Synthesis of sulfur-containing analogues of αGalNAc (Tn-antigen) and βGal1,3αGalNAc (T-antigen)

Article abstract of DOI:10.1007/s007060200026

A method for the preparation of thio-analogues of the T- and Tn-antigen was developed. Thus, starting from a known N-acetamido-glucoside derivative, the epidithio analogue of the Tn-antigen was accessible in a four-step reaction sequence. The corresponding epidithio analogue and the thioanhydro derivative of the T-antigen were synthesized starting from a disaccharide derivative. For the preparation of the epidithio analogue the sulfur atoms were introduced via thiocyanates in a stepwise fashion, using mesylate as the leaving group at C-6 and triflate as the leaving group at C-4 in the reducing carbohydrate moiety. The synthesis of the thioanhydro analogue was achieved by introducing a thiocyanate group at C-6 into the glucose moiety, followed by subsequent displacement of a mesylate group at C-4 under inversion of configuration utilizing sodium methoxide.

Full text of DOI:10.1007/s007060200026

Monatshefte fuÈr Chemie 133, 531±540 ꢀ2002)
Synthesis of Sulfur-Containing
Analogues of aGalNAc ꢀTn-Antigen)
and bGal1,3aGalNAc ꢀT-Antigen)
Irmgard Wenzl1, Norbert Neuwirth1, Alexander G. Hedenetz1,
1
2
3
È
Christian Fiedler , Hansjorg Streicher , Frank M. Unger ,
and Walther Schmid1;Ã
1

Institut fur Organische Chemie, Universitat Wien, A-1090 Wien, Austria
Fakultat fur Chemie, Universitat Konstanz, D-78457 Konstanz, Germany
2
3






Zentrum fur Ultrastrukturforschung, Universitat fur Bodenkultur, A-1180 Wien, Austria
Summary. A method for the preparation of thio-analogues of the T- and Tn-antigen was developed.
Thus, starting from a known N-acetamido-glucoside derivative, the epidithio analogue of the Tn-
antigen was accessible in a four-step reaction sequence. The corresponding epidithio analogue and
the thioanhydro derivative of the T-antigen were synthesized starting from a disaccharide derivative.
For the preparation of the epidithio analogue the sulfur atoms were introduced via thiocyanates in a
stepwise fashion, using mesylate as the leaving group at C-6 and tri¯ate as the leaving group at C-4
in the reducing carbohydrate moiety. The synthesis of the thioanhydro analogue was achieved by
introducing a thiocyanate group at C-6 into the glucose moiety, followed by subsequent displacement
of a mesylate group at C-4 under inversion of con®guration utilizing sodium methoxide.
Keywords. Carbohydrates; T-Antigen; Tn-Antigen; Thio analogues; Glycosides.
Introduction
The development of synthetic strategies towards sulfur-containing carbohydrates
remains a challenging task in organic synthesis [1]. Thiosugar derivatives are
produced by the introduction of sulfur in the place of oxygen atoms in de®ned
positions of carbohydrate molecules. Due to speci®c interactions with enzymes and
other proteins they are of interest as enzyme inhibitors or pharmaceutical lead
structures [1±3]. Here we report on the synthesis of thio analogues of the Tn-
ꢀꢀGalNAc) and T-antigen ꢀꢁGal1,3ꢀGalNAc) prepared as the allyl glycosides.
The T-antigen, ®rst described by Thomsen [4] and Friedenreich [5], has been
identi®ed to be responsible for erythrocyte agglutination with serum antibodies [6].
Subsequently, this antigen has been discovered on the surface of various carcinoma
cells [7, 8]. Therefore, the synthesis of thio analogues of the T- and Tn-antigen is
of interest for medicinal chemistry as well as immunological studies. These
Ã
Corresponding author. E-mail: walther.schmid@univie.ac.at

532
I. Wenzl et al.
compounds may potentially act as photooxidants [9] and immunomodulators [10].
Furthermore, they should be suitable for studies of various lectin binding sites
[11, 12]. Indeed, thiosugar derivatives of the T-antigen have been shown to
speci®cally bind to legume lectins [13].
Results and Discussions
Recently we have reported on the synthesis of the allyl N-acetyl-4,6-epidithio-
galactosamine analogue 4 [14], which was prepared following essentially a pro-
cedure developed by Hill et al. [15]. Here we ®rst present an improved synthesis in
terms of overall yield of the target compound 4. We started the reaction sequence
with the well known 2-acetamido-3-O-benzoyl-2-deoxy-ꢀ-D-glucopyranoside 1
[16], which upon treatment with 1.1 equivalents of toluenesulfonyl chloride gave
the monotosylate 2 ꢀScheme 1). Esteri®cation of the hydroxyl group on C-4
afforded its corresponding tri¯ate. This was followed by double displacement of
the leaving groups by thiocyanate to yield the galacto derivative 3. Conversion of
the dithiocyanate derivative 3 to the 4,6-epidithio analogue 4 was achieved by
applying the Zemplꢀen saponi®cation procedure, providing 4 in 27% overall yield
from 1.
The synthesis of the epidithio analogue 12 of the T-antigen was performed
starting from the known disaccharide 7 [17, 18], which was easily prepared by
Helferich glycosylation [19] of the saccharide 6 with acetobromogalactose ꢀ5) [20]
ꢀScheme 2). The sulfur atoms of the target 10 were introduced in a stepwise fashion
as thiocyanate groups, a strategy signi®cantly superior over the one-step sub-
stitution procedure described above for the Tn-analogue. Thus, mesylating the
primary hydroxyl group of the disaccharide 7 yielded the monomesylate 8. The
leaving group at the primary position was subjected to a thiocyanate displacement
in dimethoxy ethane ꢀDME) at elevated temperature to yield the derivative 9.
Alcohol 9 was transformed to the corresponding tri¯ate 10, which in a second
displacement step using potassium thiocyanate was converted into the dithiocya-
nato disaccharide derivative 11. For con®rmation of the change in con®guration at
Scheme 1

Synthesis of Analogues of T- and Tn-Antigen
533
Scheme 2
C-4 of derivative 9, various two dimensional NMR techniques were employed
[21, 22]. Thus, all 1H and 13C signals were assigned, and small coupling constants
J3,4 and J4,5 ꢀ4.0 and 1.1 Hz, respectively) indicate the galacto-con®guration of the
dithiocyanato moiety of 11. Conversion into the epidithio analogue 12 was achieved
by treatment of 11 with sodium methoxide in dry methanol. The presence of the
intramolecular disul®de bridge in the T-antigen thio analogue 12 was assigned
unambiguously on the basis of NMR and electrospray mass spectroscopy. To facil-
itate structural assignment, compound 12 was also transformed into its per-
acetylated derivative 13.
The synthesis of the 4,6-thioanhydro derivative 16 was achieved starting
from disaccharide 7 and applying a slightly modi®ed strategy ꢀScheme 3). Thus,
mesylation of both hydroxyl groups of derivative 7 followed by monosubstitu-
tion of the primary mesylate leaving group using potassium thiocyanate gave
compound 15. Treatment of 15 with sodium methoxide in methanol at elevated
temperature followed by reacetylation of the crude thioanhydro analogue for
simpli®cation of structural assignments afforded compound 16 in moderate yields.
In conclusion, various sulfur-containing analogues of the T- and Tn-antigen
were synthesized in a straightforward way. These compounds show selective
binding to plant lectins [13] and are interesting candidates for further bioorganic
studies. Investigations aiming at the exploration of carbohydrate binding sites of
various biomolecules are under way.

534
I. Wenzl et al.
Scheme 3
Experimental
IR spectra were recorded on a Perkin Elmer 1600 FT-IR spectrometer. 1H and 13C NMR spectra were
measured in CDCl3 unless otherwise given using CHCl3 as internal standard on a Bruker DRX
400 NMR spectrometer at 400.13 and 100.61 MHz, or on a Bruker DPX 250 NMR spectrometer at
250.13 and 62.90 MHz. Primed assignments for disaccharide derivatives refer to the galactose
moiety, double primed ones to the allyl aglycon, and all others to the GlcNAc residues, respectively.
Mass spectra were recorded on a Finnigan MAT 900S spectrometer. Electrospray interface mass
spectra were measured on a LC-MS=MS PE Sciex API 365 using MeOH ꢀ10% NH4OAc) as solvent.
Melting points were determined on a Ko¯er apparatus and are uncorrected. Optical rotations were
measured on a Perkin Elmer 361 polarimeter in a 1 dm cell. TLC was performed on precoated Merck
plates ꢀsilica F254). Detection was performed using UV light, I2, and by dipping the plates into a
solution of 2% CeꢀSO4)2 Á 4H2O in 5% H2SO4 with subsequent heating. Flash chromatography was
performed with Merck silica gel 60 ꢀ230±400 mesh). Elemental analyses agreed favourably with the
calculated data. All solvents were of reagent grade. Reagents were obtained from commercial
suppliers. Compound 1 was prepared according to Ref. [16], compound 5 according to Ref. [20], and
compound 6 according to Ref. [23].
Allyl 2-acetamido-3-O-benzoyl-2-deoxy-6-p-toluenesulfonyl-ꢀ-D-glucopyranoside
ꢀ2; C25H29NO9S)
To a stirred solution of 624 mg of 1 ꢀ1.71 mmol) in 7 cm3 dry pyridine and 10 mg of dimethylamino
pyridine ꢀDMAP), a solution of 358 mg p-toluenesulfonic acid ꢀ1.88 mmol) in 2 cm3 dry pyridine was
added at 0ꢀC. The reaction mixture was kept at 0ꢀC for 5 days. The solvent was removed by
coevaporation with toluene, and the crude product was puri®ed by ¯ash chromatography ꢀgradient:
hexanes:ethyl acetate ˆ 1:1 to 1:2) to yield 80% of 2 ꢀ12% of starting material were recovered).
TLC ꢀethyl acetate): Rf ˆ 0.74; 1H NMR ꢀ400 MHz, ꢂ, CDCl3): 7.98±7.32 ꢀm, C6H4CH3, C6H5),
0
5.84 ꢀm, H2 ), 5.78 ꢀd, JNH,2 ˆ 8.86, NHAc), 5.24 ꢀm, H3), 4.80 ꢀd, J1,2 ˆ 3.45, H1), 4.34 ꢀm,
0
0
J5,6a ˆ 1.97, J6a,6b ˆ 11.32, H2, H6a, H6b), 4.13 ꢀm, H1 ), 3.93 ꢀm, H1 ), 3.88 ꢀddd, J4,5 ˆ 9.85,
J5,6b ˆ 4.92, H5), 3.78 ꢀdt, J4,OH ˆ 4.43, H4), 3.11 ꢀbr s, OH), 2.42 ꢀs, C6H4CH3), 1.81 ꢀs, NHCOCH3)
ppm; 13C NMR ꢀ100 MHz, ꢂ, CDCl3): 169.95, 167.92, 144.97, 133.59, 133.09, 132.82, 129.95,
129.82, 129.03, 128.51, 127.97, 118.32, 96.43, 74.81, 70.69, 70.08, 68.66, 68.57, 68.54, 51.36,
23.10, 21.62 ppm.

Synthesis of Analogues of T- and Tn-Antigen
535
Allyl 2-acetamido-3-O-benzoyl-4,6-di-thiocyanato-2,4,6-trideoxy-ꢀ-D-galactopyranoside
ꢀ3; C20H21N3O5S2)
To a solution of 710 mg ꢀ1.37 mmol) of derivative 2 in 18 cm3 dry pyridine, 0.35 cm3 ꢀ2.08 mmol) of
tri¯ic anhydride were added dropwise at 30ꢀC. After 10 min of stirring the reaction ¯ask was
transferred to an ice bath, and stirring was continued for 4 h. The reaction was quenched by the
addition of 14 cm3 saturated aq. NaCl solution and 25 cm3 ethyl acetate. The phases were separated,
and the aqueous phase was extracted twice with 25 cm3 of ethyl acetate. The combined organic
phases were dried over MgSO4, ®ltered, and evaporated to dryness. The crude material obtained was
dissolved in 10 cm3 DMF, and 1.33 g of KSCN ꢀ13.7 mmol) were added. The reaction mixture
was stirred for 16 h at room temperature ꢀr.t.), followed by heating to 110ꢀC for 72 h. The mixture
was cooled to r.t., diluted with 10 cm3 H2O, and extracted with three portions of 20 cm3 ethyl acetate.
The combined organic phases were dried over MgSO4, ®ltered, and the solvent was removed under
reduced pressure. After ¯ash chromatography ꢀhexanes:ethyl acetate ˆ 1:1), 360 mg of dithiocyanate
3 ꢀ59%) were obtained. Additionally, 76 mg of 2 ꢀ12%) were recovered.
20
D
TLC ꢀethyl acetate:hexanes ˆ 4:1): Rf ˆ 0.77; ‰ꢀŠ ˆ ‡33:78ꢀ ꢀc ˆ 1.19, CHCl3); IR: ꢃ ˆ 3306,
2941, 2158, 1724, 1666, 1537 cmÀ1; 1H NMR ꢀ400 MHz, ꢂ, CDCl3): 8.08±7.45 ꢀm, C6H5), 5.89 ꢀm,
0
0
H2 ), 5.77 ꢀd, JNH,2 ˆ 9.35, NHAc); 5.56 ꢀdd, J2,3 ˆ 11.32, J3,4 ˆ 3.94, H3), 5.33 ꢀm, H3 ), 4.97 ꢀd,
0
J1,2 ˆ 3.94, H1), 4.71 ꢀddd, H2), 4.60 ꢀddd, J4,5 ˆ 1.48, J5,6b ˆ 3.94, J5,6a ˆ 8.86, H5), 4.33 ꢀm, H1 ),
4.25 ꢀdd, H4), 4.09 ꢀm, H1 ), 3.28 ꢀddd, J6a,6b ˆ 13.78, H6a, H6b), 1.89 ꢀs, NHCOCH3) ppm; 13C NMR
0
ꢀ100 MHz, ꢂ, CDCl3): 170.01, 166.31, 134.01, 132.32, 130.20, 128.66, 128.21, 119.56, 110.92,
110.57, 96.65, 69.69, 69.30, 68.05, 55.58, 48.06, 35.72, 23.16 ppm; MS ꢀFI, 7 kV, 3 mA, 140ꢀC):
‡
m=z ˆ 448.3 ꢀM H), 395.2.
Allyl 2-acetamido-4,6-epidithio-2,4,6-trideoxy-ꢀ-D-galactopyranoside ꢀ4; C11H17NO4S2)
To a stirred solution of 557 mg dithiocyanate 3 ꢀ1.24 mmol) in 16 cm3 dry MeOH, 9 cm3 of a 0.1 M
solution of MeONa in dry MeOH were added. The reaction mixture was heated for 10 min to re¯ux,
‡
followed by stirring at r.t. for 1 h. The mixture was neutralized by the addition of Dowex 50 W ꢀH ).
The resin was ®ltered off, and the ®ltrate was evaporated to dryness. The crude product obtained was
puri®ed by ¯ash chromatography ꢀethyl acetate:hexanes ˆ 4:1; Rf ˆ 0.32) to yield 200 mg ꢀ55%) 4 as
yellowish crystals.
20
D
M.p.: 195ꢀC ꢀdecomposition); ‰ꢀŠ ˆ ‡202:58ꢀ ꢁc ˆ 1:24; CH3OH†; IR: ꢃ ˆ 3294.3, 1648.2,
1
1549.4 cmÀ1; H NMR ꢀ250 MHz, ꢂ, D2O=CD3OD): 5.94 ꢀm, H2 ), 5.26 ꢀm, H3 ), 4.88 ꢀd, J1,2 ˆ 3.34,
0
0
0
H1), 4.85 ꢀm, H5), 4.45 ꢀdd, J2,3 ˆ 10.96, J3,4 ˆ 4.79, H3), 4.35 ꢀdd, H2), 4.22 ꢀm, H1 ), 4.10 ꢀdd,
0
J4,5 ˆ 2.74, H4), 4.02 ꢀm, H1 ), 3.43 ꢀdd, J6a,5 ˆ 4.57, J6a,6e ˆ 11.88, H6a), 3.24 ꢀdd, J6b,5 ˆ 1.37, H6b),
1.99 ꢀs, NHCOCH3) ppm; 13C NMR ꢀ62.9 MHz, ꢂ, D2O=CD3OD): 135.37, 117.73, 99.08, 75.82,
69.70, 66.80, 66.76, 51.57, 44.45, 22.61 ppm; MS ꢀFI, 7 kV, 3 mV, 120ꢀC): m=z ꢀrel.
‡
intensity) ˆ 291.1 ꢀ100, M -H), 267.2 ꢀ18).
Allyl tetra-O-acetyl-ꢁ-D-galactopyranosyl-+1-3)-2-acetamido-2-deoxy-ꢀ-D-glucopyranoside
ꢀ7; C25H37NO15)
To 80 cm3 of a mixture of toluene and nitromethane ꢀ1:1), 800 mg glucopyranoside 6 ꢀ2.29 mmol)
were added in one portion and dissolved by heating to re¯ux. The solution was transferred under Ar
into a ¯ask containing 580 mg HgꢀCN)2 ꢀ2.30 mmol), 83 mg HgBr2 ꢀ0.23 mmol), and 2.7 g molecular
ꢀ
Ê
sieves ꢀ3 A). After stirring for 1 h at 60 C, 940 mg galactosylbromide 5 ꢀ2.29 mmol) dissolved
in 4 cm3 nitromethane were added dropwise over a period of 2 h. After stirring for 16 h at 60ꢀC,
further additions were made of 580 mg of HgꢀCN)2 ꢀ2.30 mmol), 83 mg HgBr2 ꢀ0.23 mmol), and
940 mg bromide 5 dissolved in 4 cm3 nitromethane, and heating was continued until TLC
ꢀtoluene:acetone ˆ 3:1) showed that all starting material 6 had been consumed ꢀ2 h). The suspension

536
I. Wenzl et al.
obtained was ®ltered over Celite1, and the solvent was removed by evaporation. The residue was
redissolved in CH2Cl2 ꢀ50 cm3) and washed with a 5% solution of NaHCO3. The organic phase was
dried over MgSO4, ®ltered, and the solvent was removed under vacuum. After ¯ash chromatography
ꢀtoluene:acetone ˆ 3:1), 1.37 g of benzylidene protected disaccharide were obtained. The product
obtained was dissolved in 120 cm3 of 80% acetic acid and heated to 90ꢀC for 45 min. The solvent
was removed by coevaporation with toluene ꢀ3 times); the crude product obtained ꢀ1.16 g; 86%) was
pure enough for further conversions. The physical and spectroscopic data were in full agreement
with those reported in Refs. [17] and [18].
Allyl tetra-O-acetyl-ꢁ-D-galactopyranosyl-+1-3)-2-acetamido-2-deoxy-
6-methanesulfonyl-ꢀ-D-glucopyranoside ꢀ8; C26H39NO17S)
To a solution of 460 mg ꢀ0.78 mmol) of compound 7 in 6 cm3 dry pyridine, 0.06 cm3 ꢀ0.78 mmol)
methanesulfonyl chloride were added dropwise at 0ꢀC. After 2 h of stirring the solvent was removed
by coevaporation with toluene, and the crude product obtained was puri®ed by ¯ash chromatography
ꢀethyl acetate:hexanes ˆ 9:1) to yield 461 mg of 8 ꢀ88%).
20
M.p.: 92ꢀC; ‰ꢀŠ ˆ ‡56:2ꢀ ꢁc ˆ 0:87; CH2Cl2†; IR: ꢃ ˆ 3468.1, 2938.2, 1751.9, 1654.7 cmÀ1
;
D
1
00
0
0
0
H NMR ꢀ400 MHz, ꢂ, CDCl3): 5.87 ꢀm, H2 ), 5.53 ꢀd, JNH,2 ˆ 9.85, NHAc), 5.35 ꢀd, J3 ,4 ˆ 2.95, H4 ),
00
0
0
0
0
0
0
0
0
5.27 ꢀm, H3 ), 5.20 ꢀdd, J1 ,2 ˆ 7.88, J2 ,3 ˆ 10.34, H2 ), 4.97 ꢀdd, J3 ,4 ˆ 3.44, H3 ), 4.80 ꢀd, J ˆ 3.94,
0
H1), 4.52 ꢀd, H1 ), 4.50 ꢀdd, J ˆ 1.48), 4.38 ꢀdd, J ˆ 5.42, J ˆ 10.83), 4.28 ꢀddd, J2,3 ˆ 9.85, H2), 4.13
00
00
0
ꢀm, H1 ), 3.97 ꢀm, H1 ), 3.80 ꢀm, 1H), 3.71 ꢀs, 1H, OH), 3.56 ꢀm, H6 ), 3.03 ꢀs, SO2CH3), 2.14, 2.06,
2.04, 1.99, 1.96 ꢀ5s, COCH3) ppm; 13C NMR ꢀ100 MHz, ꢂ, CDCl3): 170.40, 170.10, 169.40, 169.34,
133.02, 118.78, 101.58, 96.62, 82.72, 71.08, 70.66, 69.81, 68.62, 68.58, 68.51, 68.32, 66.79, 61.23,
51.17, 37.56, 23.40, 20.63, 20.59, 20.56, 20.48 ppm; MS ꢀFI, 7 kV, 3 mA, 230ꢀC): m=z ꢀrel.
intensity) ˆ 670.2 ꢀ82), 612.0 ꢀ18), 347.0 ꢀ20), 331.0 ꢀ36), 166.9 ꢀ36), 95.8 ꢀ63), 59.9 ꢀ100).
Allyl tetra-O-acetyl-ꢁ-D-galactopyranosyl-+1-3)-2-acetamido-2,6-dideoxy-
6-thiocyanato-ꢀ-D-glucopyranoside ꢀ9; C26H36N2O14S)
A suspension of 445 mg ꢀ0.66 mmol) of monomesylate 8 and 276 mg ꢀ2.84 mmol) of KSCN in
10 cm3 1,2-dimethoxyethane ꢀDME) was heated at 70ꢀC for 12 h. Additional 280 mg ꢀ2.88 mmol) of
KSCN and 10 cm3 DME were added, and heating at re¯ux was continued for 20 h. The solvent was
removed under vacuum, and the residue was dissolved in CH2Cl2 ꢀ35 cm3) and washed 3 times with
20 cm3 portions of H2O. The organic layer was dried over MgSO4, ®ltered, and the solvent was
evaporated. The crude product obtained was puri®ed by ¯ash chromatography ꢀtoluene:acetoneˆ
3:1) to yield 381 mg ꢀ91%) of 9 as colorless crystals.
20
D
M.p.: 177ꢀC; ‰ꢀŠ ˆ ‡76:6ꢀ ꢁc ˆ 1:23; CH2Cl2†; IR: ꢃ ˆ 3468.0, 2933.9, 2156.4, 1752.6,
1
1658.6 cmÀ1; H NMR ꢀ400 MHz, ꢂ, CDCl3): 5.89 ꢀm, H2 ), 5.53 ꢀd, JNH,2 ˆ 9.53, NHAc), 5.35 ꢀdd,
00
0
0
0
0
0
00
0
0
0
0
0
J4 ,5 ˆ 1.00, J3 ,4 ˆ 3.51, H4 ), 5.28 ꢀm, H3 ), 5.20 ꢀdd, J1 ,2 ˆ 8.03, J2 ,3 ˆ 10.54, H2 ), 4.97 ꢀdd,
Ã
0
0
00
H3 ), 4.79 ꢀd, J1,2 ˆ 4.02, H1), 4.51 ꢀd, H1 ), 4.30 ꢀddd, J2,3 ˆ 10.04, H2), 4.25 ꢀm, H1 ), 4:12 ꢁm; H60 †,
Ã
Ã
00
4:00 ꢁm; H50 ; H1 †, 3.84 ꢀddd, J5,6a ˆ 2.51, H5), 3.73 ꢀs, OH), 3.56 ꢀdd, J3,4 ˆ 8.53, H3 ), 3.51 ꢀdd,
J6a,6b ˆ 14.06, H6a), 3.43 ꢀt, J ˆ 9.03, H4), 3.01 ꢀdd, J5,6b ˆ 8.53, H6b), 2.13, 2.06, 2.05, 2.00, 1.95
ꢀ5s, COCH3) ppm; 13C NMR ꢀ100 MHz, ꢂ, CDCl3): 170.41, 170.09, 169.42, 169.30, 132.87, 119.10,
112.17, 101.61, 96.41, 82.60, 71.21, 71.16, 70.65, 70.45, 68.61, 68.45, 66.82, 61.23, 51.18, 35.95,
23.42, 20.62, 20.59, 20.47 ppm; assignments marked with an asterisk may be interchanged.
Allyl tetra-O-acetyl-ꢁ-D-galactopyranosyl-+1-3)-2-acetamido-2,6-dideoxy-6-thiocyanato-
4-tri¯uoromethanesulfonyl-ꢀ-D-glucopyranoside ꢀ10; C27H35F3N2O16S2)
To a solution of 237 mg ꢀ0.37 mmol) of 9 in 6 cm3 dry pyridine, 0.11 cm3 ꢀ0.65 mmol) tri-
¯uoromethanesulfonic anhydride were added dropwise at À15ꢀC. After 15 min the temperature

Synthesis of Analogues of T- and Tn-Antigen
537
was raised to 0ꢀC, and stirring was continued for 3 h. The reaction mixture was diluted with
30 cm3 CH2Cl2 and washed twice with saturated aqueous NaHCO3. The organic layer was dried
over MgSO4, ®ltered, and evaporated to dryness. The crude product obtained could be puri®ed
by ¯ash chromatography ꢀhexanes:ethyl acetate ˆ 1:2) to yield 178 mg ꢀ63%) of 10 as colorless
crystals.
20
D
M.p.: 139±140ꢀC; ‰ꢀŠ ˆ ‡45:67ꢀ ꢁc ˆ 0:9; CH2Cl2†; IR: ꢃ ˆ 3369.2, 2938.0, 2158.8, 1750.1,
1683.2 cmÀ1; H NMR ꢀ400 MHz, ꢂ, CDCl3): 5.91 ꢀm, H2 ), 5.59 ꢀd, JNH,2 ˆ 10.04, NHAc), 5.35 ꢀm,
1
00
0
00
0
0
0
0
0
0
0
0
H4 , H3 ), 5.10 ꢀdd, J1 ,2 ˆ 8.03, J2 ,3 ˆ 10.54, H2 ), 4.92 ꢀdd, J3 ,4 ˆ 3.51, H3 ), 4.80 ꢀd, J1,2 ˆ 4.02,
0
00
H1), 4.64 ꢀd, H1 ), 4.57 ꢀt, J ˆ 9.53, H5), 4.47 ꢀddd, J2,3 ˆ 10.54, H2), 4.30 ꢀm, H1 ), 4.20 ꢀdd,
J ˆ 6.03, J ˆ 11.55), 4.15 ꢀdt, J5a,6 ˆ 2.51, H5), 4.05 ꢀm, 3 H), 3.85 ꢀt, J ˆ 7.03), 3.40 ꢀdd, J6a,6b
ˆ
14.06, H6a), 3.07 ꢀdd, J5,6b ˆ 9.04, H6b), 2.10, 2.05, 2.03, 1.95 ꢀ4s, COCH3) ppm; 13C NMR
ꢀ100 MHz, ꢂ, CDCl3): 170.12, 169.61, 169.27, 132.24, 120.00, 111.31, 100.41, 96.03, 83.16, 72.82,
70.82, 70.45, 69.29, 68.35, 68.27, 66.52, 60.69, 52.40, 35.12, 23.36, 20.65, 20.52, 20.34 ppm.
Allyl tetra-O-acetyl-ꢁ-D-galactopyranosyl-+1-3)-2-acetamido-4,6-di-thiocyanato-
2,4,6-trideoxy-ꢀ-D-galactopyranoside ꢀ11; C27H35N3O13S2)
A suspension of 73 mg ꢀ0.095 mmol) of 10 and 46 mg ꢀ0.473 mmol) of KSCN in 10 cm3 DME was
heated to re¯ux for 12 h. Then the solvent was removed under vacuum. The crude material obtained
was redissolved in 30 cm3 of CH2Cl2 and washed 3 times with 20 cm3 portions of H2O. The organic
layer was dried over MgSO4, ®ltered, and evaporated. After puri®cation by ¯ash chromatography
ꢀtoluene:acetone ˆ 3:1), 45 mg ꢀ83%) of 11 were obtained as colorless crystals.
20
M.p.: 142ꢀC; ‰ꢀŠ ˆ ‡86:30ꢀ ꢁc ˆ 0:52; CH2Cl2†; IR: ꢃ ˆ 3306.4, 2936.2, 2157.6, 1750.3,
D
1659.9 cmÀ1; H NMR ꢀ600 MHz, ꢂ, CDCl3): 5.91 ꢀm, H2 ), 5.58 ꢀd, JNH,2 ˆ 9.5, NHAc), 5.37 ꢀdd,
1
00
0
0
0
0
0
00
0
0
0
0
0
0
J4 ,5 ˆ 0.8, J3 ,4 ˆ 3.3, H4 ), 5.34 ꢀm, H3 ), 5.32 ꢀdd, J1 ,2 ˆ 8.4, J2 ,3 ˆ 10.4, H2 ), 5.01 ꢀdd, H3 ),
0
4.90 ꢀd, J1,2 ˆ 3.8, H1), 4.74 ꢀd, H1 ), 4.41 ꢀm, J4,5 ˆ 1.1, J5,6a ˆ 3.9, J5,6b ˆ 8.8, H5), 4.38 ꢀddd,
0
0
0
00
J2,3 ˆ 9.7, H2), 4.30 ꢀm, J6a ,6b ˆ 11.5, H6a , H1 ), 4.18 ꢀdd, J4,5 ˆ 1.1, J3,4 ˆ 4.0, H4), 4.14 ꢀdd,
0
0
0
00
0
J5 ,6b ˆ 5.6, H6b ), 4.06 ꢀm, H3, H1 ), 3.89 ꢀm, H5 ), 3.29 ꢀm, J6a,6b ˆ 14.4, H6a, H6b), 2.15
ꢀs, COCH3), 2.02 ꢀs, 2 Â COCH3), 2.00, 1.98 ꢀ2s, COCH3) ppm; 13C NMR ꢀ150 MHz, ꢂ, CDCl3):
170.63, 170.58, 170.31, 169.37, 169.33, 132.55 ꢀC200), 119.72 ꢀC300), 111.29 ꢀ±SCN), 110.94
ꢀ±SCN), 101.91 ꢀC10), 96.70 ꢀC1), 74.96 ꢀC3), 71.80 ꢀC50), 70.65 ꢀC30), 69.29 ꢀC100), 68.90 ꢀC20),
68.03 ꢀC5), 67.17 ꢀC40), 61.86 ꢀC60), 57.75 ꢀC4), 48.55 ꢀC2), 35.87 ꢀC6), 23.37, 20.93, 20.74, 20.67,
20.64 ppm; MS ꢀFI, 7 kV, 3 mA, 190ꢀC): m=z ꢀrel. intensity) ˆ 674.5 ꢀ78), 621.6 ꢀ100), 589.6 ꢀ19).
Allyl ꢁ-D-galactopyranosyl-+1-3)-2-acetamido-4,6-epidithio-2,4,6-trideoxy-
ꢀ-D-galactopyranoside ꢀ12; C17H27NO9S2)
To a solution of 101 mg ꢀ0.15 mmol) of dithiocyanate derivative 11 in 50 cm3 dry MeOH, 0.45 cm3 of
a 1 M methanolic MeONa solution were added. After 4 h of stirring the mixture was neutralized by
addition of solid CO2. The solvent was concentrated under vacuum to a volume of 2 cm3, ®ltered over
silica gel ꢀ2 g), and the silica gel layer was eluted with 10 cm3 of MeOH. The product was obtained
by removing the solvent under vacuum as a colorless glass.
1
Yield: 54 mg ꢀ79%); IR: ꢃ ˆ 3319.5, 2948.3, 2162.7, 1629.0, 1538.1 cmÀ1; H NMR ꢀ400 MHz,
00
00
ꢂ, D2O): 6.03 ꢀm, H2 ), 5.37 ꢀm, H3 ), 5.09 ꢀs, H5), 5.01 ꢀd, J1,2 ˆ 3.01, H1), 4.59 ꢀm, J2,3 ˆ 10.54, H2,
0
0
0
00
00
0
0
0
H3, H4), 4.48 ꢀd, J1 ,2 ˆ 8.0, H1 ), 4.31 ꢀm, H1 ), 4.13 ꢀm, H1 ), 3.95 ꢀd, J3 ,4 ˆ 3.51, H4 ), 3.81 ꢀm,
0
0
0
0
0
0
H6 ), 3.71 ꢀdd, J5,6a ˆ 4.51, J5,6b ˆ 7.53, H5 ), 3.65 ꢀdd, J2 ,3 ˆ 10.03, H3 ), 3.52 ꢀm, H2 , H6a), 3.42
ꢀdd, J5,6b ˆ 1.01, J6a,6b ˆ 12.55, H6b), 2.06 ꢀs, NHCOCH3) ppm; 13C NMR ꢀ100 MHz, ꢂ, D2O):
175.13, 133.91 ꢀC200), 118.58 ꢀC300), 105.43 ꢀC10), 97.80 ꢀC1), 75.54 ꢀC50), 75.29 ꢀC30), 75.11 ꢀC5),
72.95, 70.93 ꢀC20), 69.24 ꢀC100), 69.01 ꢀC40), 63.06 ꢀC4), 61.37 ꢀC60), 48.81 ꢀC2), 43.70 ꢀC6),
22.38 ppm; ES-MS ꢀnegative mode): m=z ˆ 452.0 ꢀcalcd.: 452.5; M-HÀ).

538
I. Wenzl et al.
Allyl tetra-O-acetyl-ꢁ-D-galactopyranosyl-+1-3)-2-acetamido-4,6-epidithio-
2,4,6-trideoxy-ꢀ-D-galactopyranoside ꢀ13; C25H35NO13S2)
To a solution of 50 mg ꢀ0.11 mmol) of 12 in 4 cm3 dry pyridine, 0.4 cm3 acetic anhydride were added,
and the reaction mixture was stirred at r.t. for 16 h. The solvents were removed under vacuum, and
the crude product was puri®ed by ¯ash chromatography ꢀethyl acetate) to yield 65 mg ꢀ96%) of 13.
20
M.p.: 223±224ꢀC; ‰ꢀŠ ˆ ‡87:65ꢀ ꢁc ˆ 0:85; CHCl3†; IR: ꢃ ˆ 3306.8, 2931.0, 1751.1,
D
1654.2 cmÀ1; H NMR ꢀ400 MHz, ꢂ, CDCl3): 5.84 ꢀm, H1 ), 5.51 ꢀd, JNH,2 ˆ 9.03, NHAc), 5.31
1
00
0
00
0
0
0
0
0
0
0
0
ꢀd, H4 ), 5.22 ꢀm, H3 ), 5.08 ꢀdd, J1 ,2 ˆ 8.03, J2 ,3 ˆ 10.54, H2 ), 4.91 ꢀdd, J3 ,4 ˆ 3.51, H3 ), 4.87 ꢀd,
Ã
Ã
Ã
0
J1,2 ˆ 3.52, H1), 4:72 ꢁm; H5 †, 4.58 ꢀm, H2, H1 ), 4.27 ꢀdd, J ˆ 5.02, J ˆ 10.5, H3 ), 4.14 ꢀm, H6a0
,
Ã
Ã
Ã
00
00
H1 ), 4.05 ꢀm, H4 , H6b0 ), 3.96 ꢀm, H1 ), 3.83 ꢀdt, J ˆ 6.53, H50 ), 3.24 ꢀm, H6a, H6b), 2.11, 2.01, 1.98,
1.93, 1.90 ꢀ5s, COCH3) ppm; 13C NMR ꢀ100 MHz, ꢂ, CDCl3): 170.35, 170.24, 170.15, 169.51,
169.36, 133.33, 118.34, 101.45, 98.04, 74.68, 73.34, 70.88, 70.73, 68.74, 68.50, 66.86, 62.88,
61.18, 48.23, 43.51, 23.40, 20.66, 20.64, 20.61, 20.50 ppm; MS ꢀFI, 7 kV, 3 mA, 130ꢀC): m=z
‡
ꢀrel. intensity) ˆ 621.36 ꢀ100; M ), 530.63 ꢀ18); assignments marked with an asterisk may be
interchanged.
Allyl tetra-O-acetyl-ꢁ-D-galactopyranosyl-+1-3)-2-acetamido-2-deoxy-
4,6-dimethanesulfonyl-ꢀ-D-glucopyranoside ꢀ14; C27H41NO19S2)
To a solution of 15 mg ꢀ0.144 mmol) of disaccharide 7 in 3 cm3 dry pyridine, 0.06 cm3 ꢀ0.775 mmol)
methanesulfonyl chloride and 10 mg DMAP were added at 0ꢀC. After stirring for 20 min the reaction
mixture was allowed to warm to r.t. Stirring was continued for 6 h; then the reaction was quenched
by addition of 3 cm3 H2O. The solvents were removed by coevaporation with toluene. The residue
was redissolved in 10 cm3 of H2O and extracted 3 times with 10 cm3 portions of ethyl acetate. The
combined organic phases were dried over MgSO4, ®ltered, and evaporated to dryness. The dime-
sylated derivative 14 was obtained after ¯ash chromatography ꢀethyl acetate:hexanes ˆ 5:1).
20
Yield: 90 mg ꢀ84%); colorless glass; ‰ꢀŠ ˆ ‡45:45ꢀ ꢀc ˆ 0.77, CHCl3); IR: ꢃ ˆ 3386.9, 2940.0,
D
1
1752.6, 1680.0 cmÀ1; H NMR ꢀ400 MHz, ꢂ, CDCl3): 5.88 ꢀm, H2 ), 5.60 ꢀd, JNH,2 ˆ 10.04, NHAc),
00
0
00
0
0
0
0
0
0
0
0
5.36 ꢀd, H4 ), 5.30 ꢀm, H3 ), 5.10 ꢀdd, J1 ,2 ˆ 8.03, J2 ,3 ˆ 10.54, H2 ), 4.93 ꢀdd, J3 ,4 ˆ 3.51, H3 ),
0
4.75 ꢀd, J1,2 ˆ 3.51, H1), 4.64 ꢀd, H1 ), 4.46 ꢀdd, J ˆ 2.51, J ˆ 12.04), 4.37 ꢀdt, J2,3 ˆ 10.54, H2), 4.32
ꢀm, 2 H), 4.20 ꢀdd, J ˆ 6.03, J ˆ 11.55), 4.14 ꢀm, H100), 3.98 ꢀm, 4 H), 3.85 ꢀt, J ˆ 7.02), 3.22, 3.04
ꢀ2s, SO2CH3), 2.10, 2.09, 2.04, 2.02, 1.95 ꢀ5s, COCH3) ppm; 13C NMR ꢀ100 MHz, ꢂ, CDCl3):
170.37, 170.05, 170.01, 169.56, 169.29, 132.65, 119.46, 100.86, 96.36, 76.54, 74.63, 70.53, 70.40,
68.96, 68.13, 67.92, 67.63, 66.88, 60.61, 52.15, 38.69, 37.58, 23.38, 20.62, 20.55, 20.47 ppm; MS
‡
ꢀFI, 7 kV, 3 mA, 175ꢀC): m=z ꢀrel. intensity) ˆ 748.4 ꢀ79; M ), 712.7 ꢀ64), 687.4 ꢀ100), 651.4 ꢀ30).
Allyl tetra-O-acetyl-ꢁ-D-galactopyranosyl-+1-3)-2-acetamido-2,6-dideoxy-
4-methanesulfonyl-6-thiocyanato-ꢀ-D-glucopyranoside ꢀ15; C27H38N2O16S2)
A suspension of 64 mg ꢀ0.086 mmol) of 14 and 82 mg ꢀ0.84 mmol) of KSCN in 10 cm3 DME was
heated to 100ꢀC for 15 h. Subsequently, the solvent was removed under vacuum. The residue
was redissolved in 10 cm3 H2O and extracted 3 times with 15 cm3 portions of ethyl acetate. The
combined organic phases were dried over MgSO4, ®ltered, and evaporated to dryness. The crude
product obtained was puri®ed by ¯ash chromatography ꢀethyl acetate:hexanes ˆ 5:1) to yield 50 mg
ꢀ82%) of 15 as a colorless glass.
20
D
‰ꢀŠ ˆ ‡68:67ꢀ ꢀc ˆ 1.20, CHCl3); IR: ꢃ ˆ 3350.7, 3019.8, 2938.1, 2157.6, 1753.8,
1
1681.8 cmÀ1; H NMR ꢀ400 MHz, ꢂ, CDCl3): 5.90 ꢀm, H2 ), 5.60 ꢀd, JNH,2 ˆ 10.04, NHAc), 5.33
00
0
00
0
0
0
0
0
0
0
0
ꢀm, H4 , H3 ), 5.01 ꢀdd, J1 ,2 ˆ 8.04, J2 ,3 ˆ 10.54, H2 ), 4.93 ꢀdd, J3 ,4 ˆ 3.51, H3 ), 4.79 ꢀd,
0
J1,2 ˆ 4.02, H1), 4.62 ꢀd, H1 ), 4.44 ꢀdt, J2,3 ˆ 10.54, H2), 4.27 ꢀm, 3 H), 4.05 ꢀm, 3 H), 4.00 ꢀdd,
J3,4 ˆ 9.53, H3), 3.89 ꢀt, 1H), 3.47 ꢀdd, J5,6a ˆ 2.51, J6a,6b ˆ 14.06, H6a), 3.22 ꢀs, SO2CH3), 3.10 ꢀdd,

Synthesis of Analogues of T- and Tn-Antigen
539
J5,6b ˆ 8.03, H6b), 2.10, 2.09, 2.05, 2.02, 1.95 ꢀ5s, COCH3) ppm; 13C NMR ꢀ62.9 MHz, ꢂ, CDCl3):
00
170.04, 169.53, 169.29, 132.43 ꢀC2 ), 119.75 ꢀC3 ), 112.03 ꢀSCN), 100.91 ꢀC1 ), 96.20 ꢀC1), 78.62,
00
0
00
74.54, 70.47, 70.39, 69.02 ꢀC1 ), 68.90, 67.87, 66.86, 60.59 ꢀC6 ), 52.17 ꢀC2), 38.56 ꢀSO2CH3), 35.23
0
‡
ꢀC6), 23.38, 20.64, 20.55, 20.47 ppm; MS ꢀFI, 7 kV, 3 mA): m=z ꢀrel. intensity) ˆ 711.3 ꢀ48, M ),
650.3 ꢀ100).
Allyl tetra-O-acetyl-ꢁ-D-galactopyranosyl-+1-3)-2-acetamido-4,6-thioanhydro-
2,4,6-trideoxy-ꢀ-D-galactopyranoside ꢀ16; C25H35NO13S)
To a solution of 27 mg ꢀ0.038 mmol) of 15 in 3 cm3 dry MeOH, 0.08 cm3 of a 1 M methanolic
MeONa solution were added at r.t. The reaction mixture was heated at re¯ux for 2 h and sub-
‡
sequently neutralized by addition of Dowex 50 W ꢀH ). The resin was ®ltered off, and the solvent
was removed under vacuum. The crude product obtained was redissolved in 2 cm3 dry pyridine, and
0.5 cm3 acetic anhydride as well as 5 mg DMAP were added. The reaction mixture was stirred for
20 h at r.t. The solvents were removed by coevaporation with toluene, and the product obtained
was puri®ed by ¯ash chromatography ꢀethyl acetate:hexanes ˆ 6:1).
Yield: 8 mg ꢀ36%); IR: ꢃ ˆ 3308.0, 2938.0, 1750.1, 1654.2, 1544.2 cmÀ1; 1H NMR ꢀ400 MHz, ꢂ,
00
0
0
0
0
0
CDCl3): 5.84 ꢀm, H2 ), 5.52 ꢀd, JNH,2 ˆ 8.95, NHAc), 5.34 ꢀdd, J3 ,4 ˆ 3.53, J4 ,5 ˆ 0.95, H4 ), 5.23
00
0
0
0
0
0
0
ꢀm, H3 ), 5.06 ꢀdd, J1 ,2 ˆ 8.01, J2 ,3 ˆ 10.36, H2 ), 4.99 ꢀd, J1,2 ˆ 2.83, H1), 4.94 ꢀdd, H3 ), 4.72 ꢀddd,
Ã
Ã
0
00
J2,3 ˆ 10.13, H2), 4.59 ꢀd, H1 ), 4.56 ꢀt, J5,6a ˆ 4.71, H5), 4.40 ꢀt, H4 ), 4.14 ꢀm, H60 , H1 ), 3.97 ꢀm,
Ã
00
0
H1 ), 3.91 ꢀdd, J ˆ 6.12, H3 ), 3.85 ꢀdt, H5 ), 3.42 ꢀdd, H6a), 2.59 ꢀd, J6a,6b ˆ 10.12, H6b), 2.19, 2.10,
2.08, 2.03, 1.99 ꢀ5s, COCH3) ppm; 13C NMR ꢀ100 MHz, ꢂ, CDCl3): 170.37, 170.27, 170.18, 169.54,
00
133.54 ꢀC2 ), 117.94 ꢀC3 ), 100.95 ꢀC1 ), 97.21 ꢀC1), 72.96, 72.04, 70.86, 70.84, 68.63 ꢀC1 ), 68.55,
00
0
00
0
66.92, 61.27 ꢀC6 ), 49.54, 43.45, 28.48 ꢀC6), 23.45, 20.68, 20.60, 20.55 ppm; MS ꢀFI, 7 kV, 3 mA,
‡
120±170ꢀC): m=z ꢀrel. intensity) ˆ 590.2 ꢀ100, M ), 488.3 ꢀ10), 142.1 ꢀ20); assignments marked
with an asterisk may be interchanged.
Acknowledgements
This work was supported by the Jubilaumsfonds der Oesterreichischen Nationalbank ꢀProject Nos.
6011 to F. M. Unger and 4663 to W. Schmid ).
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I. Wenzl et al.: Synthesis of Analogues of T- and Tn-Antigen

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Received October 16, 2001. Accepted November 7, 2001