.99
(s, 9H, 3 ꢁ CH3CO); 13C, d 20.8, 20.9, 21.0 (3 ꢁ CH3CO), 61.5 (C-6), 67.7
(C-3), 67.8 (C-4), 68.2 (C-2), 69.4 (C-5), 93.6 (C-1), 118.2–122.5 (aromatic C),
161.0 (C ¼ NH) 170.1, 170.3, 170.5 (3 ꢁ CH3CO); HR MALDI-TOF MS: m/z:
Calc for C21H20Cl3F2NO10: 589.0121; found 612.0019 [M þ Na]þ.
Phenyl 2-Azido-4,6-O-benzylidene-2-deoxy-3-O-(3,4,6-tri-O-acetyl-2-
O-(2,5-difluorobenzoyl)-b-D-galactopyranosyl)-1-thio-b-D-galactopyra-
noside (25). Trimethylsilyl trifluoromethane sulfonate (7 mL, 38.7 mmol) was
added, at 2208C and under argon, to a stirred mixture of 10 (35 mg, 91.1 mmol),
˚
23 (75 mg, 128 mmol), and 4A molecular sieves in CH2Cl2 (5 mL). The reaction
was allowed to slowly return to rt and was quenched with triethylamine.
The reaction was diluted with CH2Cl2 (50 mL), filtered through Celite, and
evaporated to dryness. The residue was purified by silica gel chromatography
(hexane/EtOAc 2 : 1) to furnish 25 (55 mg, 67.4 mmol, 74%); TLC (hexane/
EtOAc 1 : 1) Rf ¼ 0.65; [a]D þ 10.6 (c 2 mg/mL, CHCl3); NMR data (CDCl3):
1H, d 7.70–7.05 (m, 13H, dFBz, 2Ph), 5.51 (s, 1H, PhCH), 5.48 (t, 1H, J1,2
10.1 Hz, H-20), 5.42 (d, 1H, J3,4,5 3.0 Hz, H-40), 5.15 (dd, 1H, J2,3 10.4 Hz, J3,4
3.3 Hz, H-30), 4.91 (d, 1H, J1,2 8.0 Hz, H-10), 4.42–4.34 (m, 2H, H-50, H-1),
4.26 (d, 1H, J3,4,5 2.5 Hz, H-4), 4.17–3.94 (m, 4H, H-5, H-6, 2H-60), 3.73
(t, 1H, J1,2,3 9.9 Hz, H-2), 3.54 (dd, 1H, J2,3 10.2 Hz, J3,4 3.0 Hz, H-3), 3.46
(m, 1H, H-6), 2.14, 2.05, 1.93 (s, 9H, 3 ꢁ CH3CO); 13C, d 20.7, 20.9, 21.0
(3 ꢁ CH3CO), 60.0 (C-2), 61.7 (C-6), 67.2 (C-40), 70.0 (C-20), 70.1 (C-50) 71.2
(C-30), 74.9 (C-4), 81.1 (C-3), 86.0 (C-1), 101.0 (PhCH), 102.3 (C-10), 126.6–
137.9 (aromatic C), 170.3, 170.4, 170.5 (3 ꢁ CH3CO); HR MALDI-TOF MS:
m/z: Calc for C38H37F2N3O13S: 813.2015; found 836.1913 [M þ Na]þ.
N-(9-Fluorenylmethyloxycarbonyl)-O-[2-Azido-4,6-O-benzylidene-
2-deoxy-3-O-(3,4,6-tri-O-acetyl-2-O-(2,5-difluorobenzoyl)-b-D-galacto-
pyranosyl)-a-D-galactopyranosyl]-L-threonine Benzylester (26). To a
solution of compound 25 (64 mg, 79 mmol), Ph2SO (45 mg, 221 mmol), and 2,5-
di-tert-butyl-3-methylpyridine (49 mg, 235 mmol) in dry CH2Cl2 (4 mL) was
added trifluoromethanesulfonic anhydride (19 mL, 112 mmol) at 2608C. The
mixture was stirred for 10 min, after which a solution of acceptor 6 (68 mg,
158 mmol) in CH2Cl2 (1.5 mL) was added. The reaction was stirred at 2608C
for 1 hr and then it was slowly warmed to rt and quenched by the addition of
saturated aqueous NaHCO3 (2 mL). The organic phase was washed with
brine, dried (MgSO4), and concentrated. Purification by silica gel chromato-
graphy (hexane/EtOAc 3 : 1) gave 26 (74 mg, 65 mmol, 82%); TLC (hexane/
EtOAc 1 : 1) Rf ¼ 0.67; [a]D þ 21.0 (c 2 mg/mL, CHCl3); NMR data (CDCl3):
1H, d 7.60–6.97 (m, 21H, dFBz, Ph, Bn, Fmoc), 5.71 (d, 1H, J 9.3 Hz, NH),
5.54 (t, 1H, J1,2,3 9.8 Hz, H-20), 5.45 (d, 1H, J4,5 2.5 Hz H-40), 5.18 (dd, 1H, J2,3
10.3 Hz, J3,4 2.9 Hz, H-30), 5.13 (s, 1H, PhCH), 4.93 (d, 1H, J1,2 7.8 Hz H-10),

D. Cato, T. Buskas, and G.-J. Boons
514
4.89 (d, 1H, J1,2 2.9 Hz, H-1), 4.50–4.13 (m, 10 H, Fmoc 3H, Ha, Hb, H-4, H-5,
H-6ax, H-60), 4.02–3.97 (m, 2H, H-3, H-50), 3.73 (dd, 1H, J1,2 2.9 Hz, J2,3 10.3 Hz,
H-2), 3.63 (m, 1H, H-6eq), 2.18, 2.03, 1.93 (s, 9H, 3 ꢁ CH3CO), 1.27 (d, 3H, CH3);
13C, d 19.2 (CH3), 20.8, 20.9, 21.0 (3 ꢁ CH3CO), 59.3 (Ca), 59.8 (C-2), 63.6 (C-6),
69.2-66.2 (C-40, CH2-Fmoc), 70.1 (C-20), 71.3 (C-50, C-30), 75.8 (C-3), 75.6 (C-4),
76.3 (Cb), 99.2 (C-1), 101.0 (CHPh), 102.4 (C-10), 117.9–130.2 (aromatic C),
169.2–171.1 (5 ꢁ C ¼ O); HR MALDI-TOF MS: m/z: Calc for
C58H56F2N4O18: 1134.3558; found 1157.3450 [M þ Na]þ.
ACKNOWLEDGEMENTS
The authors are grateful for financial support from the National Cancer
Institute of the National Institutes of Health (Grant No. RO1 CA88986).
REFERENCES
[1] Strous, G.J.; Dekker, J. Mucin-type glycoproteins. Crit. Rev. Biochem. Mol. Biol.
1992, 27, 57–92.
[2] Baldus, S.E.; Engelmann, K.; Hanisch, F.-G. MUC-1 and the MUCs: A family of
human mucins with impact in cancer biology. Crit. Rev. Clin. Lab. Sci. 2004, 41,
189–231.
[3] Springer, G.F. T and Tn, general carcinoma auto-antigens. Science 1984, 224, 1198.
[4] Kuduk, S.D.; Schwarz, J.B.; Chen, S.-T.; Glunz, P.W.; Sames, D.; Ragupathi, G.;
Livingston, P.O.; Danishefsky, S.J. Synthetic and immunological studies on clus-
tered modes of mucin-related Tn and TF O-linked antigens: The preparation of a
glycopeptide-based vaccine for clinical trials against prostate cancer. J. Am.
Chem. Soc. 1998, 120, 12474–12485.
[5] Dziadek, S.; Kunz, H. Synthesis of tumor-associated glycopeptide antigens for the
development of tumor-selective vaccines. The Chemical Record 2004, 3, 308–321.
[6] Danishefsky, S.J.; Allen, J.R. From the laboratory to the clinic: A retrospective on
fully synthetic carbohydrate-based anticancer vaccines. Angew. Chem. Int. Ed.
2000, 39, 836–863.
[7] Seitz, O. Glycopeptide synthesis and the effects of glycosylation on protein struc-
ture and activity. Chem. Bio. Chem. 2000, 1, 214–246.
[8] Arsequell, G.; Valencia, G. O-Glycosyl a-amino acids as building blocks for glyco-
peptide synthesis. Tetrahedron: Asymmetry 1997, 8, 2839–2876.
[9] Taylor, C.M. Glycopeptides and glycoproteins: Focus on the glycosidic linkage.
Tetrahedron 1998, 54, 11317–11362.
[10] Mizuno, M. Recent trends in glycopeptide synthesis. Trends in Glycoscience and
Glycotechnology 2001, 13, 11–30.
[11] Meldal, M.; St Hilaire, P.M. Synthetic methods of glycopeptide assembly and bio-
logical analysis of glycopeptide products. Current Opinion in Chemical Biology
1997, 1, 552–563.
[12] Yule, J.E.; Wong, T.C.; Gandhi, S.S.; Qiu, D.; Riopel, M.A.; Koganty, R.R. Steric
control of N-acetylgalactosamine in glycosidic bond formation. Tetrahedron Lett.
1995, 36, 6839–6842.

Stereospecific Preparation of Tn and TF Building Blocks
515
[13] Qiu, D.; Gandhi, S.S.; Koganty, R.R. bGal(1-3)GalNAc block donor for the synthesis
of TF and asialyl(2-6)TF as glycopeptide building blocks. Tetrahedron Lett. 1996,
37, 595–598.
[14] Qiu, D.; Koganty, R.R. A novel glycosyl donor for the synthesis of cancer specific
core 5 and sialyl core 5 as glycopeptide building blocks. Tetrahedron Lett. 1997,
38, 961–964.
[15] Liebe, B.; Kunz, H. Solid-phase synthesis of a tumor-associated sialyl-T-N antigen
glycopeptide with a partial sequence of the “tandem repeat” of the MUC-1 mucin.
Angew. Chem. Int. Ed. 1997, 36, 2830–2832.
[16] Paulsen, H.; Bielfeldt, T.; Peters, S.; Meldal, M.; Bock, K. Eine neue strategie zur
festphasensynthese von O-glycopeptiden uber 2-adido-glycopeptide. Liebigs Ann.
1994, 369–379.
[17] Paulsen, H.; Holck, J.-P. Synthese der glycopeptide O-b-D-galactopyranosyl-
(1 ! 3)-O-(2-acetamido-2-desoxy-a-D-galactopyranosyl)-(1 ! 3)-L-serin und-L-threonin.
Carbohydr. Res. 1982, 109, 98–107.
[18] Kunz, H.; Birnbach, S.; Wernig, P. Synthesis of glycopeptides with the Tn and T
antigen structures, and their coupling to bovine serum albumin. Carbohydr. Res.
1990, 202, 207–223.
[19] Schmidt, R.R.; Kinzy, W. Anomeric-oxygen activation for glycoside synthesis—The
trichloroacetimidate method. Adv. Carbohydr. Chem. Biochem. 1994, 50, 84.
[20] Chen, X.-T.; Sames, D.; Danishefsky, S.J. Exploration of modalities in building a-O-
linked systems through glycal assembly: A total synthesis of mucin-related F1a
antigen. J. Am. Chem. Soc. 1998, 120, 7760–7769.
[21] Winans, K.A.; King, D.S.; Rao, V.R.; Bertozzi, C.R. A chemically synthesized
version of the insect antibacterial glycopeptide, Diptericin, disrupts bacterial
membrane integrity. Biochemistry 1999, 38, 11700–11710.
[22] Nakahara, Y.; Iijima, H.; Shibayama, S.; Ogawa, T. Stereoselective total synthesis
of glycopeptides bearing the dimeric and trimeric sialosyl-Tn epitope. Carbohydr.
Res. 1991, 216, 211–225.
[23] Rademann, J.; Schmidt, R.R. Solid-phase synthesis of a glycosylated hexapeptide
of human sialophorin, using the trichloroacetimide method. Carbohydr. Res.
1995, 269, 217–225.
[24] Paulsen, H.; Peters, S.; Bielfeldt, T.; Meldal, M.; Bock, K. Synthesis of the glycosyl
amino acids Na-Fmoc-Ser[Ac4-b-D-Galp-(1 ! 3)-Ac2-a-D-GalN3p]-OPfp and Na-
Fmoc-Thr[Ac4-b-D-Galp-(1 ! 3)-Ac2-a-D-GalN3p]-OPfp and the application in the
solid-phase peptide synthesis of multiply glycosylated mucin peptides with Tn
and T antigenic structures. Carbohydr. Res. 1995, 268, 17–34.
[25] Nakahara, Y.; Nakahara, Y.; Ogawa, T. Solid-phase synthesis of an O-linked glyco-
peptide based on a benzyl-protected glycan approach. Carbohydr. Res. 1996, 292,
71–81.
[26] Svaravsky, S.A.; Barchi, J.J.J. Highly efficient preparation of tumor antigen-
containing glycopeptide building blocks from novel pentenyl glycosides. Carbohydr.
Res. 2003, 338, 1925–1935.
[27] Jiaang, W.-T.; Chang, M.-Y.; Tseng, P.-H.; Chen, S.-T. A concice synthesis of the
O-glycosylated amino acid building block using phenyl selenoglycoside as a
glycosyl donor. Tetrahedron Lett. 2000, 41, 3127–3130.
[28] Paulsen, H.; Rauwald, W.; Weichert, U. Glycosidierung mit thioglycosiden von
oligosacchariden zu segmenten von O-glycoprotein. Liebigs Ann. 1988, 75–86.

D. Cato, T. Buskas, and G.-J. Boons
516
[29] Eberling, J.; Kowalczyk, D.; Schultz, M.; Kunz, H. Chemoselective removal of pro-
tecting groups from O-glycosyl amino acid and peptide(methoxyethoxy)ethyl esters
using lipases from Papain. J. Org. Chem. 1996, 61, 2638–2646.
[30] Elofsson, M.; Kihlberg, J. Synthesis of Tn and sialyl Tn building blocks for solid
phase glycopeptide synthesis. Tetrahedron Lett. 1996, 36, 7499–7502.
[31] Elofsson, M.; Lourdes, A.S.; Kihlberg, J. Preparation of Tn building blocks used in
Fmoc solid-phase synthesis of glycopeptide fragments from HIV gp120. Tetrahe-
dron 1997, 53, 369–390.
[32] George, S.K.; Schwientek, T.; Holm, B.; Reis, C.A.; Clausen, H.; Kihlberg, J.
Chemoenzymatic synthesis of sialylated glycopeptides derived from mucins and
T-cell stimulating peptides. J. Am. Chem. Soc. 2001, 123, 11117– 11125.
[33] Crich, D.; Smith, M. Benzenethiosulfinate (MPBT)/Trifluoromethanesulfonic
anhydride: A convenient system for the generation of glycosyl triflates from thio-
glycosides. Org. Lett. 2000, 2, 4067–4069.
[34] Crich, D.; Smith, M. 1-Benezesulfinyl piperidine/trifluoromethanesulfonic anhy-
dride: A potent combination of shelf-stable reagents for the low-temperature con-
version of thioglycosides to glycosyl triflates and for the formation of diverse
glycosidic linkages. J. Am. Chem. Soc. 2001, 123, 9015–9020.
[35] Codee, J.D.C.; Litjens, R.E.J.N.; den Heeten, R.; Overkleeft, H.S.; van Boom, J.H.;
van der Marel, G.A. Ph2SO/Tf2O: A powerful promoter system in chemo-selective
glycosylations using thioglycosides. J. Org. Lett. 2003, 5, 1519–1522.
[36] Codee, J.D.C.; van den Bos, L.J.; Litjens, R.E.J.N.; Overkleeft, H.S.;
Overkleeft, H.S.; van Boom, J.H.; van der Marel, G.A. Chemoselective glycosyla-
tions using sulfonium triflate activator systems. Tetrahedron 2004, 60, 1057–1064.
[37] Lemieux, R.U.; Ratcliffe, R.M. The azidonitration of tri-O-acetyl-D-galactal. Can. J.
Chem. 1979, 57, 1244–1251.
[38] Luning, B.; Norberg, T.; Tejbrant, J. Synthesis of mono- and di-saccharide amino-
acid derivatives for use in solid phase peptide synthesis. Glycoconjugate J. 1989, 6,
5–19.
[39] Tanaka, H.; Adachi, M.; Takahashi, T. Efficient synthesis of core 2 class glycosyl
amino acids by one-pot glycosylation approach. Tetrahedron Lett. 2004, 45,
1433–1436.
[40] Hanessian, S.; Banoub, J. Chemistry of the glycosidic linkage. An efficient syn-
thesis of 1,2-trans-di-saccharides. Carbohydr. Res. 1977, 53, C13–C16.
[41] Sjolin, P.; Kihlberg, J. Use of fluorobenzoyl protective groups in synthesis of glyco-
peptides: b-elimination of O-linked carbohydrates is suppressed. J. Org. Chem.
2001, 66, 2957–2965.
[42] Chittenden, G.J.F.
A simplified synthesis of a-D-galactopyranose 1,3,4,6-
tetraacetate. Carbohydr. Res. 1988, 183, 140–143.
[43] Schmidt, R.R. New methods for the synthesis of glycosides and oligosaccharides—
Are there alternatives to the Koenings-Knorr method? Angew. Chem. Int. Ed. Engl.
1986, 25, 212–235.