Total synthesis of a sialylated and sulfated oligosaccharide from O- linked glycoproteins

Article abstract of DOI:10.1016/S0040-4039(00)00261-6

The total synthesis of a sialylated and sulfated oligosaccharide 1 representative of a structure occurring in respiratory mucins has been accomplished. Our strategy depends upon the employment of 2-naphthymethyl (NAP) protection for hydroxyl functions. Ch

Full text of DOI:10.1016/S0040-4039(00)00261-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 2773–2776
Total synthesis of a sialylated and sulfated oligosaccharide from
O-linked glycoproteins
Jie Xia, Conrad F. Piskorz, James L. Alderfer, Robert D. Locke and Khushi L. Matta ∗
Molecular & Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
Received 1 November 1999; revised 8 February 2000; accepted 9 February 2000
Abstract
The total synthesis of a sialylated and sulfated oligosaccharide 1 representative of a structure occurring in
respiratory mucins has been accomplished. Our strategy depends upon the employment of 2-naphthymethyl (NAP)
protection for hydroxyl functions. Choice of the well-defined sialyl donor 15 was made because of its enhanced
reactivity over the parent compound 14 for glycosylation. © 2000 Elsevier Science Ltd. All rights reserved.
Glycoproteins and glycolipids are major components of the outer surface of mammalian cells. The
hydrophilic oligosaccharide head groups of these glycoproteins and glycolipids are recognized as playing
a significant role in many important biological processes, including fertilization, immune defense,
parasitic infection, cell growth, cell–cell adhesion, and inflamation.1 There is tremendous interest in
structural studies of the sulfated carbohydrate chains of O-linked mucinous glycoproteins, such as CF
respiratory mucin,2a colonic tumor associated glycoproteins2b and the natural ligands for selectin.2c
The chemical synthesis of well-defined oligosaccharides which occur as a part of these mucinous
glycoproteins is receiving increased attention.3
Consideration of the different reactivities of the sugar hydroxyl groups, in combination with detailed
structural information available from two-dimensional homonuclear (1H–1H) (DQF-COSY, ROESY,
TOCSY) and heteronuclear (13C–1H) (HMQC, or g-HSQC and HMBC) NMR correlation experiments,
has afforded the development of a new strategy of regioselective and stereoselective glycosylation
through the employment of unprotected or partially-protected acceptors.4 Advancement of this new
approach may overcome the traditional tedious multi-step protection–deprotection schemes and offer
a much shorter and easier route to complex biologically active oligosaccharide molecules. Herein, we
describe the total synthesis of complex oligosaccharide 1.
Treatment of compound 45 with bis(tributyltin)oxide6 in refluxing toluene followed with naphthyl-
methyl bromide in the presence of tetrabutylammonium iodide gave 5 in 69% yield. Regioselective
glycosylation of the 4-hydroxyl group of diol 5 with 2,3,4,6-tetra-O-acetyl-β-galactopyranosyl fluoride
67 was successfully achieved using SnCl2–AgOTf8 as a catalyst. Apparently, the presence of a 6-naphthyl
methyl group at HO-6 enhanced the reactivity of the relatively inactive HO-4 of diol 5 and disaccharide
∗
Corresponding author.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00261-6
tetl 16541

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7 was obtained in 75% yield. A strong NOE cross peak between Hb-1 and Ha-4 of disaccharide 7 was
indicative of a β(1→4) interglycoside linkage. A similar glycosylation of acceptor 8 with donor 6 also
afforded the β(1→4) linked disaccharide 9, the structure of which was established by a combination of 2D
NMR (DQF-COSY and ROESY) and X-ray structural analysis.9 Disaccharide 7 was further fucosylated
with the known methyl tri-O-benzyl-thio-β-L-fucoside 1010 under catalysis by CuBr2–n-Bu4NBr to give
11 in excellent yield (88%) (Scheme 1).
Scheme 1. (a) n-Bu2SnO–benzene, refluxing, 4 h, Bu4NBr, NAPBr, 80–85°C, 48 h, 69%; (b) 6, SnCl2–AgOTf, CH2Cl2–toluene,
4A-MS, −15 to 0°C, 12 h, 75%; (c) CuBr2–n-Bu4NBr, ClCH2CH2Cl:DMF (5:1), 4A-MS, rt, N2, 16–24 h, 87%; (d)
SnCl2–AgOTf/CH2Cl2–toluene, 4A-MS, −15 to 0°C, 12 h, 73%
The preparation of 17 was performed as outlined in Scheme 2. Selective removal of the O-acetyl
groups of 1211 in the presence of 6-O-pivaloyl was successfully achieved through treatment with a
sodium methoxide solution at −20 to −15°C giving 13 in an excellent yield (81%). We were then able to
achieve the regioselective and stereoselective α-sialylation of acceptor 13 with new sialyl donor 15 (β-
Scheme 2. (a) 1 M CH3ONa–CH3OH/CH3OH–CH2Cl2, −20 to −15°C, 20 min, 81%; (b) NIS–TfOH/CH2Cl2–CH3CN (1:1)
−45 to −40°C, 12 h, 45%; (c) Ac2O:pyridine (1:1), rt, 12 h; (d) 60% HOAc, 60–65°C, 2 h, 75%

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configuration was determined by X-ray structural analysis)9 which was prepared according to literature
protocol.12 The (2→3) linkage of 16 was confirmed by observation of a weak NOE cross peak between
H-3 of the galactose residue and H-3a of the sialic acid residue.13a An α-configuration of the glycoside
was deduced from an observation of the chemical shifts of H-4 (5.56–5.47pm), H-7 (5.25 ppm) and a
large coupling constant of J=7,8 (7–8 Hz) in the sialic acid residue.13b,13c
Compound 16 was treated with 60% HOAc to give 17 in 75% yield. Target oligosaccharide 1 was
prepared according to Scheme 3. Regioselective glycosylation of HO-6 of 17 with 11 was performed
under controlled reaction conditions resulting in β(1→6)-linked 18 in 79% yield. The presence of a
β(1→6) linkage in oligosaccharide 18 was confirmed by observation of NOE cross peaks between H-6a
and H-6b of the N-acetyl-galactose residue and H-1 of NPhth-protected glucose residue. Compound 18
was treated with pyridine:Ac2O (1:1) and a catalytic amount of DMAP to give 19 in 85% yield. Removal
of 2-naphthymethyl (NAP) protection from 19 was effected by treatment with DDQ.14 Conversion of
20 into 21 was obtained by treatment with SO3·pyridine in dry pyridine. Compound 21 was converted
into 1 in four steps: (a) removal of benzyl with Pd–C (10%) under a H2 atmosphere; (b) removal of
methyl from the carboxyl group with lithium iodide3b in refluxing pyridine under a N2 atmosphere; (c)
removal of the N-phthalimido group with methanol:NH2–NH2·xH2O (5:1), followed by pyridine:Ac2O
(1:1) in the presence of catalytic amounts of DMAP; (d) O-deacetylation with 1 M sodium methoxide in
a methanol–water solution at room temperature. The structure and purity of 1 was established by two-
dimensional homonuclear correlation experiments (1H–1H DQF-COSY, ROESY, TOCSY), 13C NMR
experiments and FABMS spectroscopy.15
Scheme 3. (a) NIS–TfOH/CH2Cl2, 4A-MS, −65 to −60°C, 2 h, 79%; (b) Ac2O–pyridine, DMAP, rt, 85%; (c)
DDQ/CH2Cl2:CH3OH (4:1), rt, 12 h, 80%; (d) SO3·pyridine/pyridine, 0–5°C, 6 h, 86%, then Na+–resin, rt, 4 h; (e) Lil–pyridine,
120–125°C, 6–8 h; (f) NH2–NH2·H2O:CH3OH (1:5), 80–85°C, then Ac2O:pyridine (1:1), rt, 12 h; (g) 1 M CH3ONa–CH3OH,
rt, 12 h; (h) Pd–C (10%)/CH3OH:HOAc (1:1), H2, 12 h, 35% in four steps

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In conclusion, we have described an efficient route towards the total synthesis of complex sialylated
and sulfated oligosaccharide. Our strategy takes advantage of the electron-rich NAP protecting group,
which can be readily removed with DDQ, and the sialyl donor 15.
Acknowledgements
This work was supported by Grant No. CA 35329, and in part by Grant No. CA63218 and Grant No.
P30CA16056, all awarded by the National Cancer Institute.
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15. Selected physical properties of compound 1: 1H NMR (D2O, 600 MHz) δ 5.12 (d, 1H, J1,2=3.6 Hz, Hf-1), 4.85–4.81 (dd,
1H, Hf-5), 4.78–4.77 (d, 1H, J1,2=3.0 Hz, Ha-1), 4.61–4.59 (d, 1H, J1,2=8.4 Hz, Hd-1), 4.56–4.55 (d, 1H, J1,2=7.8 Hz, He-1),
4.54–4.53 (d, 1H, J1,2=7.8 Hz, Hb-1), 4.39 (s, 1H, He-4), 4.32–4.30 (m, 1H, Ha-2), 4.21– 4.15 (s, 3H, Hd-6b, Hd-6a, Ha-4),
4.10–4.00 (m, 5H, Ha-5, Hb-3, Ha-6b, Hd-4, Ha-3), 3.95–3.56 (m, 25H, Hd-2, Hf-3, Hd-3, Ha-6a, Hc-6, Hf-4, Hc-5, Hc-4,
Hf-2, He-3), 3.57–3.48 (m, 2H, He-2, Hb-2), 3.37 (s, 3H, OCH3), 2.78–2.75 (dd, 1H, J=3.0 Hz, J=11.4 Hz, Hc-3e), 2.04 (s,
3H, Ac), 2.02 (s, 3H, Ac), 2.01 (s, 3H, Ac), 1.82–1.80 (t, 1H, Jgem=12.0 Hz, Hc-3a), 1.20 (d, 3H, J=7.6 Hz, CHf3); 13C NMR
(D2O, 100.6 MHz) δ 173.3 (C_O), 173.1 (C_O), 173.0 (C_O), 172.9 (C_O), 103.4, 100.5, 100.4, 98.7, 97.5, 97.0, 76.2,
74.6, 73.9, 73.7, 73.6, 72.2, 71.9, 71.7, 71.4, 70.9, 70.7, 69.9, 69.6, 68.2, 68.1, 68.0, 67.7, 67.4, 67.6, 67.0, 66.7, 66.3, 65.6,
f
64.9, 62.2, 60.3, 59.8, 54.6, 53.9, 50.6, 47.5, 38.6 (CH2), 21.2 (Ac), 21.0 (Ac), 21.0 (Ac), 14.1 (CH3 ); FABMS (positive ion
mode) (m/z) calcd for C46H76O36N3SNa2 (M++Na): 1324.5; found: 1324.9 (M++Na).