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Biometals (2021) 34:259–275
DFOANI: Yield: 26.7% m.p: 180–182 8C. Rf:
0.52. H-NMR (500 MHz; DMSO) d(ppm) 9.92 (s;
ESIMS (m/z): 786 (M ? H)?. Elemental Formula
C39H59N7O10 Calculated (%): C 59.6 H 7.6 N 12.5
Found (%): C 59.9 H 7.9 N 12.0 Experimental
Formula: C39H59N7O10. 0.1CH3OH.
1
1H), 9.66 (s; 1H), 9.61 (s; 2H), 7.84 (t; J = 5.4 Hz;
1H); 7.78 (t; J = 5.3 Hz; 2H), 7.57 (d; J = 7.7 Hz;
2H); 7.27 (t; J = 7.9 Hz; 2H), 7.01 (t; J = 7.4 Hz; 1H),
3.45 (t; J = 6.4 Hz; 6H), 3.05–2.96 (m; 6H), 2.58 (t;
J = 7.1 Hz; 3H), 2.54 (dd; J = 9.4; 5.1 Hz; 3H), 2.39
(t; J = 7.3 Hz; 2H), 2.27 (t; J = 7.2 Hz; 4H), 1.97 (d;
J = 5.0 Hz; 3H), 1.51 (dd; J = 22.7; 16.1 Hz; 6H);
1.37 (d; J = 6.8 Hz; 6H), 1.22 (d; J = 6.7 Hz; 6H). 13C
RMN (126 MHz., DMSO) d (ppm) 172.44, 171.78,
171.49, 170.96, 139.80, 129.11, 123.33, 119.37, 47.55,
47.25, 38.90, 32.23, 30.80, 30.37, 29.28, 28.03, 26.49,
23.96, 20.82. ESIMS (m/z): 736 (M ? H)?. Elemen-
tal Formula C35H57N7O10, Calculated (%): C 57.1,
H 7.8, N 13.3, Found (%): C 55.6, H 7.9, N 12.9,
Experimental Formula: C35H57N7O10.0.4CH3-
OH.0.95H2O. e [DFOANI(Fe)] complex (kmax-
DFOQUN: Yield: 24.3% m.p: 143–145 8C. Rf: 0.5
1H-NMR (500 MHz; DMSO) d(ppm) 10.38 (s, 1H),
9.63 (s, 2H), 8.85 (dd, J = 4.4, 1.5 Hz, 1H), 8.52–8.37
(m, 2H), 8.01 (d, J = 9.1 Hz, 1H), 7.85 (dd, J = 9.1,
2.3 82 Hz, 2H), 7.77 (t, J = 5.4 Hz, 2H), 7.60 (dd,
J = 8.4, 4.5 Hz, 1H), 3.01 (dq, J = 13.2, 6.7 Hz, 8H),
2.67–2.53 (m, 8H), 2.42 (q, J = 6.9 Hz, 4H), 2.26 (t,
J = 7.1 Hz, 5H), 1.95 (s, 3H), 1.54–1.44 (m, 6H),
1.41–1.33 (m, 6H), 1.26–1.12 (m, 6H).13C RMN
(126 MHz., DMSO) d (ppm) 172.43, 171.79, 171.67,
171.42, 147.89, 138.31, 129.11, 128.42, 128.00,
124.84, 122.32, 114.99, 47.53, 47.24, 38.89, 32.25,
30.64, 30.39, 29.28, 28.02, 26.48, 23.94, 20.79.
ESIMS (m/z): 787 (M ? H)?. Elemental Formula
C38H58N8O10 Calculated (%): C 58.0 H 7.4 N 14.2
Found (%): C 58.3 H 7.5 N 14.1 Experimental
Formula: C38H58N8O10 e [DFOQUN(Fe)] complex
= 430 nm): 2037.3 L mol-1 cm-1
.
DFOBAN: Yield: 24.9% m.p: 184–185 8C. Rf:
0.0.25 1H-NMR (500 MHz; DMSO) d(ppm) 10.28 (s,
1H), 9.66 (s, 1H), 9.61 (s, 2H), 7.85 (t, J = 5.5 Hz,
1H), 7.77 (t, J = 5.3 Hz, 2H), 7.74 (d, J = 9.5 Hz, 4H),
7.23 (s, 2H), 3.45 (t, J = 7.0 Hz, 6H), 3.06–2.95 (m,
6H), 2.63–2.53 (m, 6H), 2.43–2.36 (m, 2H), 2.27 (t,
J = 7.1 Hz, 4H), 1.96 (s, 3H), 1.55–1.44 (m, 6H),
1.42–1.34 (m, 6H), 1.22 (d, J = 6.8 Hz, 6H). 13C
RMN (126 MHz., DMSO) d (ppm) 172.44, 171.61,
171.37, 142.68, 138.44, 127.13, 118.84, 47.55, 47.25,
32.21, 30.51, 30.37, 29.28, 28.03, 26.50, 23.96, 20.82.
ESIMS (m/z): 815 (M ? H)?. Elemental Formula
C35H58N8O12S Calculated (%): C 51.8, H 7.2, N 13.8
Found (%): C 50.8, H 7.2, N 13.2 Experimental
(kmax = 430 nm): 1231.8 L mol-1 cm-1
.
Calcein competition studies
The ability of the new chelators to scavenge iron was
studied by competition against the fluorescent chelator
´
calcein, according to published protocols (Esposito
et al. 2002a). Aliquots of 190 lL of ferric calcein
(CAFe; 2 lM) in HBS/Chelex (Hepes Buffered
Saline; hepes 20 mM, NaCl 150 mM, Chelex 1 g/
100 mL; pH 7.4) were added to transparent 96-well
microplates. Next, 10 lL of conjugates (final concen-
trations: 0–15 lM) in DMSO were added to each well.
Fluorescence was recorded after 2 h incubation at
37 °C in a BMG FluoStar Optima microplate reader
(kexc/kem = 485/520 nm). Two independent assays
were performed, each one in duplicate.
Formula: C35H58N8O12S.0.55CH3OH.0.45H2O
e
[DFOBAN(Fe)] complex (kmax = 430 nm): 1642.7
L mol-1 cm-1
.
DFONAF: Yield: 43.8% m.p: 180–182 Rf: 0.6
1H-NMR (500 MHz; DMSO) d(ppm) 10.14 (s, 1H),
9.65 (s, 1H), 9.61 (s, 2H), 8.29 (s, 1H), 7.89–7.75 (m,
6H), 7.59–7.54 (m, 1H), 7.49–7.42 (m, 1H), 7.41–7.35
(m, 1H), 3.45 (t, J = 7.0 Hz, 6H), 3.01 (td, J = 13.2,
6.6 Hz, 6H), 2.64–2.52 (m, 6H), 2.43 (t, J = 7.2 Hz,
2H), 2.27 (t, J = 6.6 Hz, 4H), 1.97 (s, 3H), 1.49 (d,
J = 6.3 Hz, 6H), 1.39 (dq, J = 14.8, 7.5 Hz, 6H), 1.22
(d, J = 6.2 Hz, 6H). 13C RMN (126 MHz., DMSO) d
(ppm) 172.44, 171.78, 171.49, 171.28, 137.38, 133.93,
130.05, 128.74, 127.89, 127.68, 126.82, 124.87,
120.33, 115.28, 47.55, 47.25, 38.92, 38.89, 32.29,
30.79, 30.37, 29.28, 28.03, 26.50, 23.96, 20.82.
DHR antioxidant activity
The ability of the new chelators to block iron-
dependent oxidation of DHR was studied according
´
to previous methods (Esposito et al. 2003). Aliquots of
10 lL of ferric nitrilotriacetate (Fe(NTA)) were added
to transparent 96 wells microplates followed by 10 lL
of the conjugates (0–300 lM) in DMSO. Next, 180 lL
of fluorogenic mixture (DHR 50 lM and ascorbic acid
40 lM in HBS/Chelex) were transferred to the wells.
123