3.71 (m, 12H, 6 ] NCH of Apr), 7.85 (t, J \ 5.5, 3H,
denote, respectively, absorbances at the initial time, at the
Ðnal time and at time t, and the Ðrst-order rate law was fol-
lowed in most cases for 4 half-lives. The rate was obtained
with an error of ^5% by averaging at least two determi-
2
3 ] NH), 8.37 (s, 3H, C H ), 8.64 (t, J \ 5.5 Hz, 3H, 3
6
3
] C H ÈCONH). Anal. calc. for C
H
N
O
É H O: C,
6
3
63 102 12 24
2
52.93; H, 7.33; N, 11.76%. Found: C, 52.78; H, 7.54; N,
11.46%.
nations. In the case of the consecutive reactions for Fe -2, two
2
C H [COÈAheÈ(HO)AprÈAheÈ(HO)AprÈAheÈ(HO)AprÈ
pseudo-Ðrst-order rate constants (kup and klow) were obtained
6
3
1
1
OH] (ligand 3) (0.0316 g, 86%) was obtained from 18 (0.0515
by curve Ðtting the data (40 data points) to eqn. 4 within the
indicated error limit. DMF (2.5%) in the solution had no
e†ect on the rates.
3
g, 0.0183 mmol) as an amorphous solid. HPLC: R 2.8 min.
t
IR (KBr): l
CJO
(d6-DMSO):
1723 (carboxylic acid), 1635 (amide) cm~1. 1H
NMR
d
1.23È1.53
[m,
54H,
9
Equilibrium determinations. Each of the equimolar reactions
was carried out under the conditions shown in Table 3, by
following the decrease in the absorbance of Fe(L) at 420 nm
periodically, without the use of any bu†er. As the reaction
progressed toward equilibrium, the pH of the solution
changed and was adjusted periodically, to 5.4 with 0.1 M
KOH solution. More than 3 weeks was needed for the equili-
bration. A value of e \ 52 M~1 cm~1 (420 nm) was used for
correcting the spectral intensity for the presence of
Fe(EDTA)~.
] NHCH (CH ) CH CO], 2.28È2.41 (m, 30H, 9 ] CH CO
2 3
of Ahe and 6 ] CH CO of Apr), 2.41 (t, 6H, 3 ] CH CO H),
2
2
2
2
2
2
2.99 (m, 12H, 6 ] NCH of Ahe), 3.16 (m, 6H, 3 ] NCH of
2
2
COÈAhe), 3.64È3.68 (m, 18H, 9 ] NCH of Apr), 7.89 (br t,
2
6H, 6 ] NH), 8.36 (s, 3H, C H ), 8.67 (br t, 3H,
3
6
3
] C H ÈCONH). Anal. calc. for C
H
N
O
É H O: C,
6
3
90 150 18 33
2
53.24; H, 7.54; N, 12.41%. Found: C, 53.28; H, 7.49; N,
11.98%.
Iron(III) complex formation
A solution of ferric nitrate (3.60 ] 10~3 M) in 0.1 M nitric
acid was prepared by diluting a commercially available ferric
nitrate standard solution with 0.1 M nitric acid. A ligand solu-
tion of 0.60 mM was prepared with doubly distilled deionized
water. An aqueous stock solution of ligand 3 contained 15%
DMF (v/v). Each iron(III) complex solution (2.0 ] 10~4 M, a
Ðnal volume of 3.0 mL, I \ 0.1) was prepared by mixing the
ligand solution (1.0 mL) and the ferric nitrate solution in
Biological assay
Growth promotion tests were performed by the standard
paper-disc procedure using a mutant, E. coli K-12 RW 193
(ATCC 33475).12 The bacto nutrient agar medium (10 mL)
contained 2.0 mM ethylenediamine di(o-hydroxyphenylacetic
acid) and two drops of the test strain. Filter paper discs (6 mm
diameter) were impregnated with 10 lL of each ligand (1.0
mM) or their iron complex (0.1 mM) solution (50% DMF in
water) and placed on the plate. Water was used as a blank.
The diameter of the growth response zone was checked
against a reference provided with desferrichrome (0.5 mM)
and desferrioxamine B after 48 h at 37 ¡C. With the former
and the latter as references, growth promotion diameter zones
of 20 and 16 mm were observed, but no growth zone was
detected with the ligands and their iron(III) complexes.
water with KNO (1.0 M; 0.3 mL) (pH meter reading, 2.2) and
3
neutralizing to pH 7.0 with 0.1 or 1.0 M KOH. (In some cases
precipitates appeared after mixing of the ligand and the acidic
iron solution for a while, but disappeared as the complexation
reaction proceeded). These complex-containing solutions were
used for further study after 2-fold dilution (1.0 ] 10~4 M,
I \ 0.1). The temperature was maintained at 25.0 ^ 0.1 ¡C
during measurements of UV-vis spectra.
Determinations of UV-vis spectral changes vs. pH were
made by serial addition of HNO (0.1 or 0.01 M) or KOH (0.1
or 0.01 M) to a neutral iron(III) complex solution and any
Acknowledgements
We thank JASCO International Co., Ltd. for recording the
3
changes in its volume due to the addition of acid or base were
corrected. The presence of DMF (2.5%) had no practical e†ect
on the pH-meter reading for every solution in the entire range
(a shift of less than ]0.02 pH unit).
Schwarzenbach plots were made using the spectral data
that exhibited isosbestic points during gradual acidiÐcation.
The presence of 2.5% DMF had no signiÐcant e†ect on the
ESI mass spectra of the iron(III) complexes.
References
1
Iron T ransport in Microbes, Plants and Animals, ed. G. Win-
kelmann, D. van der Helm and J. B. Neilands, VCH Publishers,
Weinheim, 1987.
2
3
J. B. Neilands, J. Biol. Chem., 1995, 45, 26723.
Handbook of Microbial Iron Chelates, ed. G. Winkelmann, CRC
Press, Boca Raton, FL, 1991.
electrode calibration. However, the accuracy of K
less than 30 should be considered with care because of the
limitations of electrode function.
values
Fe(HL)
4
B. F. Matzanke, G. Muller-Matzanke and K. N. Raymond, in
Iron Carriers and Iron Proteins, ed. T. M. Loehr, Physical Bio-
inorganic Chemistry Series, vol. 5, VCH Publishers, New York,
1989, p. 3.
Mass spectrometry of iron complexes
The formation of intramolecular iron(III) complexes was
further conÐrmed by the ESI mass spectra using samples pre-
pared by mixing the ligand and ferric hydroxide made in situ
in water; Fe -2 (H OÈMeCN, 1 : 1 v/v): m/z 757.3
5
B. F. Matzanke, in Encyclopedia of Inorganic Chemistry, ed. R. B.
King, John Wiley & Sons, Chichester, 1994, vol. 4, p. 1915.
R. C. Hider, Struct. Bonding, 1984, 58, 25.
T. Emery, in Metal Ions in Biological Systems, ed. H. Sigel,
Marcel Dekker, New York, 1978, vol. 7, ch. 3, p. 77.
W. KellerÈSchierlein, V. Prelog and H. Zahner, Fortschr. Chem.
Org. Naturst., 1964, 22, 279.
D. van der Helm, M. A. F. Jalal and M. B. Hossain, in Iron
T ransport in Microbes, Plants and Animals, ed. G. Winkelmann,
D. van der Helm and J. B. Neilands, VCH Publishers, Weinheim,
1987, p. 135.
6
7
2
2
[M [ 2H`]2~; Fe -3 (H OÈMeCN, 1 : 1 v/v): m/z 1084.4
3
2
[M [ 2H`]2~ and 722.6 [M [ 3H`]3~.
8
9
Iron(III) exchange with EDTA
Rate determinations. Each of these exchange reactions was
performed at 25 ¡C in a 10-mm cell containing a solution of
2.5 mL of a pH 5.4 AcOHÈAcONa bu†er (40 mM) maintained
10 A. Zalkin, J. D. Forrester and D. H. Templeton, J. Am. Chem.
at ionic strength 0.1 with KNO . The iron(III) complex con-
Soc., 1966, 88, 1810.
3
centration was 1.0 ] 10~4 M, and the reaction was initiated
11 D. van der Helm and M. Poling, J. Am. Chem. Soc., 1976, 98, 82.
12 J. B. Neilands, Struct. Bonding, 1984, 58, 1.
13 G. Muller and K. N. Raymond, J. Bacteriol., 1984, 160, 304.
14 V. Braun and G. Winkelmann, Prog. Clin. Biochem. Med., 1987,
5, 67.
by adding a 50 lL EDTA solution (0.1 M); Ðnal EDTA con-
centration was 2.0 ] 10~3 M. The reaction was monitored by
following the decrease in absorbance at 420 nm and it was
found to obey a Ðrst-order rate law. The pseudo-Ðrst-order
rate constant was determined from the slope of the plot of
ln[(A [ A )/(A [ A )] vs. time, where A , A and A
15 S. A. Leong and G. Winkelmann, in Metal Ions in Biological
Systems, ed. A. Sigel and H. Sigel, Marcel Dekker, New York,
1998, vol. 35, p. 147.
t
=
0
=
0
=
t
New J. Chem., 2001, 25, 275È282
281
Tsubouchi
