Synthesis and characterization of an unsymmetrical cobalt(III) active site analogue of nitrile hydratase

Article abstract of DOI:10.1007/s00775-011-0794-7

The design, synthesis, and characterization of an unsymmetrical diamidato-dithiol ligand (H₄ 1, where the hydrogen atoms represent deprotonatable amide and thiol protons) and its cobalt(III) complex, a synthetic analogue of the cobalt-containin

Full text of DOI:10.1007/s00775-011-0794-7

J Biol Inorg Chem (2011) 16:937–947
DOI 10.1007/s00775-011-0794-7
ORIGINAL PAPER
Synthesis and characterization of an unsymmetrical cobalt(III)
active site analogue of nitrile hydratase
•
Jennifer K. Angelosante Lauren M. Schopp Breia J. Lewis Amber D. Vitalo Dustin T. Titus
•
•
•
•
•
•
•
•
Rebecca A. Swanson April N. Stanley Brendan P. Abolins Michelle J. Frome Lisa E. Cooper
•
•
David L. Tierney Curtis Moore Arnold L. Rheingold Christopher J. A. Daley
•
•
Received: 18 January 2011 / Accepted: 19 May 2011 / Published online: 3 June 2011
Ó SBIC 2011
Abstract The design, synthesis, and characterization of
an unsymmetrical diamidato-dithiol ligand (H41, where the
hydrogen atoms represent deprotonatable amide and thiol
protons) and its cobalt(III) complex, a synthetic analogue
of the cobalt-containing nitrile hydratase enzyme family,
are reported. The ligand was prepared in 24% yield from an
overall eight-step synthetic pathway following a modified
protocol established in our laboratory that includes two
peptide couples using O-(1H-benzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate as the coupling
agent. The ligand and all precursors were characterized by
NMR spectroscopy and elemental analysis. The cobalt
nitrile hydratase synthetic analogue complex [NBu4][Co(1)]
was prepared on deprotonating ligand H41 to [1]4- on
addition of 5 equiv of NaH in N,N-dimethylformamide and
adding 1 equiv of CoCl2 at -40 °C under a N2 atmosphere
followed by oxidizing the complex by stirring it overnight
open to dry air. The complex [NBu4][Co(1)] was isolated
after counterion exchange with 1 equiv of NBu4Cl fol-
lowed by crystallization from MeCN/Et2O in 71% yield.
The structure of the complex was confirmed by X-ray
diffraction analysis. Cyclic voltammetry studies on
[NBu4][Co(1)] in a 0.1 M [NBu4][PF6]/MeCN solution
showed a quasi-reversible reduction potential at -1.1 V
(vs. Ag/AgCl), and magnetic susceptibility investigations
indicated the complex is paramagnetic in both the solid and
the solution states as determined from inverse-Gouy and
Evans NMR methods, respectively.
J. K. Angelosante ꢀ B. J. Lewis ꢀ D. T. Titus ꢀ
R. A. Swanson ꢀ B. P. Abolins ꢀ M. J. Frome ꢀ L. E. Cooper
Department of Chemistry,
Western Washington University,
516 High Street,
Bellingham, WA 98225, USA
Keywords Cobalt nitrile hydratase ꢀ Metal-amidato ꢀ
Synthetic analogue ꢀ X-ray structure ꢀ Electrochemistry
L. M. Schopp ꢀ A. D. Vitalo ꢀ A. N. Stanley ꢀ
C. J. A. Daley (&)
Department of Chemistry and Biochemistry,
University of San Diego,
´
5998 Alcala Park, San Diego,
CA 92110, USA
Introduction
e-mail: cjdaley@sandiego.edu
Although metals commonly bind to enzymes through
Lewis basic side-chain functional groups of the amino acids
[e.g., thiolates of cysteine], amidato binding [M-NC(O)R]
of deprotonated backbone carboxamido nitrogen atoms is
also known and is of significant interest in biological and
nonbiological systems. Outside medicinal [1] and bioinor-
ganic chemistry [2–4] metal–amidato complexes have
impacted numerous fields of science, including catalysis
[5–11] and polymer chemistry [12, 13]. Examples of metal–
amidato complexes of biological importance include the
P cluster of nitrogenase [14] that is believed to participate in
C. Moore ꢀ A. L. Rheingold
Department of Chemistry and Biochemistry,
University of California,
San Diego,
9500 Gilman Drive,
MC 0332, La Jolla, CA 92093, USA
D. L. Tierney
Department of Chemistry and Biochemistry,
Miami University,
701 East High Street,
Oxford, OH 45056, USA
123

938
J Biol Inorg Chem (2011) 16:937–947
the transfer of electrons required to reduce N2 to NH3 [15],
the A cluster of carbon monoxide dehydrogenase/acetyl
coenzyme A synthase [16] that is the active site responsible
for interconversion of C1 carbon species such as CO2 and
CO via redox reactions [17, 18], technetium–amidato and
rhenium–amidato complexes used in radiopharmaceutical
chemistry [19–21], vanadium–amidato complexes used as
insulin mimetics for diabetes [22, 23], and the copper–
amidato bonding in prions which has potential implications
in Alzheimer’s disease [24] and spongiform encephalopathy
(mad cow disease) [25]. Another enzyme that has received a
great deal of attention in the last decade is the metalloen-
zyme nitrile hydratase (NHase) owing to its use as a bio-
catalyst in the kiloton production of nicotinamide [26] and
other applications [27–29] as well as to its intriguing active
site coordination sphere. NHases contain mononuclear iron-
or cobalt-centered active sites that are coordinated to the
peptide through three cysteine–sulfur and two backbone
amidato bonds (Fig. 1) [30–32]. Of note, two of the three
cysteine–sulfur bonds are found posttranslationally modi-
fied to sulfenic (SOH) and sulfinic (SO2H) bonds and have
been shown to be imperative for proper function of the
enzyme [33–37]. The significance of the active site is
highlighted by the amidato bonding, the unique trio of
thiolate, sulfinic, and sulfenic sulfur-bound moieties, and the
Lewis acid nature of the active site, with no redox activity
observed under native conditions. The latter aspect is par-
ticularly interesting as most other thiolate-bonded iron or
cobalt enzyme active sites are redox centers.
synthesis, and characterization of a diamidato-dithiol
ligand (H41, where the hydrogen atoms represent depro-
tonatable amide and thiol protons) and its four-coordinate
cobalt(III) complex [NBu4][Co(1)] that is an accurate
structural model of the six-, five-, five-membered ring
square planar chelation of the cobalt(III) center in NHase.
We also report on the electrochemistry (cyclic voltamme-
try) and the magnetochemistry (inverse-Gouy and Evans
NMR methods) of [NBu4][Co(1)].
Materials and methods
Reagents and techniques
All reactions were performed using standard Schlenk
techniques or in a N2-filled MBraun glove box. Solvents
were purified using an Innovative Technologies solvent
purification system and were deoxygenated with N2 bub-
bling prior to use (CH2Cl2, Et2O, and MeCN). Senecioic
acid (2; 98%, Alfa Aesar), benzyl mercaptan (3; 99%, Alfa
Aesar), dimethylglycine (H-Aib-OH, 9; Novabiochem),
O-(1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate (HBTU; Advanced ChemTech), and
N,N-dimethylformamide (DMF; 99%, DriSolv EMD) were
purchased from suppliers and used without further purifi-
cation unless stated otherwise.
Physical methods
Synthetic analogues of NHase have been reported and
have yielded significant information about the nature of the
enzyme, including insight into the stability of the cobal-
t(III) center to reduction owing to the amidato bonding in
the active site [38–40]. Several models have been prepared,
but only a couple of unsymmetrical diamidato-dithiol
models have been reported and only one with the accu-
rately modeled unsymmetrical six-, five-, five-membered
ring chelations about the metal center found in the native
active site. Herein we report on our design, complete
NMR data were obtained using Varian Mercury 300 MHz,
Mercury 400 MHz, or INOVA 500 MHz NMR spectrome-
ters. The chemical shifts for proton and carbon were refer-
enced to tetramethylsilane at 0.00 ppm. Electrospray mass
spectrometry was performed at Western Washington Uni-
versity using a Thermo Finnigan LCQ Advantage ESMS.
Elemental analyses were performed at Micro Analysis. The
IR spectra were recorded using a JASCO FT/IR 480 Plus
spectrometer as KBr pellets. UV–vis spectra were recorded
using a Unicam UV 5000 spectrometer. X-ray crystal
structure analysis was performed by Curtis Moore and
Arnold Rheingold at the University of California, San Diego.
Synthesis
Synthesis of 3-(benzylthio)-3-methylbutanoic acid
The synthesis was modified slightly from that reported by
Trost et al. [41]. 3-(Benzylthio)-3-methylbutanoic acid (4)
was prepared by adding senecioic acid (2: 10.06 g,
100.5 mmol), benzyl mercaptan (3: 12.0 mL, 102 mmol),
and piperidine (20 mL) to a 100-mL round-bottom flask in
air. The flask was placed under a N2 atmosphere, stirred, and
Fig. 1 ChemDraw representations of the mononuclear metal active
site of nitrile hydratase (NHase) in the proposed active form (left) and
in the inactive form (right Fe-NHase only)
123

J Biol Inorg Chem (2011) 16:937–947
939
the contents were refluxed overnight (14 h). The solution
appeared amber and viscous after refluxing. The solution was
cooled in an ice bath, acidified with 6 M HCl(aq) (pH \ 4),
and then Et2O was added (75 mL). The mixture was placed
in a separatory funnel, the organic layer was isolated, the
aqueous layer was further extracted with Et2O (2 9 25 mL),
and then the organic layers were combined. The organic
layer was washed with saturated sodium bicarbonate
(3 9 50 mL) and deionized water (3 9 50 mL) and then it
was dried over MgSO4 (16 h). The mixture was filtered, the
MgSO4 was rinsed with Et2O (3 9 20 mL), and the solvent
was removed under a high vacuum. The product 4 was iso-
lated as an oil (16.20 g, 72.22 mmol) in 72% yield. On
standing over days, the trace solvents evaporated and the
product crystallized into a solid. The product was confirmed
by comparison with the reported 1H NMR spectrum and was
used without further purification. 1H NMR (500 MHz,
CDCl3): d 7.21–7.36 (m, 5H, phenyl-H), 3.81 (s, 2H,
SCH2Ph), 2.65 (s, 2H, CCH2COOH), 1.49 (s, 6H, CH3CCH3).
and the contents were stirred in an ice/acetone bath. To a
separate 250-mL round-bottom flask was added 7 (9.4 g,
42 mmol) in THF (100 mL). The solution of 7 was trans-
ferred to the 500-mL flask via a syringe over a period of
1 h as the reaction is highly exothermic. The solution was
warmed to room temperature and further stirred at room
temperature overnight (14 h). The solution was cooled in
an ice/acetone bath and a 20% potassium sodium tartrate
(200 mL) solution was added slowly via a syringe. After
the addition, a white emulsion was present at the bottom of
the aqueous layer. The aqueous layer was transferred to a
separatory funnel and extracted with Et2O (2 9 100 mL).
The organic phase was collected and dried over MgSO4
for 18 h. The solution was filtered and the solvent was
removed under a vacuum to give 2-(benzylthio)-2-meth-
ylpropan-1-amine (8) (7.5460 g, 38.634 mmol) in 93%
yield. Compound 8 was used without further purification.
1H NMR (CDCl3, 500 MHz): d 7.18–7.40 (m, 5H, phenyl-
H), 3.68 (s, 2H, –CH2Ph), 2.62 (s, 2H, –CH2NH2), 1.29 [s,
6H, –C(CH3)2)]. 13C NMR (125 MHz, CDCl3) d 138.18,
128.69, 128.39, 126.79, 51.38, 48.54, 32.46, 26.28.
Synthesis of benzyl(2-methyl-1-nitropropan-2-yl)
sulfane
Synthesis of H-Aib-OBnꢀHTos
Nitromethane (6: 5.4 mL, 100 mmol) was added to acetone
(5: 7.3 mL, 99 mmol) with stirring using a syringe under a
N2 atmosphere in a dry 100-mL round-bottom flask. After
10 min, benzyl mercaptan (3: 5.8 mL, 50 mmol) was added
using a syringe, followed by addition of piperidine (5.0 mL,
50 mmol). The flask was then quickly opened to air and
25 mL of benzene was added to the reaction mixture and a
Dean-Stark trap with 12 mL of benzene was attached along
with a water-cooled reflux condenser. The reaction flask was
put back under a N2 atmosphere and the solution was
refluxed for 10 h. The previously yellow solution was then
an orange color. The reaction mixture was washed with
30 mL of 2 M HCl(aq) in a 125-mL separatory funnel and the
aqueous layer was discarded. The organic layer was washed
twice more with 25 mL of brine and then the aqueous layer
was once again discarded. The resulting organic layer was
dried over anhydrous MgSO4. Solvent was removed under a
high vacuum, leaving the product benzyl(2-methyl-1-nitro-
propan-2-yl)sulfane (7) as an orange oil (9.4 g, 42 mmol) in
84% yield. The product was used without further purifica-
tion to make 2-(benzylthio)-2-methylpropan-1-amine (8).
1H NMR (CDCl3, 500 MHz): d 7.20–7.40 (m, 5H, phenyl-
H), 4.42 (s, 2H, –CH2Ph), 3.82 (s, 2H, –CH2NO2), 1.52 [s,
6H, –C(CH3)2]. 13C NMR (125 MHz, CDCl3) d 136.95,
128.93, 128.72, 127.39, 84.98, 44.41, 33.56, 26.45.
H-Aib-OH (9: 4.0030 g, 38.800 mmol), p-toluenesulfonic
acid monohydrate (11: 7.5019 g, 43.600 mmol), and ben-
zyl alcohol (10: 16 mL, 16 mmol) in toluene (35 mL) were
added to a 100-mL round-bottom flask. A Dean-Stark trap
and a condenser were attached to the flask and the solution
was heated to reflux and left to react for 16 h. On cooling, a
white precipitate formed. The solid was collected by fil-
tration, washed with Et2O (3 9 10 mL), and dried under a
vacuum to yield C-benzyl-protected dimethylglycine
(H-Aib-OBnꢀHTos, (12) (13.66 g, 37.48 mmol) as a white
solid in 97% yield. The product was confirmed by com-
parison with the 1H NMR spectrum of commercially
available 12 and was used without further purification.
1H NMR (400 MHz, dimethyl sulfoxide, DMSO) d 7.91
(d, 2H, phenyl-H), 7.77 (m, 5H, phenyl-H of –CH2Ph),
7.53 (d, 2H, phenyl-H), 5.64 (s, 2H, –CH2Ph), 2.69 (s, 3H,
p-CH3), 1.88 [s, 6H, –NHC(CH3)2C(O)]. 13C NMR
(125 MHz, DMSO) d 172.23, 146.07, 138.76, 136.10,
129.38, 129.19, 129.00, 128.72, 126.31, 68.10, 56.76,
24.19, 21.63.
Synthesis of benzyl 2-(3-(benzylthio)-3-
methylbutanamido)-2-methylpropanoate
Solid H-Aib-OBnꢀHTos (12; 13.0325 g, 35.6605 mmol)
was added to a 250-mL round-bottom flask along with 4
(7.9886 g, 35.6125 mmol), N,N-diisopropylethylamine
(19.0 mL, 109 mmol), and DMF (75 mL) with stirring
under a N2 atmosphere. In a separate 100-mL round-bottom
Synthesis of 2-(benzylthio)-2-methylpropan-1-amine
Tetrahydrofuran (THF; 200 mL) and LiAlH4 (4.7545 g,
125.28 mmol) were added to 500-mL round-bottom flask
123

940
J Biol Inorg Chem (2011) 16:937–947
flask, HBTU (14.8665 g, 39.1995 mmol) was dissolved in
DMF (75 mL) under a N2 atmosphere. The HBTU solution
was transferred to a 250-mL reaction flask and the reaction
solution was stirred at room temperature for 12 h. The
previously clear yellow solution turned a gold-brown color
over this period. The solution was transferred to a 1-L
separatory funnel with ethyl acetate (100 mL) and 10%
HCl(aq) (100 mL). The aqueous layer was extracted and the
organic layer was further washed with 10% HCl(aq)
(3 9 50 mL), saturated sodium bicarbonate (3 9 50 mL),
brine (2 9 50 mL), and deionized water (2 9 50 mL). The
organic layer was dried over MgSO4 (14 h), filtered, and the
solvent was removed under a vacuum leaving, the product
benzyl 2-(3-(benzylthio)-3-methylbutanamido)-2-methyl-
propanoate (13) (10.80 g, 27.03 mmol) in 76% yield. Ele-
mental analysis, Calc. (Obs.): %C 69.14 (69.04), %H 7.32
(7.36), %N 3.51(3.42). 1H NMR (400 MHz, CDCl3) d
7.45–7.06 (m, 10H, aromatics), 6.92 (br s, 1H, N–H), 5.15
(s, 2H, –OCH2Ph), 3.76 (s, 2H, –SCH2Ph), 2.38 [s, 2H,
–C(O)CH2–], 1.51 [s, 6H, –C(CH3)2], 1.39 [s, 6H,
–C(CH3)2]. 13C NMR (101 MHz, CDCl3) d 174.14, 169.42,
137.90, 135.78, 128.95, 128.67, 128.50, 128.20, 128.17,
127.13, 67.06, 56.21, 48.40, 44.73, 33.30, 28.90, 24.88.
(14) (5.6474 g, 18.251 mmol) in 92% yield. Elemental
analysis, Calc. (Obs.): %C 62.11 (62.16), %H 7.49 (7.51),
1
%N 4.53(4.46). H NMR (400 MHz, CDCl3) d 7.35–7.22
(m, 5H, phenyl-H), 7.10 (s, 1H, –NH), 3.79 (s, 2H,
–SCH2Ph), 2.43 [s, 2H, –C(O)CH2–], 1.52 [s, 6H,
–C(CH3)2], 1.43 [s, 6H, –C(CH3)2]. 13C NMR (101 MHz,
CDCl3) d 177.76, 170.91, 137.79, 128.91, 128.75, 127.24,
56.64, 48.10, 44.74, 33.33, 29.01, 24.77.
Synthesis of N-(2-(2-methyl-2-
(benzylthio)propylcarbamoyl)propan-2-yl)-3-methyl-3-
(benzylthio)butanamide
A solution of 14 (5.99771 g, 19.384 mmol) and 8 (3.7872 g,
19.390 mmol) in DMF (250 mL) was prepared in a 500-mL
round-bottom flask under N2. To this was added N,N-diiso-
propylethylamine (10.2 mL, 58.5 mmol) via a syringe. In a
separate 250-mL flask, HBTU (8.0911 g, 21.332 mmol) was
dissolved in DMF (125 mL) under an N2 atmosphere. The
HBTU solution was transferred by a syringe to the 500-mL
round-bottom flask and the reaction mixture was stirred at
room temperature for 18 h. The previously yellow solution
turned golden. The reaction solution was transferred to a 1-L
separatory funnel along with ethyl acetate (350 mL). The
organic layer was washed with 10% HCl(aq) (1 9 200 mL,
3 9 40 mL), with the first wash requiring 200 mL of aque-
ous solution to allow separation of the aqueous and organic
phases. The organic layer was further washed with saturated
NaHCO3(aq) (3 9 30 mL), saturated NaCl (3 9 30 mL),
and deionized water (1 9 50 mL), and the organics were
then dried over MgSO4 for 16 h. The mixture was filtered,
rinsed with CH2Cl2 (4 9 40 mL), and the solvent was
removed under a vacuum to give N-(2-(2-methyl-2-(ben-
zylthio)propylcarbamoyl)propan-2-yl)-3-methyl-3-(benzyl-
thio)butanamide (15) (7.1588 g, 14.708 mmol) as a solid in
76% yield. Elemental analysis, Calc. (Obs.): %C 66.06
(66.36), %H 7.68 (7.89), %N 5.93(5.77). 1H NMR
(400 MHz, CDCl3) d 7.29 (m, 10H, aromatic-H), 6.78 (br t,
1H, –NHCH2–), 6.69 [br s, 1H, –C(O)NHC(Me)2–], 3.77 (s,
2H, –SCH2Ph), 3.70 (s, 2H, –SCH2Ph), 3.26 (d, 2H,
–NHCH2–), 2.38 (s, 2H), 1.49 [s, 6H, –C(CH3)2–], 1.44 [s,
6H, –C(CH3)2–], 1.28 [s, 6H, –C(CH3)2–]. 13C NMR
(101 MHz, CDCl3) d 174.22, 169.78, 138.25, 137.99,
128.92, 128.86, 128.75, 128.57, 127.20, 127.01, 57.41,
49.06, 47.49, 46.98, 44.79, 33.40, 32.76, 29.19, 26.62, 25.33.
Synthesis of 2-(3-(benzylthio)-3-methylbutanamido)-2-
methylpropanoic acid
To a 500-mL round-bottom flask was added 13 (7.9068 g,
19.789 mmol), methanol (110 mL), and deionized water
(60 mL). While the mixture was being stirred vigorously,
solid K2CO3 (4.7841 g, 34.615 mmol) was added. After
15 min the previously cloudy yellow-orange solution
became clear and another portion of K2CO3 (4.7810 g,
34.136 mmol) was added. A reflux condenser was attached
to the flask and the solution was refluxed for 12 h. The
solution was cooled to room temperature and the methanol
was removed under a vacuum, leaving a cloudy yellow
aqueous solution which was transferred to a 250-mL se-
paratory funnel with deionized water (25 mL). The solu-
tion was washed with CH2Cl2 (3 9 35 mL) to remove
leftover benzyl alcohol from the reaction mixture. The
aqueous layer was transferred to a 600-mL beaker, to
which 4 M HCl(aq) was added until the solution pH was
approximately 1, as determined by pH paper. This left an
oil and a cloudy aqueous solution to which CH2Cl2
(100 mL) was added until both the organic and the aqueous
layers were clear. The mixture was transferred to a 500-mL
separatory funnel, organic layer was separated, and the
aqueous layer was extracted with CH2Cl2 (3 9 35 mL),
with all the organic layers being combined. The organic
solution was dried over Na2SO4 for 18 h, filtered, and
the solvent was removed under a vacuum, leaving 2-(3-
(benzylthio)-3-methylbutanamido)-2-methylpropanoic acid
Synthesis of N-(2-(2-mercapto-2-
methylpropylcarbamoyl)propan-2-yl)-3-mercapto-3-
methylbutanamide
In a dry 250-mL three-neck flask under an N2 atmosphere,
immersed in a dry ice/2-propanol bath, and fitted with a
condenser which was filled with dry ice and 2-propanol,
123

J Biol Inorg Chem (2011) 16:937–947
941
ammonia (100 mL) was condensed. A solution of 15
(7.8563 g, 14.745 mmol) in THF (80 mL) was prepared
under N2 and transferred to the three-neck flask via a syr-
inge. Over the course of 2 h, sodium (approximately 3 g)
was added to the three-neck flask in small portions with
stirring until the solution turned deep blue and remained
blue for 30 min without the addition of more sodium. The
reaction mixture was then quenched by the addition of
NH4Cl (3.0482 g, 56.986 mmol), leaving a light-yellow
solution and a white precipitate. The flask was removed
from the dry ice/2-propanol bath and a stream of N2 was
blown over the surface of the solution to facilitate the
evaporation of ammonia. The remaining solution was
inverse-filtered into a 250-mL side-arm flask and the
remaining white solid residue was washed with THF
(4 9 50 mL) and inverse-filtered into the 250-mL side-arm
flask. The solvent was removed under a high vacuum,
leaving N-(2-(2-mercapto-2-methylpropylcarbamoyl)pro-
pan-2-yl)-3-mercapto-3-methylbutanamide (H41) as an off-
white solid (4.1609 g, 13.5654 mmol) in 92% yield. Ele-
mental analysis, Calc. (Obs.): %C 50.94 (51.08), %H 8.55
(8.62), %N 9.14 (8.86). 1H NMR (500 MHz, CDCl3) d 6.96
(br t, 1H, –NHCH2–), 6.41 [br s, 1H, –C(O)NHC(Me)2–],
3.35 (d, 2H, –NHCH2–), 2.48 [s, 2H, –C(O)CH2C(Me)2–],
2.31 (s, 1H, –SH), 1.71 (s, 1H, –SH), 1.61 [s, 6H,
–C(CH3)2–], 1.51 [s, 6H, –C(CH3)2–], 1.36 [s, 6H,
–C(CH3)2–]. 13C NMR (125 MHz, CDCl3) d 174.26, 170.25,
57.62, 53.12, 52.33, 45.65, 42.73, 32.98, 29.91, 25.47.
The solvent was removed under a high vacuum and the
residual solid was dried under a high vacuum for 2 days. The
green solid was dissolved in minimal MeCN and layered
with Et2O to crystallize in the fridge. After 4 days, large
cubic dark-green crystals of [NBu4][Co(1)] (251.2 mg,
0.4259 mmol) were collected by filtration. The filtrate was
transferred to a 250-mL round-bottom flask and placed in a
freezer for 2 days to precipitate a second batch of product
yielding [NBu4][Co(1)] in an overall 71% yield. Elemental
analysis, Calc. (Obs.): %C 57.02 (56.85), %H 9.57 (9.46),
%N 7.12 (6.84). 1H NMR (500 MHz, CDCl3) d 14.81 (br s, 6
H), 12.65 (br s, 6 H), 7.08 (br s, 2 H), 4.60 (br s, 6 H), 3.60 (br
s, 8 H), 2.38 (br s, 2 H), 1.91 (br s, 8 H), 0.29 (br s, 8 H), -0.19
(br s, 12 H). ES-MS: [Co(7)]? m/z expected 361.05;
m/z observed 361.1. UV–vis (MeCN, 22 1°C): kmax
(e) = 628 nm (589), 441 nm (352), 313 (908). Fourier
transform IR (KBr disc): mC=O = 1,608, 1,575 cm-1
.
Crystallography measurements on [NBu4][Co(1)]
The crystal data for [NBu4][Co(1)] are collected in
Table 1. Crystals of [NBu4][Co(1)] suitable for X-ray
structure determination were obtained by vapor diffusion
of diethyl ether into
a
concentrated solution of
[NBu4][Co(1)] in MeCN. Data for [NBu4][Co(1)] were
Table 1 Crystallographic data for [NBu4][Co(1)]
Parameter
Synthesis of [NBu4][Co(1)]
Formula
C29H58CoN3O2S2
603.83
Formula weight (g/mol)
Space group
In a glove box, NaH (109 mg, 4.54 mmol) was added to a
200-mL side-arm flask along with DMF (10 mL). Ligand
H41 (249 mg, 0.812 mmol) was added to a sample vial and
dissolved in DMF (10 mL) with stirring. The DMF solution
of H41 was added slowly to the 200-mL side-arm flask and
stirred for 30 min. In a separate vial, CoCl2 (111 mg,
0.855 mmol) was dissolved in DMF (10 mL) and the vial
was sealed with a septum. Both the side-arm flask and the
CoCl2 sample vial were brought out of the glove box and
placed on a Schlenk line under N2. The side-arm flask was
cooled to approximately -40 °C and then the CoCl2 solu-
tion was added dropwise over 10 min. Upon complete
addition of the CoCl2 solution, the solution immediately
turned dark emerald green. The solution was left stirring
under N2 for 1 h and then it was stirred for a further 16 h
with the flask slowly warming to room temperature and
being opened to air via a drying tube. To the flask was added
NBu4Cl (276.4 mg, 0.9945 mmol), then the flask was
placed under a N2 atmosphere again and the contents were
stirred for 40 min. The solvent was removed under a high
vacuum and the green residue was redissolved in MeCN
(100 mL) and inverse-filtered into a 200-mL side-arm flask.
P21/n
Crystal system
Monoclinic
11.964 (4)
18.589 (6)
16.033 (6)
107.555 (4)
3,399 (2)
4
˚
a (A)
˚
b (A)
˚
c (A)
b (deg)
3
V (A )
˚
Z
qcalcd (g/cm3)
1.180
T (K)
150 (2)
l (Mo Ka) (mm-1
Total reflections
2h range (deg)
Unique reflections
)
0.655
49,025
1.72–27.16
7,432
Parameters/restraints
R1, wR2 [I [ 2r(I)]
R1, wR2 (all reflections)
GOF (on F2)
344/0
0.0398, 0.0970
0.0549, 0.1102
1.054
-3
)
˚
Residual density (e A
-0.743/0.721
See the CCDC-806229 structure file for more information
GOF goodness of fit
123

942
J Biol Inorg Chem (2011) 16:937–947
collected using a Bruker APEXII equipped with a charge-
coupled-device area detector and a graphite monochro-
mator utilizing Mo Ka radiation at -123 °C. Absorption
corrections were performed by multiscan using SADABS
[42]. The structures were solved by the direct methods in
SHELXS97 and refined using full-matrix least squares of
F2 with SHELXL97 [43]. The non-hydrogen atoms were
treated anisotropically, whereas the hydrogen atoms were
calculated in ideal positions and were riding on their
respective carbon atoms. A total of 344 parameters were
refined in the final cycle of refinement using 7,432 reflec-
tions with I [ 2r(I) to yield R1 and wR2 values of 3.98 and
9.70%, respectively. Crystallographic data for this structure
have been deposited with the Cambridge Crystallographic
Data Centre (CCDC) as supplementary publication no.
CCDC-806229. Copies of the data can be obtained free of
charge from the CCDC (12 Union Road, Cambridge CB2
1EZ, UK; Tel.: ?44-1223-336408; Fax: ?44-1223-336003;
e-mail: deposit@ccdc.cam.ac.uk; Web site http://www.
ccdc.cam.ac.uk).
3-methylbutanamide (Scheme 1; H41, where H4 repre-
sents the four protic hydrogens of the thiol and carbox-
amido nitrogen groups of the ligand) was designed to
accurately model the planar six-, five-, five-membered
chelate ring system that is found as part of the first
coordination sphere of the metal center in the NHase
native enzyme. The ligand was synthesized following our
earlier established protocol [44] with some modifications
(Scheme 1, reaction 1). First, senecioic acid (2) was
converted to 3-(benzylthio)-3-methylbutanoic acid (4), via
a Michael addition [45] of benzyl mercaptan (3), in 72%
yield (Scheme 1, reaction 2). The thiol-protected acid 4
was then coupled with C-benzyl-protected dimethylgly-
cine (commonly referred to as H-Aib-OBnꢀHTos, 12),
which was prepared by reaction of a 1:1:1 mixture of
dimethylglycine (H-Aib-OH, 9), benzyl alcohol (10), and
p-toluenesulfonic acid (11) in toluene in a Dean-Stark
trap (Scheme 1, reaction 4). Reaction of 4 with 12 was
the first of two peptide couples using HBTU as the
peptide bond coupling agent. After workup, benzyl
2-(3-(benzylthio)-3-methylbutanamido)-2-methylpropanoate
(13) was obtained in 76% yield. The C terminus was then
deprotected by deesterification with K2CO3 in a 1:2.25
water/methanol mixture under reflux. The product
2-(3-(benzylthio)-3-methylbutanamido)-2-methylpropanoic
acid (14) was obtained as a white solid on acid workup in
92% yield. We note, similar to what was reported by
Kaestle et al. [46], if one increases the amount of water
in the water/methanol mixture to more than 30%, dee-
sterification fails, with 13 being recovered. To complete
the ligand synthesis, the protected thiol amine 2-(ben-
zylthio)-2-methylpropan-1-amine (8) was required. To
prepare the protected thiol amine 8, 2-(benzylthio)-2-
methyl-1-nitropropane (7) was first synthesized using a
modification of a literature preparation [47, 48] where
2 equiv of acetone (5) was reacted with 1 equiv each of
nitromethane (6) and benzyl mercaptan (3) in toluene
(Scheme 1, reaction 3). The water by-product was
removed using a Dean-Stark trap and after workup, 7 was
obtained as an oil in 84% yield. The thiol protected nitro
compound 7 immediately converted to 8 in 93% yield
under reducing conditions [49] with LiAlH4 in diethyl
ether. The thiol-protected ligand (15) was prepared in
76% yield on coupling 8 and 14 under conditions similar
to those used to form 13. The desired ligand H41 was
then obtained as a white solid by deprotecting 15 under
reducing metal conditions (Na, NH3, -78 °C) in 92%
yield after acid workup. The structure of H41 was con-
firmed by elemental analysis and NMR analysis with the
absence of the signals for the methylene and aromatic
protons of the benzyl protecting groups and the appear-
ance of the broadened singlets at d 1.71 and 2.31 that
correspond to the thiol protons.
Cyclic voltammetry measurements on [NBu4][Co(1)]
Measurements were conducted at 20 2 °C using a stan-
dard three-electrode arrangement with a platinum wire as the
counter electrode, and a Ag/AgCl double-junction reference
electrode in MeCN containing 0.1 M (Bu4N)(PF6) support-
ing electrolyte. All potentials are reported versus Ag/AgCl
and were further referenced to ferrocene as an internal
standard. Cyclic voltammograms (500 mV/s) were recorded
with a PINE potentiostat using a platinum working electrode.
Oxygen was removed from each sample by purging the
solutions with high-purity nitrogen before analysis.
Magnetic susceptibility measurements
on [NBu4][Co(1)]
Solid-phase measurements were conducted using the
reverse-Gouy method and a Mathey MSB-1 balance using
two separate batches of freshly recrystallized [NBu4][Co(1)]
sample (crushed to required consistency) at 23.2 0.2 °C.
Solution-phase measurements were conducted using the
Evans method with a 500 MHz NMR spectrometer at
20.1 0.1 °C. The sample concentration was 33.9 mM in
CDCl3 with 2% CH2Cl2 used as the reference signal.
Results and discussion
Synthesis and characterization of ligand
The unsymmetrical ligand framework of N-(2-(2-mer-
capto-2-methylpropylcarbamoyl)propan-2-yl)-3-mercapto-
123

J Biol Inorg Chem (2011) 16:937–947
943
Scheme 1 Synthetic pathways
used to produce the nitrile
hydratase (NHase) analogue
ligand H41. HBTU O-(1H-
benzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium
hexafluorophosphate, DIPEA
N,N-diisopropylethylamine,
DMF N,N-dimethylformamide
Synthesis and characterization of cobalt(III) complex
changes to an emerald green solution immediately on
addition of the CoCl2 solution. The reaction mixture was
stirred for 0.5–1 h and then allowed to react overnight with
oxygen through a drying tube placed on the flask open to
air. At this point the desired tetraalkyl ammonium salt
(1 equiv) was added to perform a counterion exchange
with the sodium of Na[Co(1)]. We found the tetra-n-butyl
ammonium counterion to produce the best results, yielding
the desired complex as a pure crystalline solid on crystal-
lization as confirmed by mass spectral and elemental
analyses as well as X-ray crystallography. Attempts with
other counterions were less successful, yielding fine pow-
ders or oily products. The product [NBu4][Co(1)] is
obtained as an emerald green solid in 71% yield. Electro-
spray mass spectral analysis identified the anion [Co(1)]-
(m/z expected 361.05; m/z observed 361.1), and UV–vis
[kmax(e) = 628 nm (5,890), 441 nm (3,520), and 313 nm
(9,080)] and Fourier transform IR (mC=O = 1,608 and
1,575 cm-1) spectroscopic analyses corroborated the tet-
radentate diamidato-dithiolate coordination. The latter is
supported by the average lowering of the amide C=O
Two methods can be used to obtain the desired cobalt(III)
synthetic analogue complex: (1) following the method of
Tyler et al. [50] (Scheme 2, method A) using a direct
substitution of the ligand onto the cobalt(III) precursor
[Co(NH3)5Cl]Cl2 in DMF under refluxing conditions or (2)
following a similar procedure reported by Ellison et al. [51]
(Scheme 2, method B) and Chatel et al. [52] (Scheme 2,
method C) by coordinating the ligand first to a cobalt(II)
precursor followed by oxidation of the product via addition
of 1 equiv [FeCp2][BF4] (where Cp is cyclopentadienyl) or
dry air to obtain the desired cobalt(III) oxidation state.
Each method was used successfully, but the method C
produced the best yields and easiest workup in our hands
and is thus our method of choice for the preparation of
[NBu4][Co(1)]. The procedure involves the preliminary
deprotonation of H41 using an excess of NaH (5 equiv) in
DMF followed by the slow addition of a DMF solution of
CoCl2 (1 equiv) at –40 °C, all under inert-atmosphere
conditions. The pale golden yellow DMF solution of Na41
123

944
J Biol Inorg Chem (2011) 16:937–947
Scheme 2 Successful synthetic pathways to the unsymmetrical NHase analogue [NR4][Co(1)]. Cp cyclopentadienyl
stretching frequencies in H41 (mC=O = 1,653 and
1,530 cm-1) indicating increased resonance to the amide
nitrogen that is expected on coordination and which is
consistent with other reported systems [38–40]. Unambig-
uous confirmation of the coordination sphere was con-
firmed by cobalt extended X-ray absorption fine structure
EXAFS analyses of [NEt4][Co(1)], indicating coordination
˚
of only two nitrogen (Co–NAvg = 1.93 A) and two sulfur
˚
(Co–SAvg = 2.10 A) atoms to the cobalt(III) metal center
(unpublished results). The structural characterization of
[NBu4][Co(1)] in the solid state was completed through
single-crystal X-ray diffraction analysis and is presented in
the next section. The complex [NBu4][Co(1)] is consider-
ably air and water stable as indicated by an unchanging
UV–vis spectrum in MeCN left in air for over 24 h as well
1
as an unchanged H NMR spectrum after storing the solid
Fig. 2 ORTEP view of [NBu4][Co(1)] showing atom labeling with
30% probability thermal ellipsoids. Hydrogen atoms and the coun-
terion have been removed for clarity
complex for over 1 week in air.
Single-crystal X-ray crystallographic analysis
˚
[53] [Co–N, 1.903(4) A; Co–N, 1.890(4) A; Co–S,
˚
˚
2.141(1) A; Co–S, 2.145(1)] for the similar glycine-back-
Crystals suitable for single-crystal X-ray analysis were
obtained from the vapor diffusion of diethyl ether into a
concentrated solution of [NBu4][Co(1)] in MeCN at
-25 °C. The X-ray data are presented in Fig. 2 and Tables 1
and 2. The ligand H41 binds in a slightly distorted square
planar configuration about the cobalt(III) as confirmed by
the summation of the bond angles between the coordinating
atoms [sum of S(1)–Co–N(1) ? N(1)–Co–N(2) ? N(2)–
Co–S(2) ? S(2)–Co–S(1) bond angles 360° ideally for
square planar; [NBu4][Co(1)] sum of bond angles
361.4(2)°]. The structure is slightly distorted from square
planar geometry,with the sulfur atom of the six-membered
bone-derived complex (16; Fig. 3), with only the Co–N1
bond length of [NBu4][Co(1)] being shorter.
However, the Co–Namide and Co–Sthiolate bond lengths of
[NBu4][Co(1)] lie in the range, within error (3r), observed
for other reported similar square planar amidato-thiolate-
˚
based cobalt(III) complexes (Co–Namide, 1.86–1.92 A;
˚
Co–S, 2.13–2.14 A) and are only slightly shorter that the
˚
those reported by Miyanaga et al. [54] (Co–N1, 1.96 A;
˚
Co–N2, 2.09 A, Co–S1, 2.28 A, Co–S2, 2.14 A) for the
˚
˚
˚
native enzyme structure at 1.8-A resolution. As expected,
the bond angles of [NBu4][Co(1)] are similar to those of
16, with an N1–Co–N2 bond angle of 85.53° [85.1(2)°], an
N1–Co–S1 bond angle of 87.05° [86.7(4)°], an N2–Co–S2
bond angle of 100.88° [100.5(1)°], and an S1–Co–S2 bond
angle of 87.92° [87.81(5)°], where the angles for 16 are
listed in brackets. The N1–Co–N2 and N2–Co–S2 angles
are smaller and larger, respectively, than those of other
reported amidato-thiolate cobalt(III) complexes but they
correspond more closely with the native active site struc-
ture (N1–Co–N2, 84.7°; N1–Co–S1, 86.3°; N2–Co–S2,
97.2°; S1–Co–S2, 91.2°). Overall, [NBu4][Co(1)]
˚
ring (S2) being offset by 0.65 A from the plane set by S1–
˚
N1–N2–Co, where the atoms all are within 0.08 A of the
˚
˚
˚
˚
plane (0.04 A for S1, 0.01 A for N1, 0.05 A for N2, 0.08 A
for Co). Within the complex, the Co–N1 and Co–N2 bond
lengths are similar but with the Co–N2 bond being slightly
elongated as compared with the Co–N1 bond [1.8528
˚
(18) A and 1.8957 (18) A, respectively]. The Co–S1 and
˚
Co–S2 bond lengths are also found to be similar to each
˚
other [2.1591(9) A and 2.1495(8) A, respectively]. These
˚
bond lengths are similar to those reported by Yano et al.
123

J Biol Inorg Chem (2011) 16:937–947
945
˚
Table 2 Selected bond distances (A) and angles (deg) for [NBu4][Co(1)]
Bond
Bond length
Angle
Bond angle
Bond
Bond length
Angle
Bond angle
Co–N1
Co–N2
Co–S1
Co–S2
1.8528 (18)
1.8957 (18)
2.1591 (9)
2.1495 (8)
N1–Co–N2
S2–Co–S1
N2–Co–S2
N1–Co–S1
85.52 (8)
87.92 (3)
100.88 (6)
87.05 (6)
C5–O1
C5–N1
C9–O2
C9–N2
1.236 (3)
1.339 (3)
1.247 (3)
1.348 (3)
N1–Co–S2
N2–Co–S1
165.89 (6)
169.81 (6)
Fig. 3 Unsymmetric NHase analogue reported by Yano et al. [53]
represents a good-quality structural model of the native
enzyme active site.
Electrochemical analysis
Fig. 4 Cyclic voltammogram of [NBu4][Co(1)] in MeCN with 0.1 M
[NBu4][PF6] as the electrolyte at room temperature under an argon
atmosphere. Scans were run at various scan rates to determine
reversibility. A run at 500 mV/s referenced to Ag/AgCl is shown with
arrows indicating the sweep direction
Studies of active site synthetic analogues of NHase and the
native NHase have shown that the coordination of carbox-
amido nitrogens protects the cobalt (III) [and iron(III)]
mononuclear metal center from reduction even in the pres-
ence of coordinated thiolate groups [38–40]. In contrast,
higher oxidation state transition metal complexes with
coordinated thiolate ligands often undergo autoreduction
[38]. Furthermore, carboxamido nitrogen coordination has
been shown to stabilize the higher oxidation states of other
metals as well. The reason for the increased stability has
been associated with the strong r-donor nature of the car-
boxamido nitrogen anion. As such, it was expected that
[NBu4][Co(1)] would have a high reduction potential, sim-
ilar to that of the native cobalt nitrile hydratase. Cyclic
voltammetry studies on [NBu4][Co(1)] dissolved in 0.1 M
[NBu4][PF6] in MeCN under an argon atmosphere showed
(Fig. 4) a single quasi-reversible reduction at -1.1 V versus
Ag/AgCl. This value is in agreement with values reported for
similar amidato-containing synthetic analogues and indi-
cates a high stability of the cobalt(III) center to reduction as
required for the complex to be redox inactive under physi-
ological conditions, possessing only Lewis acid character.
signals observed between -0.5 and 20 ppm (Fig. 5). This
paramagnetic behavior was confirmed by the measurement
of the molar susceptibility of [NBu4][Co(1)] using the
Evans method [55]. On the basis of the chemical shift
difference of reference CH2Cl2 in 0.3 M [NBu4][Co(1)] in
CDCl3 as compared with that in pure CDCl3, the molar
susceptibility was determined to be 3.64 9 10-3 cm3 mol-1,
corresponding to a magnetic moment of 2.93 lB. The
magnetic moment is in agreement with the complex having
spin S = 1, corresponding to two unpaired electrons. The
result is not in agreement with the native cobalt nitrile
hydratase, which is diamagnetic with a low-spin configu-
ration. However, our results parallel those reported for
other similar cobalt nitrile hydratase analogues, where
square planar S = 1 complexes were observed [38–40]. To
corroborate the measured magnetic moment of the complex
in solution, we calculated it in the solid state using the
reverse-Gouy method. In the solid state, [NBu4][Co(1)]
retains its paramagnetic nature, with an average magnetic
moment of 3.36 0.23 lB being determined for multiple
trials. The magnetic moment is consistent with an S = 1
species (two unpaired electrons, taking into account
angular momentum contributions) and further corroborates
the high-spin nature of the four-coordinate complex
[NBu4][Co(1)].
Magnetic susceptibility
The electronic structure of [NBu4][Co(1)] was investigated
through magnetic susceptibility measurements in the solu-
1
tion and solid states. H NMR analysis of [NBu4][Co(1)]
indicates the complex is paramagnetic, with broadened
123

946
J Biol Inorg Chem (2011) 16:937–947
Fig. 5 1H NMR spectrum of
[NBu4][Co(1)] in CDCl3
recorded at room temperature.
The assignments shown are
based on analysis of integration,
chemical shift, and comparisons
with similar complexes
Conclusions
active site. Ligand binding studies with nitriles, cyanide,
isocyanide, and other biologically relevant molecules are
also being conducted.
We have reported the synthesis and characterization of
[NBu4][Co(1)], a cobalt(III) nitrile hydratase synthetic
analogue, that is an accurate structural model of the
unsymmetrical tetradentate plane found in the native
enzyme. The complex has been characterized in solution
and in the solid state and its electronic and magnetic
properties have been investigated. X-ray analyses of
[NBu4][Co(1)] confirmed the desired coordination structure
is obtained, whereas electrochemical analysis (cyclic
voltammetry) supported the Lewis acid character of the
complex, analogous to the native enzyme, showing a quasi-
reversible reduction potential that lies outside that expected
under physiological conditions. Finally, analysis of the
electronic structure of [NBu4][Co(1)] via magnetic suscep-
tibility measurements demonstrated that the complex is
paramagnetic (S = 1) in both the solution and the solid
states. The diamagnetic state is native to the enzyme and
thus modification of [NBu4][Co(1)] through thiolate oxi-
dation and/or electronic and steric derivatization of the
parent ligand (H41) may induce the desired diamagnetic spin
state. Current work on the oxidation of the thiolate moieties
is under way using strong oxidants, as [NBu4][Co(1)] is
relatively air and water stable, to more accurately model the
posttranslational modification observed in the native NHase
Acknowledgments We are pleased to acknowledge the financial
support of the National Science Foundation (NSF-RUI), the Research
Corporation (Cottrell College #CC6100), the Dreyfus Foundation
(Faculty start-up), the American Philosophical Society (Franklin
Research Grant), and the Offices of Sponsored Research at both the
University of San Diego and Western Washington University.
References
1. Intini FP, Lanfranchi M, Natile G, Pacifico C, Tiripicchio A
(1996) Inorg Chem 35:1715–1717
2. Ghosh A, de Oliveira FT, Yano T, Nishioka T, Beach ES,
Kinoshita I, Mu¨nck E, Ryabov AD, Horowitz CP, Collins TJ
(2005) J Am Chem Soc 127:2505–2513
´
3. Meyer M, Fremond L, Espinosa E, Guilard R, Ou Z, Kadish KM
(2004) Inorg Chem 43:5572–5587
4. Henderson W, Oliver AG, Rickard CEF (2000) Inorg Chim Acta
307:144
5. Patten TE, Troeltzsch C, Olmstead MM (2005) Inorg Chem
44:9197–9206
6. Pelagatti P, Carcelli M, Calbiani F, Cassi C, Elviri L, Pelizzi C,
Rizzotti U, Rogolino D (2005) Organometallics 24:5836–5844
7. McPherson LD, Drees M, Khan SI, Strassner T, Abu-Omar MM
(2004) Inorg Chem 43:4036–4050
8. Battacharya S, Snehalatha K, Kumar VP (2003) J Org Chem
68:2741–2747
123

J Biol Inorg Chem (2011) 16:937–947
947
9. Lee BY, Bu X, Bazan GC (2001) Organometallics 20:5425–5431
10. Nishino S, Kunita M, Kani Y, Ohba S, Matsushima H, Tokii T,
Nishida Y (2000) Inorg Chem Commun 3:145–148
11. Ishikawa Y, Ito S, Nishino S, Ohba S, Nishida Y (1998) Z Na-
turforsch C J Biosci 53:378–382
33. Stevens JM, Belghazi M, Jaouen M, Bonnet D, Schmitter JM,
Mansuy D, Sari MA, Artaud I (2003) J Mass Spectrom 38:955–961
34. Murakami T, Nojiri M, Nakayama H, Odaka M, Yohda M,
Dohmae N, Takio K, Nagamune T, Endo I (2000) Protein Sci
9:1024–1030
12. Rojas RS, Wasilke JC, Wu G, Ziller JW, Bazan GC (2005)
Organometallics 24(23):5644–5653
13. Kwon HY, Lee SY, Lee BY, Shin DM, Chung YK (2004) Dalton
Trans 6:921–928
14. Mayer SM, Lawson DM, Gormal CA, Roe SM, Smith BE (1999)
J Mol Biol 292:871–891
15. Jeannine MC, Christiansen J, Dean DR, Seefeldt LC (1999)
Biochemistry 38:5779–5785
35. Piersma SR, Nojiri M, Tsujimura M, Noguchi T, Odaka M,
Yohda M, Inoue Y, Ambe F, Endo I (2000) J Inorg Biochem
80:283–288
36. Nojiri M, Nakayama H, Odaka M, Yohda M, Takio K, Endo I
(2000) FEBS Lett 465:173–177
37. Odaka M, Tsujimura M, Endo I (2001) RIKEN 41:58–60
38. Kovacs JA (2004) Chem Rev 104:825–848
39. Harrop TC, Mascharak PK (2004) Acc Chem Res 37:253–260
40. Yano T, Ozawa T, Masuda H (2008) Chem Lett 37(7):672–677
41. Trost BM, Van Vranken DL, Bingel C (1992) J Am Chem Soc
114:9327–9342
16. Loew GH, Harris DL (2000) Chem Rev 100:407–419
17. Harrop TC, Mascharak PK (2005) Coord Chem Rev 249(24):
3007–3024
18. Riordan CG (2004) J Biol Inorg Chem 9:509–510
19. Aloj L, Panico M, Caraco C, Del Vecchio S, Arra C, Affuso A,
Accardo A, Mansi R, Tesauro D, De Luca S, Pedone C, Visentin
R, Mazzi U, Morelli G, Salvatore M (2004) Cancer Biother
Radiopharm 19(1):93–98
20. Jurisson SS, Lydon JD (1999) Chem Rev 99:2205–2218
21. Liu S, Edwards DS (1999) Chem Rev 99:2235–2268
22. Garribba E, Lodyga-Chruscinska E, Micera G, Panzanelli A,
Sanna D (2005) Eur J Inorg Chem 7:1369–1382
23. Wang D, Behrens A, Farahbakhsh M, Gatjens J, Rehder D (2003)
Chem Eur J 9(8):1805–1813
42. Bruker AXS (2003) SADABS Bruker Nonius area detector
scaling and absorption correction—version 2.10. Bruker AXS,
Madison
43. Sheldrick GM (2008) Acta Crystallogr A 64:112–122
44. Angelosante JK, Lewis BJ, Cooper LE, Swanson RA, Daley CJA
(2006) Phosphorous Sulfur Silicon Relat Elem 181:2263–2376
45. Pattenden G, Shuker AJ (1992) J Chem Soc Perkin Trans
1:1215–1221
46. Kaestle KL, Anwer MK, Audhya TK, Goldstein G (1991)
Tetrahedron Lett 32:327–330
47. Lin WW, Jang YJ, Wang Y, Liu JT, Hu SR, Wang LY, Yao CF
(2001) J Org Chem 66:1984–1991
24. Brown DR, Kozlowski H (2004) Dalton Trans 13:1907–1917
25. Millhauser GL (2004) Acc Chem Res 37:79–85
26. Yamada H, Kobayashi M (1996) Biosci Biotechnol Biochem
9:1391–1400
48. Carroll FI, White JD, Wall ME (1963) J Org Chem 28:1240–1243
49. Zienkiewicz J, Kaszynski P, Young VG Jr (2004) J Org Chem
69:2551–2561
27. Kobayashi S, Shimizu S (1998) Nat Biotechnol 16:733–736
28. Wyatt JM, Knowles KC (1995) Int Biodeterior Biodegrad
35:227–248
29. Maddrell SJ, Turner NJ, Crosby J (1996) Tetrahedron Lett
37:6001–6004
30. Hourai S, Miki M, Takashima Y, Mitsuda S, Yanagi K (2003)
Biochem Biophys Res Commun 312:340–345
31. Miyanaga A, Fushinobu S, Ito K, Wakagi T (2001) Biochem
Biophys Res Commun 288:1169–1174
50. Tyler LA, Noveron JC, Olmstead MO, Mascharak PK (2000)
Inorg Chem 39:357–362
51. Ellison JJ, Nienstedt A, Shoner SC, Barnhart D, Cowen JA,
Kovacs JA (1998) J Am Chem Soc 120:5691–5700
52. Chatel S, Rat M, Dijols S, Leduc P, Tuchagues JP, Mansuy D,
Artaud I (2000) J Inorg Biochem 80:239–246
53. Yano T, Wasada-Tsutsui Y, Kajita Y, Shibayama T, Funahasi Y,
Ozawa T, Masuda H (2008) Chem Lett 37:66–67
54. Miyanaga A, Fushinobu S, Ito K, Wakagi T (2001) Biochem
Biophys 288:1169–1174
32. Nagashima S, Nakasako M, Dohmae N, Tsujimura M, Takio K,
Masafumi O, Yohda M, Kamiya N, Endo I (1998) Nat Struct Biol
5:347–351
55. Schubert EM (1992) J Chem Ed 69(1):62
123