Nitrile Hydratase Model Complex
2
were prepared as previously reported. Thallium triflate was
prepared from thallium carbonate and triflic acid according to
2
7
published protocols.
N,N′-Bis-(2′-methyl-2′-mercaptopropyl)-1-thia-4,7-diazacy-
clononane iron(III) Triflate ([LFe]OTf ·0.5CH Cl ). To a suspen-
2
2
sion of 100 mg (0.24 mmol) of LFeCl in 100 mL of dry acetonitrile
was added dropwise via cannula a solution of 86 mg (0.24 mmol)
of thallium triflate in 15 mL of acetonitrile. After stirring for 15 h,
the solvent was removed under vacuum, and the product extracted
into 60 mL of dichloromethane followed by filtration through a
fritted tube. Removal of solvent from the filtrate yielded
Figure 1. Representation of the active site of nitrile hydratase.
[
6
LFe]OTf ·0.5CH
3%). Electronic absorption (dichloromethane (22 °C)): λmax(ε):
274(7601), 317(6740), 427(2570), 504(1400), 623(1600). IR (KBr
2 2
Cl as a dark blue solid. Yield: 81 mg (0.15 mmol,
4
,8,19-21
catalytic mechanisms have been proposed.
Despite
intensive efforts, it is still uncertain whether water, nitrile,
or both substrates coordinate at the active site during
catalysis.
The substrate binding affinities of active site models have
A high-spin (S ) 5/2),
five-coordinate iron complex with deprotonated amides and
-1
pellet), cm : 3436 (br), 2949 (m), 2912 (m), 2880 (m), 2843 (m),
450 (m), 1262 (s), 1135 (m), 1102 (m), 1074 (m), 1029 (s), 804
m), 636 (m). MS-ESI, m/z calcd. For C14 Fe, [M+] 376.08;
FeCl([LFe]OTf·
1
(
28 2 3
H N S
2
2,23
31 2 4 3 3
Found, 376.05. Anal. Calcd. for C15.5H N S O F
not provided consistent results.
0
.5CH
N, 4.88.
(LFe)
2
Cl
2
): C, 31.48; H, 4.95; N, 4.59. Found: C, 31.17; H, 4.92;
thiolate donors coordinates water but shows no affinity for
[
2
OH]Tf. A solution of (LFe) O (50 mg, 65 µmol) in 100
2
2
2
nitriles. A closely related low-spin (S ) 1/2), five-
coordinate iron complex featuring imine nitrogen donors and
thiolates exclusively binds nitriles at low temperature but
mL of acetonitrile was cooled to -15 °C in a dry ice/ethylene glycol
bath. A solution of 5.7 µL (9.7 mg, 65 µmol) of 98% triflic acid in
1
min. period. The solution was stirred for 2 h as it gradually warmed
to room temperature. The solution was filtered through a fritted
tube and concentrated to 15 mL. Diethyl ether addition led to
0 mL of acetonitrile was added dropwise via cannula over a 30
2
3
undergoes ligand degradation upon exposure to water. To
our knowledge, a single model complex that coordinates both
nitrile and water has not yet been reported. In this manuscript
we report the five-coordinate iron dithiolate, N,N′-bis-(2′-
methyl-2′-mercaptopropyl)-1-thia-4,7-diazacyclononane)iro-
n(III) triflate ([LFe]OTf). [LFe]OTf binds water, nitriles,
and amides allowing for the first time a direct comparison
of substrate (and product) binding affinities in a single model
2
precipitation of [(LFe) OH]Tf as a purple solid. Yield: 45 mg (49
µmol, 75%). X-ray quality crystals were obtained by vapor diffusion
of diethyl ether into a methanol solution of 2 at 4 °C under an
argon atmosphere. Electronic absorption (acetonitrile) λmax(ε):
-1
311(3600), 433(700), 602(1600). IR (KBr pellet), cm : 3490 (br),
958 (m), 2913 (m), 2880 (m), 2847 (m), 1454 (m), 1254 (s), 1147
m), 1131 (m), 1074 (m), 1029 (s), 976 (m), 641 (s), 518 (m). Anal.
Calcd for C29 Fe ([(LFe) OH]OTf·HOTf): C, 36.24;
2
(
a
complex. Additionally, the pK of the water and hydroxide
H
58
N
4
8
S O
7
F
6
2
2
bound derivatives of [LFe]OTf have been evaluated and
offer insight regarding the need for low-spin iron at the
enzyme active site.
H, 5.88; N, 5.64. Found: C, 36.92; H, 5.77; N, 5.57.
Physical Methods. Elemental analyses were obtained from
Midwest Microlab (Indianapolis, IN). Infrared spectra were recorded
-1
on a Thermo Nicolet Avatar 360 spectrometer at 4 cm resolution.
1
H NMR spectra were obtained on a Varian Inova500 500 MHz
Experimental Section
spectrometer. Mass spectrometry (ESI-MS) was performed by the
Laboratory for Biological Mass Spectrometry at Texas A&M
University. The room temperature magnetic moment of [LFe]OTf
was determined by the Evan’s method in dichloromethane with use
Materials and Methods. All reagents were obtained from
commercially available sources and used as received unless
otherwise noted. All solvents were dried and freshly distilled using
standard techniques under a nitrogen atmosphere and degassed using
All reactions were conducted
using standard Schlenk techniques under an argon atmosphere or
of residual CH
2
Cl
2
peak, and its shifted counterpart in calculation
2
8
2
4,25
of µ . Cyclic voltammetry (CV) was performed using a PAR
2
freeze-pump-thaw techniques.
eff
73 potentiostat with a three electrode cell (glassy carbon working
26
electrode, platinum wire counter electrode, and a Ag/AgCl pseudo
reference electrode) at room temperature in an argon filled glovebox.
All potentials were scaled to a ferrocene/ferrocenium standard using
an internal reference. Catalytic trials (see Supporting Information)
were monitored by gas chromatography using an HP 5890 series
II chromatograph fitted with a flame ionization detector using
compressed air as a carrier gas with an RTX-1 column (60 m length,
i.d. 32, serial number 469083) from Restek Corporation.
Electronic absorption spectra were recorded with an Agilent 8453
diode array spectrometer with air free 1 cm path length quartz cell
or in a custom-made Dewar flask with a 1 cm path length quartz
sample compartment. The equilibrium constant for acetonitrile
binding was determined as described in the Supporting Information
in an argon-filled glovebox unless otherwise noted. The complexes
(bmmp-TASN)FeCl], LFeCl, and [(bmmp-TASN)Fe] O, (LFe) O,
[
2
2
(
(
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(
(
(
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(
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Inorganic Chemistry, Vol. 48, No. 5, 2009 2301