G. Serratrice, J.-L. Pierre et al.
7 Hz, 2H; CH2N), 7.36 (d, 4J
A
(H,H)=
10ꢀ3 m). Samples of the ferric complex of La ([La]tot =510ꢀ4 m, [Fe3+
] =
tot
2 Hz, 1H); 13C NMR (75 MHz, CD3OD, 188C): d=15.2 (CH3), 24.5, 28.8,
31.0, 31.2 and 31.4 (CH2), 33.8 (CH2CH2N), 41.7 (CH2N), 117.0 (Cqarom ),
117.7 and 117.9 (CHarom), 137.1, 148.1 and 152.6 (Cqarom ), 171.5 ppm (C=
O); MS (DCI, NH3/iC4H10): m/z (%): 374 (100) [M+H]+; elemental anal-
ysis calcd (%) for C17H27NO6S·H2O: C 52.16, H 7.47, N 3.58; found: C
52.08, H 7.51, N 3.52.
2.510ꢀ4 m) were prepared in buffer solution (MOPS, 0.05m, pH 7.4)
with methanol (5% v/v) and also with octanol (1% v/v, 610ꢀ3 m). The
samples were prepared 8 h before the first experiment and the second ex-
periment was performed on the samples 8 d later. The samples for cryo-
TEM were prepared by the following procedure:[13] a 3 mL drop of the
sample solution was applied to a grid covered with a holey carbon/Pt-
carbon film, the excess liquid was blotted with Whatman 40 filter paper,
and the remaining film was vitrified in liquid ethane held just above
freezing point. The grid was then mounted in a Gatan 626 cryoholder
(Gatan, Pleasanton CA) and observed at ꢀ1808C with a Philips CM200
electron microscope operating at 80 kV. Micrographs were recorded in
low-dose mode on Kodak SO-163 film (Eastman Kodak, Rochester NY)
at a direct magnification of 50000 and ꢃ1 mm defocus, and developed
in full-strength D19 developer (Kodak) for 12 min. Contrast enhance-
ments and intensity gradient removal of the images were performed with
Scion Image software.
Spectrophotometric studies: The solutions were prepared with boiled de-
ionised water, which was deoxygenated and flushed continuously with
argon (purified by a Sigma Oxiclear cartridge) to exclude CO2 and O2.
An ionic strength of 0.1m was maintained with NaClO4 (Prolabo, Puriss
or Merck, p.a.) and all measurements were carried out at (25.0ꢁ0.2)8C.
The free hydrogen concentrations were measured with a glass-Ag/AgCl
combined electrode (Metrohm or Tacussel High Alkalinity, filled with
0.1m aqueous NaCl and saturated with AgCl). The electrode was cali-
brated to measure [H] by the classical method of titrating HClO4 (0.01m)
with NaOH (0.02m).[11] Spectrophotometric measurements were carried
out by using a Varian Cary 50 UV/Vis spectrophotometer equipped with
a Peltier thermostatting accessory and a PC for data collection and evalu-
ation. The spectrophotometric titrations were recorded in aqueous solu-
tions with an ionic strength of 0.1 m (adjusted with 0.1 m aqueous NaClO4
for LS10 or NaClO4+HClO4 for the complex) at 258C between pH 3.5
and 12.6 for LS10 (1.010ꢀ4 m) and between pH 1 and 10 for the ferric
complex (ligand=3.3610ꢀ4 m, Fe3+ =1.010ꢀ4 m). The pH was adjusted
with NaOH. The stoichiometry of the FeIII complex at pH 7.4 was also
monitored by titrating a solution of ligand with a solution of FeIII in a
buffer solution (MOPS=0.05m, NaClO4 =0.05m).
Acknowledgements
We gratefully acknowledge Professors Marguerite Rinaudo and Michel
Milas for help in performing the surface tension measurements in their
laboratory, Dr. Anabelle Varrot for assistance with the scattering meas-
urements (Centre de Recherches sur les MacromolØcules VØgØtales
(CERMAV-UPR CNRS 5301, Grenoble), and Gisle Gellon for the syn-
thesis of LS10
.
The spectrophotometric data were processed with the SPECFIT/32
Global Analysis system (Spectrum Software Associates).[12] This program
performs a global analysis of system equilibria, and by using a nonlinear
regression model given by the Levenberg–Marquardt method we can cal-
culate the thermodynamic constants and spectra of absorbing species.
Calculating the protonation constants of the ligand was achieved by using
two spectral sets. For pKa2, readings were taken between pH 2 and 8 in
the 200 to 400 nm spectral region with a 1 nm step, and for pKa1, readings
were taken between pH 8 and 12.6 in the 300 to 400 nm spectral region
with a 1 nm step. Calculating the complexation stability constant was ach-
ieved by using the spectral set of the iron complex between pH 1 and 10
in the 400 to 800 nm spectral region with a 1 nm step.
[1] J. B. Neilands, J. Biol. Chem. 1995, 270, 26723–26726.
[2] a) J. S. Martinez, G. P. Zhang, P. D. Holt, H. T. Jung, C. J. Carrano,
[3] a) D. Imbert, P. Baret, D. Gaude, I. Gautier-Luneau, G. Gellon, F.
1100; b) L. Yun-Ming, M. J. Miller, U. Mçllmann, Biometals 2001,
[5] a) J. S. Martinez, J. N. Carter-Franklin, E. L. Mann, J. D. Martin,
Surface tension studies: The surface tension was measured at 258C by
using an automatic drop tensiometer (Tracker, I.C. Concept, Longessange
France) in rising drop mode. The surface tension was calculated by a
mathematical analysis of the axial symmetric shape of the drop (Lapla-
cian profile). The CMC was estimated from the change in surface tension
due to the concentrations of the ligand (110ꢀ6–310ꢀ3 m) and complex
(310ꢀ7 m–110ꢀ3 m; [Fe3+ [LS10
]tot/ACHTREUNG ]tot =1:3). All experiments were per-
formed at 258C in a buffer solution (MOPS, 0.05m, pH 7.4).
DLS studies: DLS measurements were performed by using a Zetasizer
Nano Series ZS (Malvern Instruments) instrument equipped with a
633 nm laser. The non-invasive back-scatter detection method was used
in which the incident beam does not have to pass through the sample,
but instead the light scattered 1738 from the incident beam was mea-
sured. Data were fitted by using Dispersion Technology Software v5.00
(DTS; Malvern Instruments). The DTS software uses algorithms to ex-
tract the decay rates of the correlation function for a range of particle
sizes. This information is then used to produce a size distribution of the
hydrodynamic diameter. The basic intensity distribution is obtained from
the DLS measurements, from which all other distributions are generated
by using Mie theory. The results are presented as an estimate of the size
distribution of the most numerous particles. Experiments were performed
in buffer solution (MOPS, 0.05m, pH 7.4) for the ligand (210ꢀ3 m) and
the ferric complex (110ꢀ3 m) at 258C. The solutions were passed
through a filter (Pall, Acrodisc CR 13 mm Syringe Filter with a 0.45 mm
PTFE Membrane) prior to analysis to exclude dust particles.
Cryo-TEM studies: Experiments were performed with the ferric com-
[6] M. Apostol, P. Baret, G. Serratrice, J. Desbrieres, J.-L. Putaux, M.-J.
[7] S. J. H. Hickford, F. C. Kupper, G. Zhang, C. J. Carrano, J. W. Blunt,
Inorg. Chem. 1989, 28, 128–133.
[9] P. D. T. Huibers, V. S. Lobanov, A. R. Katritzky, D. O. Shah, M. Kar-
[11] A. E. Martell, R. J. Motekaitis, Determination and Use of Stability
Constants, VCH, Weinheim, 1988, Chapter 1, pp. 7–19.
[12] a) H. Gampp, M. Maeder, C. J. Meyer, A. D. Zuberbꢂhler, Talanta
berbꢂhler, Talanta 1985, 32, 257–264.
[13] J. Dubochet, M. Adrian, J. J. Chang, J. C. Homo, J. Lepault, A. W.
McDowall, P. Schultz, Q. Rev. Biophys. 1988, 21, 129–228.
plexes of La and LS10. Samples of the ferric complex of LS10 ([LS10
10ꢀ4 m, [Fe3+ tot =0.8310ꢀ3 m) were prepared in buffer solution (MOPS,
0.05m, pH 7.4) in the absence and presence of octanol (1% v/v, 6
]tot =5
]
Received: October 17, 2007
Published online: February 21, 2008
3686
ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 3680 – 3686