Bis(nitrilotriacetic acid) Ligands
FULL PAPER
(CDCl3, 200 MHz): d=8.35 (s, 1H), 8.17 (s, 1H), 8.01 (s, 2H), 7.94 (d,
J=7.6 Hz, 1H), 7.40–6.70 (m, 7H), 5.00 (s, 2H), 3.78 (s, 8H), 3.67 (m,
2H), 3.43 (m, 4H), 2.00–1.50 ppm (m, 12H); 13C NMR (CD3OD,
75.5 MHz): d=183.6, 175.6, 174.8, 169.5, 166.5, 157.3, 157.2, 143.0, 141.1,
136.6, 132.0, 130.4, 130.1, 129.3, 128.2, 128.0, 126.0, 123.3, 117.1, 103.4,
66.9, 66.4, 55.3, 54.6, 54.5, 54.0, 40.8, 34.7, 30.7, 29.9, 26.0, 24.7 ppm; IR
(KBr): n˜ =3434, 2928, 2502, 2330, 1964, 1727, 1635, 1548, 1455, 1391,
1314, 1267, 1211, 1116, 854, 795, 701, 445 cmꢁ1; MS (ES): unknown frag-
mentation.
mixture was then quenched with a 10% aqueous solution of HCl (4 mL)
and extracted twice with CH2Cl2. The organic phase was dried over
MgSO4, filtered, and evaporated to dryness. Purification of the residue
by column chromatography on silica gel afforded the desired compound
as an orange solid (150 mg, 81.5 mmol, 53%). 1H NMR (CD3OD,
300 MHz): d=8.61 (s, 1H), 8.18 (m, 2H), 8.01 (s, 2H), 7.49 (m, 1H), 7.49
(d, J=8.7 Hz, 2H), 7.11 (d, J=8.7 Hz, 2H), 7.26 (brs, 32H), 5.03 (brs,
12H), 4.96 (s, 2H), 3.70 (s, 8H), 3.50 (t, J=7.5 Hz, 2H), 3.36 (m, 4H),
3.05 (m, 4H), 2.17 (t, J=6.9 Hz, 4H), 1.40–1.20 ppm (m, 24H); 13C NMR
(CDCl3, 75.5 MHz): d=182.9, 175.8, 173.4, 172.3, 168.8, 167.5, 160.0,
143.4, 140.6, 136.8, 136.1, 133.9, 133.8, 129.4, 129.1, 129.0, 125.9, 120.6,
117.7, 103.3, 67.4, 65.9, 53.7, 40.8, 40.1, 36.8, 30.6, 29.8, 29.5, 27.3, 26.5,
24.0 ppm; IR (KBr): n˜ =3409, 2929, 1739, 1633, 1546, 1135, 768, 679,
610 cmꢁ1; MS (ES): m/z: 1861.7 [M+Na]+, 1883.7 [MꢁH+2Na]+, 942.7
[M+2Na]2+, 953.7 [MꢁH+3Na]2+, 964.6 [Mꢁ2H+4Na)+; HRMS (EI):
m/z: calcd for [M+Na]+: 1861.7414; found: 1861.7414; calcd for
[MꢁH+Na]+ =1883.72234; found: 1883.7116.
6-(6-Aminohexanoylamino)-2-[bis(benzyloxycarbonylmethylamino)]hex-
anoic benzyl ester (27): The corresponding NH-tert-butoxycarbonyl
(Boc)-protected compound (100 mg, 134 mmol, 1 equiv) was dissolved in
anhydrous CH2Cl2 (5 mL) containing TFA (725 mL, 9.4 mmol, 70 equiv).
The reaction mixture was then stirred under argon for 12 h at room tem-
perature and concentrated under vacuum. Excess TFA was removed by
azeotropic distillation with toluene, thereby affording the desired com-
pound as a TFA salt (117 mg, 0.13 mmol, 100%). 1H NMR (CDCl3,
200 MHz): d=7.59 (s, large, 2H), 7.40 (brs, 15H), 5.17 (s large, 6H), 3.73
(brs, 4H), 3.58 (t, J=6.8 Hz, 1H), 3.24, 3.08 (2m, 4H), 2.36 (m, 2H),
1.90–1.30 ppm (m, 12H); 13C NMR (CDCl3, 50 MHz): d=172.7, 171.7,
135.7, 135.5, 128.7, 128.6, 128.5, 128.3, 77.8, 64.6, 53.2, 39.9, 39.6, 35.3,
29.8, 28.1, 26.6, 25.3, 24.7, 22.9 ppm; MS (ES): m/z (%): 646 (100)
[M+H]+.
Compound 8:
A 1m aqueous solution of KOH (294 mL, 294 mmol,
18 equiv) was added to a solution of compound 26 (30 mg, 16.3 mmol,
1.0 equiv) in methanol (1 mL). The reaction mixture was then stirred
under argon in the dark for 18 h at room temperature and concentrated
under vacuum. The residue was dissolved in water (0.5 mL) and the de-
sired product was precipitated by slow addition of a 1m aqueous solution
of HCl. After high-speed centrifugation (13400 rpm) at 08C for 15 min,
the supernatant was removed and the precipitate was then washed with
MeOH and diethyl ether to afford the title compound 8 as a fine orange
powder (18 mg, 13.9 mmol, 85%). 1H NMR (CD3OD, 200 MHz): d=
8.40–7.90 (m, 4H), 7.50–6.80 (m, 8H), 5.11 (m, 2H), 3.84 (m, 2H), 3.69
(s, 8H), 3.30–3.15 (m, 8H), 2.21 (m, 4H), 1.90–1.20 ppm (m, 24H);
13C NMR (CD3OD, 50 MHz): d=183.4, 176.1, 174.3, 173.6, 169.3, 167.5,
157.7, 143.1, 141.2, 136.6, 132.5, 130.4, 129.6, 129.3, 128.3, 128.0, 125.9,
123.8, 117.7, 103.4, 66.1, 65.2, 54.4, 53.8, 53.4, 52.1, 40.9, 40.1, 37.0, 30.7,
30.1, 29.9, 27.6, 26.7, 24.3 ppm; IR (KBr): n˜ =3413, 2927, 1727, 1629,
1548, 1448, 1197, 1172, 1119, 801, 690, 439 cmꢁ1; MS (ES): unknown frag-
mentation.
6-{6-[3-(5-{5-Benzyloxycarbonyl-5-[bis(benzyloxycarbonylmethylamino)]-
pentylcarbamoyl}pentylcarbamoyl)-5-(tert-butoxycarbonylaminomethyl)-
benzoylamino]hexanoylamino}-2-[bis(benzyloxycarbonylmethylamino]-
hexanoic benzyl ester (24): Compound 20 (164 mg, 0.34 mmol, 1.0 equiv)
was added to a solution of 27 (645 mg, 738 mmol, 2.2 equiv) in anhydrous
CH2Cl2 (5 mL) in the presence of diisopropylethylamine (323 mL,
1.85 mmol, 5.5 equiv). The resulting mixture was stirred at room temper-
ature under an argon atmosphere for 12 h, quenched with a saturated
aqueous solution of ammonium chloride, dried over MgSO4, filtered, and
evaporated to dryness. Purification of the residue by column chromatog-
raphy on silica gel afforded the desired compound as a white foam
1
(405 mg, 0.26 mmol, 78%). H NMR (CDCl3, 300 MHz): d=8.09 (s, 1H),
H6SRC1 expression and purification: The PQE30-SRC1570–580 plasmid re-
quired for the SRC1570–580 expression was kindly provided by C. Royer
(CBS, Montpellier, France). The H6-tagged H6SRC1570ꢁ580 protein (desig-
nated by H6SRC1) was over-expressed in Escherichia coli and purified by
Ni2+–NTA agarose beads under native conditions (Qiagen, Courtaboeuf,
France).
7.85 (s, 2H), 7.27 (m, 30H), 6.11 (m, 2H), 5.37 (m, 1H), 5.02 (s, 12H),
4.28 (m, 2H), 3.64 (s, 8H), 3.40 (m, 6H), 3.07 (m, 4H), 2.10 (m, 4H),
1.80–1.10 ppm (m, 33H); 13C NMR (CDCl3, 100 MHz): d=173.6, 172.4,
171.1, 167.0, 135.6, 135.5, 134.9, 129.0, 128.5, 128.0, 127.2, 126.8, 79.5,
66.3, 64.7, 64.4, 52.7, 50.1, 39.8, 39.1, 36.0, 33.7, 29.7, 28.7, 28.3, 26.3, 25.1,
22.9 ppm; IR (CsI film): n˜ =3045, 2934, 2862, 1742, 1648, 1543, 1455,
1367, 1259, 1163, 990, 743, 699, 581 cmꢁ1; MS (ES): m/z: 225, 247, 471,
684; unknown fragmentation.
Direct-binding assay: Binding assays were performed using a Beacon
2000 polarization instrument (Panvera, Madison, WI, USA) regulated at
208C, using filters for fluorescein at an H6SRC1 concentration of 30 mm.
All solutions were freshly prepared using the buffer solution P (10 mm
Tris, 150 mm NaCl, 10% glycerol, pH 8.0). Buffer P (50 mL), 90 mm
H6SRC1 (50 mL), and 30 nm Ni2+–NTA–fluorescein (50 mL; or 50 mL of
60 nm Ni2–7 or Ni2–8) were successively added in a borosilicate tube.
Each point in the titration curve was then obtained by removing aliquots
of 100 mL from the starter solution and replacing them by 100 mL of
15 nm Ni2+–1 probe (or 100 mL of 30 mm Ni2–7 or Ni2–8). Tubes were
equilibrated at 208C for 4 min prior to measurement and the polarization
was measured successively until stabilized. The reported values are the
average of three measurements after stabilization.
6-{6-[3-Aminomethyl-5-(5-{5-benzyloxycarbonyl-5-[bis(benzyloxycarbo-
nylmethylamino)]pentylcarbamoyl}pentylcarbamoyl)benzoylamino]hexa-
noylamino}-2-[bis(benzyloxycarbonylmethylamino)]hexanoic benzyl ester
(25): The NH-Boc-protected compound 24 (200 mg, 129 mmol, 1.0 equiv)
was dissolved in anhydrous CH2Cl2 (5 mL) containing TFA (700 mL,
9.03 mmol, 70 equiv). The reaction mixture was then stirred under argon
for 12 h at room temperature and concentrated under vacuum. Excess
TFA was removed by azeotropic distillation with toluene, thereby afford-
ing the desired compound as a TFA salt (231 mg, 129 mmol, 100%).
1H NMR (CDCl3, 300 MHz): d=8.58 (m, 2H), 8.15 (s, 1H), 8.02 (s, 2H),
7.45–7.10 (m, 30H), 7.00 (m, 2H), 5.08 (m, 12H), 4.19 (m, 2H), 3.67 (s,
8H), 3.47 (t, J=7.5 Hz, 2H), 3.34 (m, 4H), 3.13 (m, 4H), 2.24 (m, 4H;
H), 1.90–1.10 ppm (m, 24H); 13C NMR (CDCl3, 50 MHz): d=173.0,
172.0, 167.4, 136.0, 135.5, 131.6, 131.6, 126.4, 129.0, 128.8, 67.0, 65.1, 53.5,
45.2, 40.5, 39.8, 36.3, 33.7, 30.2, 28.8, 26.6, 25.6, 23.3 ppm; IR (film CsI):
n˜ =3358, 3036, 2933, 1742, 1646, 1551, 1457, 1202, 992, 802, 747, 700,
598 cmꢁ1; MS (ES): m/z (%): 1451 (82) [M+H]+.
In parallel, additional experiments were performed under the same con-
ditions but in the absence of nickel. The obtained values were then sub-
tracted from the previous ones to give the specific polarization values.
Competition binding assays (displacement curve of Ni2+–1 by bis-NTA–
(Ni2+
) ligands): The polarization measurements have been carried out
2
using an AD Analyst apparatus (Molecular Devices, Sunnyvale, USA)
equipped with a dichroic filter (485 nm/530 nm) using Dynex Microfluor
96 polystyrene well plates (8 rows and 12 columns). Polarization values
were obtained by reading the microplates at 208C at half-height of the
wells. All solutions were freshly prepared using the buffer solution P
(10 mm Tris, 150 mm NaCl, 10% glycerol, pH 8.0). Each well of the plate
was preincubated with a 30 mm solution of H6SRC1 (50 mL) and a 5 nm
solution of Ni–1 (50 mL). After a 20 min period of incubation at room
temperature, each well of the first column was then rapidly treated with
6-{6-[3-(5-{5-Benzyloxycarbonyl-5-[bis(benzyloxycarbonylmethylamino)]-
pentylcarbamoyl}pentylcarbamoyl)-5-[(3-fluoresceinethioureido)methyl]-
benzoylamino]hexanoylamino}-2-[bis(benzyloxycarbonylmethylamino)]-
hexanoic benzyl ester (26): Compound 25 (278 mg, 155 mmol, 1.0 equiv)
was dissolved in anhydrous DMF (4.5 mL) in the presence of diisopropy-
lethylamine (108 mL, 619 mmol, 4.0 equiv). FITC (78 mg, 201 mmol,
1.3 equiv) was then added to the reaction mixture and the resulting solu-
tion was stirred at room temperature in the dark overnight. The reaction
Chem. Eur. J. 2009, 15, 12689 – 12701
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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