◦
of 10. Yield: 308 mg, 35%: mp 129–130 C. [a]2D0 −26.9 (c 0.5,
ꢀꢀ
J
4,“3”a + J4,“5” = 20.0, J4,“3”e = 4.5, H-4ꢀꢀ), 5.03 (d, 1 H, JNH,5 = 10.0,
N–H), 5.21 (dd, 1 H, J7,“8” = 10.0, J7,“6” = 2.0, H-7ꢀꢀ), 5.55 (ddd, 1 H,
1
CHCl3), H NMR (500 MHz, CDCl3) d: 1.05 (s, 9 H, C(CH3)3),
J8,“7” = 10.0, J8,“9”a = 7.5, J8,“9”b = 3.0, H-8ꢀꢀ), 5.64 (d, 1 H, J1,2 = 8.0,
1.90, 2.00, 2.12, 2.16, (4 × s, 12 H, CH3), 2.03–2.06 (m, 4 H, H-3ꢀꢀa,
ꢀꢀ ꢀꢀ
H-1), 5.82 (dd, 1 H, J3,2 = 10.5, J3,4 = 3.5, H-3), 5.95 (dd, 1 H, J2,3
=
CH3), 2.39 (s, 3 H, CH3), 2.76 (bs, 1 H, OH), 2.82 (dd, 1 H, J3
=
e,3
a
= 4.5, H-3ꢀꢀe), 2.98 (bs, 1 H, OH), 3.76–3.81 (m, 4 H,
ꢀꢀ ꢀꢀ
10.5, J2,1 = 8.5, H-2), 6.06 (d, 1 H, J4,3 = 3.5, H-4), 6.09 (s, 1 H, H-
13.0, J3
e,4
ꢀ
3ꢀ), 6.94 (dd, 1 H, J6 ,5 = 8.5, J6 ,8 = 2.5, H-6 ), 7.18–8.03 (m, 17 H,
H-5ꢀ, H-8ꢀ, Ar–H). 13C NMR (125 MHz, CDCl3) d: 18.9 (CH3),
21.0, 21.1 (2 × C), 21.3, 23.4, 29.9, 38.1 (C-3ꢀꢀ), 49.5 (C-5ꢀꢀ), 53.2
(–OCH3), 63.5 (C-6), 63.8 (C-9ꢀꢀ), 67.5 (C-7ꢀꢀ), 67.8 (C-8ꢀꢀ, C-4), 68.8
(C-4ꢀ), 69.8 (C-2), 71.9 (C-3), 72.6 (C-5), 73.1 (C-6ꢀꢀ), 98.9 (C-1),
100.1 (C-2ꢀꢀ), 104.4, 113.1, 114.4, 115.4, 125.7, 128.5, 128.6, 128.8,
129.3, 129.5, 129.8, 130.0 (2 × C), 130.2, 133.3, 133.5 (2 × C),
152.4, 155.3, 159.9, 161.2, 165.4, 165.5 (2 × C), 168.1, 170.3, 170.8,
171.2 (2 × C), 171.3. Anal. calcd for C57H57NO23: C, 60.9, H, 5.1,
N, 1.25; found: C, 60.7, H, 5.3, N, 1.5.
ꢀ
ꢀ
ꢀ ꢀ
H-6a, CH3), 3.86–3.89 (m, 2 H, H-6b, H-4), 3.95 (dd, 1 H, J5,6a
=
10.5, J5,6b = 6.5, H-5), 3.99–4.06 (m, 3 H, H-2, H-5ꢀꢀ, H-9ꢀꢀa), 4.12
(dd, 1 H, J6,“5” = 11.0, J6,“7” = 2.0, H-6ꢀꢀ), 4.25 (dd, 1 H, J3,2 = 12.5,
= 3.0, H-9ꢀꢀb),
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
b,8
J3,4 = 2.5, H-3), 4.29 (dd, 1 H, J9
= 13.0, J9
4.98 (ddd, 1 H, J4,“3”a = 12.0, J4,“5” =a10.0, J4,“3”e = 4.5, H-4ꢀꢀ), 5.22
b,9
ꢀꢀ
(d, 1 H, JNH,5 = 10.0, N–H), 5.24 (d, 1 H, J1,2 = 8.0, H-1), 5.34
(dd, 1 H, J7,“8” = 9.5, J7,“6” = 2.0, H-7ꢀꢀ), 5.49 (m, 1 H, H-8ꢀꢀ), 6.15
(d, 1 H, J3 ,Me = 1.0, H-3ꢀ), 7.03 (d, 1 H, J8 ,6 = 2.5, H-8ꢀ), 7.04 (dd,
ꢀ
ꢀ ꢀ
1 H, J6 ,5 = 9.0, J6 ,8 = 2.5, H-6ꢀ), 7.25–7.65 (m, 11 H, H-5ꢀ, Ar–
H). 13C NMR (125 MHz, CDCl3) d: 18.9, 19.4, 21.0 (2 × C), 21.1,
21.5, 23.4, 26.9, 27.0, 29.9, 38.2 (C-3ꢀꢀ), 49.7 (C-5ꢀꢀ), 53.5 (C-6), 62.6
(C-9ꢀꢀ), 63.0 (C-5), 67.1 (C-7ꢀꢀ), 68.1 (C-8ꢀꢀ, C-4), 68.7 (C-2), 68.9
(C-4ꢀꢀ), 72.9 (C-6ꢀꢀ), 74.2 (–OCH3), 77.0 (C-3), 97.6 (C-2ꢀꢀ), 100.2
(C-1), 104.3, 112.7, 114.2, 114.9, 125.6, 127.9, 128.0, 130.0, 130.1,
132.8, 133.0, 135.7, 135.8, 152.8, 155.0, 160.1, 161.5, 168.4 (C-1ꢀꢀ),
170.2, 170.4, 170.6, 170.8, 171.1. Anal. calcd for C52H63NO20Si: C,
59.5, H, 6.05, N, 1.3; found: C, 59.3, H, 5.9, N, 1.6.
ꢀ
ꢀ
ꢀ ꢀ
4-Methylumbelliferyl(5-acetamido-3,5-dideoxy-D-glycero-a-D-
galactonon-2-ulopyranosylonic acid]-(2→6)-b-D-
galactopyranoside (1)
To a solution of 10 (280 mg, 0.25 mmol) in dry methanol (10 cm3)
was added a methanolic sodium methoxide solution (25 cm3) and
stirred for 30 min at rt. Then the solution was neutralised by
adding Amberlite IR-120 (H+ form). The resulting solution was
filtered and the resin was washed several times with methanol. The
solutions were combined and evaporated to give a solid residue
that was subsequently dissolved in an aqueous solution of LiOH
(1 M, 15 cm3) and THF (15 cm3) that was maintained at 0 ◦C. The
resultant solution was stirred for 30 min at rt. The solution
was then neutralised by adding Amberlite IR-120 (H+ form) and
filtered. The resin was washed several times with methanol. The
solutions were combined and evaporated to give a solid residue
that was purified by flash column chromatography using ethyl
acetate: methanol–water (10 : 2 : 1). The fractions that contained
the product were combined and concentrated. The concentrated
aqueous solution was then lyophilised to obtain a solid. (120 mg,
4-Methylumbelliferyl(5-acetamido-3,5-dideoxy-D-glycero-a-D-
galactonon-2-ulopyranosylonic acid]-(2→3)-b-D-
galactopyranoside (2)
Compound 11 (250 mg, 0.24 mmol) was treated with sodium
methoxide in methanol and then with LiOH (H2O–THF) as
described above for the synthesis of 1. The crude product obtained
was purified by flash column chromatography using ethyl acetate–
methanol–water (10 : 2 : 1) as the eluent. The fractions that
contained the product were combined and concentrated. The
concentrated aqueous solution was then lyophilised to obtain a
solid (70 mg, 47%): mp 215 ◦C (decomp.). [a]D20 −14.5 (c 0.2, H2O),
1H NMR (500 MHz, D2O) d: 1.82 (t, 1 H, J3 a,3 + J3
= 25.0,
ꢀ
ꢀꢀ
ꢀꢀ ꢀꢀ
e
a,4
◦
1
76%): mp 205 C (decomp). [a]2D0 −80.8 (c 0.2, H2O), H NMR
H-3ꢀꢀa), 2.01 (s, 1 H, CH3), 2.41 (d, 3 H, JMe,3 = 1.0, CH3), 2.78 (dd,
ꢀ
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
a,4
(500 MHz, D2O) d: 1.68 (t, 1 H, J3
= J3
= 12.0, H-3ꢀꢀa),
a,3
e
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
e,4
1 H, J3
= 12.5, J3
= 5.0, H-3ꢀꢀe), 3.57–3.62 (m, 2 H, H-8ꢀꢀ,
e,3
a
ꢀ
1.98 (s, 1 H, CH3), 2.39 (d, 3 H, JMe,3 = 1.0, CH3), 2.78 (dd, 1 H,
H-9ꢀꢀa), 3.64 (dd, 1 H, J7 = 10.5, J7 = 2.0, H-7ꢀꢀ), 3.69 (ddd,
ꢀꢀ ꢀꢀ
,8
ꢀꢀ ꢀꢀ
,6
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
e,4
J3
1 H, J9
= 12.0, J3
= 4.0, H-3ꢀꢀe), 3.49 (dd, 1 H, H-8ꢀꢀ), 3.57 (dd,
e,3
a
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
,3 e
1 H, J4
= 12.5, J4 = 10.0, J4
= 4.5, H-4ꢀꢀ), 3.77 (m, 2 H,
,3
a
,5
= 12.5, J9
= 6.5, H-9ꢀꢀa), 3.63–3.72 (m, 4 H, H-4,
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
a,9
b
a,8
H-6a, H-6b), 3.81–3.93 (m, 5 H, H-2, H-5, H-5ꢀꢀ, H-6ꢀꢀ, H-9ꢀꢀb),
“H-6”, H-5, H-6a), 3.78–3.85 (m, 4 H, H-9ꢀꢀb, H-7, “H-2, H-3),
4.04 (d, 1 H, J4,3 = 3.0, H-4), 4.24 (dd, 1 H, J3,2 = 9.5, J3,4 = 3.0,
3.95–4.02 (m, 3 H, H-6b, H-4, H-5”), 5.10 (d, 1 H, J1,2 = 7.0, H-1),
H-3), 5.24 (d, 1 H, J1,2 = 8.0, H-1), 6.21 (d, 1 H, J3 ,Me = 1.0, H-3ꢀ),
ꢀ
6.17 (d, 1 H, J3 ,Me = 1.0, H-3ꢀ), 7.06 (d, 1 H, J8 ,6 = 2.5, H-8ꢀ),
ꢀ
ꢀ ꢀ
7.07 (d, 1 H, J8 = 2.5, H-8ꢀ), 7.11 (dd, 1 H, J6 ,5 = 9.0, J6 ,8
=
ꢀꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
,6
7.08 (dd, 1 H, J6 ,5 = 9.0, J6 ,8 = 2.5, H-6ꢀ), 7.66 (d, 1 H, J5 ,6
=
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
2.5, H-6ꢀ), 7.71 (d, 1 H, J5 ,6 = 9.0, H-5ꢀ). 13C NMR (125 MHz,
D2O) d: 18.0 (CH3), 22.2 (CH3), 39.8 (C-3ꢀꢀ), 51.9 (C-5ꢀꢀ), 60.8 (C-6),
62.6 (C-9ꢀꢀ), 67.4 (C-4), 68.1 (C-8ꢀꢀ), 68.5 (C-4ꢀꢀ), 68.9 (C-2), 71.9
(C-6ꢀꢀ), 73.0 (C-7ꢀꢀ), 75.4 (C-5), 75.7 (C-3), 100.1 (C-1, C-2ꢀꢀ), 103.3
(C-8ꢀ), 111.0 (C-3ꢀ), 114.1 (C-6ꢀ), 114.7 (C-4ꢀ), 126.5 (C-5ꢀ), 153.5
ꢀ
ꢀ
9.0, H-5ꢀ). 13C NMR (125 MHz, D2O) d: 18.2 (CH3), 22.1 (CH3),
40.5 (C-3“), 52.0 (C-4”), 62.7 (C-9ꢀꢀ), 63.1 (C-6), 68.3 (C-8,“C-5),
68.5 (C-5”), 70.5 (C-3), 71.7 (C-7ꢀꢀ), 72.5 (C-2), 72.7 (C-6ꢀꢀ), 74.1
(C-4), 100.4 (C-1, C-2ꢀꢀ), 103.6 (C-8ꢀ), 111.1 (C-3ꢀ), 114.1 (C-6ꢀ),
114.9 (C-4ꢀ), 126.6 (C-5ꢀ), 153.6 (C-9ꢀ), 156.0 (C-10ꢀ), 159.7 (C-7ꢀ),
(C-10ꢀ), 155.9 (C-9ꢀ), 159.5 (C-7ꢀ), 164.1 (C-2ꢀ), 174.1 (C-1ꢀꢀ),175.1
ꢀ
ꢀꢀ
=
164.2 (C-2 ), 173.4 (C-1 ), 175.1 (C O, amide). HRMS (FAB) m/z
+
=
(C O, amide). HRMS (FAB) m/z (M–H ), C27H34NO16 requires
(M–H+), C27H34NO16 requires 628.1883, found 628.1891.
628.1883, found 628.1872.
4-Methylumbelliferyl[methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-D-glycero-a-D-galactonon-2-ulopyranosyl)onate]-
(2→3)-6-O-(tert-butyldiphenylsilyl)-b-D-galactopyranoside (11)
Kinetic protocols
Michaelis–Menten parameters were measured under identical
conditions for both wild-type and the Y370G mutant. Each
0.4 cm3 reaction was performed at 37 ◦C by equilibrating the buffer,
Donor 926 (500 mg, 0.84 mmol) and the acceptor 6 (726 mg,
1.26 mmol) were coupled and purified as described in the synthesis
4458 | Org. Biomol. Chem., 2006, 4, 4453–4459
This journal is
The Royal Society of Chemistry 2006
©