690
A. K. Pathak et al. / Carbohydrate Research 339 (2004) 683–691
column chromatography (2:1 cyclohexane–AcOEt) as a
light-sensitive solid (513 mg, 61%). It was stored in a
cool, dark dry place until further reaction. H NMR
C24H32N4O13S 639.1578 (M + Na)þ. Anal. Calcd for
C24H32N4O13SÆ2.5H2O: C, 43.57; H, 5.64; N, 8.46.
Found: C, 43.69; H, 5.25; N, 8.31.
1
(600 MHz, CDCl3): d 8.47 (d, 1H, J 8.8 Hz, Ar), 8.39 (d,
1H, J 8.5 Hz, Ar), 8.33 (dd, 1H, J 1.1, 7.4 Hz, Ar), 7.97–
7.96 (m, 4H, Ar), 7.93–7.91 (m, 2H, Ar), 7.68 (dd, 1H, J
7.6, 8.8 Hz, Ar), 7.56 (dd, 1H, J 7.4, 8.5 Hz, Ar), 7.53–
7.48 (m, 3H, Ar), 7.40–7.33 (m, 6H, Ar), 7.30 (d, 1H, J
7.6 Hz, Ar), 5.80 (dd, 1H, J3;4 9.8 Hz, J4;5 10.0 Hz, H-4),
5.64 (dd, 1H, J2;3 3.3 Hz, J3;4 9.8 Hz, H-3), 5.45 (t, 1H, J
6.2 Hz, NH), 5.41–5.39 (m, 2H, H-20, H-30), 5.23 (dd,
3.1.13. 5-Azidonaphthalene-1-sulfonamidoethyl a-
D-
mannopyranosyl)-(1 ! 2)-a- -mannopyranoside (2). To
D
a solution of disaccharide 15 (394 mg, 0.36 mmol) in dry
MeOH (2 mL) was added 7 N NH3–MeOH (10 mL)
dropwise, and the reaction mixture was stirred at room
temperature for 12 h. Concentration in vacuo and flash
chromatography (2:1 CHCl3–MeOH) gave 2 as a light-
sensitive, light-yellow oil. The product was dissolved in
deionized water (5 mL), passed through a small column
of Bio-Beadse SM-4 (20-50 mesh), and lyophilized to a
fluffy yellow solid (180 mg, 81%) that was stored in a
1H, J3 4 ¼ J4 ;5 ¼ 9:8 Hz, H-40), 4.91 (d, 1H, J1;2 1.6 Hz,
0
0
0
0
H-1), 4.88 (d, 1H, J1 ;2 1.5 Hz, H-10), 4.47 (dd, 1H, J5 ;6 a
0
0
0
0
3.2 Hz, J6 a;6 b 12.2 Hz, H-60a), 4.41 (dd, 1H, J5 ;6 b 5.5 Hz,
0
0
0
0
J6 a;6 b 12.2 Hz, H-60b), 4.22–4.19 (m, 2H, H-6a, H-50),
4.09–4.06 (m, 2H, H-5, H-6b), 4.05 (dd, 1H, J1;2 1.6 Hz,
J2;3 3.3 Hz, H-2), 3.79–3.76 (m, 1H, OCH2), 3.51–3.47
(m, 1H, OCH2), 3.27–3.20 (m, 2H, CH2 NH), 2.06, 2.05,
2.02, 1.99 (s, each 3H, 4 · OCH3). 13C NMR (75 MHz,
CDCl3): d 170.71, 169.79, 169.47, 169.38 (C@O), 166.87,
165.53, 165.16 (C@O), 137.81 (C), 134.80 (C), 133.51,
133.33, 133.01 (CH), 130.44 (CH), 129.89, 129.81,
129.64 (CH), 129.57, 129.52, 129.15 (C), 128.94, 128.59,
128.42, 128.34 (CH), 127.19 (C), 124.37, 120.82, 114.85
(CH), 99.25 (C-10), 98.54 (C-1), 76.17 (C-2), 70.48 (C-3),
69.14, 68.89, 68.69 (C-5, C-20, C-30, C-40), 67.35 (C-4),
66.98 (OCH2), 66.49 (C-50), 63.52 (C-60), 62.88 (C-6),
42.84 (CH2NH), 20.73, 20.69, 20.64, 18.73 (OCH3).
ESIMS: m=z 1119.2855 (M + Na)þ, calcd for C53H52-
N4O20S 1119.2787 (M + Na)þ.
0
0
1
cool, dark, dry place until needed. H NMR (600 MHz,
CD3OD): d 8.50 (dd, 1H, J 8.7 Hz, Ar), 8.42 (dd, 1H, J
1.1, 8.5 Hz, Ar), 8.26 (dd, 1H, J 1.1, 7.3 Hz, Ar), 7.73 (dd,
1H, J 7.7, 8.7 Hz, Ar), 7.62 (dd, 1H, J 7.4, 8.5 Hz, Ar),
7.50 (d, 1H, J 7.4 Hz, Ar), 4.89 (d, 1H, J1 ;2 1.4 Hz, H-10),
4.86 (unresolved, H-1), 3.95 (dd, 1H, J1;2 1.8 Hz, J2;3
3.2 Hz, H-2), 3.82–3.79 (m, 1H, H-60a), 3.73 (dd, 1H, J5;6
2.4 Hz, J6a;6b 11.9 Hz, H-6a), 3.68–3.49 (m, 10H, H-3, H-
4, H-20, H-30, H-40, H-6b, H-60b), 3.37–3.30 (unresolved
m, OCH2), 3.15–3.05 (m, 2H, CH2NH). 13C NMR
(75 MHz, CD3OD): d 138.86 (C), 137.18 (C), 130.94
(CH), 130.50 (C), 129.13 (2 · CH), 128.42 (C), 125.56,
122.63, 116.11 (3 · CH), 104.10 (C-10), 99.94 (C-1), 80.29
(C-2), 74.90, 74.59 (C-20, C-3), 72.40 (C-4), 71.88, 71.84
(C-5, C-30), 68.89, 68.80 (C-40, C-50), 67.31 (OCH2),
63.12, 62.84 (C-6, C-60), 43.72 (CH2NH). ESIMS: m=z
639.1562 (M + Na)þ, calcd for C24H32N4O13S 639.1578
(M + Na)þ. Anal. Calcd for C24H32N4O13SÆ2.0H2O: C,
44.05; H, 5.58; N, 8.56. Found: C, 44.07; H, 5.43; N,
8.19.
0
0
3.1.12. 5-Azidonaphthalene-1-sulfonamidoethyl a-
D-
mannopyranosyl-(1 ! 6)-a- -mannopyranoside (1). To a
D
solution of disaccharide 8 (225 mg, 0.36 mmol) in dry
MeOH (2 mL) was added 7 N NH3–MeOH (10 mL)
dropwise, and the reaction mixture was stirred at room
temperature for 12 h. Concentration in vacuo and flash
chromatography (2:1 CHCl3–MeOH) gave 1 as a light-
sensitive, light-yellow oil. It was dissolved in 5 mL of
deionized water (5 mL), passed through a small column
of Bio-Beadse SM-4 (20–50 mesh), and lyophilized to a
white fluffy solid (90 mg, 71%) that was stored in cool
3.2. Mannosyltransferase assay16
The mycobacterial membranes from M. smegmatics mc2
155 and M. Bovis BCGwere prepared and used for
compound 1 and compound 2, respectively. Compounds
1 (SRI 20723) and 2 (SRI 20731), at a range of con-
centrations from 0.1 to 4.0 mM (which were stored as
100 mM ethanol stocks), were dried under a stream of
argon in a microcentrifuge tube (1.5 mL). The materials
were placed in a vacuum desiccator for 15 min to remove
any residual solvent and were then resuspended in 8 lL
of a 1% aqueous solution of Igepal. The remaining
components of the mannosyltransferase assay were ad-
ded, consisting of 50 mM MOPS (adjusted to pH 8.0
with KOH), 5 mM b-mercaptoethanol, 10 mM MgCl2,
1 mM ATP, membranes (250 lg), and GDP-[U-
14C]mannose (0.25 lCi, 11 GBq/mmol, Amersham) were
added to a final reaction volume of 80 lL. The reaction
mixtures were then incubated at 37 °C for 1 h. A CHCl3–
CH3OH (1:1, 533 lL) solution was then added to the
1
dark dry place until further use. H NMR (300 MHz,
CD3OD): d 8.49 (d, 1H, J 8.8 Hz, Ar), 8.41 (d, 1H, J
8.5 Hz, Ar), 8.26 (dd, 1H, J 1.1, 7.3 Hz, Ar), 7.72 (dd,
1H, J 7.7, 8.8 Hz, Ar), 7.61 (dd, 1H, J 7.3, 8.5 Hz, Ar),
0
0
7.49 (dd, 1H, J 0.7, 7.7 Hz, Ar), 4.75 (d, 1H, J1 ;2 1.7 Hz,
H-10), 4.39 (d, 1H, J1;2 1.4 Hz, H-1), 3.82–3.77 (m, 3H),
3.71-3.37 (m, 10H), 3.23–3.17 (m, 1H, OCH2), 3.13–3.05
(m, 2H, CH2NH). 13C NMR (75 MHz, CD3OD): d
138.86, 137.29 (C), 130.94 (CH), 130.48 (C), 129.16
(CH), 128.42 (C), 125.57, 122.57, 116.12 (CH), 101.72
(C-10), 101.37 (C-1), 74.31 (C-2), 73.11, 72.59, 72.07,
71.66 (C-3, C-4, C-5, C-20, C-30), 68.59, 68.38 (C-40,
C-50), 67.20, 67.17 (C-6, OCH2), 62.86 (C-60), 43.71
(CH2NH). ESIMS: m=z 639.1569 (M + Na)þ, calcd for