Stereoselective synthesis of sialylated tumor-associated glycosylamino acids

Article abstract of DOI:10.1021/ol402845e

Suitably protected sialyl T_N and 2,6-sialyl T tumor-associated carbohydrate antigen-derived amino acids have been prepared stereoselectively using an oxazolidinone-derived sialoside donor. These glycosylamino acids can be employed directly in t

Full text of DOI:10.1021/ol402845e

ORGANIC
LETTERS
2013
Vol. 15, No. 22
5794–5797
Stereoselective Synthesis of Sialylated
Tumor-Associated Glycosylamino Acids
Leo Corcilius and Richard J. Payne*
School of Chemistry, The University of Sydney, New South Wales 2006, Australia
richard.payne@sydney.edu.au
Received October 2, 2013
ABSTRACT
Suitably protected sialyl TN and 2,6-sialyl T tumor-associated carbohydrate antigen-derived amino acids have been prepared stereoselectively
using an oxazolidinone-derived sialoside donor. These glycosylamino acids can be employed directly in the solid-phase synthesis of
glycopeptides, as demonstrated by the efficient preparation of tumor-associated MUC1 glycopeptide fragments.
Aberrant glycosylation of cell surface glycoproteins
is a common feature on numerous tumor cell types.1 These
often truncated carbohydrate structures, called tumor-
associated carbohydrate antigens (TACAs), are a result
of the downregulation of glycosyltransferase enzymes
which occurs with concomitant overexpression of sialyl-
transferases upon tumor progression (Figure 1).1,2 Four
common TACAs include N-acetylgalactosamine (GalNAc)
and Gal-β-1,3-GalNAc, commonly termed the TN and T
antigens, as well as the sialylated antigens, sialyl TN (STN)
and 2,6-sialyl T (ST).1,3 An example of a protein that is
highly overexpressed on epithelial tumor cells and bears
these TACAs is the mucin glycoprotein MUC1. Specifi-
cally, the extracellular domain of MUC1 displays numerous
copies of these truncated glycans on serine and threonine
residues within a 20 amino acid repeat called the vari-
able number tandem repeat (VNTR) region.2,4 This leads
to the exposure of peptide epitopes which can be
targeted by the immune system, a property that has been
exploited in the development of synthetic glycopeptide
cancer vaccines.3,4
Figure 1. (A) Structures of TACAs and (B) the primary sequence
of the VNTR of MUC1 (* indicates glycosylation sites).
One strategy for the synthesis of vaccine candidates (and
other glycopeptides) involves the preparation of suitably
protected glycosylamino acid cassettes followed by incor-
poration into targets through Fmoc-strategy solid-phase
(1) Dube, D. H.; Bertozzi, C. R. Nat. Rev. Drug Discovery 2005, 4,
477.
(2) Taylor-Papadimitriou, J.; Burchell, J.; Miles, D. W.; Dalziel, M.
Biochim. Biophys. Acta Mol. Basis Dis. 1999, 1455, 301.
(3) (a) Gaidzik, N.; Westerlind, U.; Kunz, H. Chem. Soc. Rev. 2013,
42, 4421. (b) Wilson, R. M.; Danishefsky, S. J. J. Am. Chem. Soc. 2013,
135, 14462.
(5) (a) Brocke, C.; Kunz, H. Bioorg. Med. Chem. 2002, 10, 3085. (b)
Davis, B. G. Chem. Rev. 2002, 102, 579. (c) Payne, R. J.; Wong, C. H.
Chem. Commun. 2010, 46, 21. (d) Sames, D.; Chen, X. T.; Danishefsky,
S. J. Nature 1997, 389, 587.
(4) Tarp, M. A.; Clausen, H. Biochim. Biophys. Acta 2008, 1780, 546.
r
10.1021/ol402845e
Published on Web 10/25/2013
2013 American Chemical Society

Scheme 1. Synthesis of Sialyl TN and 2,6-Sialyl T-Derived Ser and Thr Residues
peptide synthesis (Fmoc-SPPS).5 Despite the enormous
interest and activity in the areas of glycosylamino acid
synthesis3a and sialylated cancer vaccines,3 as well as the
wealth of literature available on sialylation chemistry,
there remains only a small number of methods for acces-
sing sialyl TN and sialyl T-bearing glycosylamino acids for
incorporation into SPPS.3a,5a This is due, in major part, to
difficulties in both accessing a single anomer and compet-
ing 2,3-elimination reactions on the sialyl donor during
activation.6
A common method for the construction of sialylated
glycosylamino acids makes use of a stereodirecting nitrile
solvent to promote R-selectivity with a range of sialyl
donors.7 However, in most cases these reactions do not
exclusively afford the R-anomer. A number of chemoenzy-
matic methods employing sialyltransferases have also been
utilized in glycosylamino acid and glycopeptide assembly,8
but these often prove prohibitively expensive to scale up.
An alternative strategy for the induction of complete
R-selectivity in sialylation reactions involves the use of
directing auxiliaries within the sialyl donor.9 A recently
employed class of auxiliaries are the unsubstituted and
N-acylated 5N,4O oxazolidinone moieties10 which have
provided R-sialosides exclusively in the Neu5Ac-R(2,6)-
GalNAc linkage found in TACAs.11,10i Herein, we report
the first use of an oxazolidinone directing auxiliary in the
stereoselectivesynthesisof glycosylamino acidsbearing the
STN and ST antigens, as well as their rapid and efficient
incorporation into glycopeptides.
Synthesis of suitably protected STN-derived amino acid
building blocks began with 3,4,6-tri-O-acetyl-2-azido-2-
deoxy-D-galactose R-linked to Fmoc-protected serine
(Ser, 1) and threonine (Thr, 2) which were prepared in four
linear steps from D-galactosamine, as described previously
(Scheme 1).12 The glycosylamino acids were first deacety-
ꢀ
lated under Zemplen conditions before regioselective 6-silyl
(9) (a) Okamoto, R.; Souma, S.; Kajihara, Y. J. Org. Chem. 2008, 73,
3460. (b) Nakahara, Y.; Iijima, H.; Shibayama, S.; Ogawa, T. Carbo-
hydr. Res. 1992, 216, 211.
(10) (a) Tanaka, H.; Nishiura, Y.; Takahashi, T. J. Am. Chem. Soc.
2006, 128, 7124. (b) Crich, D.; Li, W. J. Org. Chem. 2007, 72, 2387. (c)
Farris, M. D.; De Meo, C. Tetrahedron Lett. 2007, 48, 1225. (d) Tanaka,
H.; Nishiura, Y.; Takahashi, T. J. Am. Chem. Soc. 2008, 130, 17244. (e)
Crich, D.; Wu, B. Org. Lett. 2008, 10, 4033. (f) Kancharla, P. K.;
Navuluri, C.; Crich, D. Angew. Chem., Int. Ed. 2012, 51, 11105. (g)
Noel, A.; Delpech, B.; Crich, D. Org. Lett. 2012, 14, 4138. (h) Liao,
H.-Y.; Hsu, C.-H.; Wang, S.-C.; Liang, C.-H.; Yen, H.-Y.; Su, C.-Y.;
Chen, C.-H.; Jan, J.-T.; Ren, C.-T.; Chen, C.-H.; Cheng, T.-J. R.; Wu,
C.-Y.; Wong, C.-H. J. Am. Chem. Soc. 2010, 132, 14849. (i) Hsu, C.-H.;
Chu, K.-C.; Lin, Y.-S.; Han, J.-L.; Peng, Y.-S.; Ren, C.-T.; Wu, C.-Y.;
Wong, C.-H. Chem.;Eur. J. 2010, 16, 1754. (j) Chu, K.-C.; Ren, C.-T.;
Lu, C.-P.; Hsu, C.-H.; Sun, T.-H.; Han, J.-L.; Pal, B.; Chao, T.-A.; Lin,
Y.-F.; Wu, S.-H.; Wong, C.-H.; Wu, C.-Y. Angew. Chem., Int. Ed. 2011,
50, 9391.
(6) (a) Boons, G. J.; Demchenko, A. V. Chem. Rev. 2000, 100, 4539.
(b) Ress, D. K.; Linhardt, R. J. Curr. Org. Synth. 2004, 1, 31. (c) Ando,
H.; Imamura, A. Trends Glycosci. Glyc. 2004, 16, 293–303.
(7) (a) Iijima, H.; Ogawa, T. Carbohydr. Res. 1988, 172, 183. (b)
Liebe, B.; Kunz, H. Tetrahedron Lett. 1994, 35, 8777. (c) Elofsson, M.;
Kihlberg, J. Tetrahedron Lett. 1995, 36, 7499. (d) Dziadek, S.;
Griesinger, C.; Kunz, H.; Reinscheid, U. M. Chem.;Eur. J. 2006, 12,
4981. (e) Qiu, D.; Gandhi, S. S.; Koganty, R. R. Tetrahedron Lett. 1996,
37, 595. (f) Qiu, D.; Koganty, R. R. Tetrahedron Lett. 1997, 38, 961. (g)
Schwarz, J. B.; Kuduk, S. D.; Chen, X.-T.; Sames, D.; Glunz, P. W.;
Danishefsky, S. J. J. Am. Chem. Soc. 1999, 121, 2662. (h) Winterfeld,
G. A.; Schmidt, R. R. Angew. Chem., Int. Ed. 2001, 40, 2654.
(8) (a) Suzuki, K.; Matsuo, I.; Isomura, M.; Ajisaka, K. J. Carbo-
hydr. Chem. 2002, 21, 99. (b) George, S. K.; Schwientek, T.; Holm, B.;
Reis, C. A.; Clausen, H.; Kihlberg, J. J. Am. Chem. Soc. 2001, 123,
11117. (c) Blixt, O.; Allin, K.; Pereira, L.; Datta, A.; Paulson, J. C. J. Am.
Chem. Soc. 2002, 124, 5739.
(11) Sahabuddin, S.; Chang, T.-C.; Lin, C.-C.; Jan, F.-D.; Hsiao,
H.-Y.; Huang, K.-T.; Chen, J.-H.; Horng, J.-C.; Ho, J. A.; Lin, C.-C.
Tetrahedron 2010, 66, 7510.
(12) Wu, Z.; Guo, X.; Guo, Z. Chem. Commun. 2010, 46, 5773.
Org. Lett., Vol. 15, No. 22, 2013
5795

protection via treatment with tert-butyldiphenylsilyl
chloride (TBDPS-Cl) and imidazole to form 3 and 4.
Isopropylidene protection of the 3- and 4-hydroxyl groups,
followed by buffered fluoridolysis of the silyl ether, af-
forded acceptors 5 and 6 in good yield over the two steps.
Gratifyingly, glycosylation of both acceptors with a stoi-
chiometric quantity of N-acetyl-5N,4O-carbonyl protected
phenylthiosialoside 710b under N-iodosuccinimide/triflic
acid (NIS/TfOH) promotion conditions at À38 °C furn-
ished the sialylated glycosyl amino acids 8 and 9 in 94%
and 80% yield, respectively, with only the R-sialoside pro-
duced in both cases. The R-stereochemistry was confirmed
through extraction of the 3JC1,H3ax coupling constants (see
Supporting Information (SI)).13 It is important to note that
only 0.4 equiv of TfOH was employed, so as to prevent
acidolysis of the isopropylidene. Rather than removing the
oxazolidinone auxiliary at this stage, we anticipated that it
could be removed following solid-phase assembly of the
glycopeptides. Accordingly, hydrolysis of the isopropyli-
dene acetals of 8 and 9 with aqueous AcOH, followed by
reductive acetylation of the C2-azido moiety with nano-
particle Zn, Ac2O, and AcOH, and acetylation of the free
hydroxyl groups at the 3- and 4-positions with Ac2O,
pyridine, and DMAP, gave acetamides 10 and 11 in 87%
and 81% yield over 3 steps. Finally, acidolysis of the tert-
butyl ester with TFA, triisopropylsilane, and water pro-
vided the peracetylated STN-derived Ser and Thr cassettes
12 and 13 in good overall yields.
Next, we turned our attention to the synthesis of suitably
protected 2,6-ST Ser and Thr cassettes. Deacetylation
of 1 and 2 followed by treatment with benzaldehyde
dimethylacetal/TsOHprovided benzylidene acetals 14
and 15. Schmidt glycosylation between acceptors 14 and
15 and tetraacetyl-D-galactose trichloroacetimidate (16)
afforded T-antigen core structures 17 and 18 in good yield
with complete β-selectivity due to the neighboring group
effect. The benzylidene acetal was next removed by hydro-
lysis with aqueous AcOH to provide diols 19 and 20.
Regioselective glycosylation of the 6-hydroxyl of acceptors
19 and 20 with a stoichiometric quantity of donor 7 under
NIS/TfOH promotion conditions afforded the correspond-
ing 2,6-ST core structures 21 and 22 in 81% and 80% yield,
respectively. Small quantities of the doubly sialylated tetra-
saccharides were detected by LC-MS (<5%); however,
these could be easily separated from the desired products by
flash chromatography. The stereo- and regiochemistry of
the ST glycosylamino acids 21 and 22 were confirmed by
NMR spectroscopic analysis (see SI). From here, reductive
acetylation of the C2-azide was achieved by treatment
with nanoparticle Zn in a mixture of AcOH and Ac2O
at elevated temperature (40 °C). The crude N-acetylated
glycosylamino acids were subsequently treated with Ac2O,
pyridine, and DMAPto acetylate the remaining 4-hydroxyl
group to provide 23 and 24 in 72% and 73% yield,
respectively. Finally, acidolytic cleavage of the tert-butyl
ester with TFA/iPr3SiH/H2O gave the target 2,6-ST amino
acids 25 and 26 in excellent yields.
Having synthesized the requisite glycosyl-Ser and
glycosyl-Thr building blocks we were next interested in
demonstrating their utility in the synthesis of glycopep-
tides via direct incorporation into Fmoc-SPPS protocols.
Specifically, we targeted four tumor-associated MUC1
glycopeptides 27À30 which were chosen with a view to
incorporation into synthetic cancer vaccine candidates in
future work (Scheme 2). Glycopeptides 27 and 28 possess
an STN- or ST-derived Ser moiety, respectively, within
the amino acid sequence GSTAPPAHGVT which embo-
dies the immunostimulatory GSTA epitope of MUC1.14
In contrast, glycopeptide targets 29 and 30 correspond
to an entire copy of the MUC1 VNTR region
(SAPDTRPAPGSTAPPAHGVT) with a key Thr residue
within the PDTRP immunodominant epitope14b,c,15 deri-
vatized with an STN (29) or ST (30) moiety. Synthesis of 27
and 28 began from preloaded 2-chlorotrityl chloride resin
31 which was elongated via Fmoc-SPPS to provide resin
bound nonapeptide 32. From here glycosyl-Ser building
blocks 12 and 25, bearing either the STN or ST antigen
respectively, were coupled to the resin bound peptide. It is
worth noting that due to the propensity of glycosyl-
Ser residues to epimerize upon coupling, a slight excess
of the precious glycosylamino acid (1.2 equiv) was used
in the presence of 2-(1H-7- azabenzotriazol-1-yl)-1,1,3,3-
tetramethyl uronium hexafluorophosphate (HATU, 1.2
equiv), sym-collidine (1.2 equiv), and 1-hydroxy-7-azaben-
zotriazole (HOAt, 1.5 equiv).16 Pleasingly, these condi-
tions almost completely prevented epimerization (<5%
detected by LC-MS) and facilitated quantitative coupling
to afford resin bound 33 and 34. Upon N-terminal Fmoc-
deprotection we discovered that the oxazolidinone moiety
was susceptible to nucleophilic attack with piperidine,
generating resin-bound N-acetyl piperidyl urea and piper-
idylurea byproducts (see SI). Assuch, weset outtoidentify
improved conditions for the en bloc removal of the oxazo-
lidinone moiety prior to further elongation of the glyco-
peptide chain. The optimized conditions involved treating
the resin bound peptides 33 and 34 with DTT and DBU in
DMF which led to clean opening of the oxazolidinone and
concomitant Fmoc-deprotection (see SI). The resulting
peptide was subsequently elongated by a further glycine
residue to provide 35 and 36. Having prepared the desired
resin bound target, the glycopeptides were deprotected and
cleaved from the resin by treating with an acidic cocktail.
ꢀ
The crude glycopeptides were next subjected to Zemplen
deacetylation conditions followed by saponification of the
(14) (a) Tarp, M. A.; Sørensen, A. L.; Mandel, U.; Paulsen, H.;
Burchell, J.; Taylor-Papadimitriou, J.; Clausen, H. Glycobiology 2007,
€
17, 197. (b) Westerlind, U.; Schroder, H.; Hobel, A.; Gaidzik, N.;
Kaiser, A.; Niemeyer, C. M.; Schmitt, E.; Waldmann, H.; Kunz, H.
Angew. Chem., Int. Ed. 2009, 48, 8263.
(15) Burchell, J.; Taylor-Papadimitriou, J.; Boshell, M.; Gendler, S.;
Duhig, T. Int. J. Cancer 1989, 44, 691.
(13) (a) Haverkamp, J.; Spoormaker, T.; Dorland, L.; Vliegenthart,
J. F. G.; Schauer, R. J. Am. Chem. Soc. 1979, 101, 4851. (b) Hori, H.;
Nakajima, T.; Nishida, Y.; Ohrui, H.; Meguro, H. Tetrahedron Lett.
1988, 29, 6317. (c) Prytulla, S.; Lauterwein, J.; Klessinger, M.; Thiem, J.
Carbohydr. Res. 1991, 215, 345.
(16) (a) Zhang, Y.; Muthana, S. M.; Farnsworth, D.; Ludek, O.;
Adams, K.; Barchi, J. J.; Gildersleeve, J. C. J. Am. Chem. Soc. 2012, 134,
6316. (b) Zhang, Y. L.; Muthana, S. M.; Barchi, J. J.; Gildersleeve, J. C.
Org. Lett. 2012, 14, 3958.
5796
Org. Lett., Vol. 15, No. 22, 2013

Scheme 2. Solid-Phase Assembly of STN- and ST-Derived MUC1 Glycopeptides
remaining methyl ester on the sialic acid moiety. Gratify-
ingly, following purification by reversed-phase HPLC, the
desired MUC1 glycopeptides 27 and 28 were isolated in
excellent yields (59% and 41%, respectively) based on the
original resin loading. Having successfully demonstrated
the utility of the sialylated glycosyl-Ser residues in the
solid-phase synthesis of glycopeptides, we next turned
our attention to the synthesis of MUC1 VNTR glycopep-
tides 29 and 30 bearing STN and ST antigen-derived Thr
residues, respectively. Synthesis of resin bound peptide
37 was achieved by Fmoc-SPPS. Coupling of sialylated
glycosyl-Thr residues 13 and 26 was carried out using
slightly modified conditions than those employed for 12
and 25 (HATU, HOAt, N,N-diisopropylethylamine in
DMF) to afford resin bound glycopeptides 38 and 39,
respectively. The oxazolidinone and N-terminal Fmoc
group of 38 and 39 were removed using the optimized
DTT and DBU deprotection conditions before the peptide
chain was elongated by a further four amino acids by
Fmoc-SPPS to provide resin bound eicosaglycopeptides40
and 41. Acidolytic side chain deprotection and cleavage
of these peptides from the resin, followed by deacetyla-
tion of the carbohydrate units and saponification of
the C-1 methyl ester, provided the crude glycopeptides.
Following purification by reversed-phase HPLC the
MUC1 VNTR glycopeptides 29 and 30 were isolated in
38% and 25% yields respectively, based on the original
resin loading.
In summary, we have successfully developed high yield-
ing and stereoselective routes to suitably protected Ser
and Thr residues bearing the STN and ST antigens. These
glycosylamino acid cassettes containing the R-directing
N-acyl oxazolidinone group could be utilized directly in
the Fmoc-SPPS of glycopeptides as highlighted by the
synthesis of four MUC1 tumor-associated targets. Future
work in our laboratories will focus on the use of this
synthetic methodology to assemble a range of glycopep-
tides for incorporation into cancer vaccine candidates.
Acknowledgment. The authors would like to acknowl-
edge the Australian Research Council for Discovery
Project funding (DP120100194). We would also like to
acknowledge a John A. Lamberton Scholarship and the
Bruce Veness Chandler Research Scholarship for PhD
funding (L.C.).
Supporting Information Available. Fullcharacterization
1
of all novel compounds, H and 13C NMR spectra, and
analytical HPLC chromatograms. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 22, 2013
5797