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(
200 mL), centrifuged (3 min, 48C) and the clear supernatant was
25-H), 3.91 (d, J=4.2 Hz, 1H, 23-OH), 3.71 (s, 1H, 21-OH), 3.49 (d,
J=9.5 Hz, 1H, 21-H), 3.01 (dd, J=9.0, 3.4 Hz, 1H, 27-H), 2.96 (ddd,
J=10.1, 4.3, 1.7 Hz, 1H, 23-H), 2.78 (s, 3H, 37-H), 2.37 s, 3H, 14-H),
screened by UPLC-ESI-MS.
2
1
1
.28–2.24 (m, 1H, 20-H), 1.90–1.87 (m, 1H, 26-H), 1.79 (s, 3H, 30-H),
.60 (s, 3H, 13-H), 1.59 (s, 3H, 36-H), 1.50 (m, 1H, 22-H), 1.47 (m,
H, 24-H), 0.99 (d, J=7.0 Hz, 3H, 32-H), 0.64 (d, J=7.3 Hz, 3H, 31-
Mutasynthetic experiments
Rifamycin W 1a: Rifamycin W 1a was obtained as a yellow solid
after supplementing a culture of the blocked mutant of A. mediter-
ranei (strain HGF003) with 3-amino-5-hydroxybenzoic acid 5 (fed
H), 0.45 (d, J=7.1 Hz, 3H, 33-H), 0.23 ppm (d, J=7.1 Hz, 3H, 34-H);
13
C NMR (125 MHz, C D , C D =128.06 ppm): d=190.7 (s, C11),
6
6
6
6
5
0 mg, 0.33 mmol per 125 mL; 10 mg per plate) carried out on
1
85.4 (s, C1), 181.8 (s, C4), 173.5 (s, C35), 172.5 (s, C6), 169.2 (s,
C15), 167.0 (s, C8), 146.3 (d, C29), 142.5 (d, C19), 139.8 (s, C2), 133.1
d, C17), 131.5 (s, C16), 131.5 (s, C10), 124.7 (d, C18), 117.1 (d, C3),
15.2 (d, C28), 115.1 (s, C7), 111.5 (s, C5), 111.4 (s, C9), 109.1 (s, C12),
2.8 (d, C27), 78.0 (d, C23), 73.9 (d, C21), 73.9 (d, C25), 56.3 (q,
C37), 40.4 (d, C20), 37.7 (d, C26), 37.4 (d, C24), 33.1 (d, C22), 22.8
À1
agar plates (isolated amount=1.6 mg; 12.8 mgL ). 1st HPLC:
preparative HPLC (C18): solvent A=water+0.1% v/v formic acid,
solvent B=MeOH+0.1% v/v
1
9
(
1
8
formic
acid;
flow
rate=
À1
5.0 mLmin ; gradient (t [min]/solvent B [%]): 0/20; 5/20; 50/55;
0/100; 100/100; t =67.0 min. 2nd HPLC: semi-preparative HPLC
R
(
2
9
C18-ISIS): solvent A=water, solvent B=MeOH; flow rate=
.5 mLmin ; gradient (t [min]/solvent B [%]): 0/20; 5/20; 70/55;
(
(
q, C13), 20.6 (q, C36), 20.3 (q, C30), 16.6 (q, C31), 12.4 (q, C34), 11.8
À1
q, C32), 8.9 (q, C33), 7.5 ppm (q, C14); UPLC-MS (MeOH); t =
R
+
0/100; 100/100; t =74.0 min. The analytical data are in accord-
R
3.0 min; HRMS-ESI: m/z calcd for C H NO : 696.3020 [M+H] ;
found: 696.3022.
37
46
12
[6,26] 1
ance with those reported previously:
H NMR (500 MHz, CD OD,
3
CHD OD=4.87 ppm): d=8.40 (bs, 1H, 2-NH), 7.57 (s, 1H, 3-H), 6.50
2
Rifamycin B 1c: Rifamycin B 1c was obtained as an orange solid
after supplementing a culture of the blocked mutant of A. mediter-
(
(
(
dd, J=16.0, 11.0 Hz, 1H, 18-H), 6.41 (d, J=10.0 Hz, 1H, 29-H), 6.25
d, J=11.0 Hz, 1H, 17-H), 6.08 (dd, J=16.0, 6.7 Hz, 1H, 19-H), 4.61
bs, 1H, 6-OH), 4.39 (bs, 1H, 8-OH), 4.04 (dd, J=10.0, 1.0 Hz, 1H,
ranei (strain HGF003) with 3-hydroxy-5-nitrobenzoic acid 48 (fed
À1
2
29 mg, 1.25 mmolL ; HRMS, LC-MS/MS), 3-azido-5-hydroxybenzo-
2
1
6
7
1-H), 4.01 (dd, J=10.0, 1.0 Hz, 1H, 23-H), 3.58 (dd, J=10.9, 7.9 Hz,
H, 34a-H), 3.48 (dd, J=10.0, 2.0 Hz, 1H, 25-H), 3.44 (dd, J=10.8,
.5 Hz, 1H, 34b-H), 3.21–3.13 (m, 1H, 28-H), 2.63 (dd, J=16.6,
.3 Hz, 1H, 27-H), 2.38–2.32 (m, 1H, 20-H), 2.16 (s, 3H, 14-H), 2.09
ic acid 47 (fed 168 mg, 0.94 mmol per 750 mL; isolated amount=
À1
1
1
.5 mg; 2 mgL
)
and 3-amino-5-hydroxybenzoic acid
5
(fed
À1
À1
91 mg, 1.25 mmolL ; isolated amount=20 mgL ). 1st HPLC:
preparative HPLC (C18): solvent A=water+0.1% v/v formic acid,
(
bs, 3H, 13-H), 2.08 (bs, 3H, 30-H), 1.88–1.85 (m, 1H, 22-H), 1.82–
solvent B=MeOH+0.1% v/v
formic
acid;
flow
rate=
1
3
3
.79 (m, 1H, 24-H), 1.44–1.38 (m, 1H, 26-H), 1.06 (d, J=7.1 Hz, 3H,
3-H or 34-H), 0.92 (d, J=7.0 Hz, 3H, 31-H), 0.73 (d, J=6.8 Hz, 3H,
3-H or 34-H), 0.43 ppm (d, J=7.0 Hz, 3H, 32-H); C NMR
À1
1
9
5.0 mLmin ; gradient (t [min]/solvent B [%]): 0/20; 5/20; 50/55;
0/100; 100/100; t =82.0 min. 2nd HPLC: preparative HPLC (C18):
13
R
À1
solvent A=water, solvent B=MeOH; flow rate=15.0 mLmin ; gra-
(
125 MHz, CD OD, CD OD=49.0 ppm): d=201.2 (s, C11), 187.1 (s,
3 3
dient (t [min]/solvent B [%]): 0/40; 5/40; 20/50; 50/50; 100/100; t =
R
C4), 183.4 (s, C1), 172.2 (s, C15), 164.7 (s, C8), 164.1 (s, C6), 142.6 (d,
C19), 141.5 (d, C29), 140.9 (s, C12), 140.8 (s, C2), 135.2 (d, C17),
7
5.0 min. 3rd HPLC: preparative HPLC (C18): solvent A=water, sol-
À1
vent B=MeOH; flow rate=15.0 mLmin ; gradient (t [min]/sol-
1
32.1 (s, C16), 129.9 (s, C10), 126.3 (d, C18), 122.2 (s, C5), 119.0 (s,
vent B [%]): 0/10; 5/10; 85/100; 100/100; t =69.0 min. UPLC-MS
R
C7), 118.0 (d, C3), 107.5 (s, C9), 79.0 (d, C27), 74.8 (d, C23), 71.0 (d,
C21), 69.1 (d, C25), 64.5 (t, C34a), 49.6 (d, C28), 44.1 (d, C26), 39.1
(
MeOH) t =3.2 min; HRMS-ESI: m/z calcd for C H NO : 756.3231
R 39 50 14
+
[M+H] ; found: 756.3228.
(
1
d, C20), 37.9 (d, C24), 34.3 (d, C22), 20.2 (q, C30), 18.0 (q, C13),
2.7 (q, C34), 11.7 (q, C32), 11.2 (q, C31), 8.9 (q, C33), 8.5 ppm (q,
Tetraketide 18: Tetraketide 18 was obtained after supplementing
a culture of the blocked mutant of A. mediterranei (strain HGF003)
C14); UPLC-MS (MeOH) t =2.9 min; HRMS-ESI: m/z calcd for
R
À1
+
with 3-hydroxybenzoic acid 14 (fed 173 mg, 1.25 mmolL ). The
C H NO : 656.3071 [M+H] ; found: 656.3068.
35
46
11
fermentation was carried out in liquid medium providing tetrake-
tide 18 as a colorless foam (isolated amount=118.0 mg). HPLC:
preparative HPLC (C18): solvent A=water+0.1% v/v formic acid,
Rifamycin S 1b: Rifamycin S 1b was obtained as a yellow solid
after supplementing a culture of the blocked mutant of A. mediter-
ranei (strain HGF003) with 3-hydroxy-5-nitrobenzoic acid 48 (fed
À1
solvent B=MeOH+0.1% v/v
formic
acid;
flow
rate=
2
29 mg, 1.25 mmolL ; HRMS), 3-azido-5-hydroxybenzoic acid 47
À1
1
5.0 mLmin ; gradient (t [min]/solvent B [%]): 0/20; 5/20; 50/55;
(
1
1
fed 168 mg, 0.94 mmol per 750 mL; isolated amount=1.0 mg;
À1
90/100; 100/100; t =44.0 min. The analytical data are in accord-
R
.3 mgL ) and 3-amino-5-hydroxybenzoic acid 5 (fed 191 mg,
[5] 1
À1
À1
ance with those reported previously: H NMR (400 MHz, CD OD,
3
.25 mmolL ; isolated amount=1.1 mgL ). Additionally, fermen-
CD HOD=3.31 ppm): d=7.15 (t, J=8.1 Hz, 1H, H-12), 6.82–6.76
2
tation with AHBA 5 on agar plates (fed 50 mg, 0.33 mmol per
À1
(m, 2H, H-11 or H-13, H-9), 6.73–6.66 (m, 1H, H-11 or H-13), 6.06 (s,
1
1
25 mL; 10 g/plate) yielded 0.3 mg of rifamycin S 1b (2.4 mgL ).
st HPLC: preparative HPLC (C18): solvent A=water+0.1% v/v
1
H, H-4), 4.67 (d, J=8.8 Hz, 1H, H-7), 2.83 (dq, J=14.1, 7.1 Hz, 1H,
1
H-6), 1.88 (s, 3H, H-14), 0.98 ppm (d, J=7.0 Hz, 3H, H-15); H NMR
(400 MHz, [D ]THF, [D ]THF=1.73 ppm): d=7.06 (t, J=7.8 Hz, 1H,
formic acid, solvent B=MeOH+0.1% v/v formic acid; flow rate=
À1
8
8
1
9
5.0 mLmin ; gradient (t [min]/solvent B [%]): 0/20; 5/20; 50/55;
H-12), 6.75–6.73 (m, 2H, H-11 or H-13, H-9), 6.62 (ddd, J=8.0, 2.3,
.0 Hz, 1H, H-11 or H-13), 5.85 (s, 1H, H-4), 4.64 (d, J=8.4 Hz, 1H,
H-7), 2.71 (dq, J=15.0, 7.1 Hz, 1H, H-6), 1.83 (s, 3H, H-14),
0/100; 100/100; t =77.0 min. 2nd HPLC: semi-preparative HPLC
R
1
(
2
9
C18-ISIS): solvent A=water, solvent B=MeOH; flow rate=
.5 mLmin ; gradient (t [min]/solvent B [%]): 0/20; 5/20; 70/55;
À1
13
0
.93 ppm (d, J=7.1 Hz, 3H, H-15); C NMR (100 MHz, [D ]THF,
8
0/100; 100/100; t =84.0 min. 3rd HPLC: semi-preparative HPLC
R
[
D ]THF=25.5 ppm): d=166.0 (s, C3), 165.4 (s, C5), 165.2 (s, C1),
8
(
2
C18-ISIS): solvent A=water, solvent B=MeOH; flow rate=
.5 mLmin ; gradient (t [min]/solvent B [%]): 0/20; 5/20; 50/60;
À1
158.6 (s, C10), 146.0 (s, C8), 129.6 (d, C12), 118.6 (d, C13), 115.2 (d,
C11), 114.6 (d, C9), 100.9 (s, C2),98.7 (d, C4), 76.2 (d, C7), 47.4 (d,
1
00/100; t =68.0 min. The analytical data are in accordance with
R
[27]
1
C6), 15.3 (q, C15), 8.7 ppm (q, C14); UPLC-MS (MeOH) t =2.4 min;
R
those reported previously:
H NMR (500 MHz, C D , C D H=
6 6 6 5
+
HRMS-ESI: m/z calcd for C H O : 277.1076 [M+H] ; found:
15
17
5
7
.16 ppm): d=12.73 (s, 1H, 8-OH), 8.34 (s, 1H, 3-H), 8.29 (s, 1H, 2-
2
77.1075.
NH), 6.34 (d, J=12.6 Hz, 1H, 29-H), 6.28 (dd, J=15.7, 8.3 Hz, 1H,
1
1
8-H), 5.86 (d, J=8.3 Hz, 1H, 17-H), 5.77 (dd, J=15.7, 8.3 Hz, 1H,
9-H), 5.27 (dd, J=12.6, 8.6 Hz, 1H, 28-H), 4.63 (d, J=1.5 Hz, 1H,
Tetraketide 19: Tetraketide 19 was obtained after supplementing
a culture of the blocked mutant of A. mediterranei (strain HGF003)
Chem. Eur. J. 2015, 21, 19231 – 19242
19238
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim