First total synthesis of sialylated and sulfated Lewis(x) mucin Core 2 structures as potential tumor associated antigens

Article abstract of DOI:10.1016/S0040-4039(00)00975-8

Two branched Core 2 structures, (3-O-SO₃Na)Galβ1→4(Fucα1→3)GlcNAcβ1→6(NeuAcα2→3Galβ1→3)Ga1NAcαOMe (1) and its positional isomer NeuAcα2→3Galβ1→4(Fucα1→3)GlcNAcβ1→6(3-OSO₃Na-Galβ1→3)GalNAcαOMe (2), were chemically synthesized for the first time as potential tumor associated antigens. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00975-8

Tetrahedron Letters 41 (2000) 6279±6284
First total synthesis of sialylated and sulfated Lewisx mucin
Core 2 structures as potential tumor associated antigens
Bao-Guo Huang, Rakesh K. Jain, Robert D. Locke, James L. Alderfer,
Walter A. Tabaczynski and Khushi L. Matta*
Molecular & Cellular Biophysics, Roswell Park Cancer Institute, Elm & Carlton Streets, Bu€alo, NY 14263, USA
Received 7 March 2000; accepted 2 June 2000
Abstract
Two branched Core
2
structures, (3-O-SO3Na)Galb1!4(Fuca1!3)GlcNAcb1!6(NeuAca2!
3Galb1!3)GalNAcaOMe (1) and its positional isomer NeuAca2!3Galb1!4(Fuca1!3)GlcNAcb1!
6(3-OSO3Na-Galb1!3)GalNAcaOMe (2), were chemically synthesized for the ®rst time as potential
tumor associated antigens. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: antigens; glycosylation; Core 2 structure; oligosaccharides.
In practice, knowledge of well de®ned carbohydrate antigens has enabled chemists to
undertake their synthesis.1 4 During our studies of enzymes involved in the assembly of tumor
associated antigens, we realized that knowledge acquired on their speci®city could provide valu-
able information regarding the structures of unknown antigens. We hypothesize that a study of
sulfotransferases combined with knowledge of enzyme speci®cities, such as those of a-fucosyl-
transferase and sialyltransferase, can indicate the potential existence of novel sulfated glyco-
conjugates in, e.g. breast, colon and ovarian tumor tissue sources. For example, our laboratory
was the ®rst to examine the speci®city of a(1!3/4)-l-fucosyltransferase from ovarian tumor
tissues and cell lines. We postulated that some sulfated glycoproteins could contain sulfated
Lewisa type structures.5 Feizi et al.6 subsequently reported the existence of such 3-O-sulfated
Lewisx and Lewisa structures as part of ovarian adenocarcinoma glycoproteins. We have
examined sulfotransferase speci®city using Core
2
[Galb1!4GlcNAcb1!6(Galb1!3)
GalNAca!OR] acceptors. It is striking that in colon tissue and cell lines sulfotransferase acts
upon the C-3 position of Gal in the Galb1!4GlcNAc arm of this Core 2 acceptor, whereas in
breast tumor tissue C-3 of Gal in the Galb1!3GalNAca1! sequence is sulfated.7,8 We have
now synthesized the branched Core 2 oligosaccharide structures, (3-O-SO3Na)Galb1!
4(Fuca1!3)GlcNAcb1!6 (NeuAca2!3Galb1!3)GalNAcaOMe (1) and its positional isomer
* Corresponding author. Tel: (716) 845-2397; fax: (716) 845-3458; e-mail: klmatta@yahoo.com
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00975-8

6280
NeuAca2!3Galb1!4(Fuca1!3)GlcNAcb1!6(3-OSO3Na-Galb1!3)GalNAcaOMe (2), as
novel epitopes for colon and breast carcinoma, respectively. During our studies, Kim and co-
workers reported that a sulfated Lewisx determinant is a major structural moiety of O-linked
glycans obtained from human colon carcinoma mucin.9 These ®ndings provide support for our
above-mentioned hypothesis.
Compounds 1 and 2 were synthesized from three key building blocks (3±5, Fig. 1).10,11 Our
synthetic strategy for 1 (Scheme 1) was based on the employment of a novel glycosyl donor (6),
having the 3,4-isopropylidene galactopyranosyl moiety, which was easily obtained from 3 in two
steps. The core oligosaccharide 8 was chosen as the key glycosyl acceptor for the synthesis of 1.
Compound 8 has the C-2, C-3 and C-4 hydroxy groups of galactose in Galb1,3GalNAc free for
regioselective sialylation at the C-3 position and 3,4-isopropylidene protection on Gal in the
Lewisx moiety, which upon acetylation followed by deisopropylidenation provided the site for
selective sulfation at C-3. On the other hand, compound 15 was employed as a key acceptor for
the construction of the target molecule 2 (Scheme 2). Compound 15 has C-2, C-3 and C-4
hydroxy groups on Gal of Lewisx free for selective sialylation at the C-3 position and 3,4-
isopropylidene protection on Gal of Galb1,3GalNAc for further selective sulfation under a
similar sequence of reactions.
Selective deacetylation of Lewisx donor 3 followed by isopropylidene formation produced the
key glycosyl donor 6. Glycosylation of 6 with disaccharide acceptor 4 in the presence of NIS±
tri¯ic acid12 led to the formation of exclusively 1,6-b-linked pentasaccharide 7 in 50% yield. The
1H NMR spectrum of 7 displayed characteristic signals at ꢀ 1.15 and 1.22 (2s, 18H, 2ÂPiv) and ꢀ
1.86, 1.93, 1.99 and 2.10 (4s, 12H, 4ÂCH3CO). Treatment of 7 with hydrazine hydrate in ethanol
(v/v, 1:9) at 80ꢀC for 2 h resulted in the selective removal of the phthalimido group and acetates
to produce the free amine, which was converted to compound 8 in 75% yield. Only two N-acetyl
1
groups are seen in the H NMR spectrum (ꢀ 1.72, 1.98), which is consistent with the structure.
Under these conditions for conversion of 7 to 8, the 6-O-pivaloyl group remained intact (ꢀ 1.16, s,
9H and ꢀ 1.19, s, 9H). Reaction of 8 with sialic acid donor 5 promoted by NIS±tri¯ic acid in
propionitrile at ^75ꢀC gave the sialylated oligosaccharide 9 in 48% yield. The NeuAca2-3Galb
1
linkage was evidenced by 13C NMR (ꢀ 98.7, C-2 for NeuAc and ꢀ 74.6, C-3 for Gal) and H
NMR (ꢀ 1.74±2.12, 7s, 21H, 7ÂCOCH3). The low reaction temperature was very important in
order to avoid the loss of fucosyl residue during glycosylation.13 Acetylation of 9 a€orded 10,
which was hydrolyzed with 60% acetic acid to produce 3,4-diol 11. Selective sulfation of 11 with
SO3±pyridine complex in pyridine gave 3-O-sulfo compound 12. The sulfation at C-3 of Gal was
con®rmed by 13C NMR (ꢀ 79.2, C-3 for Gal, with ꢀ 68.1 for C-2 and 65.7 for C-4). Removal of
the benzyl groups in 12 by hydrogenation followed by deacetylation and hydrolysis of methyl
ester produced the target molecule 1 (48% from 12).
For the synthesis of compound 2, disaccharide 13 was chosen as a key glycosyl acceptor.
Compound 13 was prepared in 39% overall yield from compound 4. Silylation of 4 with
diphenyl-t-butylsilyl chloride in DMF followed by selective deacetylation, 3,4-isopropylidene
formation and desilylation gave 13. Condensation of the resulting glycosyl acceptor 13 with
Lewisx donor 3 in the presence of NIS±tri¯ic acid a€orded the pentasaccharide 14. Selective
deacetylation of 14 with sodium methoxide in methylene chloride and methanol (v/v, 1:1) gave
1
compound 15. This was further sialylated to produce compound 16. The H NMR spectrum of
compound 16 gave signals at ꢀ 1.84±2.12 (6s, 18H, 6ÂCOCH3) and ꢀ 1.22, 1.23 (2s, 18H, 2ÂPiv).
The site of sialylation was evidenced by 13C NMR (ꢀ 76.5, C-3, Gal). Acetylation of 16 with acetic
anhydride a€orded compound 17. Removal of 3,4-isopropylidene with 60% acetic acid gave the

6281
Figure 1. The building blocks 3, 4 and 5 used in the synthesis of compounds 1 and 2
diol 18. Selective sulfation of 18 with SO3±pyridine complex gave compound 19. Deprotection of
19 with LiI in pyridine14 followed by hydrazine hydrate produced a free amine intermediate,
which was treated with acetic anhydride in methanol and triethylamine, deacetylation and
sodium resin sequentially to furnish the ®nal product 2 (40% from 19). The structures of the
newly synthesized molecules 1, 2 and some intermediates were characterized by NMR and mass
spectra.15
1
Application of 1D and 2D NMR techniques allowed unambiguous assignment of all H and
1
1
13C resonances of compounds 1 and 2. H COSY spectra were used to sort H resonances into
spin systems. These were assigned to particular residues using structural reporter groups. 13C
assignments were made using a combination of HMQC and HMBC spectra. Glycosidic linkages
and substitution sites were con®rmed using the HMBC data. Details of the NMR assignments
will be published elsewhere.

6282
Scheme 1. Reagents and conditions: (a) NaOMe, MeOH:CH2Cl2 (1:1, v/v), 0ꢀC, 5 h, 80%; (b) CSA in DMP, rt, 2 h,
82%; (c) 1.05 equiv. 6, NIS±TfOH, CH2Cl2, ^75ꢀC, 2 h, 50%; (d) hydrazine hydrate:EtOH (1:9, v/v), 80ꢀC, 2 h;
MeOH:Et3N:Ac2O (4:2:1, v/v/v), rt, 2 h, 75%; (e) 2.5 equiv. 5, NIS±TfOH, CH3CH2CN, ^75ꢀC, 2 h, 48%; (f) Ac2O,
pyr., rt, 12 h; (g) 60% HOAc, 55ꢀC, 3 h, 54% for steps f and g; (h) 10 equiv. SO3±pyridine in pyr., 0ꢀC, 12 h, 75%; (i)
10% Pd/C, H2, MeOH, 12 h; NaOMe, MeOH, rt, 48 h; H2O, 5 h, Na+ resin, 48% from 12

6283
Scheme 2. Reagents and conditions: (a) Ph2SitBuCl, imidazole, DMF, 0ꢀC, 2 h; (b) NaOMe, MeOH:CH2Cl2 (1:1, v/v),
0ꢀC, 2 h; (c) CSA in DMP, rt, 2 h; (d) TBAF in THF, rt, 30 min, 39% for four steps; (e) 1.05 equiv. 3, NIS±TfOH,
CH2Cl2, ^75ꢀC, 2 h, 32%; (f) NaOMe, MeOH:CH2Cl2 (1:1, v/v), 0ꢀC, 3 h, 70%; (g) 2.5 equiv. 5, NIS±TfOH,
CH3CH2CN, ^75ꢀC, 3 h; (h) Ac2O, pyr., rt, 16 h, 32% for two steps; (i) 60% HOAc, 65ꢀC, 1.5 h, 84%; (j) SO3±pyridine
complex, pyr., 0ꢀC, 16 h, 90%; (k) LiI, pyr., 120ꢀC, 4 h; hydrazine hydrate:MeOH (5:1, v/v), 80ꢀC, 3 h, MeOH
Et3N:Ac2O (4:2:1, v/v/v), rt, 2 h, NaOMe, MeOH, rt, 48 h; Na+ resin, 40% from 19

6284
In summary, novel procedures for the total synthesis of the sulfated hexasaccharides 1 and 2
have been developed. These procedures will also provide a practical approach toward the
synthesis of a series of branched Core 2 type linked oligosaccharides, which contain allyl or other
functional groups at the anomeric position, suitable for attachment to protein for immunological
studies.
Acknowledgements
These investigations were supported by Grant Nos. CA-63218 and CA-35329 (K.L.M.), and in
part by CA-16056 (RPCI-NMR facility) awarded by the National Cancer Institute.
References
1. Stults, C. L. M.; Sweeley, C. C.; Macher, B. A. Methods Enzymol. 1989, 179(14), 167.
2. Livingston, P. O.; Zhang, S.; Lloyd, K. O. Cancer Immunol. Immunother. 1997, 45, 1.
3. Toyokumi, T.; Singhal, A. K. Chem. Soc. Rev. 1995, 231.
4. Seeberger, P. H.; Bilodeau, M. T.; Danishefsky, S. J. Aldrichim. Acta, 1997, 30, 75.
5. Chandrasekaran, E. V.; Jain, R. K.; Matta, K. L. J. Biol. Chem. 1992, 267, 23806.
6. Yuen, C.; Lawson, A. M.; Chai, W.; Larking, M.; Stoll, M. S.; Stuart, A. C.; Sullivan, F. X.; Ahern, T. J.; Feizi, T.
Biochemistry 1992, 31, 9126.
7. Chandrasekaran, E. J.; Jain, R. K.; Matta, K. L. Glycobiology 1997, 7, 753.
8. Chandrasekaran, E. V.; Jain, R. K.; Rhodes, J. M.; Chawda, R.; Piskorz, C.; Matta, K. L. Glycoconjugate J. 1999,
16, 523±536.
9. Capon, C.; Wieruszeski, J. M.; Lemoine, J.; Byrd, J. C.; Le‚er, H.; Kim, Y. S. J. Biol. Chem. 1997, 272, 31957.
10. Huang, B.-G.; Locke, R. D.; Jain, R. K.; Matta, K. L. Bioorg. Med. Chem. Lett. 1997, 7(9), 1157.
11. Koenig, A.; Jain, R.; Vig, R.; Norgard-Sumnicht, K. E.; Matta, K. L.; Varki, A. Glycobiology 1997, 7, 79.
12. Konradason, P.; Mootoo, D. R.; McDevitt, R. E.; Fraser-Reis, B. J. Chem. Soc., Chem. Commun. 1990, 270.
13. Jain, R. K.; Vig, R.; Rampal, R.; Chandrasekaran, E. V.; Matta, K. L. J. Am. Chem. Soc. 1994, 116, 12123.
14. Nicolaou, K. C.; Hummel, C. W.; Iwabuchi, Y. J. Am. Chem. Soc. 1992, 114, 3126.
15. NMR spectra were recorded at 30ꢀC with Bruker AM-400 and AMX-600 spectrometers (1H frequencies are 400
and 600 MHz respectively). Selected data. For 1: [ꢁ]D +48 (c 0.70; H2O); 1H NMR (D2O): ꢀ 4.80 (d, J=3.6 Hz, H-
1, GalNAca-OMe), 4.55 (d, 1H, J=7.8 Hz, H-10, Galb1-3GalNAc), 4.59(d, 1H, J=8.3 Hz, H-1000, GlcNAc), 5.14
(d, 1H, J=4.0 Hz, H-10000, Fuc), 4.60 (d, 1H, J=7.8 Hz, H-100000, Galb1-4GlcNAc), 3.38 (s, 3H, OMe), 2.78 (dd,
1H, J=4.7, 12.4 Hz, H-300e), 2.03, 2.04, 2.06 (3s, 9H, 3ÂNAc), 1.81 (dd, 1H, J=11.8, 12.4 Hz, H-300a), 1.20 (d, 3H,
J=6.7 Hz, H-60000); 13C NMR (D2O): ꢀ 173.6, 173.1, 174.0 (3ÂNHCOCH3), 172.8 (C-100, NeuAc), 97.1 (C-1, Gala-
OMe), 103.4(C-10, Galb1-3GalNAc), 98.7 (C-200, NeuAca), 100.4 (C-1000, GlcNAc), 97.6 (C-10000, Fuc), 100.4 (C-
100000, Galb1-4GlcNAc), 76.2 (C-3), 69.2 (C-6), 74.6 (C-30, site of sialylation), 73.8 (C-3000), 72.4 (C-4000), 79.2 (C-300000
,
site of sulfation on Galb1-4GlcNAc), 53.9 (OMe), 54.7 (C-2000), 21.0, 21.1, 21.3 (3ÂNHCOCH3), 14.2(C-60000, Fuc);
MS m/z: 1300.4 (M^Na)^. For 2: [ꢁ]D +75 (c 0.50; H2O); 1H NMR (D2O): ꢀ 4.79 (d, 1H, H-1, J=3.7 Hz,
GalNAca-OMe), 4.58 (d, 1H, H-10, J=7.9 Hz, Galb1-3GalNAc), 4.59 (d, 1H, H-100, J=8.2 Hz, GlcNAcb1-
6GalNAc), 5.13 (d, 1H, J=4.0 Hz, H-1000, Fuc), 4.55 (d, 1H, J=7.9 Hz, H-10000, Galb1-4GlcNAc), 3.38 (s, 3H,
OMe), 2.79 (dd, 1H, J=4.7, 12.4 Hz, H-300000e), 2.03, 2.04, 2.06 (3s, 9H, 3ÂNHCOCH3), 1.82 (dd, 1H, J=12.1,
12.4 Hz, H-300000a), 1.20 (d, 3H, H-6000, Fuc); 13C NMR (D2O): ꢀ 173.1, 173.6, 174.0 (3ÂNHCOCH3), 172.8 (C-100000
,
NeuAc), 97.2 (C-1, GalNAca-OMe), 103.3 (C-10, Galb1-3GalNAc), 100.4 (C-100, GlcNAcb1-6GalNAc), 97.6 (C-
1000, Fuc), 100.6 (C-10000, Galb1-4GlcNAc), 98.7 (C-200000, NeuAc), 76.5 (C-3), 69.2 (C-6), 79.2 (C-30, site of
sulfation), 73.8 (C-300), 72.4 (C-400), 74.7 (C-30000, site of sialylation), 53.9 (OMe), 21.0, 21.0, 21.3 (3ÂNHCOCH3),
14.3 (C-6000); MS m/z: 1300.0 (M^Na)^.