Seleno-auranofin (Et3PAuSe-tagl): Synthesis, spectroscopic (EXAFS, 197Au Moessbauer, 31P, 1H, 13C, and 77Se NMR, ESI-MS) characterization, biological activity, and rapid serum albumin-induced triethylphosphine oxide generation

Article abstract of DOI:10.1021/ic902335z

Seleno-auranofin (SeAF), an analogue of auranofin (AF), the orally active antiarthritic gold drug in clinical use, was synthesized and has been characterized by an array of physical techniques and biological assays. The Moessbauer and extended X-ray absorption fine structure (EXAFS) parameters of the solid compound demonstrate a linear P-Au-Se coordination environment at a gold(I) center, analogous to the structure of auranofin. The ³¹P, ¹³C, and ¹H NMR spectra of SeAF in chloroform solution closely resemble those of auranofin. The ⁷⁷Se spectrum consists of a singlet at 481 ppm, consistent with a metal-bound selenolate ligand. The absence of ²J_PSe coupling in the ³¹P and ⁷⁷Se spectra may arise from dynamic processes occurring in solution or because the ²J_PSe coupling constants are smaller than the observed bandwidths. Electrospray ionization mass spectrometry (ESI-MS) spectra of SeAF in 50:50 methanol-water exhibited strong signals for [(Et ₃P)₂Au]⁺, [(Et₃PAu) ₂-μ-Se-tagl]⁺, and [Au(Se-tagl)₂] ^-, which arise from ligand scrambling reactions. Three assays of the anti-inflammatory activity of SeAF allowed comparison to AF. SeAF exhibited comparable activity in the topically administered murine arachadonic acid-induced and phorbol ester-induced anti-inflammatory assays but was inactive in the orally administered carragenan-induced assay in rats. However, in vivo serum gold levels were comparable in the rat, suggesting that differences between the in vivo metabolism of the two compounds, leading to differences in transport to the inflamed site, may account for the differential activity in the carrageenan-induced assay. Reactions of serum albumin, the principal transport protein of gold in the serum, demonstrated formation of AlbSAuPEt₃ at cysteine 34 and provided evidence for facile reduction of disulfide bonds at cysteine 34 and very rapid formation of Et₃P=O, a known metabolite of auranofin.

Full text of DOI:10.1021/ic902335z

Inorg. Chem. 2010, 49, 7663–7675 7663
DOI: 10.1021/ic902335z
Seleno-Auranofin (Et3PAuSe-tagl): Synthesis, Spectroscopic (EXAFS, 197Au
31
1
13
77
€
Mossbauer, P, H, C, and Se NMR, ESI-MS) Characterization, Biological Activity,
and Rapid Serum Albumin-Induced Triethylphosphine Oxide Generation
David T. Hill,*,‡ Anvarhusein A. Isab,§,|| Don E. Griswold,^ Michael J. DiMartino,^ Elizabeth D. Matz,z Angel L. Figueroa,z
Joyce E. Wawro,z Charles DeBrosse,‡ William M. Reiff,# Richard C. Elder,††,r Benjamin Jones,r James W. Webb,†
and C. Frank Shaw III*,†,§
†Department of Chemistry, Illinois State University, Normal Illinois 61790-4160, ‡Department of Chemistry, Temple
University, Philadelphia, Pennsylvania 19122, §Department of Chemistry, University of Wisconsin-Milwaukee,
Milwaukee, Wisconsin 53201, ||Department of Chemistry, King Fahd University of Petroleum and Minerals,
Dhahran 31261, Saudi Arabia, ^Departments of Pharmacology, and zAnalytical Chemistry, GlaxoSmithKline
Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania 19406-0939, #Department of Chemistry,
Northeastern University, Boston, Massachusetts 02115, and rDepartment of Chemistry, University of Cincinnati,
††
Cincinnati Ohio 45221-0172. Deceased.
Received November 25, 2009
Seleno-auranofin (SeAF), an analogue of auranofin (AF), the orally active antiarthritic gold drug in clinical use, was
€
synthesized and has been characterized by an array of physical techniques and biological assays. The Mossbauer and
extended X-ray absorption fine structure (EXAFS) parameters of the solid compound demonstrate a linear P-Au-Se
coordination environment at a gold(I) center, analogous to the structure of auranofin. The 31P, 13C, and 1H NMR spectra of
SeAF in chloroform solution closely resemble those of auranofin. The 77Se spectrum consists of a singlet at 481 ppm,
consistent with a metal-bound selenolate ligand. The absence of 2JPSe coupling in the 31P and 77Se spectra may arise from
dynamic processes occurring in solution or because the 2JPSe coupling constants are smaller than the observed bandwidths.
Electrospray ionization mass spectrometry (ESI-MS) spectra of SeAF in 50:50 methanol-water exhibited strong signals for
[(Et3P)2Au]þ, [(Et3PAu)2-μ-Se-tagl]þ, and [Au(Se-tagl)2]-, which arise from ligand scrambling reactions. Three assays of
the anti-inflammatory activity of SeAF allowed comparison to AF. SeAF exhibited comparable activity in the topically
administered murine arachadonic acid-induced and phorbol ester-induced anti-inflammatory assays but was inactive in the
orally administered carragenan-induced assay in rats. However, in vivo serum gold levels were comparable in the rat,
suggesting that differences between the in vivo metabolism of the two compounds, leading to differences in transport to the
inflamed site, may account for the differential activity in the carrageenan-induced assay. Reactions of serum albumin, the
principal transport protein of gold in the serum, demonstrated formation of AlbSAuPEt3 at cysteine 34 and provided evidence
for facile reduction of disulfide bonds at cysteine 34 and very rapid formation of Et3PdO, a known metabolite of auranofin.
Introduction
matoid arthritis (RA).2 Because of its immunopharmacological
properties,3 AF (1) has also been clinically evaluated for the
treatment of juvenile arthritis,4 psoriasis,5 and steroid-depen-
dent asthma.6 In the latter disease, certain chrysotherapeutic
agents, such as the oligomeric thiolates, gold thioglucose, and
gold sodium thiomalate, which are administered by injection,
Auranofin (1) (AF, Et3PAuS-tagl), a triethylphosphine-co-
ordinated gold(I) thioglucose, first described in 1972,1 has been
used clinically for over three decades in the treatment of rheu-
*Corresponding authors. E-mail: cfshaw@ilstu.edu (C.F.S) and hill@
temple.edu (D.T.H).
(1) Sutton, B. M.; McGusty, E.; Walz, D. T.; DiMartino, M. J. J. Med.
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Baum, J.; Bernstein, B. H.; Kredich, D. W.; Jacobs, J. C.; Zemel, L. S.; Gibbas, D.
J. Rheumatol. 1991, 18, 1240–2.
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r
2010 American Chemical Society
Published on Web 08/12/2010
pubs.acs.org/IC

7664 Inorganic Chemistry, Vol. 49, No. 17, 2010
Hill et al.
have had a long tradition of use, particularly in Japan.7 AF (1),
on the other hand, is effective when given orally, both in the
treatment of RA and psoriatic arthritis.8 The biological me-
chanism of action of gold-based drugs remains undeciphered in
its totality. However, its behavior in biological systems has been
the object of intense study,9 and significant progress has been
made. A key aspect of the biochemical behavior of gold agents
is the displacement of a coordinating sulfur ligand by naturally
occurring sulfhydryl compounds.10-14 Intheexchangereaction
with albumin, the tetraacetylthioglucose portion of AF is
displaced by the SH of cysteine-34,11,13 the only reduced cys-
teine residue, to form AlbSAuPEt3.11-14 This is followed by
displacement of the phosphine ligand and the generation of
Et3PO.11-14 The rate of in vitro generation of Et3PO from
AlbSAuPEt3 is fastest for the thiolate ligands that have the
greatest affinity for gold.11 AF also reacts in vivo and in whole
blood to form Et3PO as a metabolic product.12 Studies of
related AF analogues suggest that for R3PAuX, increasing the
bulk and basicity of the phosphine (R = CH3 < CH3CH2 <
(CH3)2CH) retards its oxidation to R3PdO. Conversely in-
creasing the affinity of the anion for gold(I) favors oxidation
and formation of phosphine oxide.11
The reactions of auranofin and other gold(I) species
with selenoproteins are significant for their metabolism and
enzymology16-19 and in the development of treatments for
arthritis,9d,20,21 cancer,9e,21-24 and a variety of parasitic25-28
and infectious29 diseases. Numerous gold(I) model complexes
with selenium ligands have been characterized,30-36 and the
field has recently been reviewed.37
Described herein is the synthesis and spectroscopic charac-
1
terization (EXAFS, 197Au Mossbauer, and H, 13C, 31P, 77Se
€
NMR, and ESI-MS) of SeAF. The results of in vitro ligand
exchange studies with albumin and cysteine-modified albumin
using 31P NMR spectroscopy and chromatographic separation
are reported. SeAF was tested in vivo against three immuno-
logical models related to inflammation and RA. Only one
report38 and two patents39,40 describing the synthesis of SeAF
have appeared in the literature, but no information regarding
its solution chemistry, biochemistry, or biological activity has
been presented subsequently.
Experimental Methods
Materials. Sephadex G-50 and 5,50-dithiobis(2-nitrobenzoic
acid) were obtained from Sigma Biochemicals; BSA (fatty acid
free, lot no. 10740823-51) from Boehringer Mannheim Bio-
chemicals Co.; and Iodoacetamide-blocked BSA (Ac-BSA) was
prepared as described previously.11 Cysteine-blocked BSA (Cys-
BSA lot no. 11 m) from Miles Chemical Co.; D2O (99.8 d),
Since selenols have higher affinities for mercury(II) and
gold(I) than do thiols and also undergo more rapid exchange
with disulfides than do thiols,15 it was of interest to us to
prepare seleno-auranofin (2) (SeAF, Et3PAuSe-tagl) and to
compare its biological and bioinorganic behavior with AF. In
addition, we sought further insight into the chemistry of cys-
teine-34 of albumin in the reduced (thiolate) and oxidized
(disulfide with a nonprotein thiol) states with gold compounds.
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Article
Inorganic Chemistry, Vol. 49, No. 17, 2010 7665
HS-tagl, and selenourea were purchased from Aldrich Chemical
Co. and used directly as received. Et3PAuCl,1 AF,1 Et3PAuCN,41
and acetobromoglucose42 were prepared as described in the
literature.
on the wiggler side station IV-3 at the gold Lm edge, 11921.2 eV,
out to 14 A-1 and at the selenium K edge, 12660.0 eV, out to 13.5
A-1. The absorption edge energies were calibrated to the gold
Lm edge energy using a 0.01 mm gold foil as an internal
calibrant. All data were measured in the solid phase at ∼17 K
using a flowing helium cryostat (Oxford Instrument CF1208).
NMR Spectra. All the NMR data (1H, 13C, 31P, and 77Se)
reported in the characterization of SeAF were obtained using a
Bruker WM 360 spectrometer operating at 297 K and employing
CDCl3 as a solvent. Thus, the 1H chemical shifts were measured at
360 MHz relative to tetramethylsilane; the 13C{1H} spectra were
obtained at 90.56 MHz using tetramethylsilane as internal standard;
the 31P{1H} chemical shift was recorded at 145.8 MHz employ-
ing trimethylphosphate ((CH3O)3PO) in CDCl3 as the external
standard (δ 31P = 2.00 upfield from 85% phosphoric acid); the
77Se{1H} spectra were recorded at 68.75 MHz using diphenyldise-
lenide in CDCl3 as the external standard (δ 77Se = 481 relative to
dimethylselenium in CDCl3) at 29ꢀ and 55 ꢀC. Low-temperature
31P{1H} spectra were measured from 20 to -27 ꢀC in CDCl3 and
from ambient to -73 ꢀC in CD3OD.
Electrospray ionization (ESI) Mass Spectrometry. ESI mass
spectra were recorded in both positive and negative ion mode using
a Hewlett-Packard (Agilent) LC-MS (Series 1100 LC-MSD) with
40 V fragmentation voltage; 3500 V capillary voltage; N2 flow 10 L/
min @ 25 psi; ionization temperature of 250 ꢀC. Samples (10.0 μLof
5 or 10 mM SeAF in 50:50 methanol-water, MeOH:H2O, mixture)
were injected into the ESI chamber. The flow rate was set at 0.400
mL/min. The mobile-phase solvent was also 50:50 MeOH:H2O.
The solvent composition and spray parameters were fixed for the
analyses. Using the auto sampler, the delay time between mixing
and the first spectral accumulation was typically 3 min.
Biomimetic Albumin-Binding Studies. 31P{1H} NMR Mea-
surements. Preliminary to the BSA-binding experiments, 31P{1H}
NMR spectra were obtained on solutions in deuterated 100 mM
NH4HCO3 buffer, pH of 7.9, using a Bruker WM 250 multinuclear
NMR spectrometer operating at 101.3 MHz. The 31P NMR chemi-
cal shifts were measured and are reported relative to trimethylpho-
sphate, TMP, as an internal reference. Approximately 5000-10 000
scans were accumulated for each spectrum.
31P NMR Titrations of BSA with SeAF. Successive aliquots
(61, 55, and 100 μL) of SeAF (50 mM in MeOH) were added to
2950 μL of BSA (SH titer 0.56; 4.10 mM BSA in 100 mM
N4HCO3 buffer; pH of 7.9). After each addition, the NMR was
measured, and a 100 μL sample was withdrawn and chromato-
graphed to allow analysis of the protein-bound gold.
2-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)-2-selenopseu-
dourea Hydrobromide. Acetobromoglucose (33.2 g, 0.08 mol)
and selenourea (10.0 g, 0.08 mol) in acetone (150 mL) were
refluxed under argon for 2 h (precipitate appeared). After cool-
ing overnight, the resulting product was collected, washed with
cold acetone, and dried in vacuo to give 26.4 g (61%) of
selenopseudourea hydrobromide; Mp 196-197 ꢀC dec (uncor,
darkens at 185 ꢀC; lit43 Mp 187-189 ꢀC). This material was used
in the next reaction without further purification.
(l-Seleno-β-D-glucopyranose-2,3,4,6-tetraacetato-Se)(triethyl-
phosphine)gold(I) (SeAF). A solution of potassium carbonate
(1.40 g, 0.010 mol) in water (10 mL) was added with stirring to a
mixture of the selenopseudourea hydrobromide (5.34 g, 0.010 mol)
in water (30 mL) and was kept at 0 ꢀC. After 30 min, a solution of
Et3PAuCl (3.50 g, 0.010 mol) in ethanol (10 mL)/methylene
chloride (5 mL) was added and stirring continued for an additional
30 min. The mixture was poured into water (200 mL) and extracted
with methylene chloride (3 ꢀ 50 mL). The combined extracts were
dried (MgSO4), filtered, and the solvent removed at reduced
pressure to give 6.48 g of white solid (crude product). Chromatog-
raphy on silica gel, eluting with chloroform, gave 3.72 g (51%) of
product, SeAF. Treatment with anhydrous ethyl ether followed by
crystallization from aqueous methanol gave an analytical sample;
Mp 97-100 ꢀC (lit44 Mp 52 ꢀC); [R]D25 = -55.4ꢀ (1% in CH3OH).
Anal. calcd for C20H34AuO9PSe: C, 33.12; H, 4.72; P, 4.27; Se
1
10.89. Found: C, 33.06; H, 4.70; P, 4.27; Se, 10.70. H NMR
(CDCl3: 05.28 (m, 1H, H1); δ 5.09-5.05 (m, 3H, H2, H3, H4); 4.22
(dd, 1H, J = 5.1 Hz, 12.2 Hz); 4.08 (dd, 1H, dd, J = 2.4 Hz); 3.66
(m, 1H, H5); 2.06, 2.04, 1.99, 1.96 (all S, 12H, CH3CO); 1.85 (m,
6H, PCH2); 1.21 (m, 9H, CH3-). 13C[1H]NMR (CDCl3): δ 170.66
(S, CO); 170.21 (S, CO); 169.49 (S, CO); 169.44 (S, CO); 77.23 (S,
C1, or C2); 74.19 (S, C3, or C5); 73.93 (S, C3, or C5); 68.88 (S, C4);
62.70 (S, C6); 21.15 (S, CH3); 20.72 (S, CH3); 20.60 (S, CH3); 20.55
(S, CH3); (S, CH3); 17.94 (d, PCH2, J = 31.94 Hz); 8.77 (S, CH3).
31P{lH}NMR (CDCl3): δ 35.11. 77Se{1H}NMR (CDCl3): δ 417.8.
197
€
€
Mossbauer Spectroscopy. The Au Mossbauer spectrum of
SeAF (Figure 1) was obtained for 100 mg of finely powdered
crystals in a nylon holder of 12 mm diameter and maintained at
4.2 K throughout the data collection. The 197Pt Mossbauer source
€
was generated via neutron irradiation of highly isotopically enriched
(>90% 196Pt) platinum foils and was also maintained at 4.2 K. The
holder was wrapped in aluminum foil. The instrumentation used
was previously described45 and is based primarily on an integrating
counting technique46 in conjunction with a thin Tl-doped NaI
(crystal) detector. The recorded spectrum was fitted to a single
SeAF/Et3PAuCN/AF Comparison. Three BSA solutions
(4.30 mM; SH titer 0.56; 2.0 mL) were prepared in 100 mM
NH4HCO3 buffer under argon atmosphere. SeAF, Et3PAuCN,
or AF (50 mM in MeOH; 110 μL) were added to the BSA
solutions. The 31P NMR spectra were recorded within 1.5 h and
after 24 h. After the second NMR measurement, each sample
was chromatographed, and the SH titer was measured.
quadrupole doublet.47 The isomer shift was measured relative to
197
metallic gold foil. The Au Mossbauer parameters of AF were
€
measured similarly and agreed with literature values.48
X-ray Absorption Spectroscopy (XAS). XAS data were mea-
sured in transmission mode at the Stanford Synchrotron Radia-
tion Laboratory (3.0 GeV, = 50 mA). The data were collected
Comparisons of Ac-BSA and Cys-BSA Reactions with SeAF.
Ac-BSA (1.89 mM; SH titer <0.01) was prepared in 1950 μL of
100 mM NH4HCO3 buffer. SeAF (75 μL of 50 mM solution in
MeOH) was added, and 31P NMR spectrum was measured. Cys-
BSA (4.39 mM; SH titer <0.06) was prepared in 2375 μL of
buffer, and 200 μL of 50 mM SeAF in MeOH was added to it.
The 31P NMR spectrum was measured, and 100 μL of sample
was withdrawn for chromatographic analysis of Au bonding.
Successive aliquots (50, 50, and 100 μL) of Et3PAuCl (100 mM
in MeOH) were added to the Cys-BSA/SeAF mixture; after each
addition, 100 μL of sample was withdrawn for analysis of gold-
binding to albumin (vide infra). Two aliquots of tagl-SH (100
μL; 100 mM in MeOH) were added successively to the final Cys-
BSA/SeAF/Et3PAuCl reaction mixture. 31P NMR spectra were
accumulated after each addition.
(41) (a) Hormann, A. L.; Shaw, C. F., III.; Bennett, D. W.; Reiff, W. M.
Inorg. Chem. 1986, 25, 3953–3957. (b) Hormann, A. L. Ph.D. Thesis, Unversity
of Wisconsin, Milwaukee, 1988.
(42) Lemieux, R. U. Methods Carbohydr. Chem. 1963, 2, 221–222.
(43) Wagner, G.; Nuhn, P. Ann. Pharm. 1964, 297, 461–473.
(44) A Mp of 52 oC for SeAF is reported in reference 38; no Mp is
disclosed in reference 39.
(45) Viegers, M. P. A.; Trooster, J. M. Phys. Rev. B: Solid State 1977, 15,
72–83.
(46) Viegers, M. P. A.; Trooster, J. M. Nucl. Instrum. Methods 1974, 118,
257–8.
(47) Calis, G. H. M.; Trooster, J. M.; Razi, M. T.; Sadler, P. J. J. Inorg.
Biochem. 1982, 17, 139–145.
(48) Hill, D. T.; Sutton, B. M.; Isab, A. A.; Razi, T.; Sadler, P. J.;
Trooster, J. M.; Calis, G. H. M. Inorg. Chem. 1983, 22, 2936–2942.
Chromatographic Analysis of Gold Binding to BSA and Cys-
BSA. Solutions of BSA (4.3 mM; SH titer 0.56) and Cys-BSA

7666 Inorganic Chemistry, Vol. 49, No. 17, 2010
Hill et al.
(SH titer <0.06; 4.3 mM) were prepared in NH4HCO3 buffer.
To 100 μL aliquots of each were added 10, 20, 30, 40, 50, or 60 μL of
Et3PAuSe-tagl (50 mM in MeOH). After 0.5 h of gentle stirring, the
samples were chromatographed over Sephadex G-50 by using
NH4HCO3 buffer. The ratio of gold bound to albumin (Aub/BSA)
and the SH titer of albumin were determined by analysis of the
appropriate chromatographic fractions. Gold was quantitated by
flame atomic absorption spectroscopy (AAS), albumin by its
UV absorption at 278 nm (E278 =39 600 L mol-1 cm-1) and the
albumin SH titer by using DTNB,49 as previously described.10
Biological Assays of Anti-Inflammatory Activity. Phorbol
Ester-Induced Inflammation. This assay was carried out following
a previously published method used in evaluating AF.50 Thus 1 mg
of drug was dissolved in acetone (0.1 mL) and applied to the inner
and outer surfaces of the right ear of Balb/c mice which had been
treated 30 min before with 12-O-tetradecanoylphorbol acetate
(TPA, 4 μg/20 μL). After 4 h the ear edema was evaluated using a
thickness gauge and compared to the left ear which received only
acetone. After euthanizing the animals with CO2, the inflamed ear
was removed and assayed for myeloperoxidase (MPO) activity as a
measure of inflamed polymorphonuclear leukocytes.
Scheme 1
Arachidonic Acid-Induced Inflammation. Details of the meth-
odology used in this assay were described earlier.51 Arachidonic
acid (AA) (2 mg/20 μL) was applied to the inner surface of the left
ear. The thickness of both left and right ears was measured 1 h after
treatment, and the change in thickness between treated and
untreated ear was recorded. The gold compounds (1 mg/ear) in
acetone were given 15 min prior to AA. At the conclusion of the
experiment (1 h) the animals were euthanized with CO2, and the
inflamed ear was removed and assayed for myeloperoxidase as a
measure of polymorphonuclear leukocyte infiltration.
ience in chemical equations, AF and SeAF are designated as
Et3PAuS-tagl and Et3PAuSe-tagl, respectively, where tagl is
tetraacetylglucose.) In the reaction of acetobromoglucose,
however, selenourea was used in place of thiourea to give the
known acetoglucose selenopseudourea hydrobromide re-
quired as an intermediate. Hydrolysis of this compound with
aqueous potassium carbonate followed by coupling with
chloro(triethylphosphine)gold(I) and purification via chroma-
tography gave SeAF in 51% yield as a white crystalline solid.
The β configuration at the anomeric carbon of SeAF follows
from its mode of synthesis, which proceeds via the characterized
intermediate hydrobromide as well as its 1H, 13C, and 31PNMR
spectra recorded in CDCl3. All of these spectra are comparable
in key aspects to the analogous NMR spectra of AF.54
Carrageenan Rat Paw Edema Assay. The effect of SeAF on
edema in the rat paw was measured in a manner similar to that
employed earlier for the Et3PAu(imido) complexes, according to a
published procedure.52,53 Doses of drug equivalent to between 15
and 20 mg of Au/kg of body weight were administered orally in
tragacanth to male Charles River Lewis rats 1 h before subplantar
injection of carrageenan into the right hind paw. The paw volume
was determined after 3 h and compared to controls.
The proton-decoupled 31P and 77Se NMR spectra both
yielded singlets for SeAF in chloroform solution. The 31P
NMR spectra in methanol, DMSO, and 1:1 MeOH:H2O
obtained during the protein studies (vide infra) also exhib-
Gold Levels in Sera of Rats Dosed with Placebo, AF, or SeAF.
Serum gold concentrations were determined by using a Varian
Spectra AA-20 atomic absorption spectrophotometer equipped
with a GT-96 graphite furnace tube atomizer and an auto
sampler. The furnace was fitted with a plateau tube. The inert gas
used was argon. The samples were diluted 20-fold into 1% v/v
aqueous Triton X-100. The stock standard (998 ppm) was obtained
from Inorganic Ventures, Lakewood, NJ. A series of working
standards was prepared by diluting the stock standard into serum
Triton X-100 solution to obtain a matrix equivalent to the samples.
Standards were run at 0.02, 0.05, 0.10, 0.15, 0.20, 0.30, and 0.40
ppm. A blank was also prepared and analyzed. The method was
validated by spiking sera samples or a serum blank with gold
standard solution to cover the range of the standard curve. The
range covered was equivalent to 0-8 ppm in the samples. Percen-
tage recovery varied from 97 to 105% of theory.
2
ited only a singlet for SeAF. Sadler earlier observed J
coupling of 31P to 15N through gold in 15N-enriched phos-
phine gold(I) phthalimide complexes of 41 and 43 Hz for
Et3P and (C6H5)3P, respectively.53 Hormann and Shaw
2
observed JPC coupling in aliphatic R3PAu13CN com-
plexes (R = Me, Et, iPr, and Cy) at room temperature,
whereas ligand scrambling obscured the coupling in Ph3PAu-
CN at room temperature but not at 200 or 240 K in metha-
nol.41,55 However, 2JPSe coupling through gold between 31
P
and 77Se was not observed in the NMR spectra of either
nuclei at ambient temperature in chloroform or by 31P NMR
in methanol, DMSO, or 1:1 MeOH:H2O. Variable tempera-
2
ture 31P{1H} experiments likewise failed to reveal JPSe
Results and Discussion
coupling through gold(I). In CDCl3 at -27 ꢀC the single
line broadened to ca. 20 Hz at half height, and the chemical
shift moved upfield by ∼150 Hz (1 ppm). In CD3OD, the
temperature was varied incrementally to -73 ꢀC, resulting in
an upfield shift of 5.5 ppm and line broadening to 120 Hz.
Two-bond selenium-phosphorus coupling (2JPSe) may
exhibit either positive or negative values.56 Coupling across gold
Synthesis and Spectroscopic Characterization. The syn-
thesis of SeAF is shown in Scheme 1 and was carried out in a
manner similar to that used in making AF.1 (For conven-
(49) Ellman, G. Arch. Biochem. Biophys. 1959, 82, 70–77.
(50) Carlson, R. P.; O’Neill-Davis, Chang, J.; Lewis, A. J. Agents Actions
1985, 17, 197–204.
(51) Griswold, D. E.; Webb, E.; Schwartz, L.; Hanna, N. Inflammation
1987, 11, 189–199.
(52) Walz, D. T.; DiMartino, M. J.; Griffin, C. L.; Misher, A. Arch. Int.
Pharmacodyn. Ther. 1970, 185, 337–343.
(53) Berners-Price, S. J.; DiMartino, M. J.; Hill, D. T.; Kuroda, R.;
Mazid, M. A.; Sadler, P. J. Inorg. Chem. 1985, 24, 3425–3424.
(54) Razi, M. T.; Sadler, P. J.; Hill, D. T.; Sutton, B. M. J. Chem. Soc.,
Dalton Trans. 1983, 1331–1334.
(55) Hormann, A. L.; Shaw, C. F., III. Inorg. Chem. 1990, 29, 4683–4687.
(56) Duddeck, H. Prog. Nucl. Magn. Reson. Spectrosc. 1995, 27, 1–323.

Article
Inorganic Chemistry, Vol. 49, No. 17, 2010 7667
197
€
Au Mossbauer spectrum of SeAF at 4.2 K; IS = 0.0 for
Figure 1.
gold foil.
Figure 2. FT Au EXAFS spectrum (in phase-shifted angstrom space)
of SeAF. The peak was fit to a combination of P and Se scattering at 2.28
˚
and 2.41 A, respectively.
has been observed in only three cases, (2,4,6-trit-butylphe-
nylselenide)(triphenylphosphine)gold(I), 2JPSe = ( 41 Hz;30
[Ph3PAu{SeC(NH2)2}]Cl, 2JPSe=-13 Hz;31 and [(μ-dppm)-
(AuSeC(NH2)2)2]Cl2, 2JPSe =-13 Hz.32 No 2JPSe coupling
was reported for 28 additional compounds with P-AuI-
Se coordination reviewed by Molter and Mohr,37 including
Beck et al.’s38 earlier report of SeAF. The absence of coupling
may reflect scalar coupling values that are less than the line
widths of the 31P (7 Hz in both CDCl3 and CD3OD) or 77
S
(ca. 3.5 Hz) resonances observed at room temperature. The
broadening observed at low temperature may result from
viscosity effects and/or dissociation or exchange equilibria
for the phosphine and/or selenoglucose ligand. The ESI-MS
studies below address this issue.
€
Mossbauer Spectroscopy. The gold-197 Mossbauer
spectrum of SeAF was obtained at 4.2 K and is exhibited
(Figure 1) as an asymmetric quadrupole doublet. This
asymmetry is indicative of residual sample texture, i.e.,
nonrandom orientation of the principal component of the
electric field gradient tensor at the gold centers with respect to
Figure 3. FT Se EXAFS spectrum (in phase-shifted angstrom space)
of SeAF. Peaks 1-3 arise from C, Au, and, due to the linear geometry at
Au, P at distances of 1.91, 2.39, and 4.61 A.
˚
€
the direction of propagation of the 77.3 keV Mossbauerγray
Based on the peak intensity at 11 928 eV in the XANES
spectrum, SeAF appears to have a central Au atom in the
þ1 oxidation state with a single phosphorus ligand bound
of 197Au. Both the isomer shift (IS) and quadrupole split-
ting (QS) of SeAF values are fairly typical of linear AuI in the
solid state but are smaller than those obtained for AF
measured by us under the same conditions: IS 4.03 and QS
7.40 mm s-l for SeAF compared to 4.89 and 8.69 mm s-l,
respectively, for AF. The latter values for AF are comparable
to those reported in the literature.48 The smaller IS and QS
values for SeAF indicate less distortion in the Se-Au and
Au-P bonds and more covalent character than the S-Au
and Au-P bond of AF. This is consistent with the fact that
selenium is a softer Lewis base.
€
to it (consistent with the Mossbauer spectrum).
EXAFS data were measured at both the gold and
selenium absorption edge energies to determine the local
environment for both atoms. Figure 2 shows the Fourier
transform (FT) for the data measured at the gold edge. A
˚
single intense peak is present at 2.0 A in the filtered EXAFS
(Ff). Curve fitting to the Ff from this peak using a single shell
of either phosphorus or selenium was found to be inadequate.
This is due in part to the large Z difference between the
phosphorus and selenium donors. A much better fit was
achieved by assuming that the peak arises from both phos-
phorus and selenium donors. The results are 0.9 phosphorus
XANES and EXAFS. Both the gold oxidation state and
the presence of Au-P bonds produce readily identifiable
patterns in the X-ray absorption near-edge structure
(XANES) spectrum.57 Gold(I) lacks a peak for the
2p-5d transition that predominates the XANES region
of Au(III) species and does not have the recognizable
peaks at 11 945 and 11 967 eV that are seen in this region
for Au metal. Complexes which contain Au-P bonding
exhibit a peak at 11 928 eV whose magnitude is directly
proportional to the number of bound phosphorus atoms.
˚
˚
atoms at 2.28 A and 1.6 selenium atoms at 2.41 A.
Three distinct peaks are present in the FT for the data
measured at the selenium edge energy (Figure 3). The
filtered EXAFS extracted from first peak was fit as a
carbon donor. The results of this fit are 1.2 carbon atoms
˚
at 1.91 A. In accordance with the Ff from the data at the
gold edge energy, the filtered EXAFS from the main peak
was fit as a Se-Au bond. The results for the calculation
were 0.9 Au atoms at a distance of 2.39 A. The third peak
˚
(57) Elder, R. C.; Eidsness, M. K. Chem. Rev. 1987, 87, 1027–1046.

7668 Inorganic Chemistry, Vol. 49, No. 17, 2010
Hill et al.
in Figure 2 is attributed to the second-shell phosphorus
atom, which results from the colinearity of the Au-P and
Au-Se bonds. The curve fitting to the filtered EXAFS
from this peak gave 1.4 phosphorus atoms at a non-
˚
bonded distance of 4.61 A.
The results from the gold and selenium edge energy data
are in close agreement. First, the Au-Se bond distances
determined by Au and Se EXAFS differ only by 0.02 A.
˚
Second, the sum of the Au-P and Au-Se bond distances
calculated from the gold edge data is 4.69 A, which is in
˚
˚
moderate agreement with the nonbonded distance of 4.61 A
resulting from the fit to a phosphorus donor from the
selenium edge data. This also indicates that there is a nearly
linear angle —P-Au-Se, which is consistent with the pre-
€
sent Mossbauer spectrum and many known gold(I) struc-
tures.57,58 The EXAFS bond distances are in good agreement
with the average Au-Se and Au-P bond lengths, 2.418 (
˚
0.007 and 2.269 ( 0.006 A based on six structures (Ar3-
PAuSeR0) having triarylphosphine and selenol ligands with 9
independent, linear P-Au-Se groupings retrieved from the
Cambridge Crystallography Database.58 The structure im-
€
plied by the EXAFS and Mossbauer results is also in good
agreement with the crystallographically determined structure
of AF,59 which has a sulfur atom in place of the selenium.
Electrospray Ionization Mass Spectrometry. Positive-
and negative-ion spectra were obtained on 5.0 mM samples
in MeOH:H2O solvent to determine whether scrambling to
cationic and anionic species was occurring in solution. The
positive-ion spectra (Figure 4a,b) included dominant signals
for [(Et3P)2Au]þ exhibiting the parent ion (m/z = 433 and
an M þ 1 13C signal); [(Et3PAu)2Se-tagl]þ (m/z = 1041,
80Se) exhibiting the expected six component Se isotopic
signature (74Se, 76Se, 77Se, 78Se, 80Se, and 82Se at 1.8, 18.9,
15.4, 47.9, 100, and 17.6 abundances relative to 80Se with
superimposed M þ 1 13C signals, see Supporting Informa-
tion, Figure S1), and [(Et3PAu)3Se-tagl]þ (m/z = 1357).
Protonated seleno-auranofin [SeAF-H]þ and weaker so-
diated and potassiated signals (m/z = 727, 749, 765, 80Se)
were also observed (Figure 4a). In the negative-ion spec-
trum, a single peak for [Au(Se-tagl)2]- (m/z = 1019 80Se2,
pattern characteristic of a diselenium species, see Support-
ing Information, Figure S2) was observed (Figure 4c). The
signals for the [(Et3P)2Au]þ and [Au(Se-tagl)2]- are strong
and unlikely to be artifacts of the electrospray desolvation
process, unlike the weaker protonated, sodiated, and po-
tassiated seleno-auranofin signals. Thus, the mass spectra
provide evidence for scrambling of the Et3P and tagl-Se-
ligands in solution according to eq 1a:
-
2Et3PAuSe-tag1h½ðEt3PÞ Auꢁþ þ ½AuðSe-tag1Þ ꢁ
Figure 4. ESI-MS spectra of SeAF (10 mM in 1:1 MeOH:H2O):
(a) positive-ion spectrum; (b) isotope pattern for [(Et3PAu)2Se-tagl]þ
(m/z = 1041, 80Se); (c) negative-ion isotope pattern for [Au(Se-tagl)2]-
(m/z = 1019, 80Se2).
2
2
ð1aÞ
The [(Et3PAu)2(Se-tagl)]þ ion can form by a reaction of two
SeAF molecules with displacement of a seleno-tetraacetyl-
glucose ligand according to eq 1b:
2Et3PAuSe-tag1 þ Hþh½ðEt3PAuÞ2Se-tag1ꢁþ
(58) (a) SIHPOO: Jones, P. G.; Thone, C. Chem. Ber. 1990, 123, 1975–
1978. (b) LECTUI, LECVIY: Eikens, W.; Kienitz, W. C.; Jones, P. G.; Thone, C.
J. Chem. Soc., Dalton Trans. 1994, 83–90. (c) UMUJIV: Canales, S.; Crespo,
O.; Gimeno, M. C.; Jones, P. G.; Laguna, A.; Romero, P. J. Chem. Soc., Dalton
Trans. 2003, 4525–4528. (d) OKANIX: Schneider, D.; Nogai, S.; Scheier, A.;
Schmidbaur, H. Inorg. Chim. Acta 2003, 352, 179–187. (e) MESYEP: Laromaine,
A.; Teixidor, F.; Kivekas, R.; Sillanpaa, R.; Arca, M.; Lippolis, V.; Crespo, E.; Vinas,
C. J. Chem. Soc., Dalton Trans. 2006, 5240–5247.
þ tag1-SeH
ð1bÞ
Equilibrium scrambling, analogous to eq 1a, has been
reported for many classes of two-coordinate mixed-ligand
gold(I) species, [YAuX]þ/0/- that exist in equilibrium with
the related homoleptic complexes, [AuY2]( and [AuX2](.
(59) Hill, D. T.; Sutton, B. M. Cryst. Struct. Commun. 1980, 9, 679–686.

Article
Inorganic Chemistry, Vol. 49, No. 17, 2010 7669
The equilibrium constant for scrambling is defined as KS=
[AuX2][AuY2]/[YAuX]2. If the extent of scrambling is purely
statistical, KS=0.250; larger values indicate that the homo-
leptic complexes are favored, whereas a lower value indicates
that the mixed-ligand complex is favored. The initial re-
ports,60,61 beginning in 1985-1986, were primarily qualita-
tive. The cyano(thiomalato)gold(I) complex, [TmSAu-
CN]3-, characterized by 13C NMR,60,61 15N NMR,62 and
Raman spectroscopy60 in aqueous solution, exists in equili-
brium with [Au(CN)2]- and [Au(STm)2]5-; the mono- and
dicyano species were resolved in the 13C and 15N NMR
spectra.60,61 The analogous complexes of glutathione ([GtS-
AuCN]2-),60 thioglucose ([TgSAuCN]-),60,63 and thiosulfate
N)2CS}I], KS = 0.63;65,66 [Au(CN)I]-, KS = 0.50;65,67 and
[Au(SCN)I ]-, KS=0.50.65,68 DMSO dissolves solid AuCN
to form both [Au(CN)2]- and[(DMSO)AuCN],69 indicative
of scrambling by the latter compound. Selenocyanate under-
goes a complex set of reactions with gold(I) thiomalate
[{Au(STm)}n]2n-, producing inter alia [TmSAuSeCN]3- 36
,
which scrambles to form [Au(STm)2]5- and [Au(SeCN)2]-,
which slowly decomposes to selenium and [Au(CN)2]-.
Equilibrium scrambling constants for a series of cyano-
(imidazoladine-2-thione)gold(I) complexes,69 [(RR0Imt)Au-
CN], have been determined for [(Imt)AuCN] in DMSO,
KS=0.630 (0.005) and in methanol, Ks=0.47, and in DMSO
for [(MeImt)AuCN], KS=0.56 (0.02); [(EtImt)AuCN], KS=
0.62 (0.01); [(nPrImt)AuCN], KS=0.59 (0.01); [(iPrImt)Au-
CN], KS = 0.55 (0.01); [(Diaz)AuCN], KS=0.91 (0.01); and
[(Diap)AuCN], KS =0.96 (0.01).69 All are larger than the
statistical value, which indicates that formation of the ionic
products is somewhat favored in solution. The constant mea-
sured in aqueous solution for [(ErgS)AuCN] (KS =1.08)70
reflects the more-polar aqueous environment.70
([Au(CN)(S2O3)]2- 64
also equilibrate to form the dicy-
)
anoaurate(I) and bis(thiolato)aurate(I) or bis(thiosulfato)-
aurate(I) complexes. [Et3PAuCN] is a molecular solid, but
concomitant observation of the signals for [(Et3P)2Au]þ and
[Au(CN)2]- in solution by 31P and 13C NMR demonstrated
the scrambling equilibrium.41,55 ESI-MS spectra of [Et3PAu-
CN] and [Me3PAuCN] in solution exhibit strong signals for
[Au(CN)2]- and the respective [(R3P)2Auþ] cations (J.W.
Webb and C.F. Shaw III, unpublished observations). When
the mixed-ligand complex is neutral, the homoleptic com-
plexes are oppositely charged and, therefore, solvent effects
should be significant. [Et3PAuCN], the first case for which KS
values were reported, exhibits increasing extent of scrambling
in polar solvents at room temperature (with esd values):
CDCl3, KS=0.018 (0.004); C6D6, KS=0.034 (0.008); (CD3)2-
SO, KS=0.092 (0.004); and CH3OD, KS=0.20 (0.06).41,55
Yet, scrambling does occur in the less-polar solvents, benzene
and chloroform (the solvent used for SeAF NMR studies), as
well as more-polar solvents. In contrast, at 297 K, [Ph3PAu-
CN] undergoes rapid exchange with [(Ph3P)2Au]þ and [Au-
KS values were measured in DMSO for a similar series
of cyano(imidazoladine-2-selenone)gold(I) complexes, [(R,
R0ImSe)AuCN], [(ImSe)AuCN], KS=2.95 (0.05); [(MeIm-
Se)AuCN], KS = 2.90 (0.08); [(EtImSe)AuCN], KS=2.76
(0.06); [(i-PrImSe)AuCN], KS=3.12 (0.02); [(PhImSe)Au-
CN], KS = 4.28 (0.05); [(Et2ImSe)AuCN], KS = 2.84; and
[(DiazSe)AuCN], KS=3.64 (0.06).32 The KS values for the
imidazolidine-2-selenone complexes are typically 4-5 times
greater in value for the corresponding imidazolidine-2-
thione, indicating that for selenones, the scrambling equi-
librium lies farther to the right.32
Scrambling of [Cy3PSeAuCN] (KS=1.81) and [Cy3PS-
AuCN] (KS = 0.147) in methanol has been observed by 13
C
(CN)2]-, yielding only a single resonance in the 13C and 31
NMR spectra, but at 200-240 K the respective 13C and 31
P
P
NMR,33,34 but in the 31P NMR spectra, only singlets, con-
sistent with rapid exchange of the phosphine ligand between
[Au(PEt3)2]þ and the parent compound, are observed. In
this case, KS for the selenophosphine complex is more than
an order of magnitude greater than for the thiophosphine
derivative.33
NMR signals were observed for the homoleptic ionic species
(Ks = 0.112 (0.005) at 240 K). Ks values were measured for
four trialkylphosphine analogues, which in methanol exhi-
bited resolved 31P and 13CN resonances for [(Et3P)2Au]þ and
[Au(CN)2]-, respectively, at 298 and 240 K. The Ks values,
measured at 240 K for comparison to the phenyl analogue,
are [Me3PAuCN], Ks=0.37 (0.05); [Et3PAuCN], KS=0.24
(0.02); [iPr3PAuCN], KS = 0.29 (0.03); and [Cy3PAuCN],
KS = 0.49 (0.02).41,55
The equilibrium constants of four inorganic species in
aqueous solution, initially reported as KRD for the reaction in
the reverse sense (i.e., KS=KRD-1), were determined using
electronic spectroscopy for [Au(CN)(mpt)], KS=3.1665 or
calculated from related equilibrium constants for [Au{(H2-
Et3PAuSTg (deacetylated auranofin) in CH3OD and in
1:3 CH3OH:D2O exhibits 31P resonances characteristic of
[(Et3PAuSTg)] and [(Et3P)2Au]þ. The latter increased in
intensity in the more polar MeOH:H2O mixture. Aura-
nofin itself, like selenoauranofin, exhibits a single reso-
nance at ambient temperature, that was not resolved at
lower temperature.41b
Nine neutral compounds having an empirical formula,
YAuX, have been characterized crystallographically
as ionic solids consisting of homoleptic two-coordinate
gold(I) cations and anions: [{(NC(CH2)2)3P}2Au][Au-
(CN)2],71 [Au{(CH3NH)2CdS}2][Au(CN)2],72 [(THT)2-
Au][AuI2],73 [ (TPA)2Au][Au(CN)2],74 [(Imt)2Au][AuI2],75
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hedron 1996, 16, 2781–2785.
(72) Stocker, F. B.; Britton, D. Acta Crystallogr., Sect. C: Cryst. Struct.
Commun. 2000, C56, 798–800.
(73) Ahrland, S.; Noren, B.; Oskarsson, A. Inorg. Chem. 1985, 24, 1330–1333.
(74) Assefa, Z.; Omary, M. A.; McBurnett, B. G.; Mohamed, A. A.;
Patterson, H. H.; Staples, R. J.; Fackler, J. P., Jr. Inorg. Chem. 2002, 41,
6274–6280.
(75) Friedrichs, S.; Jones, P. G. Acta Crystallogr. 1999, C55, 1625–1627.

7670 Inorganic Chemistry, Vol. 49, No. 17, 2010
Hill et al.
[(Ad2BzP)2Au][Au(CN)2],76 [(Me2PhP)2Au][Au(GeCl3)2],77
[(Ph3P)2Au][Au(S(dO)2-p-Tol)2],78 and [(3-BrPy)2Au][Au-
Cl2].79 Spectroscopic evidence also supports ionic formula-
tions for two isocyanide compounds, [(RNC)2Au][AuS-
(dO)2-pTol], (R = tBu, 2,6-Me2C6H3),78 and five alkyl-sub-
stituted imidazolidine-2-selenone complexes, [(RR0ImSe)2-
Au][Au(CN)2], R, R0=Et2; Et,H; Me,H; iPr,H; and Ph,H.32
The equilibrium scrambling constant for [Au{(CH3NH)2Cd
S}2][Au(CN)2] has been measured (KS=0.98(0.03)), demon-
strating the reversibility of the scrambling process,72 i.e., the
homoleptic ionic complexes equilibrate in solution to form
the neutral species. The ionic selenones also exhibited sepa-
rate 13CN resonances for [Au(CN)2]- and [R,R0ImSeAu-
CN], further demonstrating that the scrambling process for
the gold(I) species is reversible.
Biomimetic Albumin-Binding Studies. Human serum al-
bumin is the principle gold transport agent in the blood.9
It is a microheterogeneous protein which consists principally
of mercaptalbumin (AlbS-) in which the acidic cysteine-34
residue is in the reduced state and in an oxidized, disulfide
form in which cysteine 34 is linked to a nonprotein cysteine
residue (AlbSSCy). Bovine serum albumin (BSA) was used
in preference to human albumin because it has a greater and
more reliable content of mercaptalbumin (AlbS-), which
binds gold most avidly10,83 due to the low pKSH value (∼5) of
cysteine-34. (To clarify the discussion, microheterogeneous
preparations of albumin will be designated BSA, whereas
individual molecular species will be designated as AlbS- and
AlbSSCy, etc. Two modified preparations, Ac-BSA and Cy-
BSA, in which the reduced cysteine-34 residues are converted
to a thioether and a disulfide, respectively, are described
below.) The reactions of Et3PAuSe-tagl with BSA were
examined using 31P NMR spectroscopy, size-exclusion (gel
permeation) chromatography to resolve free (i.e., low-mo-
lecular-weight) and protein-bound gold, and DTNB analysis
of the albumin thiol content. The amino-acid sequences of
mammalian albumins contain a total of 35 conserved cy-
steine residues, of which the remaining 34 form 17 “internal”
disulfide linkages. The experiments described below were
performed using a single source of BSA with a thiol titer
([AlbS-]/[BSA]total) of 0.56. This value is close to the in vivo
range of 0.6-0.7, which indicates that minimal oxidation of
Cys-34 had occurred during the preparation and storage of
the albumin. The interpretation of the reactions of Se-AF
This extensive array of gold(I) complexes known to
undergo scrambling in solution, including the thiolato
auranofin analogue [Et3PAuSTg], reinforced by the mul-
tiple examples of imidazolidine-2-selenone and triethyl-
phosphine selenide complexes that exhibit a greater extent
of scrambling, and therefore larger KS values, than the
sulfur analogues, strongly supports the ESI-MS evidence
for the equilibrium scrambling of selenoauranofin accord-
ing to eqs 1a and 1b.
The observation of [(Et3P)2Au]þ and [Au(Se-tagl)2]- in
the ESI-MS spectra, but not by NMR, suggests that the ab-
2
sence of JPSe scalar coupling in the 31P and 77Se NMR
spectra may arise in part from a labile exchange process in
solution. 2JPSe coupling is routinely observed in square planar
Pt(II) compounds and is typically larger for trans orientation
of the Se and P donor atoms than the cis orientation.56,80-82
For example, Pan and Fackler observed JPSe,trans = -100
and JPSe,cis =-10 Hz for [Pt(Se2CN-iBu2)(PPh3)Cl], and
JPSe,trans=-100.5 and JPSe,cis=-10 Hz for [(Se2CN-Et2)-
(PPh3)Cl].80 For cis-[Pt(κ2-Ph2P0-CH2-P00Ph2(dSe)(PEt3)-
with albumin depends, in part, on extensive studies9-14,83-85
31
employing P NMR (Table 1), Mossbauer, and EXAFS
€
spectroscopies, combined with protein modification and
analysis of bound gold and reduced thiol status, in order to
characterize the gold species formed by reactions of aurano-
fin, gold thiomalate, and related compounds at: (i) cysteine-
34, (ii) cysteine residues generated by reduction of internal
disulfide bonds, and (iii) the numerous histidine residues
present in albumin.11-14,83-85 Ralph et al. recently confirmed
the various gold binding modes by ESI-MS.86
Cl]þ and trans-[Pt(κ2-Ph2P0-CH2-P00Ph2(dSe)(PBun )Cl]þ
3
(cis and trans are defined by the relationship of the Se and
Cl- donor atoms)82 Berry et al. observed coupling between
the PR3 ligand and Se, JPSe,trans=( 102.9 and JPSe,cis=( 20
Hz, respectively.82 Interestingly, there are cases where the
coupling has been observed for a Pt compound but not for
the analogous Pd(II) or Ni(II) compound:80,82 For example,
Pan and Fackler80 observed no JPSe coupling for [Pd(Se2CN-
iBu2)(PPh3)Cl] or[Ni(Se2CN-Et2)(PPh3)Cl], which are ana-
logues of the Pt diselenocarbamates just described. Similarly,
Preliminary to the present albumin studies, 31P NMR
chemical shifts of Et3PAuSe-tagl were determined in DMSO
and CH3OD and, by adding a concentrated methanolic solu-
tion, in the aqueous buffer (l00 mM NH4HCO3, pH 7.9)
used for the protein chemistry (Table 1). The δP falls between
37.7 and 37.9 ppm in these solvents. The spectrum obtained
in the methanol/buffer solution is shown in Figure 5a.
Two successive aliquots of Et3PAuSe-tagl were added
to BSA to generate mixtures with Au/BSA stoichiometric
ratios of 0.25 and 0.48. 31P NMR resonances observed at
38.6 and 61.2 ppm after the first addition (Figure 5b)
increased in intensity with the second aliquot (Figure 5c).
2
Berry et al82 observed that JPSe,cis was not observed for
trans-[Pd(κ2-Ph2P0-CH2-P00Ph2(dSe)(PEt3)Cl]þ, although
the cis complex exhibits JPSe,trans =(125 Hz. Pt(II) is inert to
ligand exchange, whereas Pd(II) and Ni(II) are both labile
metal centers, thus providing a precedent for loss of obser-
vable coupling in labile systems, such as gold(I), where in only
3 of 31 instances was coupling resolved.37
11-14
The 38.6 ppm resonance is due to AlbSAu-PEt3
formed at cysteine-34 according to eq 2:
(76) Monkwius, U.; Zabel, M.; Yersin, H. Inorg. Chem. Commun. 2008,
11, 409–412.
AlbS- þ Et3PAuSe-tag1 þ Hþ h AlbSAuPEt3
(77) Bauer, A.; Schmidbaur, H. J. Am. Chem. Soc. 1996, 118, 5324–5325.
þ tag1-SeH
ð2Þ
€
(78) Rombke, P.; Schier, A.; Schmidbaur, H. J. Chem. Soc., Dalton Trans.
2001, 2482–2486.
(79) Freytag, M.; Jones, P. G. Chem. Commun. 2000, 277–278.
(80) Pan, W.-H.; Fackler, J. P., Jr. J. Am. Chem. Soc. 1978, 100, 5783–
5789.
(83) Isab, A. A.; Hormann, A. L.; Coffer, M. T.; Shaw, C. F., III. J. Am.
Chem. Soc. 1988, 110, 2278–2284.
(84) Xiao, J.; Shaw, C. F., III. Inorg. Chem. 1992, 31, 3706–3710.
(85) Kinsch, E. M.; Stephan, D. W. Inorg. Chim. Acta 1984, 91, 263–267.
(86) Talib, J.; Beck, J. L.; Ralph, S. F. J. Biol. Inorg. Chem. 2006, 11,
559–570.
€
(81) Pietschnig, R.; Moser, C.; Spirk, S.; Schafer, S. Inorg. Chem. 2005, 45,
2798–2802.
(82) Berry, E. E.; Browning, J.; Dixon, K. R.; Hilts, R. W. Can. J. Chem.
1988, 66, 1272–1282.

Article
Inorganic Chemistry, Vol. 49, No. 17, 2010 7671
Table 1. 31P NMR Chemical Shiftsa of Seleno-Auranofin, Analogues, and Reaction Products
species
solvent
δP/ppmb
refs
c
c
c
Et3PAuSe-tagl
Et3PAuSe-tagl
Et3PAuSe-tagl
Et3PAuS-tagl
Et3PAuCN
100 mM NH4HCO3 buffer
DMSO-d6
CD3OD-d4
37.8 ( 0.1
37.83
37.80
37.0 ( 0.3
35.4 ( 0.1
36.6 ( 0.01
44.0 ( 0.4
61.5 ( 0.5
38.6 ( 0.2
33-37
100 mM NH4HCO3 buffer
100 mM NH4HCO3 buffer
100 mM NH4HCO3 buffer
100 mM NH4HCO3 buffer
100 mM NH4HCO3 buffer
100 mM NH4HCO3 buffer
100 mM NH4HCO3 buffer
100 mM NH4HCO3 buffer
100 mM NH4HCO3 buffer
14
36
Et3PAuSCy
[(Et3P)Au]þ
Et3PdO
10b, 15
10b, 14, 36
10b, 14, 36, 37
10b,
37
14, 37
d
AlbSAuPEt3
(Et3PAuS)xBSA d
Et3PAuNHis-BSA d
AlbS(AuPEt3)2
23-30
35.7 ( 0.1
d
a Chemical shift values were measured and are reported vs internal TMP (2.74 ppm vs H3PO4). b Error limits reflect variations observed in protein
solutions; ranges are given for binding sites that occur in multiple protein environments. c This work. d Microheterogeneous commercial and modified
albumin samples are designated using “xx-BSA” and contain two or more different albumin species. The individual, albumin molecules dependent on the
status of cysteine-34 are abbreviated using “AlbS-xxx”.
The 61.2 ppm resonance is due to Et3PO formed via the
displacement (eq 3a or 3b) and subsequent oxidation
(eq 4) of the phosphine ligand by disulfide bonds.11
AlbS- þ Et3PAuSe-tagl h ½AlbSAuSe-taglꢁ þ Et3P
ð3aÞ
-
AlbSAuPEt3 þ tag1-SeH h ½AlbSAuSe-taglꢁ
þ Et3P þ Hþ
ð3bÞ
The transformations of eqs 2, 3a, and 3b can occur via a
three-coordinate transition state (Scheme 2), analogous to
that previously discussed for interactions of Et3PAuCN
with cyanide and AlbS- with Et3PAuCN, which also leads
to extensive displacement of Et3P and its subsequent
oxidation.83 Displacement of Et3P from AlbSAuPEt3 by
thiols has been documented previously11 and increases with
the affinity of the thiol for gold.11a Thus displacement by
tagl-Se (eqs 3a, 3b) can be reasonably anticipated, given the
greater affinity of selenium ligands for gold(I) compared to
the analogous thiols.15 Analogues of AlbSAuSe-tagl with
thiol ligands, AlbSAuSTm10a and AlbSAuS-tagl,14 were
isolated and characterized by their gold and thiol contents,
XANES, and EXAFS spectroscopy, and AlbSAuSTm was
€
also characterized by Mossbauer spectroscopy.
When the Et3PAuSe-tagl:BSA ratio reached 0.89
(Figure 5d), the cysteine-34 thiolate (0.56 per mole of
albumin) became saturated, and a resonance at 37.8 ppm
appeared due to excess Et3PAuSe-tagl. The minor reso-
nance at 43.7 ppm is assigned to [(Et3P)2Au]þ, which
forms via ligand scrambling of the excess Et3PAuSe-tagl
according to eq 1a. It did not appear in Figure 5a-c. Two
factors may contribute to its detection in Figure 5d, where
Et3PAuSe-tagl is in excess of the reduced cys-34 residue.
First, the extent of disproportionation may be greater
because the solvent for Figure 5d is more polar than that
for Figure 5a (buffer vs 1:1 MeOD/buffer). Second,
electrostatic binding of any of the equilibrium compo-
nents of eq 1a to albumin can retard the rate of exchange,
rendering it slow on the NMR time-scale and thereby
allowing resolution of the individual components.
Figure 5. 31P{1H} NMR spectra of SeAF: (a) in methanol/buffer
solution and (b-d) after incubation with BSA (4.1 mM, SH/BSA =
0.56), [Et3PAuSe-tagl]/AlbSH: (b) 0.25, (c) 0.48 (d) 0.89 in deuterated
100 mM NH4HCO3 buffer, pH = 7.9.
Because the extent of Et3PO formation in Figure 5 exceeds
that previously observed for reactions of BSA with Et3P-
AuCN83 or Et3PAuS-tagl,11,14 a comparative study of reac-
tions with Et3PAuS-tagl, Et3PAuCN, and Et3PAuSe-tagl
under identical, anaerobic conditions was carried out. 31P
NMR spectra were recorded within 1 h (Figure 6 a-c; left
column) and at 24 h (Figure 6 a0-c0; right column) after
mixing the reactants. The ratios of the Et3PO (61.4 ppm)
and AlbSAuPEt3 (38.5 ppm) peak intensities, which reflect

7672 Inorganic Chemistry, Vol. 49, No. 17, 2010
Hill et al.
Figure 6. 31P{1H} NMR spectra of BSA (4.30 mM; SH/BSA = 0.56 in
100 mM deuterated NH4HCO3 buffer, pH = 7.9) incubated with
Et3PAuX (X = S-tagl, CN, Se-tagl; [Et3PAuX]/[BSA] = 0.64) and
measured within 1 h (a-c) and after 24 h (a0-c0); Et3PAuS-tagl (a,a0),
Et3PAuCN (b,b0), and Et3PAuSe-tagl (c,c0).
Scheme 2
Figure 7. 31P{1H} NMR spectra in 100 mM deuterated NH4HCO3
buffer, pH = 7.9, of cysteine-blocked albumins: (a) 1.82 mM Ac-Cys
þ1.85 mMSeAF; (b) 4.05mMCys-BSAþ 3.88mMSeAF;(c) sampleb þ
8.04 mM Et3PAuCl; (d) 3.81 mM Cy-BSA þ 3.08 mM Et3PAuCl.
reduction of internal disulfide bonds and oxidation of
Et3P (eq 4).
Sulfhydryl-modified albumins have been used previously
to demonstrate that cysteine-34 is the principal gold binding
site and to explore reactions at other sites on albumin.10,14
Acetamide-blocked BSA (Ac-BSA) contains cysteine-34 that
has been converted to a thioether, AlbSCH2CONH2, which
is unable to displace phosphine, thiolate, or selenolate ligands
from gold(I). Ac-BSA is a heterogeneous mixture consisting
of ∼60% AlbS-CH2CONH2 and ∼40% naturally occur-
ring disulfide-blocked albumins, principally AlbSSCy (where
CySH = cysteine). Figure 7a shows the 31P NMR spectrum
obtained after the reaction between Et3PAuSe-tagl and Ac-
BSA. Resonances were observed for Et3PO (61.2 ppm),
AlbSAuPEt3 (38.5 ppm), and Et3PAuSe-tagl (37.2 ppm).
Thus, unlike auranofin, Et3PAuSe-tagl reacts with the disul-
fide bonds at Cys-34 of AlbSSCy to form AlbSAuPEt3 and
Et3PO. Both the phosphine and selenolate ligands (eqs 5 and
6) can reduce the cysteine-34 disulfide to AlbSH:
the extent of Et3P oxidation, decrease in the following order:
Et3PAuSe-tag1 > Et3PAuCN >> Et3PAuS-tagl
both at 1 h and after 24 h. No signals for Et3PAuCN
(35.4 ppm) or Et3PAuSe-tagl (37.8 ppm) were observed even
at 1 h, although unreacted auranofin was still present after
24 h. For all three complexes, the Et3PO concentrations
increased over 24 h and the AlbSAuPEt3 concentrations
decreased, consistent with eqs 3a, 3b, and 4. The resonance
of[(Et3P)2Au]þ at 43.7 ppm appeared only for the Et3PAuSe-
tagl system. The albumin thiol titers, which measure the free
sulfhydryl groups but not those coordinated to gold, were
0.18, 0.30, and 0.28 after reaction with Et3PAuS-tagl, Et3-
PAuCN, and Et3PAuSe-tagl, respectively. The presence of
sulfhydryl groups, in addition to the Cys-34 residues to which
Et3PAuþ moieties are bound, is consistent with concomitant
AlbSSR þ Et3P þ H2O f AlbSH þ RSH þ Et3PO ð5Þ
AlbSSR þ tagl-SeH h AlbSH þ tagl-SeSR
ð6Þ
The newly formed AlbSH reacts further with Et3PAuSe-
tagl to generate AlbSAuPEt3, according to eq 2. After

Article
Inorganic Chemistry, Vol. 49, No. 17, 2010 7673
chromatographic separation of the albumin from low-mo-
lecular weight species in the NMR sample, the albumin thiol
titer was 0.65, and the Aub/BSA ratio was 0.56. These values,
given the initial mole ratio of ∼0.40 AlbS-SCy present,
reflect the fact that internal disulfide bonds as well as the
cysteine-34 disulfide bonds are being reduced by Et3P
(eqs 3a, 4, 5) and/or tagl-SeH (analogous to reduction of
AlbS-SCy via eq 6). The 35.8 ppm resonances previously
observed for Et3PAuþ bound to sulfhydryl groups generated
from internal disulfides are not observed, indicating the
Et3PAuþ binds preferentially to Cys-34 or remains bound
to the selenolate, consistent with their greater affinities for
gold(I) compared to that of less acidic cysteine residues.15,84
To confirm that AlbSAuPEt3 observed in Figure 7a was
generated by reduction of the Cys-34 disulfide bonds, the
reaction between Et3PAuSe-tagl and Cys-BSA (100% disul-
fides at Cys-34, generated by reaction of BSA with cystine)
was carried out at a 0.96:1.00 mol ratio. The 31P NMR spec-
trum (Figure 7b) exhibited strong signals for Et3PO (61.2
ppm) and AlbSAuPEt3 (38.7 ppm) but not SeAF (37.8
ppm), indicating that it reacted completely. The weak reso-
nance at 36.6 ppm is reasonably assigned to a small amount
of Et3PAuSCy formed by free cysteine released from the
Cys-34 disulfide bond according to eq 5, which is consistent
with the SH titer of 0.79 measured after the reaction.
Reactions of Et3PAuCl with the protein products present
after obtaining spectrum 7b were carried out to confirm the
presence of reduced thiols and to distinguish by NMR
chemical shifts between the binding at Cys-34 (δP = 38.7
ppm) and the reduced internal disulfide sites (δP = 33-36
ppm). Two successive aliquots, each containing one equiva-
lent of Et3PAuCl, were added to the reaction mixture. After
the first addition (not shown), the resonances at 38.7 and
36.6 ppm increased in intensity, indicating that additional
reduced cysteine residues had been formed at cysteine-34
(AlbSH) and from internal disulfide bonds ((HS)2Alb) by
the initial reaction with Et3PAuSe-tagl leading to spectrum
7b. Their respective Et3PAuþ adducts, identified by their
characteristic chemical shifts, were formed by the added
Et3PAuCl as follows:
Table 2. Gold Binding to BSA and Cys-BSA
[Au]0/mM
gold/protein ratios
1.61
3.31
4.55
5.88
7.14
8.33
Au0/BSAa
0.39
0.32
0.34
0.78
0.45
0.48
1.17
0.61
0.61
1.55
0.72
0.78
1.93
0.75
0.85
2.32
0.75
0.88
Aub/BSAb
Aub/Cys-BSAb
a Initial ratios [Et3PAuSetagl]0/[BSA]0 and [Et3PAuSetagl]0/[Cys-BSA]0
used in the reaction mixtures. b Ratio of albumin-bound gold measured after
chromatographic resolution of albumin-bound and free gold.
that Et3PAuSe-tagl facilely generates new thiol sites
((HS)2BSA and Alb-SH) in Cys-BSA and, therefore,
can generate them from disulfides in native BSA, Ac-
BSA, and perhaps in vivo from other proteins.
The extent of gold binding to BSA (containing mercap-
talbumin) and to Cys-BSA (lacking mercaptalbumin) was
compared by measuring protein-bound gold after chroma-
tographic isolation of the protein from reaction mixtures
with increasing concentrations of the seleno-auranofin. The
data of Table 2 shows very little difference in the extent of
gold bound (Aub/BSA and Aub/Cys-BSA) at low Au0/BSA
ratios (<5) and slightly more bound to Cys-BSA at higher
ratios (>5). This reflects the ability of the seleno-auranofin
to generate thiol binding sites in both forms of albumin and
stands in contrast to earlier results with auranofin, in which
reaction with Cys-BSA was negligible compared to BSA.14
Scheme 2 summarizes the reactions of AlbS- and AlbSS-
Cy with Et3PAuSe-tagl and the reduction of internal disul-
fide bonds to form Et3PdO and additional, weaker thiolate
binding sites for gold. The oxidation of Et3P is more rapid
and extensive than previously observed for auranofin or
Et3PAuCN, suggesting that in vivo metabolism will proceed
more rapidly, thereby quickly reducing the concentrations
of Et3PAuSe-tagl and the metabolites containing intact
Et3PAuþ moieties.
Biological Activity. SeAF was tested in three different in
vivo assays selected in order to determine its anti-inflamma-
tory activity. For each assay, direct comparison with aur-
anofin (AF) was made. The first protocol, the phorbol ester-
induced inflammation (PEIF), utilizes the mouse ear to
measure the inhibitory effects of drugs on the inflamma-
tory response induced by phorbol ester.50 This complex
response utilizes edema or swelling mediated by cyclooxy-
genase-lipoxygenase products and inflammatory cytokines
(e.g., IL-1 and TNFR). The inhibition of swelling was deter-
mined by measuring ear thickness with a gauge after topical
administration of a drug and compared to controls. Myelo-
peroxidase (MPO) activity was assayed as a measure of poly-
morphonuclear (PMN) leukocyte (white cell) infiltration.
The data are displayed in Table 3. AF and SeAF showed
comparable activity in the inhibition of ear swelling, whereas
the PMN infiltration in this model and the next were less for
SeAF than for AF.
Alb-S- þ Et3PAuCl f AlbSAuPEt3 þ C1-
ð7aÞ
ðHSÞ2BSA þ xEt3PAuCl f ðEt3PAuSÞxBSA þ xHþ
þ xCl-
ð7bÞ
The second Et3PAuCl addition increased the Au0/BSA
ratio to nearly 3/1. The reduced SH groups became satu-
rated, and the excess Et3PAuCl reacted with some of the 17
histidine residues to form BSA-NHisAuPEt3, accounting for
the resonances between 30.3 and 25.3 ppm as shown in
Figure 7c. AlbSAuPEt3, (Et3PAuS)xBSA, BSA-NHisAu-
PEt3 have been observed previously by 31P NMR,84,85 iso-
lated, and characterized by EXAFS spectroscopy10a,14 and
more recently by ESI-MS studies of albumin reactions with
Et3PAuCl.86
Figure 7d demonstrates that the reaction between Cys-
BSA and Et3PAuCl in the absence of tagl-SeH, generates
only the resonances at 28.1 and 25.8 ppm, characteristic
of the histidine binding sites. No Et3PAu-thiolate binding
(33-38 ppm) is observed, because the oxidation-reduc-
tion processes of eqs 4-6 did not occur, demonstrating
The second protocol, the arachidonic acid (AA)-induced
inflammation assay also involves the evaluation of a drug,
topically administered, against mouse ear edema.51 This test
affords an advantage over the PEIF model in that it is sen-
sitive to 5-lipoxygenase (5-LO) inhibitors but is relatively in-
sensitive to selective inhibition of cyclooxygenase and affords
a measure of a compound’s anti-inflammatory activity.51
The induced edema has a strong vasoactive amine com-
ponent, but the MPO response appears to be 5-LO-product

7674 Inorganic Chemistry, Vol. 49, No. 17, 2010
Hill et al.
Table 3. Phorbol Ester-Induced Inflammation
showed no significant activity. In light of the similar
activity seen with AF and SeAF in the previous two
topically administered studies, the lack of oral SeAF
activity in the Carrageenan assay was surprising. Ac-
counting for these observed differences remains specula-
tive. SeAF may be more poorly absorbed across the
gastrointestinal membrane of rats than AF, which itself
is only 17-23% absorbed.87 However, serum gold levels
from AF- and SeAF-treated rats were similar (Table 5),
suggesting comparable oral bioaccumulation of gold.
Moreover, serum gold levels from AF-treated rats corre-
lated with AF’s effect on paw edema (r = 0.77; p<0.01).
Alternatively ligand exchange reactions may occur in
vivo differently with SeAF than with AF, thus preventing
the gold from getting to the “active site”. In the topical
assays, ligand exchange leading to transport of gold is less
of a problem because administration of the drug occurs
directly at the site of activity. The exchange reactions of
SeAF with serum albumin studied by 31P NMR spectros-
copy (above) demonstrate that formation of Et3PO and
reduction of protein disulfides to create new binding sites
occur more rapidly and more extensively for Se-AF than
for the comparable reactions of AF. Displacement of the
triethylphosphine ligand from gold reduces the lipophili-
city of the metabolites that form, which would in turn alter
their tissue and cellular distribution patterns, consistent
with the significantly reduced activity after oral adminis-
tration.
% inhibition
compounda
edemab
MPOb
AF
SeAF
50***
57***
59***
43**
a Dosed at 1 mg/ear; administered topically. b A double asterisk (**)
denotes p<0.01 and a triple asterisk (***) denotes p<0.001 as signifi-
cant differences from control with use of Student’s t test. Results are
based on eight mice per test group and eight mice in the control group,
which were administered vehicle only.
Table 4. Arachidonic Acid-Induced Inflammation
% inhibition
compounda
edemad
MPOc
Ab
Bc
AF
0NS
70***
54**
41***
28**
SeAF
AF
SeAF
0NS
11**
12**
a Dosed at 1 mg/ear; applied topically. b Experiment A: DMA vehicle.
c Experiment B: 30 min pretreatment using acetone vehicle. d NS = not
significant. A double asterisk (**) denotes p<0.01 and a triple asterisk
(***) denotes p<0.001 as significant differences from control with use
of Student’s t test. Results are based on 5 mice per test group and 10 mice
in the control group (administered vehicle only).
Table 5. Anti-inflammatory Activity of AF and SeAF in the Carrageenan-
Induced Rat Paw Edemaa
oral dose
mg Au/kg
serum gold
μg/mLc
Conclusions
compound
AF
% inhibitionb
Seleno-auranofin (Se-AF; Et3PAuSe-tagl) was prepared in a
two-step procedure derived from the synthesis of auranofin.1
Extended X-ray absorption fine structure (EXAFS), X-ray ab-
20
10
5
20
10
5
58***
28**
12NS
12NS
11NS
9NS
1.66 ( 0.21
1.12 ( 0.09
0.88 ( 0.02
1.90 ( 0.20
1.06 ( 0.02
0.98 ( 0.05
sorption near-edge structure (XANES), and 197Au Mossbauer
SeAF
€
spectra (Figures 1-3) confirmed a linear P-Au-Se coordina-
tion sphere in the solid state, analogous to the structure of
auranofin.59 1H and 13C NMR parameters were generally
similar to those of auranofin. The 31P resonance at 35.11
ppm and the 77Se resonance at 417.8 ppm in chloroform were
singlets, consistent with rapid equilibration via ligand scram-
bling (eqs 1a and 2) to form [Au(PEt3)2]þ, [Au(Se-tagl)2]-, and
[(Et3PAu)2Se-tagl]þ, which were identified in the Electrospray
ionization mass spectrometry (ESI-MS) spectra (Figure 4).
Binding to Cys-34, the only reduced cysteine in albumin, via
displacement of the selenolate ligand is less extensive than the
analogous reaction of AF, reflecting the greater affinity of
selenolates vs thiolates for gold(I) (Figure 6). Yet, comparative
31P NMR studies and measurement of protein-bound gold
establish that Se-AF reacts more rapidly and more extensively
than either Et3PAuCN or Et3PAuS-tagl with serum albumin to
form Et3PO, the metabolic product of the phosphine ligand in
vivo (Figure 6, Table 2). Thus, the seleno-auranofin molecule,
although selenolates have a higher affinity for gold(I) thiolates,
is more rapidly degraded under biomimetic conditions.
a Three hour assay. b Percent of inhibition of paw volume increase in
the rat carrageenan assay compared to controls. A double asterisk (**)
denotes p < 0.01 and a triple asterisk (***) denotes p < 0.001 as
significant differences from controls with use of Student’s t test. Results
are based on 6 rats per test group and 12 rats in the control group
(administered vehicle only). c By GFAA, serum from control animals
had 0 μg/mL of Au.
mediated. The data are shown in Table 4. Both AF and SeAF
showed comparable activity in this assay although the anti-
edema potency of both was considerably diminished com-
pared to that observed in PEIF. Mechanistically, these results
suggest that the gold compounds are not particularly good
inhibitors of AA-induced edema, although inhibition of
PMN infiltration remains high. This suggests that perhaps
vasoactive amine response is not effectively inhibited. Again,
AF appears to be more effective than SeAF.
Evaluation using the Carrageenan-induced rat paw
edema assay constituted the third in vivo test of seleno-
auranofin. This assay is a well-known model for inflam-
mation employed to measure the effectiveness of various
drugs as anti-inflammatory agents.52 Auranofin, earlier,
was reported to show good dose-dependent inhibition of
rat paw swelling upon oral administration.53 The data
comparing AF with SeAF are reported in Table 5. Dose-
dependent inhibition of paw edema by auranofin was also
observed in this study at oral doses of 20, 10, and 5 mg per
kg of gold. However, at the same gold-based doses, SeAF
Se-AF was comparable to AF in two topically administered
tests of anti-inflammatory activity (phorbol ester- and arachi-
donic acid-induced inflammation assays in the mouse ear,
Tables 3 and 4). Unlike AF, orally administered Se-AF was
inactive in the Carrageenan-induced rat paw edema assay
(87) Intoccia, A. P.; Flanagan, T. L.; Walz, D. T.; Gutzait, L.; Swagzdis,
J. E.; Flagiello, J.; Hwang, B.Y. H.; Dewey, R. H.; Nogochi, H. J.
Rheumatol. 1982, 9(Suppl.8), 90–98.

Article
Inorganic Chemistry, Vol. 49, No. 17, 2010 7675
DiazSe = 1,3-diazinane-2-selenone
DMSO = dimethylsulfoxide
(Table 5). The more rapid oxidation of triethylphosphine in
the presence of serum albumin, resulting from a greater extent
of ligand exchange reactions of SeAF, which in turn lead to less
lipophilic metabolites, suggests that the in vivo metabolism
and the transport of AF and SeAF will be significantly diffe-
rent. The reduced anti-inflammatory activity by oral adminis-
tration, but not by direct, topical administration of Se-AF in
comparison to AF, is consistent with the more rapid and
extensive reaction of SeAF with albumin in vitro and may
similarly be caused by more rapid metabolism in vivo.
DTNB = 5,50-dithiobis(2-nitrobenzoic acid)
ErgS = ergothionine
ESI-MS = electrospray ionization mass spectrometry
Et = ethyl
EtImSe = N-ethylimidazolidine-2-selenone
EtImt = N-ethylimidazolidine-2-thione
Et2ImSe = N,N0-diethylimidazolidine-2-selenone
Et2Imt = N,N0-diethylimidazolidine-2-thione
EXAFS = extended X-ray absorption fine structure
Ff = Fourier filtered
(HS)2AlbSX = any albumin molecule with a reduced
internal disulfide bond
ImSe = imidazolidine-2-selenone
Imt = imidazolidine-2-thione
Me = methyl
Acknowledgment. The authors thank Gerald J. Capella
for technical assistance in the preparation of this manu-
script and Edie Reich for elemental analysis, both of
GlaxoSmithKline Pharmaceuticals. A.A.I. thanks the
Chemistry Department of the King Fahd University of
Petroleum and Minerals, Dhahran, Saudi Arabia, for
a sabbatical leave. C.F.S. acknowledges the support of
Smith Kline Beecham, the NIH AR 39902, and the Illinois
State University Department of Chemistry.
MeImSe = N-methylimidazolidine-2-selenone
MeImt = N-methylimidazolidine-2-thione
mpt =1-methyl-pyridine-2-thione
Ph = phenyl
PhImSe = N-phenylimidazolidine-2-selenone
PhImt = N-phenylimidazolidine-2-thione
Pr = propyl
iPrImSe = N-(i-propyl)imidazolidine-2-selenone
iPrImt = N-(i-propyl)imidazolidine-2-thione
nPrImt = N-(n-propyl)imidazolidine-2-thione
py = pyridine
SeAF = seleno-auranofin (Et3PAuSe-tagl)
THT = tetrahydrothiophene (C4H8S)
TgS = thioglucose
TmS = thiomalate
p-tol = para-tolyl
XANES = X-ray absorption near-edge structure
Abbreviations
Ad2PzP = bis(adamantyl)benzylphosphine
AF = auranofin (Et3PAuS-tagl)
AlbS- = mercaptalbumin component of serum albu-
min
AlbSSCy = cysteine-34 disulfide to another cysteine
AlbSCH2CONH2 = iodoacetate modified albumin
BSA = microheterogeneous albumin containing mer-
captalbumin
Cys-BSA microheterogeneous albumin, primar-
ily cysteine 34 disulfide form
Ac-BSA - microheterogenous albumin, contains
AlbSCH2CONH2
iBu = iso-butyl
Cy = cyclohexyl
Diap =1,3-diazipane-2-thione
Diaz =1,3-diazinane-2-thione
Supporting Information Available: Supplemental Figures S1
and S2 show the calculated isotopic patterns for [(Et3PAu)2(Se-
tagl)]þ and [Au(Se-tagl)2]-. This material is available free of
charge via the Internet at http://pubs.acs.org.