Enterobactin Protonation and Iron Release
J. Am. Chem. Soc., Vol. 120, No. 25, 1998 6285
Fe[TRENSAM] (5). TRENSAM (0.3 mmol) was dissolved in 7
mL of MeOH and 0.2 mL of pyridine. To this was added FeCl3 (0.3
mmol). The solution instantly turned deep red, with rapid precipitation
of the product as red crystals. After the solution was stirred for 30
min, the solid was collected by filtration and dried overnight. Yield:
97%. IR (KBr) ν 1537, 1574, 1605 cm-1. (+)-FABMS m/z 560 [M+
+ H].
ArH), 7.90 (s br, 3H, NH). 13C NMR (400 MHz, DMSO-d6, 25 °C) δ
37.8 (CH2), 53.2 (CH2), 56.2 (OCH3), 115.5 (Ar), 115.7 (Ar), 118.2
(Ar), 119.0 (Ar), 148.9 (ArCO), 151.0 (ArCO), 169.8 (CdO). Anal.
Calcd (Found) for C30H36N4O9‚1.75MeOH: C, 58.42 (58.72); H, 6.64
(6.15); N, 8.58 (8.18).
Fe[TREN(3M)SAM] (10). 9 (0.16 mmol) was dissolved in 20 mL
of MeOH. To this solution was added anhydrous FeCl3 (0.14 mmol)
and 0.6 mL of pyridine. The solution turned a deep purple-red color.
The reaction mixture was stirred for 30 min. The solution was
evaporated to dryness and the remaining purple solid collected. IR
Al[TRENSAM] (6). TRENSAM (0.8 mmol) was dissolved in 13
mL of MeOH and 0.2 mL of pyridine. To this was added AlCl3 (0.8
mmol). Within minutes the product precipitated as colorless crystals.
After the solution was stirred for 30 min the solid was collected by
filtration and dried overnight. Yield: 88%. IR (KBr) ν 1537, 1615
(KBr) ν 1435, 1546, 1573, 1592 cm-1
.
Al[TREN(3M)SAM] (11). 9 (0.32 mmol) was dissolved in 20 mL
of MeOH. To this was added AlCl3 (0.30 mmol) and 1 mL of pyridine.
The reaction mixture was stirred for 30 min. The solution was
evaporated to dryness and the remaining white solid collected. IR (KBr)
cm-1 1H NMR (300 MHz, CD3OD, 25 °C) δ 3.47 (s br, 6H, NCH2),
.
3.79 (s br, 3H, CH2NHCO), 4.15 (s br, 3H, CH2NHCO), 6.62 (ddd,
3
4
3J3,4 or 5,6 ) 8.1 Hz, J4,5 ) 7.0 Hz, J4,6 or 3,5 ) 1.1 Hz, 3H, ArH4 or 5),
6.82 (dd, 3J5,6 or 3,4 ) 8.6 Hz, 4J4,6 or 3,5 ) 1.1 Hz, 3H, ArH6 or 3), 7.32
(ddd, 3J5,6 or 3,4 ) 8.6 Hz, 3J4,5 ) 7.0 Hz, 4J3,5 or 4,6 ) 1.6 Hz, 3H, ArH5
ν 1451, 1551, 1584, 1610 cm-1 1H NMR (300 MHz, DMSO-d6, 25
.
°C) δ 2.84 (br s, 6H, CH2), 3.34 (br s, 6H, CH2), 3.76 (br s, 9H, CH3),
or 4), 7.52 (dd, 3J3,4 or 5,6 ) 8.1 Hz, 4J3,5 or 4,6 ) 1.6 Hz, 3H, ArH3 or 6). 13
C
6.53 (br m, 3H, ArH), 7.26 (br d, 3H, ArH), 7.40 (br m, 3H, ArH). 13
C
NMR (400 MHz, CD3OD, 25 °C) δ 36.8 (CH2), 61.1 (CH2), 114.4
(Ar), 116.8 (Ar), 124.3 (Ar), 128.2 (Ar), 137.0 (Ar), 167.5 (ArCO),
174.1 (CdO). (+)-FABMS m/z 531 [M+ + H].
NMR (400 MHz, DMSO-d6, 25 °C) δ 34.6 (CH2), 55.6 (CH2), 56.3
(OCH3), 114.0 (Ar), 115.5 (Ar), 118.6 (Ar), 119.9 (Ar), 151.9 (ArCO),
157.1 (ArCO), 171.3 (CdO).
Structure Solution and Refinement. All X-ray structure data sets
were collected on a Siemens SMART area detector diffractometer.41
Crystals were mounted on quartz capillaries in Paratone oil and were
cooled in a nitrogen stream on the diffractometer. Peak integrations
were performed with the Siemens SAINT software package.42 Space
group determinations were done by the software XPREP. The
structures were solved by direct methods and refined with the
SHELXTL software package (PC version).43 All hydrogen atoms were
fixed at calculated positions and their thermal parameters refined
isotropically; all non-hydrogen atoms were refined anisotropically.
Fe[TRENSAM] (5). Fe[TRENSAM] crystallized as red plates from
a methanol solution of the complex diffused with pentane. The
hydrogen on the TREN scaffold was found during the refinement. One
molecule of methanol is in the asymmetric unit of the crystal.
Al[TRENSAM] (6). Al[TRENSAM] crystallized as colorless, thin
plates from a concentrated methanol solution of the complex diffused
with pentane. It is isostructural with the Fe3+ complex. The hydrogen
on the TREN scaffold was found during the refinement. One molecule
of methanol is in the asymmetric unit of the crystal.
2-Benzoxy-3-methoxybenzoic Acid (7). 3-Methoxysalicylic acid
(54 mmol), K2CO3 (153 mmol), and benzyl chloride (139 mmol) were
suspended in 140 mL of DMF. The solution was heated to 95 °C
overnight. The solution was filtered, and the filtrate was evaporated
to dryness to get a dark oil. The oil was purified through a silica plug
eluted with CH2Cl2, to liberate the benzyl ester intermediate as an amber
oil after removal of solvent. The benzyl ester was hydrolyzed by
stirring the oil in 45 mL of methanol diluted with 25 mL of 6 M NaOH.
After 3.5 h the reaction was complete. The solution was evaporated
to near dryness, diluted with 20 mL of water, and acified with 6 M
HCl, precipitating an amber, crystalline solid. Yield: 96%. IR (film
from CH2Cl2) ν 1486, 1703, 1734 cm-1 1H NMR (300 MHz, CDCl3,
.
25 °C) δ 4.04 (s, 3H, CH3), 5.26 (s, 2H, CH2), 7.30 (br m, 2H, ArH),
7.42 (br m, 5H, ArH), 7.70 (br m, 1H, ArH). 13C NMR (400 MHz,
CDCl3, 25 °C) δ 56.3 (CH3), 65.2 (CH2), 117.4 (Ar), 123.9 (Ar), 125.0
(Ar), 127.0 (Ar), 127.6 (Ar), 128.5 (Ar), 128.9 (Ar), 129.1 (Ar), 129.2
(Ar), 135.1 (Ar), 146.9 (ArCO), 152.3 (ArCO), 165.9 (CdO). Anal.
Calcd (Found) for C15H14O4: C, 69.76 (70.05); H, 5.46 (5.72).
Tris[(2-benzoxy-3-methoxybenzoyl)-2-aminoethyl]amine (8). 7
(9.4 mmol) was dissolved in 20 mL of thionyl chloride and 0.15 mL
of DMF. The reaction mixture was stirred under a nitrogen atmosphere
overnight. The solution was evaporated to dryness and the residue
coevaporated with 2 × 30 mL of CHCl3. The oily residue was
dissolved in 30 mL of dry THF and reacted dropwise (10 min) with a
solution of TREN (3.0 mmol) and 1 mL of triethylamine dissolved in
20 mL of dry THF. The resulting slurry was stirred for 10 min and
filtered, and the filtrate was evaporated to dryness. The residue was
purified by column chromatography with silica gel, eluted with 0-4%
MeOH in CH2Cl2. Evaporation of the eluant gave a brown oil.
Fe[TREN(3M)SAM] (10). Fe[TREN(3M)SAM] crystallized as
purple blocks from a methanol solution of the complex diffused with
diethyl ether. One molecule of pyridine, 2.5 (two full occupancy and
one-half occupancy) water molecules, and 5 methanol molecules were
found in the asymmetric unit. The disorder of some of the solvent
molecules limited the quality of the refinement.
Al[TREN(3M)SAM] (11). Al[TREN(3M)SAM] crystallized as
light pink blocks from a concentrated methanol solution of the complex
diffused with pentane. The hydrogen on the TREN scaffold was found
during the refinement. Two molecules of methanol and one pyridinium
chloride are in the asymmetric unit.
Yield: 53%. IR (film from CH2Cl2) ν 1538, 1657, 2934 cm-1
.
1H
3
1
NMR (300 MHz, CDCl3, 25 °C) δ 2.28 (t, J ) 6.9 Hz, 6H, CH2),
Variable-Temperature H NMR Measurements. Variable-tem-
3.12 (q, 3J ) 6.2 Hz, 6H, CH2), 3.88 (s, 9H, CH3), 5.01 (s, 6H, CH2),
perature 1H NMR experiments were carried out in the range 265-335
K on a Bruker AMX 300 spectrometer operating at 300 MHz. The
temperature was controlled by the B-VT2000 equipment of the
spectrometer that ensures a precision of (1 K. The probe temperature
was allowed to equilibrate for 10 min prior to final magnetic
homogeneity optimization on the lock signal. Variation at a given
temperature was less than (0.1 K. The spectra were recorded in
CD3OD (Aldrich, % D > 99.8). The sample concentration was
approximately 15 mM. All chemical shifts were referenced to the
solvent peak.
3
3
7.03 (d, J ) 6.5 Hz, 3H, ArH), 7.12 (t, J ) 8.0 Hz, 3H, ArH), 7.30
3
(m, 15H, ArH), 7.62 (d, J ) 7.8 Hz, 3H, ArH), 7.77 (br t, 3H, NH).
13C NMR (400 MHz, CDCl3, 25 °C) δ 37.2 (CH2), 52.4 (CH2), 56.0
(CH3), 76.1 (CH2), 115.1 (Ar), 122.5 (Ar), 124.3 (Ar), 127.5 (Ar), 128.5
(Ar), 128.6 (Ar), 128.7 (Ar), 128.8 (Ar), 136.6 (Ar), 136.7 (Ar), 146.3
(ArCO), 152.5 (ArCO), 165.3 (CdO).
Tris[(2-hydroxy-3-methoxybenzoyl)-2-aminoethyl]amine (TREN-
(3M)SAM, 9). 8 (1.7 mmol) was dissolved in 35 mL of MeOH and
0.5 mL of glacial acetic acid. To this solution was added 10% Pd/C
catalyst (10 wt % of 10). The solution was stirred under a hydrogen
atmosphere for 24 h. The solution was filtered, and the filtrate was
evaporated to dryness to give a white foam. The foam was purified
by column chromatography with silica gel, eluted with 0-5% MeOH
in CH2Cl2. Evaporation of the eluting solvent gave the product as a
white foam. Yield: 75%. IR (film from CH2Cl2) ν 1458, 1540, 1587,
1635, 2939 cm-1.1H NMR (300 MHz, CDCl3, 25 °C) δ 2.98 (br s, 6H,
Solutions Thermodynamics. General Methods. All titrant solu-
tions were prepared with distilled water that was further purified by
passing through a Millipore Milli-Q reverse osmosis cartridge system
(resistivity 18 MΩ cm) and then degassed by boiling for 1 h while
being purged by argon. Titrant solutions were stored under an
atmosphere of purified argon with Ridox Oxygen Scavenger (Fisher)
and Ascarite II (A. H. Thomas) scrubbers to prevent absorption of
oxygen and carbon dioxide. Carbonate-free 0.1 M KOH was prepared
from Baker Dilut-It concentrate and standardized by titrating against
3
CH2), 3.64 (br s, 6H, CH2), 3.82 (s, 9H, CH3), 6.53 (t, J ) 8.0 Hz,
3H, ArH), 6.83 (d, 3J ) 12.1 Hz, 3H, ArH), 7.24 (d, 3J ) 5.5 Hz, 3H,