H. Kunz et al.
FULL PAPER
based on the sialic acid donor 13) was formed as a by-product, and a
small amount of the glycosyl acceptor 11 (17 mg, 15%) was re-isolated.
83.13 (Cq-tBu), 78.15 (C3), 75.94, 75.15 (Tb, C4, C3’), 73.60 (CH2-Bn),
73.43, 72.92 (C6’’,C5’), 69.72 (C8’’, C6’), 69.07 (C6), 68.61 (C4’’), 68.22,
68.11 (CH2-Bn, C2’,C4’,C7’’), 67.25 (CH2-Fmoc), 63.64 (C9’’), 63.52 (C5),
59.24 (Ta), 49.08 (C5’’), 47.79 (C2), 47.09 (C9-Fmoc), 37.42 (C3’’), 28.06
(CH3-tBu), 23.16, 23.07 (CH3-NHAc), 21.33, 21.21, 21.01, 20.78, 20.64
(CH3-OAc), 19.47 (Tg) ppm; MS (HR-ESI-TOF): m/z:found:1512.5729
[M+Na]+; C77H91N3O27Na calcd 1512.5737.
Procedure B:Fmoc-Thr( b-6-O-Bn-Gal(1 3)-a-4,6-O-Bzn-GalNAc)-OtBu
11 (338 mg, 0.359 mmol) and a-Ac4NeuNAcCOOBnXan 15 (482 mg,
0.718 mmol, 2 equiv) were dissolved in a mixture of acetonitrile and di-
chloromethane (55 mL, 2:1). The solution was stirred for 1 h in a Schlenk
flask (brown glass) in the presence of flame-dried molecular sieves (2.1 g,
powder, 3 ä) under an argon atmosphere and with exclusion of moisture.
After the reaction mixture had been cooled to ꢀ688C, silver triflate
(200 mg, 0.78 mmol) and di-tert-butylpyridine (186 mL, 213.8 mg,
1.12 mmol) were added. A pre-cooled (ꢀ158C) solution of phenylsulfenyl
chloride[47] (90 mL, 0.78 mmol) in dry dichloromethane (0.5 mL) was
added dropwise, and stirring was continued at ꢀ688C for 3.5 h. The reac-
tion mixture was diluted with a suspension of silica gel (0.7 g) in ethyl
acetate (10 mL), filtered through Hyflo Super Cel, and washed with satu-
rated aqueous NaHCO3 (2î75 mL) and brine (75 mL). The organic layer
was dried over MgSO4 and concentrated in vacuo. Purification of the
crude product by flash chromatography (silica gel, ethyl acetate!ethyl
acetate/ethanol 65:1) yielded 14 (262 mg, 50%) as a colorless, amorphous
solid (a/b, 97:3).
b Anomer: Rf = 0.43 (ethyl acetate/ethanol 10/1); [a]2D3 = 30.9 (c = 1,
CH2Cl2); 1H NMR (400 MHz, COSY, HMQC, CDCl3): d
=
7.75 (d,
JH3,H4 = JH5,H6 = 6.3 Hz, 2H; H4-, H5-Fmoc), 7.61 (dd, JH1,H2 = JH7,H8
=
7.1 Hz, 2H; H1-, H8-Fmoc), 7.52 7.48 (m, 2H; Har-Bzn), 7.41 7.24 (m,
17H; H2-, H3-, H6-, H7-Fmoc, Har-Bzn (3H), Har-Bn (2î, 10H)), 6.40
(d, JNH,2H = 9.0 Hz, 1H; NH-GalNAc), 5.86 (d, JNH,H5’’ = 9.8 Hz, 1H;
NH-NeuNAc), 5.63 (d, JNH,Ta = 9.4 Hz, 1H; NH-T), 5.49 (s, 1H; CH-
Bzn), 5.43 (dt, JH3’’ax,H4’’ = JH4’’,H5’’ = 10.6 Hz, JH3’’eq,H4’’ = 4.7 Hz, 1H;
H4’’), 5.34 (dd, JH6’’,H7’’ = 2.0 Hz, JH7’’,H8’’ = 6.3 Hz, 1H; H7’’), 5.26 5.22
(m, 1H; H8’’), 5.21 (d, Ja,b = 12.1 Hz, 1H; CH2a-Bn), 5.12 (d, Ja,b
=
11.7 Hz, 1H; CH2a’-Bn), 4.94 (d, JH1,H2 = 3.1 Hz, 1H; H1), 4.66 3.86 (m,
16H; H2 {4.62}, H9a,b’’ {4.59, 3.95}, CH2-Bn {4.55}, CH2-Fmoc {4.45},
H6’’ {4.37, dd, JH5’’,H6’’ = 10.6 Hz, JH6’’,H7’’ = 2.0 Hz}, H1’ {4.31, d, JH1’,H2’
=
7.4 Hz}, Ta {4.27}, H4 {4.26}, H9-Fmoc {4.26}, Tb {4.18}, H6a,b {4.15,
3.94}, H5’’ {4.14}), 3.82 3.58 (m, 8H; H3 {3.78}, H3’ {3.78}, H2’ {3.70}, H4’
{3.66}, H6’a,b {3.66}, H5’ {3.65}, H5 {3.61}), 2.55 (dd, JH3’’ax,eq’’ = 13.7 Hz,
JH3’’eq,H4’’ = 4.3 Hz, 1H; H3’’eq), 2.10 (s, 3H; CH3-Ac), 2.01 (s, 3H; CH3-
Ac), 2.06 2.03 (m, 1H; H3’’ax), 1.99 (s, 3H; CH3-Ac), 1.96 (s, 3H; CH3-
Procedure C:Fmoc-Thr(
b-6-O-Bn-Gal-(1 3)-a-4,6-O-Bzn-GalNAc)-
OtBu 11 (210 mg, 0.223 mmol) and Ac4NeuNAcCOOBn-Xan 15 (375 mg,
0.558 mmol) were each separately co-evaporated with toluene (3î
10 mL) and dried under high vacuum. The glycosyl acceptor 11 and the
glycosyl donor 15 were dissolved in a mixture of dry dichloromethane
(3 mL) and dry acetonitrile (7 mL), powdered molecular sieves (600 mg,
3 ä) were added, and the reaction mixture was stirred for 1 h at ambient
temperature under argon. The suspension was cooled to ꢀ688C, and
silver triflate (143 mg, 0.558 mmol) and a solution of methylsulfenyl bro-
mide in dry 1,2-dichloroethane (0.35 mL of a 1.6m solution; the methyl-
sulfenyl bromide solution in 1,2-dichloroethane was prepared by addition
ꢀ
Ac), 1.95 (s, 3H; CH3-Ac)), 1.86 (s, 3H; CH3-Ac), 1.44 (s, 9H; CH3
tBu), 1.28 (d, JTb,g = 6.3 Hz, 3H; Tg) ppm; 13C NMR (100.6 MHz, BB,
HMQC, CDCl3): d = 172.6, 170.9, 170.7, 170.4, 170.2, 170.1, 167.7 (C=
O), 156.5 (C=O-urethane), 143.7 (C1a-, C8a-Fmoc), 141.3 (C4a-, C5a-
Fmoc), 138.1, 137.6 (Cq-Bn, Cq-Bzn), 134.7 (Cq-Bn), 128.9, 128.5, 128.4,
127.6 (Car-Bn (2î)), 128.6, 128.1, 126.4 (Car-Bzn), 127.8 (C3-, C6-Fmoc),
127.1 (C2-, C7-Fmoc), 125.0 (C1-, C8-Fmoc), 120.0 (C4-, C5-Fmoc), 105.3
(C1’), 100.9 (CH-Bzn), 100.5 (C1), 98.9 (C2’’), 83.4 (Cq-tBu), 76.8 (C3,
C3’), 75.7 (C4, Tb), 73.5 (CH2-Bn), 73.4 (C5’), 71.2 (C6’’), 70.0 (C8’’), 69.5
(C6’), 69.1 (C4’’), 68.9 (C6), 68.0 (CH2-Bn), 67.8 (C2’, C4’, C7’’), 67.3
(CH2-Fmoc), 63.7 (C5), 62.4 (C9’’), 59.0 (Ta), 48.5 (C2, C5’’), 47.2 (C9-
of bromine (410 mL, 7.99 mmol) to
a solution of dimethyl disulfide
(709 mL, 7.99 mmol) in dry 1,2-dichloroethane (10 mL) and stirring at
ambient temperature with exclusion of light for 15 h) were added slowly.
The reaction mixture was stirred for 4 h at ꢀ688C under an argon atmos-
phere with exclusion of light. It was then neutralized with Huenig×s base
(115 mL, 3 equiv) and allowed to warmed to room temperature. The sus-
pension was diluted with dichloromethane (100 mL) and filtered through
Hyflo Super Cel, and the solvents were removed from the filtrate in
vacuo. The residue was purified by flash chromatography (silica gel;
100% ethyl acetate) to afford the desired a-glycosylation product a-14
(194 mg, 58%) as a colorless, amorphous solid. In addition, a small
amount of the unreacted glycosyl acceptor 11 (17 mg, 8%) and the sialic
acid glycal (235 mg, 77% yield based on the sialic acid donor 15) formed
as a by-product were isolated.
ꢀ
Fmoc), 34.2 (C3’’), 28.1 (CH3 tBu), 23.3, 23.1 (CH3-NHAc), 21.0, 20.9,
20.8 (CH3-OAc (4î)), 19.4 (Tg) ppm; MS (MALDI-TOF, dhb, positive
ion mode):m/z:found:1513.3 [ M+Na]+; C77H91N3NaO27 calcd 1513.5;
found:1529.4 [ M+K]+; C77H91KN3O27 calcd:1529.5; MS (HR-ESI-TOF):
m/z:found:1512.5740 [ M+Na]+; C77H91N3O27Na calcd 1512.5737.
N-Fluorenylmethoxycarbonyl-O-(2-acetamido-2-deoxy-3-O-[6-O-benzyl-
3-O-{benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-d-galacto-
2-nonulopyranosyl)onat}-b-d-galactopyranosyl]-a-d-galactopyranosyl)-l-
threonine tert-butyl ester (16):A solution of trisaccharide 14 (359 mg,
0.241 mmol) in aqueous acetic acid (80%, 15 mL) was stirred at 808C for
1 h. Toluene (15 mL) was added, and the reaction mixture was concen-
trated in vacuo and subsequently co-evaporated with toluene (3î15 mL).
The resulting crude product was purified by flash chromatography (silica
gel, ethyl acetate/ethanol 15:1) to give the title compound as a colorless,
amorphous solid (308 mg, 82%). Rf = 0.12 (ethyl acetate/ethanol 10:1);
a Anomer: Rf = 0.28 (ethyl acetate/ethanol 10:1); Rt = 30.1 min (Phe-
nomenex Luna C18(2), grad.:CH 3CN/H2O (60:40)!(90:10), 40 min);
[a]2D7
= 32.0 (c =
1, CHCl3); 1H NMR (400 MHz, CDCl3, COSY,
HMQC): d = 7.74 (d, JH4,H3 = JH5,H6 = 7.8 Hz, 2H; H4-, H5-Fmoc), 7.58
(dd, JH1,H2 JH8,H7 = 7.8 Hz, 2H; H1-, H8-Fmoc), 7.49 (d, JHa,Hb
5.8 Hz, 2H; Har-Bzn), 7.42 7.35 (m, 2H; H3-, H6-Fmoc), 7.35 7.24 (m,
15H; H2-, H7-Fmoc, Har-Bzn (3H), Har-Bn (10H)), 6.59 (d, JNH,H2
=
=
[a]2D8
= 12.0 (c =
1, CHCl3); 1H NMR (400 MHz, CDCl3, COSY,
=
HMQC): d = 7.73 (d, JH4,H3 = JH5,H6 = 7.4 Hz, 2H; H4-, H5-Fmoc), 7.56
(d, JH1,H2 = JH8,H7 = 7.8 Hz, 2H; H1-, H8-Fmoc), 7.42 7.23 (m, 14H;
H2-, H3-, H6-, H7-Fmoc, Har-Bn (10H)), 6.61 (d, JNH,H2 = 9.4 Hz, 1H;
9.4 Hz, 1H; NH-GalNAc), 6.12 (d, JNH,Ta = 9.7 Hz, 1H; NH-T), 5.45 (s,
1H; CH-Bzn), 5.40 (t, JH8’’,H9’’ = 6.6 Hz, 1H; H8’’), 5.28 5.13 (m, 2H;
H7’’, NH-NeuNAc), 5.16 (s, 2H; CH2-Bn), 4.98 (d, JH1,H2 = 3.5 Hz, 1H;
H1), 4.92 4.81 (m, 1H; H4’’), 4.74 4.63 (m, 1H; H2), 4.55 (s, 2H; CH2-
Bn), 4.52 4.03 (m, 10H; H9a’’{4.46}, CH2-Fmoc {4.39, 4.34}, Tb {4.33}, H4
{4.26}, Ta {4.25}, H9-Fmoc {4.19}, H1’ {4.15}, H6a {4.13}, H5’’ {4.06}),
3.99 3.84 (m, 4H; H6’’ {3.93}, H9b’’ {3.92}, H3’ {3.90}, H6b {3.87}), 3.72
3.55 (m, 4H; H3 {3.67}, H2’ {3.61}, H6a’ {3.59}, H5 {3.57}), 3.54 3.39 (m,
2H; H6b’{3.87}, H5’{3.41}), 3.24 (s, 1H; H4’), 2,78 (sb, 1H; OH), 2.71
(dd, JH3eq’’,H3ax’’ = 12.7 Hz, JH3eq’’,H2’’ = 4.3 Hz, 1H; H3eq’’), 2.36 (s, 1H;
OH), 2.03 2.00 (m, 1H; H3ax’’), 2.09, 2.07, 2.02, 2.00, 1.92, 1.82 (6îs,
18H; 6îCH3-Ac), 1.42 (s, 9H; CH3-tBu), 1.26 (d, JTg,Tb = 5.9 Hz, 3H;
ꢀ
NH GalNAc), 5.99 (d, JNH,Ta = 9.8 Hz, 1H; NH-T), 5.49 5.41 (m, 1H;
H8), 5.23 5.07 (m, 4H; CH2-Bn1 {5.16, 5.14} H7’’ {5.10}, NH-NeuNAc),
4.94 4.88 (d, JH1,H2 = 1.6 Hz, 1H; H1), 4.87 4.77 (m, 1H; H4’’), 4.59 4.44
(m, 4H; H9a’’ {4.52}, H2 {4.52}, CH2-Bn {4.50}), 4.43 4.35 (m, 2H; CH2-
Fmoc), 4.34 4.26 (m, 1H; Tb), 4.26 4.12 (m, 4H; Ta {4.23}, H1’ {4.19},
H9-Fmoc {4.18}, H4 {4.16}), 4.11 4.01 (m, 1H; H5’’), 3.95 (dd, JH6’’,H5’’
=
10.8 Hz, JH6’’,H7’’ = 1.6 Hz, 1H; H6’’), 3.91 3.83 (m, 2H; H6a {3.87}, H3’
{3.86}), 3.82 3.63 (m, 4H; H5 {3.78}, H9b’’ {3.74}, H6b {3.73}, H2’ {3.67}),
3.61 3.51 (m, 2H; H3 {3.57}, H6a’ {3.54}), 3.49 3.35 (m, 2H; H6b’ {3.44},
Tg) ppm; 13C NMR (100.6 MHz, CDCl3, BB, HMQC):
d = 171.53,
H5’ {3.39}), 3.26 3.20 (m, 1H; H4’), 2.72 (dd, JH3eq’’,H3ax’’
= 13.1 Hz,
JH3eq’’,H4’’ 4.7 Hz, 1H; H3eq’’), 2.15 1.90 (m, 16H; 4îCH3OAc, 1î
=
170.91, 170.82, 170.24, 170.09, 169.76, 167.77 (C=O), 156.83 (C=O-ure-
thane), 143.71 (C1a-, C8a-Fmoc), 141.29, 141.15 (C4a-, C5a-Fmoc),
138.07, 137.51 (Cq-Bn), 134.21 (Cq-Bn), 128.97, 128.84, 128.78, 128.43,
127.51 (Car-Bn), 128.78, 128.04, 126.44 (Car-Bzn), 127.79 (C3-, C6-Fmoc),
127.09, 126.97 (C2-, C7-Fmoc), 125.17, 124.94 (C1-, C8-Fmoc), 120.03
(C4-, C5-Fmoc), 106.55 (C1’), 100.96 (CH-Bzn), 100.45 (C1), 97.47 (C2’’),
CH3NAc, H3ax’’ {2.02}), 1.83 (s, 3H; CH3NAc), 1.42 (s, 9H; CH3-tBu),
1.26 (d, JTg,Tb = 6.26 Hz, 3H; Tg) ppm; 13C NMR (100.6 MHz, CDCl3,
BB, HMQC): d = 171.77, 171.38, 170.86, 170.57, 170.06, 169.62 (C=O),
167.59 (C1’’), 156.73 (C=O-urethane), 144.64, 143.66 (C1a-, C8a-Fmoc),
141.24 (C4a-, C5a-Fmoc), 137.89, 134.16 (Cq (2îBn)), 128.94, 128.82,
4158
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2004, 10, 4150 4162