2448
G.-L. Huang et al. / Bioorg. Med. Chem. 14 (2006) 2446–2449
II
III
IV
3
.3. Bacterial strains, plasmids, and growth conditions
(CH CH@CH ), 102.44, 102.23 (C-1 , C-1 , C-1 ,
2
V
2
C-1 ), 102.0 (b-C-1), 80.65, 80.20, 79.95, 76.95, 75.51,
74.49, 73.46, 73.07, 71.04, 70.71, 70.25, 61.55, 61.00,
The E. coli strain BL21(DE3) containing a plasmid car-
1
2
I–V
56.58, 56.08, 54.68 (C-2 , C-3 , C-4 , C-5 , C-
I–V
I–V
I–V
rying the cloned nodC gene from M. loti strain E1R
was used as the source of NodC protein. Routine cul-
I–V
I–V
6a , C-6b ), 70.9 (CH CH@CH ), 23.11, 22.90
2
2
1
3
1
tures were grown in LB medium. Cell density cultures
1
(CH CH NHAc). H NMR (300 MHz, D O): d 6.27
3 3 2
1
were carried out as previously described in 2 or 10 L
bioreactors containing an initial culture volume of 1 or
(d, 1H, J1,2 7.9 Hz, a-H-1), 6.00–5.77 (m, 1H,
CH CH@CH ), 5.37–5.04 (m, 2H, CH CH@CH ),
4.47 (m, 4H, H-1 , H-1 , H-1 , H-1 ), 4.30–3.99 (m,
2
2
2
2
II
III
IV
V
7 L, respectively. The culture medium was slightly mod-
ified and had the following composition: 2 (1 g/L), glyc-
2H, CH CH@CH ), 3.90–3.30 (m, 46H), 1.97, 1.95 (s,
2
2
+
15H, CH NHAc). ESIMS: m/z 1096.4 [M+Na] . Anal.
erol (15 g/L), NH H PO (7 g/L), KH PO (7 g/L),
4
4
2
4
2
3
MgSO Æ7H O (1 g/L), thiamineÆHCl (4.5 mg/L), trace
Calcd for C H N O : C, 48.09; H, 6.66; N, 6.52.
43 71
Found: C, 48.10; H, 6.29; N, 6.48.
4
2
5
26
mineral solution (7.5 mL/L), citric acid (0.5 g/L), and
KOH (2 g/L). MgSO was added from a concentrated
4
solution that was autoclaved separately. Thiamine was
sterilized by filtration. The trace mineral stock solution
contained: nitrilotriacetate (70 mmol/L, pH 6.5), ferric
citrate (7.5 g/L), MnCl Æ4H O (1.3 g/L), CoCl Æ6H O
3.5. Synthesis of the target compound 5
NaIO (9 mg) was added to a solution of 3 (40 mg) in
4
100 mmol/L AcONa–AcOH (pH 5.5, 1.8 mL) and stir-
red for 2 h. After adjusting the solution pH to 9.0,
NaBH (5 mg) was added to the reaction solution and
2
2
2
2
(
0.21 g/L), CuCl Æ2H O (0.13 g/L), H BO (0.25 g/L),
2 2 3 3
ZnSO Æ7H O (1.2 g/L), and Na MoO Æ2H O (0.15 g/L).
4
2
2
4
2
4
For the cultivation of strain BL21(DE3), the medium
was supplemented with leucine (1 g/L). Antibiotic ampi-
cillin was used to ensure maintenance of the plasmid and
was prepared in concentrated stock solution as de-
further stirred for 2 h. Then 5 mL concentrated NH3-
H O was added and stirred for 1h again. All reactions
went along at room temperature. The reaction solution
was purified by Biogel P2 to afford 26 mg of the target
2
1
3
scribed by Sambrook et al. Its final concentration
was 50 mg/L for ampicillin. Unless otherwise indicated,
the feeding solution contained: glycerol (450 g/L),
MgSO Æ7H O (12 g/L), and trace mineral solution
compound 5 in total 65% yield. [a] +63.5 (c 1.1,
D
1
3
H O). C NMR (75 MHz, D O): d 175.57, 175.46
2
2
(C@O
NHAc),
135.3
(CH CH@CH ),
117.4
2
2
II
III
IV
(CH CH@CH ), 102.44, 102.20 (C-1 , C-1 , C-1 ,
4
2
2
V
2
(
25 mL/L).
C-1 ), 102.1 (b-C-1), 80.60, 80.20, 79.95, 77.01, 75.50,
4.49, 73.43, 73.00, 70.84, 70.56, 70.25, 61.73, 61.00,
56.83, 56.10, 54.68 (C-2 , C-3 , C-4 , C-5 , C-
7
I–V
I–V
I–V
I–V
Cell density cultures were inoculated at 2% (v/v) with a
culture grown in LB medium. Throughout the cultiva-
tion, the dissolved oxygen was maintained at 20% of
air saturation by manually increasing the air flow rate
and automatically adjusting the stirrer speed, the pH
was regulated at 6.8 by automatic addition of aqueous
I–V
I–V
6a , C-6b ), 70.6 (CH CH@CH ), 23.10, 22.87
2
2
1
(CH CH NHAc). H NMR (300 MHz, D O): d 6.26
3
3
2
(d, 1H, J1,2 7.9 Hz, a-H-1), 6.00–5.78 (m, 1H,
CH CH@CH ), 5.37–5.04 (m, 2H, CH CH@CH ),
4.45 (m, 4H, H-1 , H-1 , H-1 , H-1 ), 4.31–3.99 (m,
2
2
2
2
II
III
IV
V
NH (15% w/v), and the temperature was maintained
3
2H, CH CH@CH ), 3.85–3.29 (m, 47H), 1.95, 1.93 (s,
2
2
+
15H, CH NHAc). ESIMS: m/z 1079.5 [M+Na] . Anal.
at 34 ꢁC. After consumption of the initial glycerol, indi-
cated by a sudden increase in the dissolved oxygen level,
the feeding was started with an initial flow rate of 9 mL/
h/L. After 5 h of cultivation, the feeding rate was low-
ered to 4.8 mL/h/L and kept constant until the end of
the culture.
3
Calcd for C H N O : C, 48.86; H, 6.87; N, 7.95.
4
3
72
6
24
Found: C, 48.79; H, 6.53; N, 8.00.
3.6. Assay for chitinase activity
The chitinase activity was measured by determining the
amount of reducing sugar equivalents released on incu-
bation of the enzyme with the substrate 3 under the con-
dition stated. Assays were terminated by heating to
100 ꢁC, and the release of reducing sugars was shown
to be linear with respect to time and amount of enzyme
used. The chitinase (40 lg/mL), BSA (0.8 mg/mL), glyc-
erol 10% (v/v), and 5 (20 mmol/L) were incubated at
18 ꢁC in 40 mmol/L sodium acetate buffer at pH 5.0.
The residual activity was determined at 100 min
intervals.
3
.4. Purification of compound 3
After being harvested by centrifugation (20 min at
2,000g), bacterial cells were resuspended in a volume
1
of distilled water equal to that of the original culture
medium and disrupted by boiling for 45 min. After cool-
ing, 10 lL concentrated HCl was added. Cell debris and
precipitated proteins were eliminated by centrifugation
(
30 min at 12,000g) and the 1 L supernatant was mixed
with an equal quantity (125 g) of activated charcoal
Norit) and Celite. The slurry was filtered on Whatman
(
No. 4 paper and washed thoroughly with distilled water
to remove the salts. The adsorbed oligosaccharides were
eluted with 1.3 L of 55% (v/v) aqueous ethanol. This last
fraction was loaded onto a 100 · 1-cm column of Biogel
P2. The flowthrough fraction was recovered and lyoph-
References and notes
1
. Tokoro, A.; Tatewaki, N.; Suzuki, K.; Mikami, T.;
Suzuki, S.; Suzuki, M. Chem. Pharm. Bull. 1988, 36,
784–790.
2. Kendra, D. F.; Hadwiger, L. A. Exp. Mycol. 1984, 8, 276–
281.
ilized. The 1.8 g 3 was obtained in 65% yield. [a] +57.8
D
1
3
(
c 1.1, H O). C NMR (75 MHz, D O): d 175.57,
2
2
1
75.48 (C@O NHAc), 135.5 (CH CH@CH ), 117.4
2
2