3842
X. Cai et al. / Bioorg. Med. Chem. 19 (2011) 3831–3844
3.84–3.98 (m, 1H), 4.23 (d, 1H, J = 5.6 Hz), 4.29 (t, 1H, J = 7.2), 7.33
(s, 1H), 8.05 (s, 1H), 8.17 (s, 1H) and 8.66 (s, 1H); mass spectrum
(MALDI-TOF), m/z 1218.5 (M+Na)+ (theoretical 1218.6); mass
spectrum (ESI), m/z 1196.6289 (M+H)+ (C53H86N19O9S2 requires
1196.6297).
3.49 (m, 2H), 3.41–3.56 (m, 3H), 3.67–3.69 (m, 2H), 3.93 (t, 1H,
J = 5.6 Hz), 4.11 (d, 1H, J = 4.8 Hz), 7.21 (s, 1H), 7.93 (s, 1H), 8.08
(s, 1H) and 8.53 (s, 1H); mass spectrum (MALDI-TOF), m/z 1178.4
(M+Na)+ (theoretical 1178.6); mass spectrum (ESI), m/z 1156.
5995 (M+H)+ (C50H82N19O9S2 requires 1156.5984).
5.1.3.5. Deglycobleomycin analogue 6.
HPLC retention time:
5.1.3.10. Deglycobleomycin analogue 11.
HPLC retention
14.7 min; colorless solid; yield 1.4 mg (47%); 1H NMR (D2O) d 0.97
(d, 3H, J = 6.4 Hz), 1.07 (d, 3H, J = 7.2 Hz), 1.63–1.64 (m, 4H),
1.84–1.95 (m, 7H), 2.35 (t, 1H, J = 12.0 Hz), 2.56–2.60 (m, 3H),
2.73–2.76 (m, 2H), 2.94–3.01 (m, 14H), 3.15 (t, 2H, J = 5.6 Hz),
3.32–3.38 (m, 4H), 3.53 (t, 1H, J = 6.4 Hz), 3.66 (t, 1H, J = 5.6 Hz),
3.96–4.03 (m, 4H), 4.19 (d, 1H, J = 5.6 Hz), 4.55 (t, 1H, J = 6.4 Hz),
6.73 (s, 1H), 6.98 (d, 1H, J = 8.0 Hz), 7.02 (t, 1H, J = 7.2 Hz), 7.08
(d, 1H, J = 8.0 Hz), 7.91 (s, 1H), 8.06 (s, 1H) and 8.38 (s, 1H); mass
spectrum (MALDI-TOF), m/z 1212.4 (M+Na)+ (theoretical 1212.6);
mass spectrum (ESI), m/z 1190.5833 (M+H)+ (C53H80N19O9S2 re-
quires 1190.5828).
time: 17.2 min; colorless solid; yield 1.0 mg (30%); 1H NMR (D2O)
d 0.53 (d, 3H, J = 6.4 Hz), 0.93 (d, 3H, J = 6.4 Hz), 1.63–1.64 (m, 4H),
1.86–1.94 (m, 7H), 2.52–2.59 (m, 2H), 2.61 (t, 1H, J = 4.8 Hz), 2.69–
2.72 (m, 1H), 2.82–2.86 (m, 1H), 2.93–3.01 (m, 14H), 3.09–3.14 (m,
3H), 3.17–3.20 (m, 2H), 3.27–3.49 (m, 4H), 3.66–3.68 (m, 2H), 3.78
(t, 1H, J = 6.4 Hz), 3.89 (d, 1H, J = 4.8 Hz), 6.95 (d, 2H, J = 7.2 Hz),
6.99–7.00 (m, 3H), 7.23 (s, 1H), 7.92 (s, 1H), 8.07 (s, 1H) and 8.54
(s, 1H); mass spectrum (MALDI-TOF), m/z 1212.4 (M+Na)+ (theoret-
ical 1212.6); mass spectrum (ESI), m/z 1190.5823 (M+H)+
(C53H80N19O9S2 requires 1190.5828).
5.1.3.11. Deglycobleomycin analogue 12.
HPLC retention
5.1.3.6. Deglycobleomycin analogue 7.
HPLC retention time:
time: 16.4 min; colorless solid; yield 1.2 mg (36%); 1H NMR (D2O)
d 0.53 (d, 3H, J = 7.2 Hz), 0.59 (d, 3H, J = 6.4 Hz), 0.65–0.66 (m,
9H), 0.91–0.92 (m, 5H), 1.15–1.25 (m, 4H), 1.40–1.42 (m, 1H),
1.63–1.64 (m, 2H), 1.88–1.95 (m, 7H), 2.57–2.61 (m, 3H), 2.93–
3.01 (m, 12H), 3.07–3.11 (m, 1H), 3.17–3.20 (m, 2H), 3.38–3.40
(m, 2H), 3.44–3.50 (m, 2H), 3.57–3.60 (m, 1H), 3.65–3.67 (m, 1H),
3.89 (t, 1H, J = 6.4 Hz), 3.92–3.93 (m, 1H), 3.99–4.02 (m, 1H), 4.15
(d, 1H, J = 4.8 Hz), 7.23 (s, 1H), 7.97 (s, 1H), 8.09 (s, 1H) and 8.57
(s, 1H); mass spectrum (MALDI-TOF), m/z 1220.5 (M+Na)+ (theoret-
ical 1220.6); mass spectrum (ESI), m/z 1198.6442 (M+H)+
(C53H88N19O9S2 requires 1198.6454).
19.0 min; colorless solid; yield 1.2 mg (27%); 1H NMR (D2O) d 0.99
(d, 3H, J = 7.2 Hz), 1.09 (d, 3H, J = 7.2 Hz), 1.63–1.64 (m, 4H),
1.83–1.95 (m, 7H), 2.40 (t, 1H, J = 12.0 Hz), 2.53–2.61 (m, 3H),
2.66–2.69 (m, 1H), 2.94–3.01 (m, 14H), 3.15–3.20 (m, 2H), 3.31–
3.36 (m, 2H), 3.50–3.53 (m, 1H), 3.63–3.67 (m, 2H), 3.84–3.85
(m, 1H), 3.93–3.94 (m, 1H), 4.01–4.04 (m, 1H), 4.22 (d, 1H,
J = 4.8 Hz), 4.23–4.25 (m, 1H), 4.55 (t, 1H, J = 6.4 Hz), 6.82 (s, 1H),
6.87 (t, 1H, J = 8.0 Hz), 6.96 (d, 1H, J = 7.2 Hz), 7.01 (t, 1H,
J = 7.2 Hz), 7.05 (d, 1H, J = 8.0 Hz), 7.87 (s, 1H), 8.01 (s, 1H) and
8.43 (s, 1H); mass spectrum (MALDI-TOF), m/z 1290.7 and 1292.0
(M+Na)+ (theoretical 1290.5 and 1292.5); mass spectrum (ESI),
m/z 1268.4986 and 1270.4956 (M+H)+ (C53H79N19O9S2Br requires
1268.4933 and 1270.4913).
5.2. Biochemical evaluation
5.2.1. Materials and methods
5.1.3.7. Deglycobleomycin analogue 8.
HPLC retention time:
pBR322 plasmid DNA, and restriction endonucleases HindIII and
EcoRV, and T4 polynucleotide kinase were purchased from New
England Biolabs. Thermosensitive alkaline phosphatase and Gene-
15.5 min; colorless solid; yield 1.5 mg (47%); 1H NMR (D2O) d 0.98
(d, 3H, J = 6.4 Hz), 1.08 (d, 3H, J = 7.2 Hz), 1.63–1.64 (m, 4H),
1.85–1.95 (m, 7H), 2.00 (s, 3H), 2.33 (t, 1H, J = 12.0 Hz), 2.58–2.66
(m, 4H), 2.74 (dd, 1H, J = 9.6 and 5.6 Hz), 2.80 (dd, 1H, J = 11.2
and 3.2 Hz), 2.94–2.97 (m, 12H), 3.00 (t, 2H, J = 8.0 Hz), 3.31–3.37
(m, 2H), 3.51–3.59 (m, 2H), 3.67 (t, 1H, J = 7.2 Hz), 3.99 (t, 1H,
J = 5.6 Hz), 4.04–4.07 (m, 2H), 4.08–4.11 (m, 1H), 4.20 (d, 1H,
J = 4.8 Hz), 4.55 (t, 1H, J = 7.2 Hz), 6.72 (s, 1H), 6.84–6.87 (m, 2H),
6.90 (d, 2H, J = 7.2 Hz), 7.90 (s, 1H), 8.03 (s, 1H) and 8.39 (s, 1H);
mass spectrum (MALDI-TOF), m/z 1226.6 (M+Na)+ (theoretical
1226.6); mass spectrum (ESI), m/z 1204.5968 (M+H)+
(C54H82N19O9S2 requires 1204.5984).
Jet PCR purification kit were purchased from Fermentas. [c-
32P]ATP
was purchased from Perkin Elmer. Fe(NH4)2(SO4)2ꢀH2O was pur-
chased from Sigma Aldrich and used to prepare Fe2+ solutions
immediately prior to use. Chelex 100 was purchased from Sigma
Aldrich and used to remove adventitious Fe2+ from solutions prior
to experiments. The hairpin DNA was purchased from Integrated
DNA Technologies, Inc.
5.2.2. Cleavage of supercoiled pBR322 plasmid DNA by valerate-
modified deglycoBLM A6 analogues
Two hundred nanograms of supercoiled pBR322 plasmid DNA
was treated with the appropriate concentration of freshly prepared
5.1.3.8. Deglycobleomycin analogue 9.
HPLC retention time:
16.7 min; colorless solid; yield 1.8 mg (56%); 1H NMR (D2O) d 0.61
(t, 3H, J = 7.2 Hz), 0.89 (d, 3H, J = 7.2 Hz), 0.96 (d, 3H, J = 5.6 Hz),
1.03–1.06 (m, 2H), 1.38–1.40 (m, 2H), 1.53–1.54 (m, 2H), 1.64
(m, 2H), 1.86–1.95 (m, 8H), 2.35–2.36 (m, 1H), 2.60–2.64
(m, 3H), 2.94–3.01 (m, 14H), 3.09–3.18 (m, 4H), 3.37–3.41 (m,
3H), 3.52–3.56 (m, 4H), 4.02 (t, 1H, J = 5.6 Hz), 4.11 (d, 1H,
J = 4.0 Hz), 7.21 (s, 1H), 7.92 (s, 1H), 8.08 (s, 1H) and 8.53 (s, 1H);
mass spectrum (MALDI-TOF), m/z 1178.0 (M+Na)+ (theoretical
1178.6); mass spectrum (ESI), m/z 1156.5979 (M+H)+
(C50H82N19O9S2 requires 1156.5984).
Fe2+ and deglycoBLM solutions in 15
lL (total volume) of 50 mM
Tris–HCl buffer, pH 8.0. The reaction mixtures were incubated at
room temperature for 30 min and then quenched by the addition
of 3 lL of native gel loading buffer (30% glycerol, 0.125% (w/v) bro-
mophenol blue). Five microliters of each reaction mixture was
loaded onto a 1% agarose gel made in 90 mM Tris–borate, pH 8.3,
containing 1 mM sodium EDTA and run at 96 V for 2 h. The gel was
then stained in the same buffer solution containing 0.5 lg/mL ethi-
dium bromide for 1 h followed by destaining in water for 15 min.
The gels were analyzed using a UV transilluminator.
5.1.3.9. Deglycobleomycin analogue 10.
HPLC retention
5.2.3. [50-32P]-end labeling and purification of a 158-base pair
DNA restriction fragment69
Twenty-five micrograms of pBR322 plasmid DNA was digested at
37 °C for 3 h with 50 U of restriction endonuclease HindIII in a final
time: 17.6 min; colorless solid; yield 1.4 mg (43%); 1H NMR
(D2O) d 0.65 (d, 3H, J = 6.4 Hz), 0.69 (d, 3H, J = 6.4 Hz), 0.90 (d,
3H, J = 7.2 Hz), 0.95 (d, 3H, J = 6.4 Hz), 1.21–1.29 (m, 3H), 1.42–
1.48 (m, 2H), 1.64 (m, 4H), 1.87–1.95 (m, 7H), 2.41–2.44 (m, 1H),
2.57–2.67 (m, 2H), 2.91–3.03 (m, 12H), 3.06–3.18 (m, 4H), 3.38–
volume of 80
lL of 10 mM Tris–HCl, pH 7.9, containing 50 mM NaCl,
10 mM MgCl2 and 1 mM dithiothretol. The linearized DNA was