A simple competition assay to probe pentacopper(I)-thiolato cluster ligand exchange

Article abstract of DOI:10.1016/j.jinorgbio.2012.11.008

Two pentanuclear copper(I) thiolato clusters of the formula [Cu ₅(L)₃]^- (L = pyridine-2,6-dimethanethiolate (L1), (1); 4-methylpyridine-2,6-dimethanethiolate (L2), (2)) were synthesized utilizing a single-step procedure, and their structures were characterized by X-ray crystallography. The aforementioned pentanuclear complexes possess an interesting propeller-like Cu₅S₆ core where all Cu centers are three-coordinate. Electrospray ionization mass spectrometry (ESI-MS) investigation of the pentanuclear copper(I) thiolato clusters with added hetero-ligands demonstrated interesting ligand exchange behavior. The product distribution resulting from ligand exchange not only depended on the quantity of added ligand, but also was sensitive to the coordination ability of the ligand. The ESI-MS method used in this study can serve as a useful tool for probing exchange behavior in coordination metal complexes that cannot otherwise be determined by NMR.

Full text of DOI:10.1016/j.jinorgbio.2012.11.008

Journal of Inorganic Biochemistry 120 (2013) 24–31
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Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
A simple competition assay to probe pentacopper(I)-thiolato cluster ligand exchange
Yi-Hsun Chen a, Troy T.Y. Lin a, Hsuan-Ying Chen a, Chai-Lin Kao a, Hsing-Yin Chen a, Sodio C.N. Hsu a,
,
⁎
c,
James R. Carey b, , Michael Y. Chiang
⁎
⁎
a
Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan
Department of Applied Chemistry, National University of Kaohsiung, Kaohsiung 811, Taiwan
Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, Kaohsiung 804, Taiwan
b
c
a r t i c l e i n f o
a b s t r a c t
Two pentanuclear copper(I) thiolato clusters of the formula [Cu5(L)3]− (L = pyridine-2,6-dimethanethiolate
(L1), (1); 4-methylpyridine-2,6-dimethanethiolate (L2), (2)) were synthesized utilizing a single-step proce-
dure, and their structures were characterized by X-ray crystallography. The aforementioned pentanuclear
complexes possess an interesting propeller-like Cu5S6 core where all Cu centers are three-coordinate.
Electrospray ionization mass spectrometry (ESI-MS) investigation of the pentanuclear copper(I) thiolato
clusters with added hetero-ligands demonstrated interesting ligand exchange behavior. The product distribu-
tion resulting from ligand exchange not only depended on the quantity of added ligand, but also was sensitive to
the coordination ability of the ligand. The ESI-MS method used in this study can serve as a useful tool for probing
exchange behavior in coordination metal complexes that cannot otherwise be determined by NMR.
© 2012 Elsevier Inc. All rights reserved.
Article history:
Received 26 June 2012
Received in revised form 28 September 2012
Accepted 26 November 2012
Available online 30 November 2012
Keywords:
Cluster compounds
Copper
N ligands
S ligands
Tridentate ligands
1. Introduction
found in metalloproteins [18–24], and have been led to the generation
of several specific metal complexes [25–33]. The S2N coordination
Interest in copper(I)-thiolato chemistry has risen in the past several
decades due to its connection to the cysteine-rich copper(I) proteins
such as copper-metallothioneins, copper chaperones, copper regulatory
proteins, and phytochelatins [1–4]. Mononuclear and multinuclear
copper(I)-thiolato compounds have been divided into three classes
(I, II, III) according to their Cu:SR− ratio. Compounds that are polymeric
copper(I) with a [Cu(SR)x]x where x=1 belong to class I [5,6], mononu-
clear copper(I) complexes with [Cu(SR)x]1−x where x=2 or 3 belong to
class II [7–10], and oligomeric copper(I) clusters [Cu(SR)x]1−x where x
is between 2 and 3 belong to class III [2,8,9].
environment is present in the copper chaperones ScATx1, Sco1, and
Sco2 [34–38]. Interestingly, the more common copper chaperones do
not contain histidine residues. However, the importance of the histidine
ligand present in cysteine-rich bacterial metallothioneins has recently
been reported [39–41]. In light of increased importance of the S2N
motif, we decided to employ dithiolato ligands pyridine-2,6-
dimethanethiolate (L1), 4-methylpyridine-2,6-dimethanethiolate (L2)
and 1,3-phenylenedimethanethiolate (L3) (Chart 1) to examine the in-
fluence of ligand constitution (e.g. the nitrogen from histidine) to the
stability of copper thiolato cluster complexes. Herein, we report the
preparation and characterization of two homologous pentacopper
anionic complexes, [Cu5(L1)3]− (1) and [Cu5(L2)3]− (2). Most inter-
estingly, these pentacopper complexes displayed ligand exchange
when mixed with hetero-dithiolato ligands. A electrospray ioniza-
tion mass spectrometry (ESI-MS) experiment was designed to ascer-
tain the details for this type of ligand exchange (vide infra). This
work may furnish some insight to the copper trafficking mechanisms
in copper-metallothioneins and homeostasis issues in biological
systems.
For Class III, the tetracopper complexes [Cu4(SR)6]2− are the most
frequently observed homoleptic species present in the literature. Exam-
ples include but are not limited to [Cu4(SMe)6]2− [6,11], [Cu4(SEt)6]2−
[12], [Cu4(SiPr)6]2− [9], [Cu4(SPh)6]2− [13], [Cu4{μ-S(o-tBuC6H4)}6]2−
[8], [Cu4(SCH2CH2S)3]2− [14], and [Cu4(o-(SCH2)2C6H4)3]2− [15]. It has
been shown that the structures of these clusters are stabilized by both
monodentate and bidenate thiolates [2]. When sterically demanding
ligands such as StBu− or SAd− (adamantine thiolate anion) are used,
steric hindrance leads to ligand-poor complexes. For example, the
rarely observed pentacopper anion [Cu5(SR)6]−, which exhibits both
three-coordinated and two-coordinated copper(I) centers [7–9,16,17],
are obtained when using StBu− or SAd− as ligands.
2. Experimental section
The [S,N,S] donor ligand sets are reputable models for the two
cysteine, one histidine coordination environment (S2N) frequently
2.1. General information
All manipulations were carried out under an atmosphere of purified
dinitrogen using standard Schlenk techniques. Chemical reagents were
purchased from Aldrich Chemical Company Ltd., Lancaster Chemicals
⁎
Corresponding authors. Tel.: +886 7 3121101; fax: +886 7 3125339.
E-mail addresses: sodiohsu@kmu.edu.tw (S.C.N. Hsu), jcarey@nuk.edu.tw
(J.R. Carey), michael@faculty.nsysu.edu.tw (M.Y. Chiang).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.11.008

Y.-H. Chen et al. / Journal of Inorganic Biochemistry 120 (2013) 24–31
25
Chart 1. Representation of the dithiolato ligands used in this study.
Ltd., or Fluka Ltd. All the reagents were used without further puri-
fication, except for solvents that were dried over Na (Et2O, THF) or
CaH2 (CH2Cl2, CH3CN) and then thoroughly degassed before use.
2,6-Bis(mercaptomethyl)pyridine (L1) [42], 1,3-phenylenedimethane-
thiolate (L3H2) [43] and 4-methyl-2,6-bis(bromomethyl)pyridine [44]
were prepared according to the literature. IR spectra were recorded
using a Varian 640 FT-IR spectrometer. 1H NMR and 13C NMR spectra
were acquired using a Varian Gemini-200 proton/Carbon FT NMR or a
Varian Gemini-500 proton/Carbon FT NMR spectrometer. ESI mass
spectra were collected using a Waters ZQ 4000 mass spectrometer. Ele-
mental analyses were performed using a Heraeus CHN-OS Rapid Elemen-
tal Analyzer.
thiouronium salt which was then treated with a solution of NaHCO3
(1.93 g, 23.0 mmol) in water (60 mL) to give a clear solution. This
solution was then boiled for 2 h under N2, cooled to rt and extracted
with CH2Cl2 (3×50 mL). The extracts were combined, washed with a
saturated aqueous NaCl, dried over MgSO4 and the solvent removed in
vacuo to give a yellow oil. The yellow oil residue was further purified by
vacuum distillation to give a colorless oil. Yield 0.85 g (40%), 1H NMR
(CDCl3): δ 2.02 (t, 2H, SH), 2.38 (s, 3H, Py-CH3), 3.79 (d, 4H, Py-CH2-S),
7.04 (d, 2H, Py-H). 13C{1H} NMR (CDCl3): δ 20.97 (Py-CH3), 30.55
(Py-CH2-S), 121.53 (Py-Cm), 149.20 (Py-Cp), 159.66 (Py-Co). FT-IR
(cm−1): 2550 (SH). GC-Mass: 185 [C8H11NS2]+
. Anal. Calcd for
C8H11NS2: C, 51.85; H, 5.98; N, 7.56. Found: C, 51.90; H, 5.96; N, 7.52.
4-Methyl-2,6-bis(mercaptomethyl)pyridine (185 mg, 1.0 mmol) in
55 mL THF was added to 10 mL methanol solution of NaOCH3
(108 mg, 2.0 mmol). The mixed solution was stirred for 30 min and
concentrated to about 5 mL, and then 100 mL of Et2O was added to
yield a white solid. The solid was separated by filtration and dried
under vacuum. Yield: 194 mg (85%). 1H NMR (DMSO-d6): δ 2.11 (s,
3H, Py-CH3), 3.35 (s, 4H, Py-CH2-S), 6.65 (d, 2H, Py-H).
2.2. Synthesis of sodium pyridine-2,6-dimethanethiolate ligand (L1)
2,6-Bis(mercaptomethyl)pyridine (950 mg, 5.60 mmol) in 55 mL tet-
rahydrofuran (THF) was added to 10 mL methanol solution of NaOCH3
(610 mg, 11.3 mmol). The mixed solution was stirred for 30 min and
concentrated to roughly 5 mL, and then 100 mL of Et2O was added yield-
ing a white solid. The solid was separated by filtration and dried in vacuo.
Yield: 0.95 g (79%). 1H NMR (DMSO-d6): δ 3.39 (s, 4H, Py-CH2-S), 6.65
(d, 2H, Py-H), 7.24 (t, 1H, Py-H).
2.4. Synthesis of Na[Cu5(L1)3] (1−
)
A suspension of CuCl (200 mg, 2.02 mmol) in THF (15 mL) solution
was added to a stirred methanol (10 mL) solution of sodium pyridine-
2,6-dimethanethiolate (259 mg, 1.20 mmol). The resultant yellow solu-
tion was stirred for 30 min and concentrated to about 5 mL, and then
150 mL of Et2O was added to yield a yellow solid. The resulting yellow
solid was washed with 20 mL of Et2O. Yield: 260 mg (77%). 1H NMR
(DMSO-d6): δ 3.70 (m, 4H, Py-CH2-S), 7.01 (d, 2H, Py-H), δ 7.49 (t, 1H,
Py-H). 13C{1H} NMR (DMSO-d6): δ 38.63 (Py-CH2-S), 120.53 (Py-Cm),
2.3. Synthesis of sodium 4-methylpyridine-2,6-dimethanethiolate ligand
(L2)
4-Methyl-2,6-bis-(bromomethyl)pyridine (3.22 g, 11.5 mmol) and
thiourea (1.75 g, 23.0 mmol) were dissolved in EtOH (50 mL) and the
mixture was heated to reflux for 2 h, after which the solution was
evaporated to dryness in vacuo to give a white precipitate of solid
Fig. 1. Molecular structure of the anions [Cu5(L1)3]− of 1 with thermal ellipsoids drawn at the 50% level.

26
Y.-H. Chen et al. / Journal of Inorganic Biochemistry 120 (2013) 24–31
Fig. 2. Pseudo-threefold views of the anions [Cu5(L1)3]− of 1 with thermal ellipsoids drawn at the 50% level.
136.63 (Py-Cp), 161.66 (Py-Co). ESI-Mass: 825 [Cu5(C7H7NS2)3]-. Anal.
Calcd for C21H21Cu5N3NaS6: C, 29.73; H, 2.49; N, 4.95. Found: C, 29.95;
H, 2.42; N, 4.92.
whereas hydrogen atoms were placed at the calculated positions and
included in the final stage of refinements with fixed parameters. A
summary of relevant crystallographic data for complexes 1 and 2 is
provided in Table S4.
2.5. Synthesis of Na[Cu5(L2)3] (2−
)
3. Results and discussion
A suspension of CuCl (200 mg, 2.02 mmol) in THF (15 mL) was
added to sodium 4-methylpyridine-2,6-dimethanethiolate (276 mg,
1.20 mmol) solution in 10 mL of methanol. The resultant yellow
mixture was stirred for 30 min and concentrated to about 5 mL, and
then 150 mL of Et2O was added giving a yellow solid. The resulting
yellow solid was washed with 20 mL of Et2O. Yield: 285 mg (80%). 1H
NMR (DMSO-d6): δ 2.20 (s, 3H, Py-CH3), 3.65 (m, 4H, Py-CH2-S), 6.83
(s, 2H, Py-H). 13C{1H} NMR (DMSO-d6): δ 21.67 (Py-CH3), 32.86
(Py-CH2-S), 121.48 (Py-Cm), 150.90 (Py-Cp), 161.68 (Py-Co). ESI-Mass:
867 [Cu5(CH3C7H7NS2)3]−. Anal. Calcd for C24H27Cu5N3NaS6: C, 32.37;
H, 3.06; N, 4.72. Found: C, 32.35; H, 3.12; N, 4.82.
3.1. Synthesis and characterization
The yellow pentacopper anionic complexes, [Cu5(L1)3]− (1) and
[Cu5(L2)3]− (2), were synthesized by treating a suspension of CuCl in
THF with a solution of the sodium salt of the corresponding dithiolato
ligands (L1 or L2) in methanol. Complexes with higher or lower nuclearity
were not detected by spectroscopic means even when the stoichiometric
ratios were varied. Alternatively, complexes 1 and 2 can be prepared by
reaction of copper(II) in methanol with an excess of dithiols/tertiary
amine in a 1:1 ratio. At rt, the reduction of copper(II) to copper(I) using
excess dithiolato ligand occurs in less than one second to give the corre-
2.6. Ligand Competition ESI-MS monitoring experiments
sponding yellow pentacopper complexes in moderate yields. The [M+
]
peak in well-resolved ESI-MS spectra confirmed the generation of a
pentacopper core with three dithiolato ligands in complex 1 as well as
A solution of the sodium salt of the dithiolate ligands L1 (6.5 mg,
0.03 mmol) in methanol (10 mL) was added in one portion to a well-
stirred homogenous solution of [Cu5(L2)3]− (2; 12 mg, 0.01 mmol) in
methanol (10 mL) under N2. The reaction was monitored by ESI-MS
(sample concentration=10 μM). Experiments were run at least five
times for standard deviation determination.
2.7. Crystallography
Crystals suitable for X-ray analysis were obtained by diffusion of Et2O
into a CH3CN solution at −20 °C. All single-crystal X-ray diffraction data
were measured using a Bruker Nonius Kappa CCD diffractometer using
Mo Kα radiation (λ=0.71073 Å). The data collection was executed
using the SMART program. Cell refinement and data reduction were
made using the SAINT program [45]. The structure was determined
using the SHELXTL/PC program [46] and refined using full-matrix
least-squares. All non-hydrogen atoms were refined anisotropically,
Scheme 1. (a) The interpenatrating Cu5 core (trigonal bipyramid) and S6 core (trigonal
prism) of anions 1 and 2. (b) A schematic representation of the pentacopper anion 1.

Y.-H. Chen et al. / Journal of Inorganic Biochemistry 120 (2013) 24–31
27
in 2 (Fig. S1; see supplementary information). Further confirmation of the
structures came from NMR data (Fig. S2 and S3; see supplementary) and
crystallographic analysis. In the 1H NMR spectra, all diastereotopic
methylene proton resonances of L1 ligands in complexes 1 (or L2 in
2) reduced to one signal. This is consistent with the pseudo-D3 molecu-
lar structure of the anion core of 1 and 2 (Fig. 2 and Fig. S8) where all
methylene groups are equivalent. Therefore, these pentacopper core
structures were maintained in solution at ambient temperature. In
addition, the known oligomeric copper(I) clusters (ex. [Cu5(StBu)6]−
or [Cu4(SPh)6]2−) [9,13] react with L1 to also give 1. In order to under-
stand the chelating ability dithiolate ligands, we also examined the
reaction of CuCl with L3 ligand to give an insoluble yellow precipitate
3. Unfortunately, we were not able to grow single crystals for complex
3 to confirm its molecular structure. However, 3 reacts with L1 to give
1. These investigations lead us to conclude that complex 3 may be poly-
meric. These observations are consistent with the [S,N,S] ligand sets (L1
and L2) having higher substitution stabilities than the monothiolate and
dithiolate ligands.
The Cu5S6 core skeleton observed in 1 and 2 has been previously
reported for sterically demanding unidentate thiolato ligands, in
[Cu5(StBu)6]− [16], [Cu5(SAd)6]− (SAd− = adamantine thiolate
anion) [7] and [Cu5{S(o,o-Me2C6H3)}6]− [8]. In these complexes the
coordination geometries of the two axial copper centers (Cuax) and
the three equatorial copper centers (Cueq) are similar to that of 1 or 2.
A comparison of the structural parameters of 1, 2, and other known
[Cu5(SR)6]− (R=tBu, Ad or o,o-Me2C6H3) are listed in Table 1. A notable
geometric difference is found in the Cueq\S bond length (2.236 Å for 1;
2.229 Å for 2), which is longer than the Cueq\S bond lengths of all
known [Cu5(SR)6]− (R=tBu (2.165 Å), Ad (2.174 Å) and o,o-Me2C6H3
(2.178 Å) complexes. Complexes 1 and 2 both exhibit smaller S–Cueq–S
angles (average angle 140.63° for 1; 139.64° for 2) compared to other
[Cu5(SR)6]− complexes (R= tBu (169.99°), Ad (173.23°) or o,o-Me2C6H3
(164.40°). This difference may be due to angle strain originating
from the additional pyridyl group from the L1 or L2 ligand. This addi-
tional strain may cause a twisting of the ligand upon chelation to
multi-copper centers. This twisting coordination mode may also
explain why the Cueq⋯Cueq distance in 1 (3.452 Å) and 2 (3.475 Å) is
longer than that of other [Cu5(SR)6]− complexes (R=tBu (3.223 Å),
Ad (3.343 Å) or o,o-Me2C6H3 (3.288 Å).
3.2. Crystallographic characterization of pentacopper anion clusters
The pentacopper(I) complexes 1 and 2 crystallize as tetraphenyl-
phosphonium salts via metathesis between the corresponding sodium
salt and tetraphenylphosphonium chloride. The cage structure of the
anions [Cu5(L1)3]− (1) and [Cu5(L2)3]− (2) from their tetraphenyl-
phosphonium salts are presented in Fig. 1 and Fig. S8. Selected bond
lengths and bond angles for 1 and 2 are detailed in Tables S1 and S2
(see supplementary material), respectively. Both pentacopper anions 1
and 2 possess a pseudo-D3 geometric core (see Fig. 2 for 1 and Fig. S9
for 2). In the following text, the equatorial coppers are denoted as Cueq
(Cu1, Cu2 and Cu3) and the axial coppers as Cuax (Cu4 and Cu5). The
Cu5S6 core of both cage anions may be described as a trigonal bipyramidal
skeleton consisting of copper atoms with each Cuax⋯Cueq edge bridged by
a sulfur atom from a thiolato ligand (see Scheme 1). The sulfur atoms of
the six doubly-bridged thiolato groups in anions 1 and 2 are located at
the vertices of a trigonal prism. In addition, the three dithiolato ligands
form a “propeller” about the Cuax⋯Cuax axis.
Indeed, the Cu5S6 core structure is best described in terms of an
interpenetrating Cu5 core (trigonal bipyramid) and a S6 core (trigonal
prism). The spiral twist coordination mode of dithiolato ligands L1
and L2 in 1 and 2 (Fig. 2 and Fig. S9) is most peculiar, and is quite differ-
ent from the typical planar pincer type coordination mode in the litera-
ture [47–51]. Interestingly, the Cu(eq)⋯Cu(eq) edges of the Cu5 core are
not bridged by any atom. Each equatorial copper atom is however, coor-
dinated by a pyridyl nitrogen atom and two sulfur atoms, each coming
from a different ligand. The Cueq–N distances range from 2.079(3)Å to
2.144(3)Å in 1 and 2.077(8)Å to 2.105(8)Å in 2. These values are in
good agreement with values previously reported for copper(I) com-
plexes containing pyridine ligation [52–54].
3.3. Exchange behavior of dithiolate ligands
Since the copper(I) ion assumes a labile d10 configuration and there-
fore lacks crystal-field stabilization energy, complexes 1 and 2 are
expected to exhibit ligand exchange. To verify this and to gain more
information regarding the identity of the solution species that may
exist during ligand exchange, a competition assay was devised.
Attempts to monitor the exchange of chelated dithiolato ligands using
NMR failed due to the presence of overlapping signals originating
from anion complexes ([Cu5(L1)3]− (1) or [Cu5(L2)3]− (2)), dithiolato
ligands, and their corresponding exchange products ([Cu5(L1)2(L2)]−
and [Cu5(L1)(L2)2]−). In view of recent reports regarding the monitor-
ing of reaction processes using ESI-MS [55–58], we devised a competi-
tion assay that utilizes ESI-MS to monitor this ligand exchange
phenomenon. ESI is a very mild ionization technique that offers mini-
mal perturbations of the species present in solution. By the addition of
the hetero-ligand (L2) into the anionic complex 1 and monitoring the
growth of the ESI-MS signals of the ligand-exchanged products, one
can deduce the identities and ratios of these products.
As Scheme 2 (path A) illustrates, the addition of three equivalents of
the sodium salt of L1 to a methanol solution of anionic [Cu5(L2)3]− (2)
leads to a decrease in the peak of anion 2 (m/z 868) and a concomitant
growth of three new product signals (m/z 854, 840 and 826) which can
be assigned to [Cu5(L1)(L2)2]−, [Cu5(L1)2(L2)]−, and [Cu5(L1)3]− (1),
respectively (Fig. 3). Furthermore, the relative distribution of these
pentacopper anion complexes can be accessed by the intensities of their
corresponding mass signals. After 2 h, the ligand exchange process
Table 1
Dimensions of the Cu5S6 cores of selected copper(I)-thiolato clustersa.
[PPh4][Cu5(L1)3] (1)b,c
[PPh4][Cu5(L2)3] (2)b,c
[Et4N][Cu5(StBu)6]b,d
[Et4N][Cu5(SAd)6]b,e
[Et4N][Cu5{S(o,o-Me2C6H3)}6]b,f
Cuax–S
Cueq–S
Cueq–N
Cueq…Cueq
S–Cuax–S
S–Cueq–S
2.368–2.676, 2.251
2.2069–2.2686, 2.236
2.079–2.144, 2.118
3.422–3.476, 3.452
116.45–122.49, 119.87
139.62–141.66, 140.63
2.235–2.279, 2.258
2.92–2.273, 2.229
2.077–2.105, 2.088
3.428–3.509, 3.475
115.22–123.26, 119.57
138.36–141.69, 139.64
2.260–2.291, 2.250
2.132–2.201, 2.165
na
3.157–3.257, 3.223
114.52–124.62, 119.89
169.04–170.81, 169.99
2.228–2.358, 2.291
2.151–2.185, 2.174
na
3.091–3.509, 3.343
96.4–147.3, 119.93
170.7–175.7, 173.23
2.242–2.866, 2.262
2.170–2.191, 2.178
na
3.189–3.351, 3.288
111.71–130.76, 119.56
162.74–166.54, 164.40
a
Numbers given in Å or degrees.
(Range, mean).
This work.
Ref. [16].
Ref. [7].
b
c
d
e
f
Ref. [8].

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Y.-H. Chen et al. / Journal of Inorganic Biochemistry 120 (2013) 24–31
Scheme 2. Representation of a dithiolato ligand exchange assay of 1 and 2.
Fig. 3. ESI-MS spectra of a ligand exchange assay using anions [Cu5(L2)3]− (2) with L1, recorded at different times (a) 0 h, (b) 10 min and (c) 2 h.

Y.-H. Chen et al. / Journal of Inorganic Biochemistry 120 (2013) 24–31
29
Fig. 4. ESI-MS spectra of (a) MeOH solution of [Cu5(L1)3]− (1), (b) upon treatment with 3 equivalents of L2, (c) upon treatment with 9 equivalents of L2 and (d) upon treatment
with 30 equivalents of L2. (See the product distributions ratio value in Table 2).
reached equilibrium as evidenced by a lack of change in the height of the
ESI-MS signals. The product distributions in the final equilibrium mixtures
were close to 2:7:10:3 for 1: [Cu5(L1)2(L2)]−: [Cu5(L1)(L2)2]−: 2. The
theoretical ratio is 1:3:3:1 assuming equal accessibility of L1 and L2 for
each species. This unexpected ratio implies that a subtle difference in co-
ordinating ability of the dithiolato ligand may influence the product distri-
butions in ligand exchange assay.
Further corroboration of the ligand binding strength of 1 and 2
(Fig. S12 and S13; see supplementary) was accomplished using cyclic
voltametry in CH2Cl2 containing 0.1 M [n-Bu4N](PF6). The oxidation
potential of 2 contains an irreversible band corresponding to an Epa
value of 0.17 V, which is significantly lower than that of 1 (Epa
=
0.50 V). The results of this study show that oxidation of complex 2
with the 4-methyl substituent on the pyridine ring is more favorable
than that of the parent pyridine ligand in complex 1. This finding indi-
cates that the redox potentials of pentanuclear copper(I) thiolato clus-
ters are governed by the electronic nature of the 4-methyl substituent
of the pyridine ring ligand.
Additional ESI-MS experiments were carried out by treating 1 with 3,
9, or 30 equivalents of L2 (Fig. 4 and Table 2). The dithiolato ligand
exchange between 1 and L2 gave all the above-mentioned ligand
exchange products: [Cu5(L2)3]− (2), [Cu5(L1)(L2)2]−, [Cu5(L1)2(L2)]−
,
and [Cu5(L1)3]− (1), but in different ratios. For the 3 equivalent assay
(L1:L2=1:1), the product ratio was close to 2:7:10:3. This is also slightly
different from the theoretical ratio of 1:3:3:1. For the 9 equivalent L2
assay, the ratio increased to 0:15:70:100 (expected ratio 1:9:36:84)
favoring products with a higher L2 content. For the 30 equivalent L2
assay, the ratio increased to 0:0:15:100. Interestingly, two of the L1
containing products vanished. Taken together, these results indicate
that with an increasing amount of added L2, products containing L2
were obviously favored (Table 2). On the other hand, mixing 2 with 3,
9, 30 equivalents of L1 gave a similar MS pattern and product distribution
ratios as above (data not shown). Furthermore, the MS pattern and prod-
uct distribution ratios (Scheme 2) of the forward experiment (1 react
with L2, Fig. 4(b)) are almost the same as that of the backward experi-
ment (2 react with L1, Fig. 3(c)). This indicates that these ligand
exchange reactions are thermodynamically controlled rather than kinet-
ically controlled.
Table 2
The product distribution ratios of the dithiolato ligand exchange assay between 1 and
L2. ESI-MS results are shown in Fig. 4.
Molar
ratio L1
Theoretical height ratio of ESI-MS Experimental height ratio of
signal for 1:[Cu5(L1)2(L2)]−
:
ESI-MS signala for 1:
(in 1):L2 [Cu5(L1)(L2)2]−:2
[Cu5(L1)2(L2)]−:[Cu5(L1)(L2)2]−:2
1:1b
1:3:3:1
20(1):70(2):100(2):30(1) or ca.
2:7:10:3
1:3c
1:9:36:84
1:30:435:4060
0(0):150(2):702(5):998(6) or ca.
0:15:70:100
0(0):1(1):150(2):1000(4) or ca.
0:0:15:100
1:10d
a
Numbers in parentheses indicate standard deviation of experimental values.
3 equivalents of L2 to 1.
9 equivalents of L2 to 1.
b
c
d
30 equivalents of L2 to 1.

30
Y.-H. Chen et al. / Journal of Inorganic Biochemistry 120 (2013) 24–31
Fig. 5. A comparison of copper transport vs. ligand exchange mechanisms.
It is important to note that the above interpretations assume that
pentanuclear copper(I) thiolato clusters 1 and 2 may also provide
some insight into copper trafficking in biological systems.
the observations in the gas phase reliably describe the solution phase
equilibrium. However, the time-dependent chemistry of transfer of
ions from the solution phase to the gas phase and differences in trans-
fer efficiencies of the individual anions could complicate or skew the
results. Nevertheless, this technique has been applied to study these
types of transiently generated species and dynamic ligand exchange
behaviors [57]. The competition assay of the pentanuclear copper(I)
thiolato cluster shows that the exchange distribution depends on
the molar ratios and coordination ability of the dithiolato ligands.
This thermodynamic preference parallels the intracellular copper
trafficking systems where copper(I) cations can be delivered from
specific donor proteins to another particular target protein possessing
cysteine-rich domains containing histidine residues [18,20,21]. Simi-
lar preferences of the S2N coordination may be responsible for Cu
trafficking behavior in vivo. Indeed, such an assay would ultimately
have to include the control and measurement of the pH in aqueous
solution, which could accomplished using a variety of spectroscopic
techniques (including UV–vis, NMR), potentiometric studies (such
as CV or redox titrations), and molecular biology techniques (such
as site-directed mutagenesis).
The study of complexes involved in Cu transport is a formidable
task. Since proteins and Cu–S compounds of the type described in
this report are amiable to ESI, it is thought that these subtle biologi-
cally relevant equilibriums could be probed using assays of model
complexes as described above. Cu transport is known to occur
through transiently formed intermolecular protein complexes
(Fig. 5). These complexes mediate intermolecular metal–ligand ex-
change, therefore a well-designed ESI-MS assay of biological systems
and model complexes, as described above, could be useful to structur-
ally and thermodynamically probe these ephemerally formed species.
Acknowledgments
Financial support of the National Science Council (Taiwan) and the
Center of Excellence for Environmental Medicine in the KMU are
highly appreciated. We thank Mr. Ting-Shen Kuo, National Taiwan
Normal University for the X-ray structural determinations.
Appendix A. Supplementary material
Supplementary data to this article can be found online at doi:
CCDC 819194 and 819195 contain the supplementary crystallograph-
ic data for this paper. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.
uk/data_request/cif. Supplementary data to this article can be found
online at: http://dx.doi.org/10.1016/j.jinorgbio.2012.11.008.
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