128
P.D. Bonnitcha et al. / Journal of Inorganic Biochemistry 104 (2010) 126–135
CDCl3): d ppm 8.33 (1H, br s, NHC@S), 4.94 (1H, br s, NHBoc), 3.73
2.2.5. Zn-ATSM/en
3
3
(2H, dt, JHH = 5.5, 5.2 Hz, CH2NHC@S), 3.36 (2H, dt, JHH = 6.1,
5.5 Hz, CH2NHBoc), 2.53 (3H, s, CH3S), 1.39 (9H, s, Boc); MS (ES+):
m/z 273.1 {M + Na}+ (100%).
Zn(OAc)2ꢂ2H2O (0.23 g, 1.0 mmol) was added to a solution of
H2ATSM/en (0.25 g, 0.86 mmol) in methanol (10 mL) and the mix-
ture stirred at room temperature. for 1 h. Water (20 mL) and trieth-
ylamine (1 mL) were added causing the formation of a yellow
precipitate. The solution was filtered, the solid washed with water
and diethyl ether and dried in vacuo to give the product as a yellow
solid (0.29 g, 0.69 mmol, 80%). The 1H NMR (300 MHz, DMSO-d6)
gave broad peaks possibly due to protic equilibria or self-associa-
tion in solution; d ppm 7.32 (1H, br s), 7.16 (1H, br s), 3.27 (2H,
2.2.2. 4-N-(2-tbutoxycarbonylaminoethyl)-3-thiosemicarbazide
A solution of methyl-N-(2-tbutoxycarbonylaminoethyl)dithio-
carbamate (7.00 g, 27.9 mmol) and hydrazine hydrate (2.20 mL,
ca. 38.5 mmol) was dissolved in ethanol (100 mL) and heated un-
der reflux for 150 min. The solvent was removed under reduced
pressure and the residue redissolved in chloroform (20 mL). This
was passed through a plug of silica, washed with chloroform
(20 mL) and the product eluted with methanol (30 mL). The meth-
anol fraction was evaporated to provide an oil which solidified over
a few days (6.24 g, 96%, 26.8 mmol). 1H NMR (300 MHz, CDCl3): d
ppm 7.76 (1H, br s, NHNH2), 7.58 (1H, br s, CH2NHC@S), 4.98
(1H, br s, CH2NHBoc), 3.76 (2H, br s, NHNH2), 3.69 (2H, dt,
3
br m) 2.82 (3H, d, JHH = 3.5 Hz) 2.42 (2H, br m), 2.22 (3H, s),
2.14 (3H, s). HRMS (ES+): m/z (calcd) 352.0362 (352.0356)
{M + H}+; Crystals suitable for X-ray diffraction were grown by
the diffusion of H2O into a DMSO solution of the product. The com-
m.ac.uk/ (deposition number: CCDC 710238).
2.2.6. N-hydroxysuccinimide propanoate
3
3JHH = 6.3, 5.4 Hz, CH2NHC@S), 3.32 (2H, dt, JHH = 6.0, 5.4 Hz,
This procedure was adapted from the procedure of Hirata et al.
[40] DCC (8.00 g, 38.9 mmol) was added to a solution of N-
hydroxysuccinimide (4.60 g, 40.0 mmol) and propionic acid
(3.00 mL, 39.5 mmol) in THF (100 mL) and the mixture stirred at
ꢁ78 °C for 1 h. After stirring for a further 2 h at room temperature,
the solution was filtered, the solvent removed and the residue par-
titioned between dichloromethane and water. The organic layer
was washed with a sat. NaHCO3 solution (2 ꢀ 25 mL), dried with
MgSO4 and the solvent removed to leave N-hydroxysuccinimide
propanoate as a viscous oil which solidified to a white solid over
a few hours (5.13 g, 30.0 mmol, 77%). 1H NMR (300 MHz, DMSO-
CH2NHBoc), 1.38 (9H, s, Boc); 13C{1H} NMR (75.5 MHz, CDCl3): d
ppm 182.6, 156.6, 79.6, 44.4, 40.1, 28.3; HRMS (ES+): m/z (calcd)
257.1040 (257.1048) {M + Na}+.
2.2.3. H2ATSM/en-boc
4-N-(2-tbutoxycarbonylaminoethyl)-3-thiosemicarbazide
(0.750 g, 3.21 mmol) was suspended in ethanol (30 mL) and stirred
at 50 °C. Diacetyl-2-(4-methyl-3-thiosemicarbazone) (0.555 g,
3.21 mmol) was added in portions over 30 min. After the final
addition, 2 drops of conc. HCl was added and the reaction heated
under reflux for 3½ h. The cooled solution was poured into water
(70 mL) to produce a white precipitate. This was collected by filtra-
tion, washed with water (2 ꢀ 30 mL) and diethyl ether (1 ꢀ 10 mL),
and dried in vacuo to leave the product as a white solid (0.851 g,
2.07 mmol, 68%). 1H NMR (300 MHz, DMSO-d6) d ppm 10.31 (1H,
3
d6): d ppm 2.76 (4H, s, CH2–N(–O)–CH2), 2.66 (2H, q, JHH = 7.5 Hz,
O@C–CH2CH3), 1.11 (3H, t, JHH = 7.5 Hz, O@C–CH2CH3); 13C{1H}
3
NMR (75.5 MHz, DMSO-d6): d ppm 171.1, 170.8, 25.4, 24.3, 9.8;
MS (EI+): 171.1 (5%, {M}+), 87.1 (60%, {M–(O@C–CH2–CH2–
C@O)}+), 57.0 (100%, {M–(N-hydroxysuccinimide)}+).
3
s, NHN@C), 10.29 (1H, s, NHN@C), 8.45 (1H, t, JHH = 5.1 Hz,
2.2.7. N-hydroxysuccinimide 3-(4-nitroimidazol-1-yl)propanoate
EDC (520 mg, 2.80 mmol) was added to a mixture of N-
hydroxysuccinimide (320 mg, 2.78 mmol) and 3-(4-nitro-
imidazol-1-yl) propionic acid (500 mg, 2.75 mmol) in THF
(25 mL) and the mixture stirred for 1 h at ꢁ78 °C. The mixture
was stirred for a further by 15 h at room temperature before the
solvent was removed and water/methanol (1:1, 10 mL) added to
the residue. The resulting solid collected, cold water (2 mL) and
diethylether (2 ꢀ 20 mL) to leave pure N-hydroxysuccinimide 3-
(4-nitro-1H-imidazol-1-yl) propanoate as a white solid (611 mg,
2.16 mmol, 80%). 1H NMR (300 MHz, DMSO-d6): d ppm 8.44 (1H,
3
CH2NHC@S), 8.38 (1H, q, JHH = 4.5 Hz, CH3NHC@S), 7.03 (1H, t,
3
3JHH = 5.1 Hz, NHBoc), 3.59 (2H, td, JHH = 5.4, 5.1 Hz, CH2NHC@S),
3
3.17 (2H, td, JHH = 5.4, 5.1 Hz, CH2NHBoc), 3.02 (3H, d,
3JHH = 4.55 Hz, CH3NHC@S), 2.23 (3H, s, CH3C@N), 2.21 (3H, s,
CH3C@N), 1.36 (9H, s, Boc); 13C{1H} NMR (75.5 MHz, DMSO-d6): d
ppm 178.5, 178.0, 156.2, 148.3, 147.8, 77.9, 44.5, 39.1, 31.2, 28.2,
11.8, 11.7; HRMS (ES+): m/z (calcd) 412.1555 (412.1565)
{M + Na}+. HPLC (Method B): Rt 12.37 min.
3
3
2.2.4. H2ATSM/en
d, JHH = 1.4, imidazole), 7.87 (1H, d, JHH = 1.3, imidazole), 4.40
3
3
TFA (2 mL) was added to diacetyl-2-(4-N-methyl-3-thiosemi-
carbazone)-3-(4-N-tbutylethylcarbamate-3-thiosemicarbazone)
(0.750 g, 1.93 mmol) and the resulting colourless solution stirred
for 1½ h at room temperature. The solvent was removed to leave
a sticky residue and sat. aqueous NaHCO3 (10 mL) added slowly
to produce a white suspension. After 20 min stirring at room tem-
perature the solid was collected by filtration and washed with
water (3 ꢀ 20 mL) and diethyl ether (2 ꢀ 20 mL). Drying in vacuo
gave the product as off-white solid (0.431 g, 1.49 mmol, 77%). 1H
NMR (300 MHz, DMSO-d6 with added TFA) d ppm 10.53 (1H, d,
(2H, t, JHH = 6.8), 3.40 (2H, t, JHH = 6.8, O@C–CH2CH2-imidazole),
2.76 (4H, s, CH2–N(–O)–CH2); 13C{1H} NMR (75.5 MHz, DMSO-
d6): d ppm 170.8, 167.5, 147.7, 138.2, 122.3, 42.8, 32.0, 25.9; HRMS
(ES+): m/z (calc) 283.0684 (283.0679) {M + H}+. Elem. Anal.(%):
Found: C 42.5, H 3.6, N 19.8, Calc. (for C10H10N4O6): C 42.6, H 3.6,
N 19.9. HPLC (Method A): Rt 8.00 min.
2.2.8. H2L1
Triethylamine (150 lL, 1.07 mmol) was added to a solution of
H2ATSM/A (260 mg, 1.00 mmol) in DMSO (3 mL), followed by N-
hydroxysuccinimide 3-(4-nitroimidazol-1-yl)propanoate (296 mg,
1.05 mmol). The solution was stirred for 6 h, water (20 mL) added,
the solution centrifuged and the crude solid collected. The solid
was then washed with ethanol (10 mL) and diethylether
(3 ꢀ 10 mL) to leave H2L1 as an off-white solid (358 mg,
0.84 mmol, 84%). 1H NMR (300 MHz, DMSO-d6): d ppm 10.41 (H,
s, S@C–NH–NH–C@O), 10.11 (2H, br s, 2 ꢀ S@C–NH–N) 9.99 (H, s,
S@C–NH–NH–C@O), 8.37 (2H, m, NH–CH3 and imidazole), 7.83
3
3JHH = 8.21 Hz, NHN@C), 10.24 (1H, d, JHH = 12.0 Hz, NHN@C),
3
3
8.50 (1H, q, JHH = 6.7 Hz, CH3NHC@S), 8.39 (1H, t, JHH = 4.4 Hz,
CH2NHC@S), 7.87 (3H, br s, NH3+), 3.83 (2H, td, JHH = 6.1, 5.9 Hz,
3
CH2NHC@S), 3.05 (2H, tq, JHH = 6.3, 6.1 Hz, CH2NH3+), 3.02 (3H,
3
3
d, JHH = 6.3 Hz), CH3NHC@S), 2.22 (6H, br s, 2 ꢀ CH3C@N);
13C{1H} NMR (75.5 MHz, DMSO-d6): d ppm 178.4, 177.6, 147.6 (2
carbons), 46.3, 40.3, 31.2, 11.7, 11.5; HRMS (ES+): m/z (calcd)
290.1228 (290.1222) {M + H}+; HPLC (Method B, run with 0.1%
TFA): Rt 6.08 min.
3
(H, s, imidazole), 4.35 (2H, t, JHH = 6.7 Hz, O@C–CH2–CH2-imidaz-