M.G.D. Leed et al. / Journal of Inorganic Biochemistry 105 (2011) 1161–1172
1163
ethylamino)amine (1 mmol, 0.146 g) in 2 mL anhydrous methanol. The
solution was stirred with heating to 60 °C for 3 h. The solvent was
removed by rotary evaporation and the product used without
further purification. 1H NMR (500 MHz, CDCl3): 8.35 (s, 3 H), 7.57 (d,
J=7.0, 3 H), 7.42 (t, J=7.0, 3 H), 6.85 (t, J=7.5, 3 H), 5.24 (d, J=7.4, 3 H),
3.98 (t, J=4.5, 6 H), 2.99 (s, 6 H), 1.30 (s, 36 H). 11B NMR (160 MHz,
CDCl3): 14.80. HRMS (m/z) 789.5028 (Calc. 789.5027) (M+H+, 100%).
TRENBASIM (Tris[(2-(boronic acid)benzylidene)-2-aminoethyl]
amine). A portion of 2-formylphenylboronic acid (3.1 mmol, 0.480 g)
wasaddedtoa solutionof tris(2-ethylamino)amine(1 mmol, 0.149 mL)
in 3 mL anhydrous methanol. The solution was stirred with heating to
60 °C. After 2 h, the clear yellow liquid condensed to a yellow solid. A
sample was removed for characterization and the product used
without further purification. 1H NMR 500 MHz, CD3OD: 8.35 (s, 3 H),
7.56 (s, 3 H), 7.51 (t, 3 H), 7.12 (s, 3 H), 6.00 (s, 3 H), 3.75 (s, 6 H), 2.94
(s, 6 H). 11B NMR (160 MHz, CDCl3): 14.80. MS (m/z): 560.2 (Calc.
560.27) (M+OH−, 70%).
The slow evaporation of a wet chloroform solution of TRENBSIM
produced crystals suitable for X-ray diffraction which contained a water
in the crystal lattice.
Attempts to grow crystals of [FeHTS]OTf suitable for X-ray
diffraction were unsuccessful. As an alternative, [FeHTS]ClO4 was
prepared in small quantity by combining equimolar portions of TS and
[Fe(bipyridine)3](ClO4)2 in boiling acetonitrile and stirring for several
h [** Caution! Perchlorate salts of transition metals complexes are
potentially explosive and should be handled with care. Although no
explosions were encountered in this study, metal perchlorates could
detonate upon heating]. Upon cooling, cold ether was added to
precipitate residual [Fe(bipyridine)3](ClO4)2 and slow evaporation of
the filtrate yielded deep red prismatic crystals of [FeHTS]ClO4. The
asymmetric unit contains two Fe complexes along with one molecule
of acetonitrile.
Data for TRENBSAM, TRENBSIM and [FeHTS]ClO4 were collected at
298, 273 and 296 K, respectively, on a Bruker Kappa Apex II CCD
diffractometer equipped with a graphite monochromator and a Mo Kα
fine focus sealed tube (λ=0.71073 Å) operated at 1.75 kW power
(50 kV, 35 mA). The detector was placed at a distance of 5.0 cm from the
crystal. A total of 2655 frames were collected with a scan width of 0.5°
and an exposure time of 30.0 s/frame. The frames were integrated with
the Bruker SAINT v7.12A software package using a narrow-frame inte-
gration algorithm. Empirical absorption corrections were applied using
SADABS v2.10 and the structures were checked for higher symmetry
with PLATON v1.07. The structures were solved by direct methods with
refinement by full-matrix least-squares based on F2 using the Bruker
SHELXTL Software Package. All non-hydrogen atoms were refined
anisotropically. Hydrogen atoms of sp2 hybridized carbons and nitrogens
were located directly from the difference Fourier maps; all others were
calculated. A table of crystal data and structure refinement for all three
structures can be found in Table S1 of the Supplementary Information,
which also contains full X-ray crystallographic data in CIF format.
TB (Tris[(2-boronic acid-benzyl)2-aminoethyl]amine). A portion
of TRENBASIM (1 mmol, 0.548 g) was taken into a minimal amount of
methanol and cooled to 0 °C over an ice bath before stirring with the
addition of NaBH4 (15 mmol, 0.57 g). After the evolution of H2 gas was
complete, the solution was warmed to r.t. and stirred an additional
30 min. The reaction was quenched with 10 mL H2O and extracted
with 5×50 mL diethyl ether. The aqueous layer was subjected to
continuous extraction with 300 mL of diethyl ether overnight. The
organic portions were combined and evaporated to dryness to afford a
white solid in 40% yield. Purity was confirmed by reverse phase
HPLC, with a single peak at 18.5 min on a X-Bridge C18 column in a
linear 41-min gradient from 7 to 93% acetonitrile in water. 1H NMR
(400 MHz, CD3OD): 7.46 (s, 3 H), 7.20 (s, 9 H), 4.10 (s, 6 H), 3.08 (s, 6
H), 2.98 (s, 6 H). 13C (101 MHz, CDCl3): 184.64, 131.57, 128.35,
127.64, 124.00, 79.89, 55.39, 51.48, 46.05. 11B NMR (160 MHz, D2O):
9.64. IR (cm−1): 3350 (N–H), 2960 (C–H), 1110 (C–O). HRMS (m/z)
531.3140 (Calc. 531.3043) (M–H2O+, 100%). UV/Vis (PBS pH 7.4):
max(ε)–270 nm (1546 M−1 cm−1).
2.4. 11B NMR pH titration
λ
TS (Tris[2-hydroxybenzyl)2-aminoethyl]amine). In a procedure
slightly modified from what has been previously reported, [24]
TRENSIM (1 mmol, 0.458 g) was dissolved in 10 mL methanol and
cooled to 0 °C. To this was added NaBH4 (4 mmol, 0.1513 g) and the
reaction was stirred until the evolution of H2 gas was complete,
approximately 2 h. The solution was warmed to r.t. and stirred an
additional 30 min. The solvent was removed by rotary evaporation,
the remaining material taken into diethyl ether and washed with
3×10 mL water. Evaporation of the solvent produced a yellow solid
in 98% yield. 1H NMR (400 MHz, CDCl3): 7.14 (t, J=7.7, 3 H), 6.97
(d, J=7.2, 3 H), 6.76 (t, J=8.6, 6 H), 3.99 (s, 6 H), 2.73 (t, J=5.5, 6 H),
2.59 (t, J=5.4, 6 H). 13C (101 MHz, CDCl3): 158.04, 128.82, 128.60,
122.01, 119.01, 116.39, 54.04, 52.17, 45.86. IR (cm−1): 3250 (O–H),
Samples containing 20 mM TB and 25 mM HEPES were prepared
in D2O and the pH was adjusted by addition of 2 M NaOH or 2 M
HClO4. The reported pH values are corrected by +0.4 pH units to the
pH meter reading of the deuterated samples, per convention [25].
2.5. Kinetics of prochelator oxidation
The rate of oxidation of prochelator to chelator was determined
under pseudo-first order conditions of excess H2O2. TRENBSAM and
TB were dissolved in DMSO and phosphate buffered saline (PBS),
respectively, and diluted to 100 μM in PBS buffer, pH 7.4, with less
than 1% DMSO in TRENBSAM solutions. Spectra were taken every 12 s
immediately after the addition of 3–50 mM H2O2; data were collected
for 15 min or until no further spectral changes were detected. The
changes in absorbance at 276 nm for TB and 277 nm for TRENBSAM
were used to follow the reaction. The negative slope of the linear fit of
ln[(A−Af)/(Ao −Af)] vs time in s gives the observed rate constant
kobs (where Ao and Af are the absorbance of the intact prochelator and
the final chelator product, respectively). The slope of the line through
the plot of kobs vs [H2O2] provides the second-order rate constant, k
(M−1 s−1). This value k can then be used to determine the rate of
oxidation as defined in Eq. (2).
2832 (C–H), 1588 (N–H). MS (m/z): 465.2 (Calc. 465.28) (M+H+
100%). UV/Vis (PBS pH 7.4): λmax(ε)=276 nm (5920 M−1 cm−1).
,
[FeHTS]OTf. Iron (II) triflate (0.269 g, 0.76 mmol) was added to a
solution of TS (0.353 g, 0.76 mmol) in 3 mL anhydrous acetonitrile to
produce a purple solution. This solution was refluxed for 10 min before
cooling to r.t. over an hour. The solid was washed with cold diethyl ether
to give a deep purple, microcrystalline powder in 69% yield. UV/Vis (PBS
pH 7.4): λmax(ε)=483 nm (4440 M−1 cm−1). HRMS (m/z): 518.1974
(Calc. 518.1980 for [FeHTS]+, 100%). Elem. Anal. Found: C, 49.34; H,
5.10; N, 8.26. Calc. for C28H34F3FeN4O6S: C, 50.38; H, 5.13; N, 8.39.
Although the elemental analysis, especially for C, was somewhat
unsatisfactory, the mass spectral data reasonably support the formula.
Rate ¼ k½H2O2ꢀ½prochelatorꢀ
ð2Þ
2.3. Crystallization, X-ray data collection and structure solution refinement
2.6. Deoxyribose assay
Purified solutions of TRENBSAM failed to yield crystalline material;
however, a methanolic solution of unpurified material produced
cocrystals of TRENBSAM with an equivalent of methyl-2-iodobenzoate.
Deoxyribose assays were conducted on a Perkin Elmer Victor 1420
plate reader. The formation of hydroxyl radicals was measured by