Inorganic Chemistry
Article
dissolved in 10 mL of water and washed with 5 × 30 mL of
dichloromethane. The aqueous layer was carefully neutralized with
solid NaHCO3 and extracted with 4 × 30 mL of dichloromethane.
The solution was dried with MgSO4, filtered, and reduced to dryness
to yield a red-brown oil. The crude product was purified via column
chromatography with silica gel and methanol/dichloromethane/Et3N
(9/90/1) or with basic alumina and methanol/dichloromethane (10/
90) as the eluent to yield pure 1 as a yellow oil (1.68 g, 44%−50%).
1H NMR (500 MHz, CDCl3): δ 8.44 (d, 2H), 7.82 (dd, 2H), 7.74
Electronic Absorption Studies. A solution of lyophilized protein
(50−250 μM) was prepared in nanopure water. Four equivalents of
Fe complex (0.2−1 mM) in nanopure water was added to the protein
solution. Samples were diluted to their final concentrations in a final
volume of 500 μL containing 50 mM potassium phosphate or 100
mM sodium acetate buffer at the indicated pH.
EPR Studies. A solution of protein (500 μM) was prepared in
nanopure water. Four equivalents of Fe complex (2 mM) in nanopure
water was added to the protein solution. Samples were diluted to their
final concentrations in a final volume of 300 μL containing 50 mM
potassium phosphate or 100 mM sodium acetate buffer at the
indicated pH. The sample was transferred to an EPR tube and frozen
at 77 K in liquid nitrogen and the measurement run at 10 K.
XAS Studies. A solution of protein (750 μM) was prepared in
nanopure water. Four equivalents of Fe complex (3 mM) in nanopure
water was added to the protein solution. Samples were diluted to their
final concentrations in a final volume of 250 μL with nanopure water.
The sample was then poured in liquid ethane to freeze and packed as
a solid into a precooled XAS sample holder.
(dd, 2H) 7.42 (t, 2H), 7.34 (d, 2H), 7.06 (t, 2H), 3.85 (m, 6H), 2.86
(t, 2H). MS (ESI, MeOH): m/z calcd C22H20N4O2 [M + (Na+)]
395.15, found 395.09.
N,N-Bis(pyridin-2-ylmethyl)ethane-1,2-diamine (2).42 Hydra-
zine monohydrate (1.10 mL, 0.020 mol) and 1 (1.7 g, 5.0 mmol)
were dissolved in 60 mL of ethanol and refluxed under N2 for 3 h. The
phthalhydrazide byproduct precipitated as a white solid after 15 min
of reflux. The solution was filtered to remove the phthalhydrazide and
washed with 3 × 5 mL of chloroform. The solution was reduced to
dryness, and the yellow oily residue was dissolved in 40 mL of
chloroform and 40 mL of 1 M NaOH. The aqueous layer was
extracted with 3 × 40 mL of chloroform, dried over MgSO4, filtered,
and reduced to dryness. The product 2 was recovered as a yellow oil
(0.80 g, 77%). 1H NMR (500 MHz, CDCl3): δ 8.53 (d, 2H), 7.66 (t,
2H), 7.50 (d, 2H) 7.15 (t, 2H), 3.85 (s, 4H), 2.80 (t, 2H), 2.66 (t,
2H). MS (ESI, MeOH): m/z calcd C14H18N4 [M + (H+)] 243.16,
found 243.11.
Protein Crystallization. Crystallization of [FeII(biot-et-dpa-
(OH2)2(κ1-OE112)⊂2xm-S112E-Sav]. Apo-Sav protein was crystallized
by the sitting drop vapor diffusion method under an inert atmosphere.
Diffraction-quality crystals were grown at room temperature by
mixing 3.5 μL of protein solution (26 mg/mL lyophilized protein in
water) and 1.5 μL of crystallization buffer (2.0 ammonium sulfate, 0.1
M sodium acetate, pH 4). The droplet was equilibrated against a
reservoir solution of 100 μL of crystallization buffer. Single crystals of
Sav were prepared by soaking apo crystals in a soaking buffer (2.6 M
ammonium sulfate, 0.1 M sodium acetate, pH 8) with a 10 mM stock
solution of [FeII(Biot-et-DPA)(OH2)2]Br2 in nanopure water (9 μL
of crystallization buffer, 1 μL of [FeII(biot-et-dpa)(OH2)2]Br2)
overnight. After the soaking, crystals were transferred to cryopro-
tectant for 1 min (30% glycerol in crystallization buffer) and shock-
frozen in liquid nitrogen.
Biot-ethyl-dpa (3).42 A solution of biot-PFP (1.30 g, 3.01 mmol),
2 (0.85 g, 3.0 mmol), and triethylamine (0.39 g, 3.0 mmol) in 20 mL
of DMF was stirred overnight. The DMF was removed under vacuum
to yield a sticky tan residue. The residue was triturated with diethyl
ether until a free-flowing solid formed (3−7 days). The light tan solid
was filtered, washed with diethyl ether, and dried under vacuum (1.49
g, 91%). The solid 3 was stored under an inert atmosphere. 1H NMR
(500 MHz, DMSO): δ 8.44 (d, 2H), 7.75 (br, 1H), 7.71 (t, 2H) 7.50
(d, 2H), 7.21 (t, 2H), 6.40 (s, 1H), 6.33 (s, 1H), 4.27 (t, 1H), 4.07 (t,
1H), 3.72 (s, 4H), 3.16 (q, 2H), 3.04 (q, 1 H), 2.78 (dd, 1 H), 2.55
(s, 1H), 2.46 (t, 2H), 2.02 (t, 2H), 1.45 (m, 6H). MS (ESI, MeOH):
m/z calcd C24H32N6O2S [M + (Na+)] 491.22, found 491.17.
Preparation of Metal Complexes. [FeII(biot-ethyl-dpa)(OH2)2]-
Br2.60 [FeII(Biot-ethyl-DPA)(OH2)2]Br2 was prepared by addition of
3 (53 mg, 0.24 mmol) in 10 mL of acetonitrile to FeBr2. A pale yellow
solid immediately precipitated from the solution. The suspension was
stirred under N2 for 30 min, after which the pale yellow solid was
collected via filtration, washed with CH3CN/diethyl ether (1/1), and
dried under vacuum (0.125 g, 75%). The solid was stored under an
inert atmosphere. HR-MS (ESI, 1% DMF/MeCN): m/z calcd for
C24H32FeN6O2SBr [M − (Br−)] 603.08, found 603.08. Anal. Calcd
for C24H36N6SO4FeBr2: C, 40.02; H, 5.04; N, 11.67. Found: C, 39.67;
H, 4.52; N, 11.38.
Crystallization of [FeIII(biot-et-dpa(OAc)2(κ1-OE112)⊂2xm-S112E-
Sav]. Apo-Sav protein was crystallized by the sitting drop vapor
diffusion method. Diffraction-quality crystals were grown at room
temperature by mixing 3.5 μL of protein solution (26 mg/mL
lyophilized protein in water) and 1.5 μL of crystallization buffer (2.0
ammonium sulfate, 0.1 M sodium acetate, pH 4). The droplet was
equilibrated against a reservoir solution of 100 μL of crystallization
buffer. Single crystals of Sav were prepared by soaking apo crystals in a
soaking buffer (2.6 ammonium sulfate, 0.1 M sodium acetate, pH 6)
with a 10 mM stock solution of [FeIII(biot-ethyl-dpa)(OH2)3]Cl3 in
nanopure water (9 μL crystallization buffer, 1 μL [FeIII(biot-ethyl-
dpa)(OH2)3]Cl3) overnight. After the soaking, crystals were trans-
ferred to cryoprotectant for 1 min (30% glycerol in crystallization
buffer) and shock-frozen in liquid nitrogen. Crystals were prepared in
the same manner for data collected using XFEL.
Crystallization of biot-et-dpa⊂2xm-S112E-Sav. Apo-Sav protein
was crystallized by the sitting drop vapor diffusion method.
Diffraction-quality crystals were grown at room temperature by
mixing 3.5 μL of protein solution (26 mg/mL of lyophilized protein in
water) and 1.5 μL of crystallization buffer (2.0 ammonium sulfate, 0.1
M sodium acetate, pH 4). The droplet was equilibrated against a
reservoir solution of 100 μL of crystallization buffer. Single crystals of
Sav were prepared by soaking apo crystals in a soaking buffer (2.6
ammonium sulfate, 0.1 M sodium acetate, pH 6) with a 10 mM stock
solution of biot-et-dpa in nanopure water (9 μL of crystallization
buffer, 1 μL of biot-et-dpa) overnight. After the soaking, crystals were
transferred to cryoprotectant for 1 min (30% glycerol in crystallization
buffer) and shock-frozen in liquid nitrogen.
[FeIII(biot-ethyl-dpa)(OH2)3]Cl3.61 [FeIII(Biot-ethyl-DPA)(OH2)3]-
Cl3 was prepared by addition of 3 (0.09 g, 0.2 mmol) in 3 mL of
ethanol to FeCl3·6H2O (0.10 g, 0.38 mmol) in 3 mL of ethanol. A
yellow solid immediately precipitated from the solution. The
suspension was stirred under N2 for 15 min, after which the yellow
solid was isolated via filtration. The solid was washed with 10 mL of
chilled ethanol/ether (4/1) and dried under vacuum (0.10 g, 86%).
The solid was stored under an inert atmosphere. HR-MS (ESI,
MeOH): m/z calcd for C24H32Cl2FeN6O2S [M − (Cl−)] 594.10,
found 594.08. Anal. Calcd for C24H38N6SO5FeCl3: C, 42.09; H, 5.59;
N, 12.27. Found: C, 42.28; H, 5.12; N, 12.23.
Analytical and Spectroscopic Methods. HABA Titrations. To
2.4 mL of 8 μM Sav in 200 mM sodium phosphate buffer at pH 7 was
added 300 μL of 10 mM 2-(4′-hydroxyazobenzene)benzoic acid
(HABA) in 200 mM phosphate buffer pH 7. After 5 min
equilibration, the absorbance at 506 nm was recorded. A solution
of 1 mM Fe complex in nanopure water was added in 4−20 μL
portions until approximately 4 equiv had been added. The absorbance
at 506 nm was recorded until no further changes in intensity were
observed.
Crystallization of [FeIII(biot-ethyl-dpa)(OAc)(N3)(κ1-OE112)⊂2xm-
S112E-Sav]. Apo-Sav protein was crystallized by the sitting drop vapor
diffusion method. Diffraction-quality crystals were grown at room
temperature by mixing 3.5 μL of protein solution (26 mg/mL
lyophilized protein in water) and 1.5 μL of crystallization buffer (2.0
ammonium sulfate, 0.1 M sodium acetate, pH 4). The droplet was
equilibrated against a reservoir solution of 100 μL of crystallization
buffer. Single crystals of Sav were prepared by soaking apo crystals in a
G
Inorg. Chem. XXXX, XXX, XXX−XXX