Synthesis of a C-glycoside analogue of sTn: An HIV- and tumor-associated antigen

Article abstract of DOI:10.1002/anie.200351099

The nonhydrolyzable glycosidic linkage to sialic acid in the C-glycoside analogue of sTn, 1, is the key feature of the redesigned glycoconjugate. This potential antitumor and anti-HIV agent was prepared in one enzymatic and seventeen chemical steps starting from diisopropylidene galactopyranose.

Full text of DOI:10.1002/anie.200351099

Angewandte
Chemie
C-Glycosides
been recently prepared[8] and shown to stimulate an immune
response.[9]
Kishi and co-workers have demonstrated that C-glyco-
Synthesis of a C-Glycoside Analogue of sTn: An
HIV- and Tumor-Associated Antigen**
sides and O-glycosides have similar conformational charac-
teristics both in the free and bound states.[10] There are,
nevertheless, differences in the population distribution of
conformers adopted by C-glycosides and their natural coun-
terparts.[11] However, the small energy differences among the
various conformers allow them to interchange without any
major energy conflicts, thus rendering the C-glycosides ideally
suited as O-glycoside isosters for therapeutic evaluation. Our
rationale for undertaking the synthesis of the C-glycoside
Balagurunathan Kuberan, Sulthan A. Sikkander,
Hiroshi Tomiyama, and Robert J. Linhardt*
N-Acetylneuraminic acid occupies the nonreducing end of
many naturally occurring glycoconjugates on the cell surface,
including glycoproteins and glycolipids. The strategic location
of glycoconjugates on the cell surface and the
enormous structural information that they
carry account for their major role in biological
recognition in the control of both normal and
pathological processes.[1] While the participa-
tion of sialic acid in these biological events has
been established unequivocally in a large
number of studies,[1] the linkage of sialic acid
to glycoconjugates is one of the most labile
glycosidic linkages, which renders these com-
pounds less amenable to biological and bio-
chemical studies.[2] Terminal sialic acid residues
Figure 1. Tumor antigen sTn O-glycoside 1a and C-glycoside analogue 1b.
are easily cleaved in vitro under very mild
acidic conditions and in vivo by neuramini-
dases.[3] The biological importance of sialic acid coupled with
its lability towards hydrolysis prompted us to redesign the
molecular architecture of glycoconjugates to afford a non-
hydrolyzable glycosidic linkage to sialic acid. Such a stable
linkage should contribute to our understanding of biological
recognition and serve to enhance or suppress biological
events at the molecular level. The replacement of the
interglycosidic oxygen atom with a hydroxymethylene or
methylene group affords a new class of hydrolytically stable
C-glycoside analogues of glycoconjugates.[4] A method for the
preparation of C-glycosides of ulosonic acids using SmI2 was
pioneered in our laboratory.[5]
The sialyl-Tn epitope [a-d-Neu5Ac-(2!6)a-d-GalNAc-
(1!O)-Ser/Thr] (sTn, 1a, Figure 1) is found on the HIV
envelope glycoprotein gp120[6] and in tumor-associated anti-
gens present in the glycoproteins on the surface of cancer
cells, including those associated with carcinomas of the breast,
prostate, pancreas, colon, ovary, lung, and stomach.[7] Syn-
thetic vaccines based on the sTn epitope (O-glycoside) have
analogue of sTn (1b, Figure 1) was to develop and test a new
vaccine candidate against the elusive HIV or to prepare a
better candicate for a cancer vaccine with a longer half-life in
vivo.[9b]
Fully protected sTn C-glycoside analogue 2 was prepared
by C-glycosylation of the neuraminic acid sulfone donor 13
with an aldehyde acceptor 12 (Scheme 1). The donor 13 was
prepared from neuraminic acid in four steps as previously
reported.[12] The critical intermediate, aldehyde acceptor 12,
was prepared in 14 steps. Starting from the commercially
available diisopropylidene galactose derivative 3, the corre-
sponding 6-iodo derivative 3a was prepared and treated with
KCN. The modest yield (30%) of this reaction might be due
to unfavorable electronic and steric effects arising from the
ring oxygen atom and the axially oriented oxygen at C-4,
respectively. Reduction of the 6-cyano derivative 3b with
DIBAL-H afforded aldehyde 4 in moderate overall yield.[13]
Quantitative reduction of aldehyde 4 with NaBH4 in MeOH
afforded the corresponding alcohol 5. The acetal protecting
groups were removed by treatment with Amberlite IR-120
(H+) resin in water at 808C for 3 h to provide the 6-deoxy-d-
galacto-heptopyranose 6 in quantitative yield. The one-
carbon-extended galactal 7 was obtained in good yield from
6 by a one-pot, three-step procedure consisting of: a) per-
acetylation with acetic anhydride and catalytic HBr/HOAc;
b) conversion of the anomeric acetate to the corresponding
bromide with excess HBr/HOAc; and c) reductive elimina-
tion of the 1-bromo and 2-acetoxy groups with Zn/Cu.[14]
Azidonitration[15] of 7 with excess ceric ammonium nitrate
(CAN) and sodium azide in dry acetonitrile afforded
primarily the 2-azido-1-nitrate addition product, which has
the desired galacto configuration as unambiguously con-
firmed by 1H NMR spectroscopy. Indicative are a large
[*] Prof. Dr. R. J. Linhardt, B. Kuberan, S. A. Sikkander, H. Tomiyama
Division of Medicinal and Natural Products Chemistry and
Department of Chemical and Biochemical Engineering
University of Iowa
Iowa City, IA 52242 (USA)
Fax: (+1)319-335-6634
E-mail: robert-linhardt@uiowa.edu
[**] The authors thank Prof. D. H. G. Crout of the University of Warwick
for generously supplying the esterase used in this study and Dr.
Iontcho Vlahov of Endocyte Inc. for his helpful conversations
regarding this manuscript. This research was supported in part by
Kotobuki Pharmaceutical Company, Nagano, Japan.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2003, 42, 2073 – 2075
DOI: 10.1002/anie.200351099
ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2073

Communications
in quantitative yield. The site of the enzymatic deacetylation
was unequivocally established as C-7 based on the upfield
shift of H-7a and H-7b from d = 4.19–4.06 in 10 to 3.62–
3.74 ppm in 11 in the 1H NMR spectra. Swern oxidation[19] of
11 afforded aldehyde acceptor 12. Glycosylation of the
neuraminic acid sulfone donor 13 with aldehyde acceptor 12
in the presence of freshly prepared SmI2[20] afforded the fully
protected sTn a-C-glycoside 2a.
1
The H NMR spectrum of 2a indicated an R/S mixture
(1:1) at the newly formed stereogenic bridge carbon C-7.
Attempts were first made to remove the hydroxyl group at
C-7 by standard reductive radical deoxygenation protocols.
Compound 2a was treated with (imidazole)2CS or
ClCS(PhF5) then reduced with Ph3SnH and azobisisobutyr-
onitrile (AIBN). These reactions failed to yield the expected
deoxygenated product probably due to unfavorable stereo-
electronic factors. The C-7 hydroxyl group in 2a was next
converted to a triflate and subjected to nucleophilic reduction
with hydride, but this resulted in decomposition of the triflate,
presumably due to the unwanted rearrangement, followed by
elimination—the result of the electron-rich functional groups
close to the reactive center. Finally, chemoselective resolution
to compound 2a was undertaken by oxidation with DMSO/
Ac2O to keto-bridged compound 2b, followed by stereo-
selective reduction with Zn(BH4)2 to regenerate the bridge
hydroxyl function, gave 2c in > 90% de (Scheme 1). The
configuration at the bridge carbon was established by
ROESY. Chemical exchange of the proton of the hydroxyl
group of the bridge carbon and the oxygen of the C-1 carboxyl
group of Neu5Ac resulted in strong NOEs for H-3’e/H-7, H-7/
H-6(proR), OH-7/H-6(proS), and H-6(proR)/H-5, consistent
with the R configuration at C-7 in 2c (Figure 2).
Scheme 1. Synthesis of sTn C-glycoside derivatives. a) Ph3P, I2, Im;
b) KCN; c) DIBAL-H; d) NaBH4; e) H+ resin; f) Ac2O, HBr/HOAc;
g) excess HBr/HOAc; Zn/Cu; i) CAN, NaN3; j) LiBr; k) Z-Ser-OBn,
AgClO4, 4Š molecular sieves; l) AcSH/Py; m) R. toruloides, sodium
phosphate/sodium citrate buffer, pH 5; n) DMSO, ClCOCOCl, TEA;
o) SmI2; p) DMSO/Ac2O; q) Zn(BH4)2. R1 =Bn. Im=imidazole,
DIBAL-H=diisobutylalumininum hydride, Py=pyridine, TEA=triethyl-
amine, DMSO=dimethyl sulfoxide.
coupling constant between the two vicinal protons H-2 and H-
3 (J2,3 = 11 Hz) and the chemical shift of H-2 (d = 3.97 ppm).
Treatment of the crude product with LiBr[15] in dry acetoni-
trile in the darkafforded 8. Glycosylation of 8 with the Na-
benzyloxycarbonyl-protected OBn ester of l-serine, prepared
as previously reported,[16] in the presence of silver perchlorate
afforded the glycopeptide 9. Separation of glycopeptide 9
from unreacted starting material, Z-Ser-OBn, was cumber-
some since both compounds migrated with similar Rf values
on silica. However, conversion of 9 to 10 by treatment with
thioacetic acid/pyridine[17] resulted in a large change in the
glycopeptide polarity and permitted removal of the unreacted
amino acid acceptor from the desired glycopeptide product
10.
Our efforts next focused on the selective deprotection of
the C-7 primary acetyl group in 10. Since chemical techniques
are not sufficiently selective, we envisaged a selective
enzymatic deacetylation using an esterase. The esterase
from Rhodosporidium toruloides has been reported to
selectively deprotect primary acetates in the presence of
secondary acetates.[18] Treatment of 10 with this esterase at
pH 5 in a sodium phosphate/sodium citrate buffer afforded 11
Figure 2. Stereochemical assignment of sTn C-glycoside 2c at the
bridge carbon C-7.
In summary, the synthesis of the C-glycoside analogue 2c
was achieved in 1 enzymatic and 17 chemical steps starting
from diisopropylidene galactopyranose and sialic acid. Addi-
tional studies are underway aimed at conjugating the sTn C-
glycoside hapten to KLH carrier protein to obtain a vaccine
for biological evaluation.
Received: February 4, 2003 [Z51099]
Keywords: anti-HIV agents · antitumor agents · C-glycosides ·
.
glycopeptides · sialic acid
2074
ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2003, 42, 2073 – 2075

Angewandte
Chemie
[10] a) A. Wei, K. M. Boy, Y. Kishi, J. Am. Chem. Soc. 1995, 117,
[1] a) R. Schauer, Trends Glycosci. Glycotechnol. 1997, 9, 315 – 330;
b) C. Traving, R. Schauer, Cell. Mol. Life Sci. 1998, 54, 1330 –
1349; c) A. Varki, Glycobiology 1992, 2, 25 – 40.
[2] a) N. Sharon, H. Lis, Science 1989, 246, 227 – 234; b) H. Lis,
Lectins, Chapman and Hall, London, 1989.
[3] G. M. Air, W. Laver, Proteins Struct. Funct. Genet. 1989, 6, 341 –
356, and references therein.
[4] a) Y. Du, R. J. Linhardt, I. R. Vlahov, Tetrahedron 1998, 54,
9913 – 9959; b) M. Postema, C-Glycoside Synthesis, CRC, Boca
Raton, 1995.
[5] a) I. R. Vlahov, P. I. Vlahova, R. J. Linhardt, J. Am. Chem. Soc.
1997, 119, 1480 – 1481; b) H. G. Bazin, Y. Du, T. Polat, R. J.
Linhardt, J. Org. Chem. 1999, 64, 7254 – 7259; c) Y. Du, T. Polat,
R. J. Linhardt, Tetrahedron Lett. 1998, 39, 5007 – 5010.
[6] a) J. E. S. Hansen, H. Clausen, C. Nielsen, L. S. Teglbjaerg, L. H.
Hansen, C. M. Nielsen, E. Dabelsteen, L. Mathiesen, S. I.
Hakomori, J. O. Nielsen, J. Virol. 1990, 64, 2833 – 2840; b) J. E.
Hansen, B. Jansson, G. J. Gram, H. Clausen, J. O. Nielsen, S.
Olofsson, Arch. Virol. 1996, 141, 291 – 300; c) K. Miyajima, T.
Nekado, K. Ikeda, K. Achiwa, Chem. Pharm. Bull. 1997, 45,
1544 – 1546; d) K. Miyajima, T. Nekado, K. Ikeda, K. Achiwa,
Chem. Pharm. Bull. 1998, 46, 1676 – 1682; e) M. P. Carlos, D. E.
Anderson, M. B. Gardner, J. V. Torres, AIDS Res. Hum. Retro-
viruses 2000, 16, 153 – 161.
[7] a) S. H. Itzkowitz, M. Yuan, C. K. Montgomery, T. Kjeldsenen,
H. K. Takahashi, W. L. Bigbee, Y. S. Kim, Cancer Res. 1989, 49,
197 – 204; b) S. I. Hakomori, Cancer Res. 1989, 49, 257 – 331.
[8] a) M. Elofsson, J. Kihlberg, Tetrahedron Lett. 1995, 36, 7499 –
7502; b) J. B. Schwarz, S. D. Kuduk, X. Chen, D. Sames, P. W.
Glunz, S. J. Danishefsky, J. Am. Chem. Soc. 1999, 121, 2662 –
2673; c) B. Liebe, H. Kunz, Angew. Chem. 1997, 109, 629 – 631;
Angew. Chem. Int. Ed. Engl. 1997, 36, 618 – 621; d) H. Iijima, T.
Ogawa, Carbohydr. Res. 1988, 172, 183 – 193.
9432 – 9436; b) R. Ravishankar, A. Surolia, M. Vijayan, S. Lim,
Y. Kishi, J. Am. Chem. Soc. 1998, 120, 11297 – 11303.
[11] a) J. F. Espinosa, M. Bruix, O. Jarreton, T. Skrydstrup, J. M.
Beau, J. J. Barbero, Chem. Eur. J. 1999, 5, 442 – 448; b) A.
Poveda, J. L. Asensio, J. J. Barbero, T. Polat, H. Bazin, R. J.
Linhardt, Eur. J. Org. Chem. 2000, 1805 – 1813; c) L. M. Mik-
kelsen, M. J. Hernaiz, M. M. Pastor, T. Skrydstrup, J. J. Barbero,
J. Am. Chem. Soc. 2002, 124, 14940 – 14951.
[12] The corresponding 2-thiophenylglycoside was prepared by the
method of S. Cao, S. Meuneir, F. Andersson, M. Letellier, R.
Roy, Tetrahedron: Asymmetry 1994, 5, 2303 – 2312 and oxidized
to the sulfone according to A. Marra, P. Sinay¨, Carbohydr. Res.
1989, 187, 35 – 42.
[13] Y. Du, R. J. Linhardt, Carbohydr. Res. 1998, 308, 161 – 164.
[14] B. K. Shull, Z. Wu, M. Koreeda, J. Carbohydr. Chem. 1996, 15,
955 – 964.
[15] R. U. Lemieux, R. M. Ratcliffe, Can. J. Chem. 1979, 57, 1244 –
1251.
[16] M. Schultz, H. Kunz, Tetrahedron: Asymmetry 1993, 4, 1205 –
1220.
[17] T. Rosen, I. M. Lico, D. T. W. Chu, J. Org. Chem. 1988, 53, 1580 –
1582.
[18] T. Horrobin, C. H. Tran, D. Crout, J. Chem. Soc. Perkin Trans. 1
1998, 1069 – 1080; B. Kuberan, Q. Wang, M. Koketsu, R. J.
Linhardt, Synth. Commun. 2002, 32, 1421 – 1426.
[19] T. T. Tidwell, Synthesis 1990, 857 – 870.
[20] Examples and mechanistic studies of organo- and glycosyl
samarium compounds: a) J. L. Namy, J. Collin, C. Bied, H. B.
Kagan, Synlett 1992, 733 – 734; b) G. Molander, J. McKie, J. Org.
Chem. 1991, 56, 4112 – 4120; c) N. Miquel, G. Doisneau, J.-M.
Beau, Angew. Chem. 2000, 112, 4277 – 4280; Angew. Chem. Int.
Ed. 2000, 39, 4111 – 4114; d) Y. Du, R. J. Linhardt, Carbohydr.
Res. 1998, 308, 161 – 164; Reviews on the application of SmI2:
e) G. A. Molander, C. R. Harris, Chem. Rev. 1996, 96, 307 – 338;
f) G. A. Molander, C. R. Harris, Tetrahedron 1998, 54, 3321 –
3354; g) A. Krief, A.-M. Laval, Chem. Rev. 1999, 99, 745 – 777.
[9] a) G. Ragupathi, R. R. Koganty, D. Qiu, K. O. Lloyd, P. O.
Livingston, Glycoconjugate J. 1998, 15, 217 – 221; b) B. Kuberan,
R. J. Linhardt, Curr. Org. Chem. 2000, 4, 653 – 677, and
references therein.
Angew. Chem. Int. Ed. 2003, 42, 2073 – 2075
www.angewandte.org
ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2075