Formation, spectroscopic characterization, and solution stability of an [Fe4S4]2+ cluster derived from β-cyclodextrin dithiolate

Article abstract of DOI:10.1021/ic301324u

The formation and solution properties, including stability in mixed aqueous-Me₂SO media, have been investigated for an [Fe ₄S₄]²⁺ cluster derived from β-cyclodextrin (CD) dithiolate. Clusters of the type [Fe₄S₄(SAr) ₄]^2- (Ar = Ph, C₆H₄-3-F) are generated in Me₂SO by redox reactions of [Fe₄S ₄(SEt)₄]^2- with 2 equiv of ArSSAr. An analogous reaction with the intramolecular disulfide of 6^A,6 ^D-(3-NHCOC₆H₄-1-SH)₂-6 ^A,6^D-dideoxy-β-cyclodextrin (14), whose synthesis is described, affords a completely substituted cluster formulated as [Fe ₄S₄{β-CD-(1,3-NHCOC₆H₄S) ₂}₂]^2- (15). Ligand binding is indicated by a circular dichroism spectrum and also by UV-visible and isotropically shifted ¹H NMR spectra and redox behavior convincingly similar to [Fe ₄S₄(SPh)₄]^2-. One formulation of 15 is a single cluster to which two dithiolates are bound, each in bidentate coordination. With there being no proven precedent for this binding mode, we show that the cluster [Fe₄S₄(S₂-m-xyl) ₂]^2- is a single cubane whose m-xylyldithiolate ligands are bound in a bidentate arrangement. This same structure type was proposed for a cluster formulated as [Fe₄S₄{β-CD-(1,3-SC ₆H₄S)₂}₂]^2- (16; Kuroda et al. J. Am. Chem. Soc.1988, 110, 4049-4050) and reported to be water-stable. Clusters 15 and 16 are derived from similar ligands differing only in the spacer group between the thiolate binding site and the CD platform. In our search for clusters stable in aqueous or organic-aqueous mixed solvents that are potential candidates for the reconstitution of scaffold proteins implicated in cluster biogenesis, 15 is the most stable cluster that we have thus far encountered under anaerobic conditions in the absence of added ligand.

Full text of DOI:10.1021/ic301324u

Article
pubs.acs.org/IC
Formation, Spectroscopic Characterization, and Solution Stability of
an [Fe4S4]2+ Cluster Derived from β‑Cyclodextrin Dithiolate
Wayne Lo,† Ping Zhang,‡ Chang-Chun Ling,‡ Shaw Huang,† and R. H. Holm*,†
†Department of Chemistry and Chemical Biology, Harvard University, Cambridge Massachusetts 02138, United States
‡Department of Chemistry, Alberta Glycomics Center, University of Calgary, Calgary, Alberta, Canada T2N IN4
S
* Supporting Information
ABSTRACT: The formation and solution properties, including stability in mixed aqueous−Me2SO
media, have been investigated for an [Fe4S4]2+ cluster derived from β-cyclodextrin (CD) dithiolate.
Clusters of the type [Fe4S4(SAr)4]2− (Ar = Ph, C6H4-3-F) are generated in Me2SO by redox reactions
of [Fe4S4(SEt)4]2− with 2 equiv of ArSSAr. An analogous reaction with the intramolecular disulfide of
6A,6D-(3-NHCOC6H4-1-SH)2-6A,6D-dideoxy-β-cyclodextrin (14), whose synthesis is described, affords
a completely substituted cluster formulated as [Fe4S4{β-CD-(1,3-NHCOC6H4S)2}2]2− (15). Ligand
binding is indicated by a circular dichroism spectrum and also by UV−visible and isotropically shifted
1H NMR spectra and redox behavior convincingly similar to [Fe4S4(SPh)4]2−. One formulation of 15 is
a single cluster to which two dithiolates are bound, each in bidentate coordination. With there being no
proven precedent for this binding mode, we show that the cluster [Fe4S4(S2-m-xyl)2]2− is a single
cubane whose m-xylyldithiolate ligands are bound in a bidentate arrangement. This same structure type
was proposed for a cluster formulated as [Fe4S4{β-CD-(1,3-SC6H4S)2}2]2− (16; Kuroda et al. J. Am.
Chem. Soc. 1988, 110, 4049−4050) and reported to be water-stable. Clusters 15 and 16 are derived
from similar ligands differing only in the spacer group between the thiolate binding site and the CD platform. In our search for
clusters stable in aqueous or organic−aqueous mixed solvents that are potential candidates for the reconstitution of scaffold
proteins implicated in cluster biogenesis, 15 is the most stable cluster that we have thus far encountered under anaerobic
conditions in the absence of added ligand.
A recent approach in this laboratory has utilized clusters
derived from doubly deprotonated α-cyclodextrindithiol, 1 in
Figure 1.1 The hydrophilic nature of cyclodextrins (CDs), which
contain 6−8 D-glucopyranoside units linked in a toroidal
topology,14 together with [Fe4S4]2+ core shielding by a large
ligand, is expected to promote aqueous solubility and stability.
This ligand and the related α-cyclodextrin monodithiolate are
generated in solution by base hydrolysis of the corresponding
thioesters. Cluster binding occurs by ligand substitution of a
preformed cluster, for example, [Fe4S4Cl4]2− in a polar solvent
such as Me2SO, and was demonstrated by UV−visible and
circular dichroism spectra and by 1H NMR signals paramagneti-
cally shifted into the downfield range (10−16 ppm) diagnostic of
SCH2 protons in clusters with the core oxidation state [Fe4S4]2+.1
These clusters were formulated as [Fe4S4{α-CD-CH2S}4]2− and
[Fe4S4{α-CD-(CH2S)2}2]2− to emphasize complete substitution
by thiolate, but the formation of oligomeric species (more than
one [Fe4S4]2+ unit per molecule) could not be eliminated. The
clusters showed modestly improved stability compared to, e.g.,
[Fe4S4(SCH2CH2OH)4]2−, in aqueous Me2SO solvent mixtures
with no added ligand.
1. INTRODUCTION
We have described elsewhere1 the potential use of iron−sulfur
clusters in treating human diseases arising from impaired cluster
biogenesis that disrupts cellular iron homeostasis and leads to
mitochondrial failure. Friedreich’s ataxia is the most prominent
of these diseases.2−5 An untested approach involves the delivery
of intact iron−sulfur clusters to organelles for the purpose of
reconstituting scaffold proteins involved in cluster biogenesis as
part of a process that restores mitochondrial function.4 The
clusters required for implementation of this approach must be
soluble and stable in water or mixed organic−aqueous media and,
ideally, largely impervious to destructive reactions at physio-
logical dioxygen concentrations. Given that active mitochondrial
aconitase and cytosolic aconitase IRP1 both contain [Fe4S4]
clusters,6,7 the problem devolves to a search for suitable clusters
of this type.
Within the large family of known [Fe4S4(SR)4]2− clusters (R =
alkyl, aryl),8 nearly all of which have been isolated as quaternary
ammonium salts, very few have been prepared that are water-
soluble. This property is enhanced by hydrophilic substituents
such as R = CH2CH2OH,1,9 CH2CH(OH)Me,10 and
The investigation of α-cyclodextrin thiolate clusters was
motivated, in part, by the work of Kuroda et al.,15 who prepared
the β-cyclodextrin bis(3-thiophenyl-1-thiol) 2 (Figure 1) and the
11
−
CH2CH2CO2 . For stability in water, such clusters require
excess ligand to diminish the rate of solvolytic decomposition.12
Certain dendritically encapsulated clusters are described as
soluble in water and Me2SO−water media; stability information
was not reported.13
Received: June 21, 2012
Published: August 30, 2012
© 2012 American Chemical Society
9883
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Inorganic Chemistry
Article
Figure 1. Schematic structures of cyclodextrin dithiols: 6A,6D-(CH2SH)2-6A,6Ddideoxy-α-cyclodextrin (1), 6A,6D-(3-SC6H4-1-SH)2-6A,6D-dideoxy-
β-cyclodextrin (2), 6A,6D-(3-NHC(O)C6H4-1-SH)2-6A,6D-dideoxy-β-cyclodextrin (3). Abbreviations of doubly deprotonated forms: α-CD-(CH2S)2,
β-CD-(1,3-SC6H4S)2, β-CD-(3-NHC(O)C6H4-1-S)2. CD = cyclodextrin.
5.39−5.19 (m, 7H, 7 × H-3), 5.18−5.10 (m, 4H, 4 × H-1), 5.08 (d, J = 3.8
Hz, 1H, H-1), 5.04 (d, J = 3.5 Hz, 1H, H-1), 5.03 (d, J = 3.7 Hz, 1H, H-1),
4.88−4.74 (m, 7H, 7 × H-2), 4.66−4.51 (m, 5H, 5 × H-6a_CHaHbOAc),
4.34−4.00 (m, 12H, 7 × H-5 + 5 × H-6b_CHaHbOAc), 3.84−3.62 (m,
11H, 7 × H-4 + 2 × H-6a_CHaHbN3 + 2 × H-6b_CHaHbN3), 2.18−2.02
(m, 57H, 19 × OAc). 13C NMR (100 MHz, CDCl3): δ 170.86, 170.81,
170.73, 170.68, 170.56, 170.56, 170.52, 170.48, 170.43, 170.35, 170.27,
169.35 (CO), 97.11, 96.89 (×2), 96.80 (×2), 96.72, 96.41 (C-1), 77.59,
77.23 (×2), 76.74, 76.61, 76.54, 76.22 (C-4), 71.30, 71.24, 71.13 (×2),
71.05, 70.80, 70.66, 70.62, 70.54, 70.45, 70.24 (×2), 70.17 (×2), 70.11,
69.91, 69.66, 69.63, 69.57 (×2), 69.34 (C-2, C-3, and C-5), 62.71, 62.65,
62.61 (×2), 62.46 (C-6_CHaHbOAc), 50.82, 50.68 (C-6_CHaHbN3,
20.89−20.65 (19 × OAc). High-resolution MS (ESI). Calcd for
C80H106N6O52Na [(M + Na)+]: m/z 2005.57268. Found: m/z 2005.56980.
6A,6D-Diazido-6A,6D-dideoxy-β-cyclodextrin (8). A solution of
the per-O-acetylated diazide 7 (1.0 g, 0.50 mmol) in methanol (10 mL),
water (2.0 mL), and concentrated ammonia (2.0 mL) was heated to
50 °C for 24 h and then concentrated under reduced pressure. The
residue was purified by reverse-phase chromatography on C18 using a
gradient of water−methanol to afford the fully deacetylated diazide 8,
which was freeze-dried (0.573 g, 96%). [α]D20: +55° (c 0.45, MeOH).
1H NMR (400 MHz, D2O): δ 5.14−5.08 (m, 7H, 7 × H-1), 4.08−
3.80 (m, 27H, 7 × H-3 + 7 × H-5 + 5 × H-6a_CHaHbOH + 5 ×
H-6b_CHaHbOH + 2 × H-6a_CHaHbN3), 3.75−3.55 (m, 16H, 7 × H-2
+ 7 × H-4 + 2 × H-6b_CHaHbN3). 13C NMR (100 MHz, D2O): δ 101.81
(×3), 101.78 (×2), 101.56 (×2), 81.98 (×2), 81.23 (×2), 81.17, 81.16,
81.10 (C-4), 72.99, 72.94, 72.76, 71.96, 71.89, 71.70, 70.46 (C-3, C-5, and
C-2), 60.32, 60.26 (×2), 51.00 (×2, C-6). High-resoluton MS (ESI).
Calcd for C42H68N6O33Na [(M + Na)+]: m/z 1207.37195. Found: m/z
1207.37022. This compound was briefly described previously.19,20
6A,6D-Diamino-6A,6D-dideoxy-β-cyclodextrin Acetate (9). A
solution of the diazide 8 (200 mg, 0.169 mmol) and triphenylphosphine
(177 mg, 0.675 mmol) in pyridine (8.0 mL) and water (2.0 mL) was
heated to 50 °C overnight. The solvent was removed under reduced
pressure and coevaporated with water to remove the residual amount of
pyridine. The residue was redissolved in water (10 mL) and neutralized
with a few drops of acetic acid. The aqueous solution was washed with
dichloromethane (3 × 5 mL) and freeze-dried to afford the previously
known 6A,6D-diamino derivative,21 isolated as the acetate salt 9 (189 mg,
∼96% yield). 1H NMR (400 MHz, D2O): δ 5.02 (m, 3H, 3 × H-1), 4.98
(m, 4H, 4 × H-1), 3.99−3.69 (m, 24H), 3.65−3.38 (m, 14H), 3.32 (dd,
related monothiol. They proposed the formation of [Fe4S4{β-
CD-(1,3-SC6H4S)}4]2− and [Fe4S4{β-CD-(1,3-SC6H4S)2}2]2−
(16) by the reaction of [Fe4S4(SBut)4]2− with, apparently, 4
and 2 equiv of monothiol and dithiol, respectively. The UV−
visible spectrum and 2−/3− redox potential of the dithiol
reaction product are entirely consistent with the formation of a
cluster [Fe4S4(SAr)4]2−. However, the formulation 16 requires
that the dithiolate function as a bidentate chelating ligand to the
same cluster core, a rare, if not unprecedented, behavior in
[Fe4S4] cluster chemistry. The molecular weight measured by
light scattering was consistent with this formulation. It was also
reported that this cluster was remarkably stable in an aqueous
phosphate buffer (pH 7.0) with no added ligand, displaying a half-
life of greater than 120 h. In the context of water-soluble clusters,
the structural and stability features of the cluster derived from
2 have engaged our attention. Our results and conclusions for
a related anaerobic [Fe4S4]2+ cluster system based on the
β-cyclodextrin bis(3-carboxamidobenzene-1-thiol) 3 are described
here.
2. EXPERIMENTAL SECTION
Preparation of Compounds. (Et4N)2[Fe4S4(SR)4] (R = Et, Ph)16
and (Et4N)2[Fe4S4(S2-m-xyl)2]17 [S2-m-xyl = m-xylyl-α,α′-dithiolate-
(2−)] were prepared as previously described. The method of synthesis
of the desired ligand in oxidized form (14; β-cyclodextrin disulfide, an
oxidation product of dithiol 3) is given in Scheme 1.
2A,2B,2C,2D,2E,2F,2G,3A,3B,3C,3D,3E,3F,3G,6B,6C,6E,6F,6G-Nonade-
ca-O-acetyl-6A,6D-diazido-6A,6D-dideoxy-β-cyclodextrin (7). To
a solution of β-cyclodextrin (4, 10.0 g, 8.8 mmol) in anhydrous pyridine
(100 mL) was added dropwise a solution of 4,4′-biphenyldisulfonyl
chloride (3.09 g, 8.8 mmol)18 in anhydrous pyridine (30 mL), and the
reaction mixture was stirred at 50 °C for 2 h. Acetic anhydride (40 mL)
was added, and the reaction was continued overnight to cause
conversion of 5 to 6. The mixture was concentrated under reduced
pressure; the residue was dissolved in ethyl acetate (250 mL) and se-
quentially washed with aqueous HCl (2 N, 2 × 100 mL), saturated
NaHCO3 (2 × 100 mL), and 10% brine (1 × 100 mL). After being dried
with anhydrous Na2SO4, the organic solution was evaporated to afford a
mixture containing the capped disulfonate 6. The residue was dissolved in
N,N-dimethylformamide (DMF; 100 mL), and NaN3 (5.7 g, 88 mmol) was
added. After heating at 70 °C overnight, the solution was concentrated
under reduced pressure; the residue was extracted with ethyl acetate
(250 mL), and the solution was washed with saturated brine (1 × 100 mL),
dried over anhydrous Na2SO4, and evaporated. The crude mixture was
purified by column chromatography on silica gel using a mixture of 35%
ethyl acetate−hexane as the eluent to afford the diazide 7 (5.42 g, 31%) as a
white solid. [α]D20: +71° (c 0.82, MeOH). 1H NMR (400 MHz, CDCl3): δ
+
J = 13.6 and 2.7 Hz, 2H, 2 × H-6a_CHaHbNH3 ), 3.07 (dd, J = 13.7 and
+
7.6 Hz, 2H, 2 × H-6a_CHaHbNH3 ), 1.82 (s, 6H, 2 × Ac). 13C NMR
(100 MHz, D2O): δ 101.80 (×3), 101.77 (×3), 101.36 (C-1), 82.74,
82.74 (×2), 81.17 (×3), 81.09, 80.72 (C-4), 73.03, 72.91, 72.58, 72.01,
71.91, 71.79, 68.97 (C-2, C-3, and C-5), 60.41, 60.36, 60.26, 60.20 (×2),
40.42 (×2), 23.26 (OAc). High-resolution MS (ESI). Calcd for
C42H73N2O33 [(M + H)+]: m/z 1133.40956. Found: m/z 1133.40617.
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Inorganic Chemistry
Article
a
Scheme 1. Scheme for the Synthesis of β-Cyclodextrin Disulfide 14 via Intermediates 4−8, 13, and 14
a
Abbreviations: DCC = dicyclohexylcarbodiimide, EDC = N-ethyl-N′-[3-(dimethylamino)propyl]carbodiimide, NH3·H2O = concentrated ammonia,
and Piv = pivaloyl.
3-(Pivaloylthio)benzoic Acid (11). A solution of 3,3′-dithiodi-
benzoic acid (10; 3.0 g, 9.8 mmol) and triphenylphosphine (2.83 g,
10.78 mmol) in tetrahydrofuran (THF; 30 mL) and water (1.5 mL) was
stirred for 1 h at room temperature and evaporated to dryness under
reduced pressure. The residue was redissolved in a mixture of THF
(30 mL) and anhydrous pyridine (15 mL) and treated with pivaloyl
chloride (7.23 mL, 58.7 mmol) for 30 min. To hydrolyze the formed
anhydride, water (15 mL) was added and the mixture was stirred for 1 h at
50 °C. The reaction mixture was concentrated under reduced pressure, and
the residue was partitioned using a mixture of ethyl acetate (100 mL) and 2
N HCl (100mL); the organic solution was separated, dried over anhydrous
Na2SO4, and concentrated. The desired acid (3.45 g, 74%) was obtained
by column chromatography on silica gel using a mixture of 0.5%
methanol−dichloromethane as the eluent. 1H NMR (400 MHz, CDCl3):
δ 11.97 (br, 1H, COOH), 8.21−8.11 (m, 2H, H-2 + H-6), 7.65 (ddd, J =
1.6, 1.6, and 7.8 Hz, 1H, H-4), 7.50 (dd, J = 8.0 and 8.0 Hz, 1H, H-5), 1.35
(s, 9H, (CH3)3CCO). 13C NMR (100 MHz, CDCl3): δ 171.31 (CO),
147.99, 140.34, 136.59, 130.80, 130.22, 129.17 (Ar), 47.10
((CH3)3CCO), 27.35 ((CH3)3CCO). High-resolution MS (ESI). Calcd
for C12H14O3SNa [(M + Na)+]: m/z 261.05618. Found: m/z 261.05558.
2,5-Dioxopyrrolidin-1-yl 3-(pivaloylthio)benzoate (12). The
protected acid 11 (565 mg, 2.37 mmol) and N-hydroxysuccinimide
(273 mg, 2.37 mmol) were dissolved in ethyl acetate (10 mL) with
heating, and N,N-dicyclohexylcarbodiimide (689 mg, 3.28 mmol) was
added to the hot solution. After stirring for 3 h at room temperature, the
precipitate was filtered off and washed with ethyl acetate. The combined
solution was evaporated to dryness. The residue was recrystallized from
isopropyl alcohol to afford the desired ester 12 (620 mg, 78%). 1H NMR
(400 MHz, CDCl3): δ 8.14 (m, 2H, H-2 + H-6), 7.71−7.66 (ddd, J = 1.3,
1.7, and 7.8 Hz, 1H, H-4), 7.56 (dd, J = 7.8 and 7.8 Hz, 1H, H-5), 2.87
(br s, 4H, NHS), 1.31 (s, 9H, (CH3)3CCO). 13C NMR (100 MHz,
CDCl3): δ 203.27 ((CH3)3CCOS), 169.13 (ArCO), 161.20 (CO_NHS),
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Inorganic Chemistry
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a
Table 1. Crystallographic Data for (Et4N)2[Fe4S4(S2-m-xyl)2]
formula
C32H56Fe4N2S8
948.70
formula weight M
cryst syst
space group
a (Å)
monoclinic
Cc
18.2095(6)
11.2828(4)
21.8275(7)
90
b (Å)
c (Å)
α = γ (deg)
β (deg)
111.866(1)
4161.9(2)
1.514
V (Å3)
d
calc (g/cm3)
Z
4
GOF
1.256
b
c
R1 (wR2)
0.0403 (0.142)
a
b
Synchrotron radiation source (λ = 0.39364 Å); T = 95 K. R1 =
c
∑||Fo| − |Fc||/∑|Fo|. wR2 = {∑[w(Fo − Fc2)2/∑(Fo )2]}1/2
.
2
2
Figure 2. Schematic structure of β-cyclodextrin disulfide and desig-
nation of the aromatic protons and the 1H NMR spectrum (600 MHz)
of the downfield region in (CD3)2SO. Signal assignments are indicated;
solv = DMF.
141.47, 136.63, 130.98, 129.87, 129.51, 126.12 (Ar), 47.11
((CH3)3CCO), 27.30 (NHS), 25.64 ((CH3)3CCO). High-resolution
MS (ESI). Calcd for C16H17NO5SNa [(M + Na)+]: m/z 358.07196.
Found: m/z 358.07147.
group was assigned based on symmetry analysis and systematic absences
(XPREP). The structure was solved by direct methods and refined by
least squares on F2 (SHELXL-9722). All non-hydrogen atoms were
located in difference Fourier maps and refined anisotropically.
Hydrogen atoms were introduced at calculated positions using a riding
model. Crystallographic data are collected in Table 1.23
Other Physical Measurements. All measurements were made
under anaerobic conditions. Absorption spectra were measured on a Varian
Cary 50 Bio spectrophotometer and circular dichroism spectra (at 20 °C) on
a Jasco J-715 spectropolarimeter. NMR spectra were recorded on a Varian
500 or a Varian 600 MHz spectrometer. High-resolution mass (MS) spectra
(electrospray ionization time-of-flight, ESI-TOF) were obtained on an
Agilent 6520-Q-TOF spectrometer. Electrochemical measurements were
made with a Bioanalytical Systems potentiostat/galvanostat in Me2SO
solutions using a glassy carbon working electrode, a 0.1 M (Bu4N)(PF6)
supporting electrolyte, and a saturated calomel reference electrode.
β-Cyclodextrin Disulfide (14). Diazide 8 (200 mg, 0.169 mmol)
was reacted with triphenylphosphine (177 mg, 0.675 mmol) in a mixture of
pyridine (8.0 mL) and water (2.0 mL) at 50 °C overnight. The activated
N-hydroxysuccinimide ester 12 (170 mg, 0.506 mmol) was added, and
the mixture was maintained at 50 °C for 24 h. The reaction mixture
(presumably containing the intermediate diamide 13) was concentrated by
evaporation. Methanol (3.0 mL), water (1.0 mL), and concentrated
ammonia (1.0 mL) were added to remove the pivalate thioester. After
heating at 50 °C overnight, the solution was concentrated under reduced
pressure, and the residue was purified by column chromatography, first on
silica gel using a mixture of isopropyl alcohol−water−aqueous ammonia
(7:1:1, v/v/v) as the eluent, followed by reverse-phase chromatography on
a C18 Sep-Pak cartridge to provide disulfide 14, which was freeze-dried
(102 mg, 43%). [α]D20: +38.5° (c 0.19, MeOH). H NMR (600 MHz,
1
CD3OD): δ 8.08 (t, J = 1.7 Hz, 1H, H-2′_Ar), 7.85 (t, J = 1.7 Hz, 1H,
H-2′_Ar), 7.76 (m, 2H, 2 × H-6′_Ar), 7.72 (ddd, J = <1, <1, and 7.6 Hz,
1H, H-4′_Ar), 7.69 (ddd, J = <1, 1.7, and 7.8 Hz, 1H, H-4′_Ar), 7.51 (dd,
J = 7.8 and 7.8 Hz, 2H, 2 × H-5′_Ar), 5.05 (d, J = 3.8 Hz, 1H, H-1), 5.04 (d,
J = 3.6 Hz, 1H, H-1), 5.03 (d, J = 3.8 Hz, 1H), 5.01−4.99 (m, 4H, 4 × H-1),
4.42 (dd, J = <1 and 12.9 Hz, 1H, H-6a_CHaHbNH), 4.32 (dd, J = <1 and
12.4 Hz, 1H, H-6a_CHaHbNH), 4.10 (ddd, J = 2.4, 2.4, and 9.9 Hz, 1H,
H-5), 3.98 (dd, J = 12.4 and 2.8 Hz, 1H, H-6a_CHaHbOH), 3.95−3.49
(m, 33H, 7 × H-2 + 7 × H-3 + 6 × H-5 + 4 × H-6a_CHaHbOH + 4 ×
H-6b_CHaHbNH + 5 × H-4), 3.47 (dd, J = 1.5 and 12.2 Hz, 1H, H-6b_
CHaHbOH), 3.28 (dd, J = 9.3 and 9.3 Hz, 1H, H-4), 3.24 (dd, J = 9.3
and 9.3 Hz, 1H, H-4), 3.08 (dd, J = 14.0 and 10.4 Hz, 2H, 2 ×
H-6b_CHaHbNH). 13C NMR (150 MHz, CD3OD): δ 168.24 (CO),
167.94 (CO), 137.74, 137.57, 135.54, 135.02, 132.15, 129.25, 129.15,
129.05, 126.65, 126.32, 125.80, 124.99 (Ar), 102.94 (×2), 102.40, 102.31,
102.22, 102.17, 102.14 (C-1), 85.17, 84.99, 81.25, 81.11, 81.05, 80.91, 80.64
(C-4), 73.33 (×3), 73.27, 73.24, 73.18, 73.07, 72.93, 72.81, 72.65 (×2),
72.55, 72.51, 72.38, 71.98, 71.92, 71.89, 71.74 (×2), 71.17, 70.56 (C-2, C-3,
and C-5), 60.17, 60.11, 59.81, 59.79, 59.67 (C-6_CHaHbOH), 41.85, 41.58
(C-6_CHaHbNH). High-resolution MS (ESI). Calcd for C56H78N2O35S2Na
[(M + Na)+]: m/z 1425.37187. Found: m/z 1425.36793.
X-ray Structure Determination. The compound
(Et4N)2[Fe4S4(S2-m-xyl)2] was crystallized from DMF−ether. Owing
to the very small size of the crystals (ca. 0.02 × 0.02 × 0.001 mm), data
were acquired at Argonne National Laboratory utilizing the Advanced
Photon Source. Data were collected at 95 K with a high-brilliance
synchroton X-ray source and a Bruker ApexII CCD detector. The unit
cell was determined with Bruker SMART software. The crystal did not
show any significant decay during data collection (ca. 1 h). Data were
corrected for Lorentz and polarization effects (Bruker SAINT, version
7.46A), and absorption corrections were applied (SADABS). The space
Mossbauer spectra were determined with a constant-acceleration
̈
spectrometer; isomer shifts are referenced to iron metal at room temperature.
3. RESULTS AND DISCUSSION
Ligand Synthesis. Initially, we wished to reexamine the
results of Kuroda et al.15 involving Fe4S4 cluster binding to
doubly deprotonated 2. With reference to Scheme 1, the 6A,6D-
capped disulfonate 5 was first prepared by a published
procedure.18 Unfortunately, several attempts to synthesize the
previously reported 6A,6D-diarylthiol 2 from 5 by nucleophilic
attack with excess benzene-1,3-dithiol monoanion were
unsuccessful. Consequently, an alternate synthetic target 3 was
designed, which employs the carboxamide linkage to connect a
6A,6D-diamino-substituted β-CD to the arylthio groups.
We first sought the 6A,6D-diamino scaffold 9 using 5 as a key
intermediate. This compound was obtained from β-CD 4 and
4,4′-biphenyldisulfonyl chloride in pyridine and was isolated in
pure form (15−20%) by reverse-phase chromatography on C18
silica gel. We found that, if direct acylation was carried out after
sulfonylation, the presumed intermediate 6 could be directly
reacted with NaN3 in DMF (70 °C) to afford the diazide 7. This
compound was isolated by normal-phase chromatography on silica
gel (31%); the acetyl protecting groups were smoothly removed in
methanol−water by concentrated ammonia (50 °C) to provide 8
(96%). The azido groups were simultaneously reduced with Ph3P
in pyridine−water (50 °C) to obtain the diamine 9,21 which was
isolated in the acetate salt form. Unfortunately, the EDC-mediated
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Figure 4. 1H NMR spectra (600 MHz) in the downfield region for the
reaction between 14 mM [Fe4S4(SEt)4]2− and n equiv of (a) diphenyl
disulfide and (b) bis(3-fluorophenyl) disulfide in (CD3)2SO at ∼25 °C.
The signal assignments of bound arylthiolate ligands are indicated;
solv = DMF.
Figure 3. Cyclic voltammograms (100 mV/s) in Me2SO of diphenyl
disulfide (blue, upper), bis(3-fluorophenyl) disulfide (red, upper), and
β-cyclodextrin disulfide (lower). Peak potentials are indicated.
coupling of 9 with the diacid 10 to give the desired 14 was
unsuccessful despite numerous attempts. Given the possibility that
the difficulty might originate with dual reactive sites in both 9 and
10, it was decided to reduce the disulfide bond of 10 to provide a
carboxylic acid with one reactive group.
cluster formation. The cyclic voltammograms of two phenyldisulfides,
one containing electron-withdrawing substituents, and 14 are
presented in Figure 3. Irreversible reduction (2ArS− → ArSSAr +
2e−) and oxidation (reverse reaction) steps are observed, a
behavior typical of compounds of this type in aprotic solvents.25,26
The 3-fluorosubstitutents cause a positiveshiftofEpc, in agreement
with prior observations of 4-substituted compounds.27 The same
redox pattern is observed for 14 at nearly the same potentials as
those for PhSSPh, confirming the presence of an S−S bond. The
structure of 14 was further confirmed by high-resolution MS.
Thiolate Cluster Ligation. Variation of the terminal thiolate
ligation in [Fe4S4(SR)4]2− clusters is conventionally accom-
plished by the displacement of halide (e.g., in [Fe4S4Cl4]2−) with
thiolate or by the substitution of bound thiolate by reaction with
another, usually more acidic, thiol.8,28 To avoid conversion of
disulfide 14 to dithiol 3 required for either method, we have
utilized redox reaction (1), which finds a near-precedent in the
formation of [Fe4S4(SePh)4]2− from [Fe4S4(SBut)4]2− and
PhSeSePh.29 Reactions with both disulfides have been monitored
by 1H NMR, as seen in Figure 4. At n = 1 and 2 equiv, multiple
downfield signals (12.5−13.5 ppm) due to methylene protons in
the species [Fe4S4(SEt)4−n(SAr)n]2− are evident and the
characteristically contact-shifted aromatic ring proton signals
appear at 5.0−8.3 ppm. At n = 2.1−2.2 equiv and up to 36 h of
reaction time, reaction (1) is complete for both disulfides.
The disulfide linkage of 10 was reduced with Ph3P,24 and the
intermediate thiol was directly protected with pivaloyl chloride to
afford 11 (74%). This compound was converted to the activated
N-hydroxysuccinimide ester 12 (78%). Attempts to functionalize 9
with either 11 (using EDC as a reagent) or 12 in the presence of
base [Et3N, 4-(N,N-dimethylamino)pyridine] were unproductive.
The ultimately successful method utilized reduction of the diazide 8
to the diamine, followed by treatment in situ with ester 12 to
generate S-pivaloyl-protected 13 (not isolated) and deprotection
with ammonia. Normal-phase column chromatography, followed by
reverse-phase chromatography on C18 silica gel, yielded the 6A,6D-
disubstituted β-cyclodextrin disulfide 14 (43%), the oxidized form
of dithiol 3 formed under the aerobic purification conditions.
The structure of 14 was confirmed by several methods, including
1
1D H and 13C as well as 2D COSY and HSQC NMR exper-
ments.23 The two phenyl groups were found to be magnetically
inequivalent by the appearance of 8 aromatic proton and 12
aromatic carbon signals in CD3OD and (CD3)2SO solutions. The 1H
NMR spectrum in the downfield region in (CD3)2SO, displayed in
Figure 2, reveals two well-resolved NH resonances, two p-H signals
from different rings, three o-H signals (four are resolved CD3OD),
and a m-H doublet of doublets. This spectral region is relevant to
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Figure 6. Absorption spectra in Me2SO of [Fe4S4(SEt)4]2− (red), the
reaction product of [Fe4S4(SEt)4]2− and β-cyclodextrin disulfide
[reaction (2), blue], the reaction product plus 25 equiv of benzenethiol
(orange), and β-cyclodextrin disulfide (green).
Figure 7. Circular dichroism spectra of the reaction product between
0.51 mM [Fe4S4(SEt)4]2− and 1.0 equiv (blue) and 2.0 equiv (red) of
β-cyclodextrin disulfide in Me2SO. The disulfide (green, 1.4 mM) did
not produce an effect at 270−770 nm.
1
Figure 5. (a) H NMR spectra (600 MHz) in the downfield region
for the reaction between 2.3 mM [Fe4S4(SEt)4]2− and n equiv of
β-cyclodextrin disulfide at various times in (CD3)2SO at ∼25 °C. (b)
COSY spectrum of the n = 2.0 equiv product at 16 h of reaction time
showing the correlation between Hm and Hp.
solution. The reactions were followed by 1H NMR with variable
equivalents of 14. The spectra in Figure 5 are similar to those of
reaction (1); one mixed ligand cluster (13.05 ppm) was detected.
As n equiv of the disulfide is increased, the three phenyl proton
resonances emerge, and at n = 2.0 and 16 h, the limiting
spectrum, ascribed to cluster 15, is reached. Resonances of m-H
and p-H were correlated by a COSY experiment. Shifts of m-H
are close to those of [Fe4S4(SAr)4]2−, thus assuring cluster
binding. Shifts of o-H and p-H indicate smaller contact
contributions to their chemical shifts than in the arylthiolate
clusters. This arises from differences in spin delocalization
possibly due to phenyl group orientations controlled by the
ligand structure and by the carboxamide substituent. The
spectrum of 16 would be an interesting comparison, but it was
not reported.15
[Fe4S4(SEt)4]2− + 2ArSSAr
→ [Fe4S4(SAr)4]2− + 2EtSSEt
(1)
In the following sections, CD-derived clusters are designated
as indicated. See also Figure 1. These formulations are not
intended to imply molecularity.
[Fe4S4{β-CD-(1,3-NHCOC6H4S)2}2]2− 15
[Fe4S4{β-CD-(1,3-SC6H4S)2}2]2−
[Fe4S4{α-CD-(CH2S)2}2]2−
[Fe4S4{α-CD-(CH2S)}4]2−
1615
171
181
With the feasibility of reaction (1) demonstrated, reaction (2)
was conducted in order to generate cluster 15 in a Me2SO
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Figure 9. Structure of [Fe4S4(S2-m-xyl)2]2− with 50% probability
ellipsoids and the atom numbering scheme. The four short [2.253(1)−
2.268(1) Å] and eight long [2.297(1)−2.318(1) Å] Fe−S bond
distances define the indicated ranges. The mean of the Fe−Fe distances
[2.698(1)−2.756(1) Å] is 2.73(3) Å.
Figure 8. Cyclic voltammograms (100 mV/s) of [Fe4S4(SEt)4]2−
(upper) and the reaction product between 1.2 mM [Fe4S4(SEt)4]2−
and 2.1 equiv of β-cyclodextrin disulfide after 16 h (reaction 2, lower).
The inset diagrams show the redox potentials obtained by differential
pulse voltammetry (pulse width 50 ms; pulse period 200 ms).
Voltammograms were recorded in Me2SO solutions at ∼25 °C; peak
potentials are indicated.
We conclude that (barring an unlikely core dissembly/
assembly event) reaction (2) proceeds with retention of the
[Fe4S4]2+ core of the starting cluster. Note also the circular
dichroism spectra in Figure 7, where the effect is induced by 1
and 2 equiv of β-cyclodextrin disulfide. Cluster 18 with four α-
cyclodextrin monothiolate ligands also shows a strong circular
dichroism signal.1 Disulfide 14, as CD itself, does not exhibit a
circular dichroism spectrum in the same region. The circular
dichroism spectrum constitutes further evidence for the
occurrence of reaction (2), whose chromophoric product is
necessarily chiral.
[Fe4S4(SEt)4]2− + 2β‐CD‐(1,3‐NHCOC6H4S)2
→ [Fe4S4{β‐CD‐(1,3‐NHCOC6H4S)2}2]2− + 2EtSSEt
(2)
Lastly, the voltammetries of the 2−/3− redox couples of
[Fe4S4(SPh)4]2− and the product of reaction (2) are compared in
Figure 8. Note that both are chemically reversible (ipc/ipa ≈ 1)
and differ by only 30 mV, a result that requires arylthiolate
coordination in the reaction product. Alkylthiolate clusters such
as [Fe4S4(SEt)4]2− (−1.33 V) exhibit 2−/3− couples at
substantially more negative potentials.30
Structural Considerations. The results from four physical
techniques not only establish a reaction between β-cyclodextrin
disulfide and [Fe4S4(SEt)4]2− in a Me2SO solution but require
arylthiolate binding to the iron sites rather than any other form of
interaction. These results do not demonstrate the molecularity of
the reaction product, nor is that property known in the solid state
for we have not yet achieved diffraction-quality crystals. The
dianion reduction product of 14 can bind to the [Fe4S4]2+ core as
a bridging or terminal ligand in the stoichiometry of 15. The
formation of μ2-SR bridges would lead to oligomeric species, of
which there is one documented example.31 The binding of one
thiolate at an iron site would afford a single cluster with two
Because a crystalline reaction product has not been isolated
from reaction (2), additional evidence for cluster binding to
reduced 14 has been sought from other observations. Relevant
absorption spectra in Me2SO are collected in Figure 6. The
spectrum of the product of reaction (2) has a prominent band
at 440 nm, which is the counterpart of the 457 nm feature
of [Fe4S4(SPh)4]2−. These spectra distinguish arylthiolate from
alkylthiolate clusters such as [Fe4S4(SEt)4]2− (λmax = 297 and
419 nm). Treatment of the reaction product with ca. 25 equiv of
benzenethiol in reaction (3) generates a spectrum identical with
that of authentic [Fe4S4(SPh)4]2− and corresponds to 98% of the
initial cluster concentration in reaction (2).
[Fe4S4{β‐CD‐(1,3‐NHCOC6H4S)2}2]2− + 4PhSH
→ [Fe4S4(SPh)4]2− + 2β‐CD‐(1,3‐NHCOC6H4SH)2
(3)
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Figure 10. Absorption spectra of the product of reaction (2) in specified Me2SO−aqueous buffer solutions (v/v) in which the aqueous component is
3.55 mM phosphate buffer (pH 7.5−7.6). Spectra were recorded under anaerobic conditions at 0−120 or 150 h after reaction at the following
concentrations: 80% Me2SO−20% aqueous, 0.27−0.41 mM; 60% Me2SO−40% aqueous, 0.20−0.31 mM; 40% Me2SO−60% aqueous, 0.14−0.21 mM;
20% Me2SO−80% aqueous; 100% aqueous, 0.39 mM. Band maxima or shoulders are indicated.
bidentate ligands in chelate-like ligation, as proposed for 16
apparently based on a molecular weight from light scattering.15
This type of binding is found with the iron protein of nitrogenase,
which consists of a cluster ligated by two cysteinate residues from
each of the two α-subunits.32,33 We have sought an example of
this binding mode in a synthetic cluster closer to the problem at
hand.
[Fe4S4(SCH2Ph)4]2−.16 The S−S distance (6.25 Å) between
terminal ligands is smaller than the values in the latter cluster
(6.30 and 6.49 Å) because of constraints of the bidentate ligand
structure.
The structure of [Fe4S4(S2-m-xyl)2]2− provides the first
demonstration of the unidentate terminal binding mode by a
bidentate dithiolate. At the very least, it offers a precedent for a
similar binding arrangement in 15 as well as 16 and 17. We note
that the S−S separations in 15 are expected to be similar to those
of [Fe4S4(S2-m-xyl)2]2− and comparable to the cavity diameter of
β-CD (6.0−6.5 Å).34 While this size range does not strongly
constrain thiolate sulfur positions, benzenethiolate substituents
at the 6A,6D positions on the rim of the structure can
accommodate the S−S distances of a synthetic cluster.
Attention has turned to the previously reported cluster
[Fe4S4(S2-m-xyl)2]2−, formulated on the basis of absorption
1
and H NMR spectral features and redox behavior but lacking
structural proof.17,30 In the original work, the m-xylyldithiolate
ligand was selected because its bite distance appeared
appropriate to span two iron sites. We verified that the cluster
possesses the [Fe4S4]2+ oxidation state from its Mossbauer
̈
parameters (δ = 0.41 mm/s, ΔEQ = 1.05 mm/s at 90 K) whose
values are typical of that state.8 As is evident in Figure 9, the
cluster has idealized S4 symmetry. The two aromatic rings are
held above opposite Fe2S2 core faces at a dihedral angle of 20.74°;
the least-mean-squares planes of these faces are disposed at
15.00°. The shortest ring carbon distance to a face is 3.14 Å. The
core unit has distorted tetrahedral iron sites and a compressed
tetragonal arrangement with four short [2.236(5) Å] + eight long
[2.313(8) Å] Fe−S bonds, a frequently observed distortion from
Td symmetry.8 Mean bond lengths are very close to those of
Solution Stability. In the examination of the stability,
reaction (2) was conducted in a Me2SO solution, after which
there were added varying volumes of aqueous phosphate buffer
to give solutions with the indicated solvent compositions (v/v).
The spectrum in an aqueous buffer alone was obtained by
removing Me2SO and dissolving the residue in the buffer. Spectra
are assembled in Figure 10 for anaerobic solutions containing
80−0% Me2SO and no excess disulfide 14 and measured at times
up to 150 h after the initial spectra. For each system, spectra were
recorded at four to six intermediate times as well, but for clarity,
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only the initial and final spectra and those at 60 min and 12 h are
shown. Spectra in the different solvent media were measured in
the concentration ranges given in the figure. The stability is
qualitatively assessed by departure from the spectrum in pure
Me2SO (Figure 6).
ACKNOWLEDGMENTS
■
This research was supported by Friedreich’s Ataxia Research
Alliance and NIH Grant GM 28856. We thank Dr. T. Rouault for
useful discussion. C.-C.L. and P.Z. thank the Alberta Glycomics
Centre, the Government of Alberta, and the University of
Calgary for financial support. We also thank Dr. Shao-Liang
Zheng at Harvard University and Dr. Yuk-Sheng Chen at
ChemMetCARS, Advanced Photon Source, for their assistance
with X-ray data collection. ChemMetCARS Section 15 is
principally supported by the National Science Foundation/
Department of Defense under Grant NSF/CHE-0822838. Use
of the Advanced Photon Source is supported by the U.S.
Department of Defense, Office of Science, Office of Basic Energy
Sciences, under Contract DE-AC02-06CH11357.
The spectrum of cluster 15 in 80% Me2SO is virtually identical
with that in the pure solvent. The cluster is completely stable in
this medium at 72 and 150 h and evidences only ca. 6% decrease
in the intensity of the 440 nm band at 150 h. At 60% Me2SO, the
cluster is fully stable at 60 min and slowly decays at 440 nm to
80% of the original intensity after 150 h. The spectra at 40% and
20% Me2SO are further progressions but with significant
increases in the absorbance in the 470−770 nm region. Increased
visible absorbances are observed for α-cyclodextrin thiolate
clusters 17 and 18 and also for [Fe4S4(SCH2CH2OH)4]2− and
[Fe4S4(SCH2CH2CO2)4}6− at 0−50% Me2SO. However, their
appearance is a certain sign of (partial) cluster degradation.
Circular dichroism spectra at 270−770 nm (not shown)
confirm stability at 60−100% Me2SO for up to 12 h and reveal
substantial changes in the ellipticity and several new features at
higher aqueous buffer content after 1 h. A comparison of
spectra at 80−20% Me2SO indicates a somewhat improved
stability of 15 in these media at 60 min to 12 h compared to
clusters 17 and 18, with the former somewhat more stable than
the latter.1
Clusters 15 and 16, derived from β-CD ligands 3 and 2,
respectively, are potentially capable of chelate-type ligation.
Both possess essentially identical visible absorption features
having maxima at 440 nm (Me2SO) and 441 nm (DMF)15 with
εM ≈ 17500, suggestive of related structures. Conceivably, the
cluster structures in their entirety differ primarily by the
connector (−S− and −NHCO−) from the 6-CH2 group of
rings A and D to the 3-C6H4S binding sites. We have not
attempted to determine the stability half-life of 15 in an
aqueous buffer, as has been reported for 16 (120 h) on the basis
of an absorbance decrease at 420−470 nm. We do note that the
absorbance of 15 at 440 nm is unchanged at 1 h and decays by
15% over 120 h with an increase in the absorbance at longer
wavelengths. The latter aspect, not just the absorbance at
or near the band maximum, should be monitored to detect
cluster decay. The results when compared to those for
[Fe4S4(SCH2CH2OH)4]2− and [Fe4S4(SCH2CH2CO2)4}6−
disclose a moderate stability enhancement owing to the
presence of the large, apparently protective α- and β-CD
ligand platforms. Thus, we concur with Kuroda et al.15 that an
unusual property of cyclodextrin-based clusters is their aqueous
stability. In our hands, 15 is the most stable cluster produced
thus far.
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ASSOCIATED CONTENT
■
S
* Supporting Information
NMR and Mossbauer spectra. This material is available free of
̈
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: holm@chemistry.harvard.edu.
Notes
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The authors declare no competing financial interest.
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