Reactions of an organoruthenium anticancer complex with 2-mercaptobenzanilide-a model for the active-site cysteine of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1039/c1dt11189b

The organometallic anticancer complex [(η⁶-p-cymene)Ru(en) Cl]PF₆ (1, en = ethylenediamine) readily reacts with thiols and forms stable sulfenate/sulfinate adducts which may be important for its biological activity. Protein tyrosine

Full text of DOI:10.1039/c1dt11189b

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PAPER
Reactions of an organoruthenium anticancer complex with
2-mercaptobenzanilide—a model for the active-site cysteine of protein
tyrosine phosphatase 1B†
Yumiao Han,a,b Qun Luo,a,b Xiang Hao,b Xianchan Li,a,b Fuyi Wang,*a,b Wenbing Hu,a,b Kui Wu,a,b
Shuang Lu¨a,b and Peter J. Sadler*c
Received 23rd June 2011, Accepted 22nd August 2011
DOI: 10.1039/c1dt11189b
6
The organometallic anticancer complex [(h -p-cymene)Ru(en)Cl]PF6 (1, en = ethylenediamine) readily
reacts with thiols and forms stable sulfenate/sulfinate adducts which may be important for its biological
activity. Protein tyrosine phosphatase 1B (PTP1B), a therapeutic target, contains a catalytic cysteinyl
thiol and is involved in the regulation of insulin signaling and the balance of protein tyrosine kinase
activity. On oxidation, the catalytic Cys215 can form an unusual sulfenyl-amide intermediate which can
subsequently be reduced by glutathione. Here we study reactions of 1 with 2-mercaptobenzanilide, 2, a
recognized model for the active site of PTP1B. We have characterized crystallographically compound 2
and its oxidized sulfenyl-amide derivative 2-phenyl-1,2-benzisothiazol-3(2H)-one (4), which shows a
close structural similarity to the sulfenyl-amide in oxidized PTP1B. At pH 7.4 and 5.3, 1 reacted with 2,
6
affording a mono-ruthenium thiolato complex [(h -cym)Ru(en)(S-RS)]+ (7+, R = (C6H4)CONH(C6H5))
6
and a triply-S-bridged thiolato complex [((h -cym)Ru)2(m-S-RS)3]+ (8+), respectively. Coordination of
˚
Ru to the S atom in 7 allows formation of a strong H-bond (2.02 A) between the en-NH and the
carbonyl oxygen. To assess the possible effect of ruthenium coordination on the redox regulation of
PTP1B, reactions of these thiolato products with H2O2 and/or GSH were then investigated,
demonstrating that coordination to Ru largely retards both the oxidation (deactivation) of the thiol in
compound 2 by H2O2 and the subsequent reduction (reactivation) of the sulfenyl-amide by GSH,
implying that the inhibition of complex 1 on PTP1B (IC50 of 19 mM) may be attributed to coordination
to its catalytic cysteine.
6
en complexes [(h -arene)Ru(en)Cl][PF6], the cytotoxicity increases
Introduction
with the size of the coordinated arene, and the cytotoxicity of
the p-cymene complex against human ovarian cancer cell line
A2780 is comparable to that of carboplatin.10,12 DNA is a potential
target for these ruthenium(II) arene complexes, most of which bind
selectively to N7 of guanine.15–20 However, these organometallic
ruthenium anticancer complexes also have a high affinity for the
thiolate sulfur in cysteine,21 glutathione22 and human albumin,23,24
and Ru–S coordination can induce the oxidation of thiolates
to sulfenates or sulfinates,22–24 and subsequently stabilize the
sulfenato ligands23,25 which as free ligands are often unstable
and highly reactive.26,27 Moreover, glutathione was shown to be
kinetically competitive with cGMP for coordination with the
ruthenium biphenyl complex, and the oxidation of coordinated
glutathione in the resulting thiolato ruthenium complex to a
sulfenate ligand appears to provide a facile route for displacement
of S-bound glutathione by G N7.22
Organometallic ruthenium(II) complexes are a family of ruthe-
nium(II) compounds of interest as potential antitumor agents,1–9
and the type [(h -arene)Ru(YZ)(X)][PF6], where X is a halide
and YZ is a chelating diamine such as ethylenediamine (en),
have in vitro and in vivo anticancer activity, including against
cisplatin-resistant cell lines.10–13 The arene ligand occupies three
coordination sites in these pseudo-octahedral complexes and
stabilizes Ru in its +2 oxidation state.14 For the family of chlorido
6
aCAS Key Laboratory of Analytical Chemistry for Living Biosystems,
Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, PR
China
bBeijing National Laboratory for Molecular Sciences, Institute of Chem-
istry, Chinese Academy of Sciences, Beijing, 100190, PR China.
E-mail: fuyi.wang@iccas.ac.cn; Fax: (+86) 10-6252-9069
cDepartment of Chemistry, University of Warwick, Gibbet Hill Road,
Coventry, CV4 7AL, UK. E-mail: P.J.Sadler@warwick.ac.uk; Tel: +44-
24-76523653
The protein tyrosine phosphatase 1B (PTP1B) negatively regu-
lates insulin signaling by dephosphorylating the phosphorylated
tyrosine residues of the insulin receptor kinase, and is involved in
balancing protein tyrosine kinase (PTK) activity as part of normal
growth-regulation pathways.28–32 Increasing interest is focused on
† Electronic supplementary information (ESI) available: HPLC, UV-Vis
and LC-ESI-MS data, Figure S1–S11. CCDC reference numbers 821489–
821492. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c1dt11189b
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PTP1B as an effective target for both anti-diabetes/obesity drug
discovery33–35 and the treatment of human breast and ovarian
cancers, a significant subset of which, especially HER2/Neu
HER2(+) tumors, overexpress PTP1B.29,30,36 The catalytic site
residue Cys215 plays a central role in modulating the activity
of PTP1B, and is a key feature in the deactivation/reactivation
pathway involving the formation and the subsequent reduction
of a sulfenyl-amide intermediate mediated by the endogenous
signaling molecules hydrogen peroxide (H2O2) and glutathione
(GSH), respectively.37–40 This intriguing behavior of PTP1B led
us to explore whether ruthenium arene anticancer complexes
coordinate to the catalytic thiol of PTP1B and inhibit enzyme
activity via the induced oxidation of thiol to sulfinate/sulfenate
and the subsequent stabilization of the sulfenate.
matography (HPLC). Redox reactions of the resulting mono- and
dinuclear thiolato products, which were both characterized by X-
ray diffraction analysis, were studied to mimic the effect of ruthe-
nium coordination on the inactivation and reactivation of PTP1B.
Results
Synthesis and crystallographic characterization of
2-mercaptobenzanilide
The model compound 2-mercaptobenzanilide (2) was synthesized
following the procedure described in the literatures,38,39,42 and
crystallized from a saturated solution in methanol. The X-ray
structure and atom numbering scheme are depicted in Fig. 1a,
crystallographic data are listed in Table 1, and selected bond
Indeed, in a preliminary study, we found that the ruthenium
6
arene anticancer complex [(h -p-cym)Ru(en)Cl]PF6 (1, cym =
cymene) is inhibitory towards PTP1B with an IC50 of 19 mM.41 In
the present work, therefore, the thiol 2-mercaptobenzanilide (2),
and the product of H2O2-oxidation, the sulfenyl-amide derivative
2-phenyl-1,2-benzisothiazol-3(2H)-one, have been synthesized
and characterized crystallographically for first time to mimic the
interactions between complex 1 and the catalytic cysteine site of
PTP1B. The aromatic thiol compound 2-mercaptobenzanilide is
an established chemical model for the redox regulation of PTP1B,
including the formation of the sulfenyl-amide intermediate in the
presence of H2O2 and GSH-mediated reactivation of the inert
intermediate,38,39 because the ortho-substituted benzene scaffold
provides a good model for the proximity of the amide and the
thiol group at the active site of PTP1B37–40 which is not readily
duplicated by aliphatic thiolates. Interactions between complex
1 and the model compound 2 are then investigated by mass
spectrometry (MS) coupled with high performance liquid chro-
Fig. 1 X-ray crystal structures and atom numbering schemes for a)
2-mercaptobenzanilide (2), and b) 2-phenyl-1,2-benzisothiazol-3(2H)-one
(4) at 50% probability thermal ellipsoids.
6
6
Table 1 Crystallographic data for 2-mercaptobenzanilide (2), 2-phenyl-1,2-benzisothiazol-3(2H)-one (4), [(h -cym)Ru(en)(RS)]PF6 (7) and [((h -
cym)Ru)2(RS)3]PF6 (8), R = C6H5CONHC6H4
2
4
7
8
Chemical formula
Molar mass
C13H11NOS
229.29
C13H9NOS
227.27
Monoclinic
0.48 ¥ 0.26 ¥ 0.17
P21/c
Colorless/block
5.8793(12)
14.397(3)
12.316(3)
90.00
98.23(3)
90.00
173(2)
4
0.287
3.29–27.49
472
7080
2341
2105
0.0457
0.1173
1.120
+0.284, -0.248
C26H34PF6N3OSRuCl2
753.56
Triclinic
C62H70PF6N3O6S3Ru2
1396.50
Triclinic
Crystal system
Crystal size/mm
Space group
Crystal
Orthorhombic
0.37 ¥ 0.29 ¥ 0.26
P212121
Colorless/block
9.6385(19)
11.635(2)
20.051(4)
90.00
90.00
90.00
173(2)
8
0.263
2.17–27.48
960
18 364
5149
4983
0.0355
0.0859
1.077
0.17 ¥ 0.15 ¥ 0.10
0.53 ¥ 0.46 ¥ 0.36
¯
¯
P1
P1
Yellow/block
10.811(2)
12.467(3)
12.891(3)
100.77(3)
109.54(3)
98.87(3)
173(2)
Red/block
11.0581(3)
15.6374(4)
17.9756(5)
84.0760(10)
80.4740(10)
88.8860
173(2)
˚
a/A
˚
b/A
˚
c/A
a/◦
b/◦
g /◦
T/K
Z
2
2
m/mm-1
0.851
1.71–27.46
764
19 247
7124
6774
0.0384
0.0958
1.084
0.696
2.03–27.46
1432
25 422
13 920
13 153
0.0478
0.0922
1.289
q range/◦
F(000)
Reflns collected
Indep. reflns
Refns obs. [I > 2s(I)]
R [F > 4o´ (F)]a
b
Rw
GOFc
-3
˚
Dr max and min [e A ]
+0.209, -0.185
+0.681, -0.688
+0.733, -0.800
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
a R = ꢀFo| - |Fcꢀ/ |Fo|. b Rw = [ w(Fo - Fc2)2/ wFo2)]1/2
.
c GOF = [ w(Fo - Fc2)2/(n - p)]1/2, where n = number of reflections and p = number
2
2
of parameters.
11520 | Dalton Trans., 2011, 40, 11519–11529
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Table 2 Selected bond lengths (A) and angles (◦) for 2-
˚
mercaptobenzanilide (2) and 2-phenyl-1,2-benzisothiazol-3(2H)-one
(4). For numbering schemes see Fig. 1
2
4
S1–C6
C1–C6
C5–C6
1.7650(17)
1.398(2)
1.405(2)
S1–C7
S1–N1
N1–C8
1.739(2)
1.7186(16)
1.430(2)
C4–C5
C5–C7
1.401(2)
1.495(2)
N1–C1
O1–C1
1.380(2)
1.222(2)
O1–C7
N1–C7
1.234(2)
1.349(2)
C1–C2
C2–C3
1.467(3)
1.393(3)
N1–C8
1.424(2)
97.80
C8–C9
1.390(3)
127.70(16)
90.45(8)
119.23(12)
116.08(13)
123.79(17)
124.58(15)
108.30(16)
127.89(17)
C6–S1–H1
S1–C6–C5
C6–C5–C7
C5–C7–O1
O1–C7–N1
C7–N1–C8
N1–C8–C9
N1–C8–C13
C6–C7–S1
C7–S1–N1
C8–N1–S1
C1–N1–S1
N1–C1–O1
C1–N1–C8
N1–C1–C2
O1–C1–C2
120.78(13)
121.23(15)
121.60(15)
122.66(16)
124.65(15)
118.70(16)
121.17(16)
Scheme 1 Pathways for the redox reactions and cyclization of model
compound 2 with H2O2 and GSH in phosphate buffer (pH 7.4).
5 and sulfinyl-amide compound 6 (Scheme 1), respectively. The
mass spectra (Fig. S1, ESI†) of peaks a and c showed that there
is a 2 Da difference between the masses of species contained in
these two peaks, indicating that this reaction afforded the expected
sulfenyl-amide compound (4) which is analogous to the sulfenyl-
amide intermediate formed after the H2O2-mediated inactivation
of PTP1B.37,39,40
We were also successful in crystallizing the sulfenyl-amide
compound 2-phenyl-1,2-benzisothiazol-3(2H)-one (4) from a sat-
urated ethanol solution. The X-ray structure is shown in Fig. 1b,
and the crystallographic data and the selected bond lengths and
angles are listed in Tables 1 and 2, respectively. Except for the
formation of the new S1–N1 bond, there is little change in other
bond lengths compared to 2. In compound 4, it is notable that the
C1–N1–S1–C7–C2 ring is almost coplanar with the benzene ring
(dihedral angle < 3◦).
Then the purified sulfenyl-amide compound 4 (1 mM) was
incubated with 10 mol equiv. of GSH in 50 mM phosphate buffer
(pH = 7.4) containing 30% MeCN at 298 K. The reaction was
complete within 5 min and compound 4 was completely reduced
back to the model compound 2 according to the HPLC analysis
(data not shown). This reaction is the direct analog of the GSH-
mediated reactivation of the sulfenyl-amide intermediate formed
by the catalytic thiol group of the enzyme PTP1B.37,39,40
lengths and angles in Table 2. As expected, the distance between
the sulfur atom (S1) of the thiol and the nitrogen atom (N1) of the
˚
˚
amide in compound 2 (3.8 A) is similar to that (3.5 A) between
the thiol group of Cys215 and the amide N of Ser216 at the active
site of PTP1B.37,40,43
To mimic the redox regulation of PTP1B, compound 2 (1 mM)
was incubated with 1.4 mol equiv. of H2O2 in 50 mM sodium
phosphate buffer (pH 7.4) containing 30% MeCN, which was used
to increase the solubility of compound 2 in the reaction mixture,
at 298 K. This reaction immediately gave rise to a product as
detected by HPLC analysis (peak b in Fig. 2a). Compound 2
(peak a in the HPLC traces) almost disappeared after 0.5 h of
reaction, accompanied by the formation of a second product,
corresponding to HPLC peak c with the same retention time
(11.0 min) as peak a, as evidenced by their different UV-Vis
spectra recorded by the DAD UV-Vis detector (Fig. 2b). One
hour later, the reaction afforded two further products (peaks d
and e), and the peaks c, d and e all increased in intensity until
24 h (Fig. 2a). The subsequent ESI-MS analysis of all the HPLC
fractions gave rise to singly-charged ion peaks at m/z (the most
abundant isotope mass-to-charge ratio) of 230.02, 457.01, 228.00,
278.02 and 244.01 for peaks a, b, c, d, and e (Table 3 and Fig.
S1, ESI†), assignable to compound 2, and its oxidized derivatives
disulfide compound 3, sulfenyl-amide compound 4, sulfonic acid
Reaction of [(g6-cym)Ru(en)Cl]PF6 (1) with 2-mercaptobenzanilide
(2)
To mimic the interaction of organometallic ruthenium anticancer
6
complexes with PTP1B, [(h -cym)Ru(en)Cl]PF6 (1, 5 mM) was
treated with 1 mol equiv. of compound 2 in 50 mM phosphate
buffer (pH 7.4, containing 30% CH3CN). In order to minimize the
content of O2, the solutions were purged by bubbling with argon
before and after mixing. The reaction reached equilibrium at 4 h,
and the main product was identified by ESI-MS to be a thiolato
ruthenium complex (Scheme 2), as indicated by the singly-charged
Fig. 2 a) HPLC time-course for the reaction of compound 2 (1 mM)
with 1.4 mol equiv H2O2 in 50 mM phosphate buffer (pH 7.4) at 298 K.
Peak assignments: a, 2; b, 3; c, 4; d, 5 and e, 6. The chemical structures of
oxidized products 3–6 are shown in Scheme 1. b) UV-Vis spectra of HPLC
fractions a (solid line) and c (dashed line) in (a) from the reaction mixtures
of compound 2 with H2O2. Mass spectra for all fractions are shown in Fig.
S1, ESI.†
6
ion peak at m/z 523.32 corresponding to [(h -cym)Ru(en)(RS)]+
(7+) (R = (C6H4)CONH(C6H5), calculated(calc.) m/z 523.32 for
7+) as shown in Fig. S2, ESI.† Even in the presence of a 10-
fold molar excess of GSH, the reaction of complex 1 with
compound 2 still afforded the mono-ruthenium thiolato complex 7
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Table 3 Positive ions observed by HPLC-ESI-MS from reaction mixtures of 2-mercaptobenzanilide (2) and H2O2, and Ru(II) arene thiolato complex
6
[(h -cym)Ru(en)(RS)]PF6 (7) with H2O2 and GSH at 298 K, R = C6H4CONHC6H5. For chemical structures of observed ions see Schemes 1–5
Reaction
pH
7.4
Reaction Time/h
<0.5 h
RTa/min
Observed Ion
Obs (Calcd)b m/z
2 + H2O2, 1 : 1.4 mM
10.95
13.78
5.03
[2 + Hc]+
[3 + H]+
230.02 (230.06)
457.01 (457.10)
278.02 (278.05)
244.01 (244.04)
228.00 (228.05)
457.01 (457.10)
294.18 (294.07)
464.20 (464.07)
230.02 (230.06)
464.20 (464.07)
464.20 (464.07)
422.16 (422.01)
463.17 (463.07)
244.01 (244.04)
464.20 (464.07)
228.00 (228.05)
422.16 (422.01)
463.17 (463.07)
464.20 (464.07)
464.20 (464.07)
228.00 (228.05)
695.64 (695.63)
422.16 (422.01)
463.17 (463.07)
535.21 (535.13)
244.01 (244.04)
464.20 (464.07)
230.02 (230.06)
413.17 (413.05)
464.20 (464.07)
230.02 (230.06)
695.64 (695.63)
656.89 (656.62)
464.20 (464.07)
617.35 (617.61)
230.02 (230.06)
1053.21 (1053.09)
24 h
[5 + H]+
5.57
[6 + H]+
10.95
13.78
5.02
8.56
11.03
8.56
[4 + H]+
[3 + H]+
7 + H2O
7.4
60 h
[1-H2O - H]+
[7 - en]+
[2 + H]+
5.3
7.4
60 h
72 h
[7 - en]+
10.71
[9 - 2H2O]+
6
+
7 + H2O2, 1 : 1.4 mM
5.05d
{10- (h -cym)}
+
{11 - en - H}
5.57
8.56
[6 + H]+
+
{7 - en}
10.95
[4 + H]+
6
+
5.3
7.4
72 h
72 h
5.05d
{10 - (h -cym)}
+
{11 - en - H}
+
8.56
{7 - en}
10.71
10.95
4.79
[9 - 2H2O]+
[4 + H]+
7 + H2O2 + GSH,e 1 : 1.4 : 10 mM
[13 + H]2+
5.05d
{10 - (h -cym)}
6
+
+
{11 - en - H}
5.31
5.57
8.56
11.03
4.79
8.56
11.03
4.79
5.07
8.56
[17f + H]+
[6 + H]+
+
{7 -en}
[2 + H]+
+
7 + GSH, 1 : 10 mM
7.4
5.3
72 h
72 h
{12 - en - Glu}
+
{7 - en}
[2 + H]+
[13 + H]2+
[14 + H]2+
+
{7 - en}
10.56
11.03
11.81
[15 + H]2+
[2 + H]+
[16g + H]+
a RT is the retention time of detected compounds in HPLC traces (Fig. 2, 4, 5, 6 and S4, ESI†). b Observed (Obs) and calculated (Calcd) mass-to-
charge ratios for the most abundant isotope of observed ions. For mass spectra see Fig. S1–S4 and S6–S7, ESI.† c H indicates gain (+) or loss (-) of
a proton. d The fraction contains a mixture of products 10 and 11. e Complex 7 was first reacted with H2O2 for 24 h, and then GSH was added into
the reaction mixture for further reaction for 72 h after removing excess H2O2 by addition of catalase. f 17 = RS–SG, R = C6H4CONHC6H5. g 16 =
6
[((h -cym)Ru)2(RS)2(CHCO2)(PO3)].
a dichloromethane/ether solution at 253 K. The crystallographic
data for 7 are listed in Table 1, and selected bond lengths and angles
in Table 4. As shown in Fig. 3, the coordination of Ru to the sulfur
atom in 2 allows the formation of an H-bond between the NH of
the en ligand and the carbonyl oxygen with a bond (NH ◊ ◊ ◊ OC)
˚
length of 2.02 A (Fig. S4, ESI†), indicative of a strong H-bond.
˚
The Ru–S bond length (2.4059(9) A) is slightly longer than that
in the thiolato compound [(h -p-cymene)Ru(en)(S-C6H6)]I.25 The
6
thiolato complex 7 is soluble and stable in aqueous solution; ca.
94% of 7 remained intact after 60 h incubation in phosphate
buffer at 298 K (Fig. S5, ESI†). However, at pH 5.3, nearly 15%
of 7 hydrolyzed after 60 h incubation. Notably, the hydrolysis
occurred via the dissociation of the en ligand instead of the
Scheme 2 Pathways for reactions of complex 1 with model compound 2
in phosphate buffer.
containing the 2-mercaptobenzanilide ligand as the main product,
and no glutathione thiolato complex was detectable in the reaction
mixture incubated at 298 K for 48 h (Fig. S3, ESI†).
The thiolato compound 7 was precipitated by adding NH4PF6 to
the equilibrium reaction mixture containing 5 mM 1 with one mol
equiv. 2 and recrystallized from dichloromethane/ether. Crystals
suitable for X-ray analysis were obtained by slow evaporation of
6
thiolate ligand, giving rise to a di-aqua adduct [(h -cym)Ru(S-
(C6H4)CONH(C6H5)S)(H2O)2]+ (9) as shown in Scheme 3 and Fig.
S5, ESI.†
The pKa of the thiol of the model compound 2 is reported to
be 5.7, similar to that of the active-site thiol in PTP1B (5.6).39 The
reaction of the protonated ligand 2 with the ruthenium complex
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Table 4 Selected bond lengths (A) and angles (◦) for [(h -
6
˚
column chromatography eluting with dichloromethane/methanol
(11 : 0.2). Slow evaporation of the dichloromethane/methanol sol-
vent gave rise to red crystals suitable for X-ray diffraction studies.
The crystallographic data are listed in Table 1, and selected bond
lengths and angles in Table 3. These results show that the di-nuclear
6
cym)Ru(en)(RS)]PF6 (7) and [((h -cym)Ru)2(RS)3]PF6 (8),
C6H4CONHC6H5. For numbering schemes see Fig. 3
R
=
7
8
6
Ru–N3
Ru–N2
Ru–S1
Ru–C16
Ru–C17
Ru–C18
Ru–C19
Ru–C20
Ru–C21
Ru–centa
2.128(2) Ru1–S1
2.147(2) Ru1–S2
2.4059(9) Ru1–S3
2.202(3) Ru1–C1
2.209(3) Ru1–C2
2.178(3) Ru1–C3
2.201(3) Ru1–C4
2.169(3) Ru1–C5
2.177(3) Ru1–C6
1.6688(4) Ru1–centa
2.4077(7) Ru2–S1
2.3940(7) Ru2–S2
2.4015(7) Ru2–S3
2.3916(7)
2.4163(7)
2.4068(7)
2.212(3)
2.210(3)
2.194(3)
2.227(3)
2.207(3)
2.219(3)
1.6956(2)
product is a triply-S-bridged thiolato complex [((h -cym)Ru)2(m-
S-RS)3]PF6 (Fig. 4), which adopts a “sandwich” configuration.
It is notable that there is an intermolecular H-bond between the
amide N3–H and the adjacent sulfur atom S3 in the thiolato ligand
2.248(3)
2.213(3)
2.215(3)
2.236(3)
2.211(3)
2.190(3)
Ru2–C11
Ru2–C12
Ru2–C13
Ru2–C14
Ru2–C15
Ru2–C16
˚
(Fig. S5, ESI†) with an N–H ◊ ◊ ◊ S bond length of 2.51 A, whereas
no H-bond is observed between the amide proton and the sulfur
atom in the other two thiolato ligands. The bond lengths of the
three thiolato ligands and the six Ru–S bonds in complex 8 differ
slightly from each another, while the angles of the three thiolato
ligands are markedly different from each another (Table 4).
1.7068(2) Ru2–centa
N3–Ru1–N2 78.87(9) C21–S1–Ru1 112.14(10) C21–S1–Ru2 115.84(10)
N2–Ru1–S1 83.68(7) C34–S2–Ru1 111.82(10) C34–S2–Ru2 112.22(9)
N3–Ru1–S1 84.16(7) C47–S3–Ru1 114.80(10) C47–S3–Ru2 106.64(9)
6
a Cent = centroid of h -p-cymene.
Fig. 3 X-ray crystal structures and atom numbering scheme for the cation
6
of [(h -cym)Ru(en)(RS)]PF6 (7) at 50% probability thermal ellipsoids.
Fig. 4 Two views of the X-ray crystal structure of the dinuclear cation
6
of [((h -cym)Ru)2(RS)3]PF6 (8) at 20% probability thermal ellipsoids,
together with the atom numbering scheme.
Scheme 3 Pathways for the hydrolysis of complex 7 in phosphate buffer.
At pH 7.4, complex 7 undergoes a slow hydrolysis, giving rise to a minor
amount of the aqua product 1-H2O.
Reactions of thiolato adduct 7 with H2O2 and GSH
was also investigated in 50 mM phosphate buffer containing 50%
methanol at pH 5.3 which is similar to the pH in endosomes (5–
6).44 Interestingly, this reaction afforded a di-ruthenium thiolato
complex (Scheme 2) as indicated by a single-charged ion peak
To provide a model for the H2O2-mediated oxidation and the
subsequent GSH-mediated reduction, the thiolato ruthenium
complex 7 (1 mM) was incubated with 1.4 mol equiv. of H2O2
in phosphate buffer (pH = 7.4, containing 30% MeCN) at 298 K,
and the reaction was followed by HPLC coupled with ESI-MS.
The HPLC time-course (Fig. 5a) showed a minor product (peak c)
after ca. 0.5 h, and then another two adducts (peaks h and e) after
1.5 h. Up to 7.5 h, peaks c, h and e slowly increased in intensity,
but no obvious changes in intensity were observed for all three
peaks after 12 h (Fig. 5a).
6
at m/z 1156.22 assignable to the cation [((h -cym)Ru)2(RS)3]+
(8+) (R = (C6H4)CONH(C6H5), calc. m/z 1156.32 for 8+) in
the mass spectrum of the product (Fig. S2, ESI†). This di-
ruthenium thiolato complex was also precipitated by a similar
procedure to that described above, washed with diethyl ether, and
dried in vacuum. The crude mixture was then purified by flash
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peak j), as indicated by the mass spectra of these fractions (Table
3, Fig. S8, ESI†), appeared in the HPLC chromatogram (Fig. 5b)
after 24 h of reaction.
At pH 5.3 thiolato complex 7 reacted with H2O2 much more
extensively than at 7.4. This reaction gave rise to a significant
amount of the sulfenyl-amide compound 4 (peak c in Fig. 6)
which resulted from the oxidation of the dissociated thiolate
ligand from 7, and a significant amount of the sulfinato Ru(II)
complex 10 and the thiolato Ru(III) complex 11 (peak h in
6
Fig. 6). Meanwhile, the diaqua adduct [(h -cym)Ru(RS)(H2O)2]+
(9, R = (C6H4)CONH(C6H5), peak g in Fig. 6) formed after the
dissociation of the en ligand became detectable after 12 h reaction
and increased in content until 72 h, when only 11.7% of 7 remained
intact (Fig. 6). Notably, due to its low extinction coefficient at 260
Fig. 5 HPLC time-courses for the reactions of a) thiolato complex 7
(1 mM) with 1.4 mol equiv. H2O2 in 50 mM phosphate buffer (pH 7.4) at
298 K, and b) complex 7 (1 mM), which has reacted with 1.4 mol equiv.
H2O2 in phosphate buffer (pH 7.4) at 298 K for 24 h, with 10 mol equiv.
GSH after removal of excess H2O2 using catalase. The inset h indicates that
the shoulder on peak h contains a mixture of two adducts as evidenced by
the distinct UV-Vis spectra corresponding to fractions eluting at 5.00 and
5.05 min shown in Fig. S5, ESI.† Peak assignments: a, 2; c, 4; e, 6; f, 7+; h,
10+ and 112+; i, 132+ and j, 17. Mass spectra for all fractions in this figure
are shown in Fig. S6 and S8, ESI.†
6
nm, the aqua Ru arene adduct [(h -cym)Ru(en)(H2O)]+ (1-H2O)
resulting from the dissociation of the oxidized thiolate ligand was
hardly detectable by HPLC (Fig. 6). Combining the above results,
the pathway for the reaction of complex 7 with H2O2 at pH 5.3
shown in Scheme 4 can be proposed.
The subsequent ESI-MS analysis identified fractions c and e
as containing the oxidized derivatives 4 (sulfenyl-amide) and 6
(sulfinyl-amide), respectively, as indicated by the singly-charged
ion peaks at m/z 244.01 and 228.00 (Fig. S6, ESI,† Table 3).
The inset in Fig. 5a shows peak h having a shoulder, suggesting
that this peak contains a mixture of products formed from the
oxidation of complex 7, as evidenced by the distinct UV-Vis spectra
of fractions eluting at 5.00 and 5.05 min (Fig. S7, ESI†). The mass
spectra of these fractions (Fig. S6, ESI,† Table 3) show two singly-
charged ion peaks at m/z 422.16 and 463.17, assignable to the
+
fragment ion {RuII(en)(RS(O)2)} (R = (C6H4)CONH(C6H5)) of
sulfinato Ru(II) arene complex [(h -cym)RuII(en)(RS(O)2)]+ (10)
6
6
6
+
(calc. m/z 422.16 for {[(h -cym)RuII(en)(RS(O)2)] - (h -cym)} )
Fig. 6 HPLC time-courses for the reaction of thiolato complex 7 (1 mM)
with 1.4 mol equiv H2O2 in 50 mM phosphate buffer at pH 5.3 and 298 K.
The inset h indicates that the shoulder on peak h contains a mixture of
two adducts as evidenced by the distinct UV-Vis spectra corresponding
to fractions eluting at 5.00 and 5.05 min shown in Fig. S5, ESI.† Peak
assignments: c, 4; e, 6; f, 7+; g, 9+; and h, 10+ and 112+. Mass spectra for all
fractions are shown in Fig. S9, ESI.†
6
2+
and a fragment ion {(h -cym)RuIII(RS)} of the thiolato Ru(III)
arene adduct [(h -cym)RuIII(en)(RS)]2+ (11) (calc. m/z 463.17 for
6
{[(h -cym)RuIII(en)(RS)] - en - H} ), respectively. After 72 h of
reaction with 1.4 mol equiv. H2O2, ca. 52% of complex 7 remained
intact.
6
+
To mimic the effect of ruthenium coordination on the re-
dox regulation of the catalytic thiol group in PTP1B, the
6
mono-ruthenium thiolato complex [(h -cym)Ru(en)(RS)]+ (R =
To investigate whether the coordinated thiolate ligand in 7 can
be released via direct substitution by GSH, the most abundant
thiol-containing biomolecule inside cells, reaction of complex 7
(1 mM) with 10 mol equiv. of GSH in 50 mM phosphate buffer
(pH = 7.4, containing 30% MeCN at 298 K) was also studied by
HPLC coupled with ESI-MS. As shown in Fig. 7a, during the early
stages (<0.5 h) no product was detectable by HPLC. A new HPLC
peak (a in Fig. 7a) appeared after 6.5 h of reaction and increased in
intensity until 72 h. This was identified as the free thiol-containing
compound 2 by subsequent MS analysis (Fig. S10, ESI,† Table
3). Meanwhile, after 12 h reaction, another product (peak k in
Fig. 7a) became detectable and was identified by MS (Fig. S10,
(C6H4)CONH(C6H5)) (7) was first allowed to react with 1.4 mol
equiv. H2O2 in phosphate buffer (pH 7.4) for 24 h, and then 10
mol equiv. GSH was added to the reaction mixture after removing
excess H2O2 by addition of catalase (260 units mL-1). The resulting
mixture was incubated at 298 K and monitored by HPLC assay.
As shown in Fig. 5b, the sulfenyl-amide compound 4 (peak c)
resulting from the oxidation of the dissociated thiolato ligand in
7 by H2O2 was quickly reduced by GSH back to its initial form,
i.e. the thiol compound 2 (peak a in Fig. 5b) which increased in
concentration until 72 h. However, the sulfinato Ru(II) complex
10 and the thiolato Ru(III) complex 11 (peak h) resulting from the
oxidation of complex 7, as well as the sulfinyl-amide compound 6
resulting from the oxidation of dissociated thiolate ligand in 7 by
H2O2, remain intact in the presence of a 10-fold excess of GSH.
The triply-glutathione-bridged diruthenium complex 13 (peak i)
and a mixed-disulfide compound GS-S(C6H4)CONH(C6H5) (17,
6
ESI†) as the glutathione thiolato complex [(h -cym)Ru(en)(GS)]+
(12). After 72 h of reaction, nearly 43% of the thiolato ligand in
7 was substituted by GSH (Fig. 7a), suggesting that GSH in 10-
fold excess can slowly displace the thiolato ligand from complex 7
(Scheme 5).
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Scheme 5 Pathways for reactions of complex 7 with GSH in phosphate
buffers of different pH. The formate and phosphate ligands in adduct 16
come from the HPLC solvents and the phosphate buffer, respectively.
Scheme 4 Pathways for the reaction of complex 7 with hydrogen peroxide
in phosphate buffers of pH 7.4 or 5.3.
In weakly acidic solution (pH 5.3), as for the reaction
of complex 7 with H2O2, the reaction of complex 7 with
excess GSH also became extensive and complicated. After 6
h incubation, this reaction gave rise to a glutathione thiolato-
Fig. 7 HPLC time-courses for the reactions of 7 (1 mM) with 10 mol
equiv. GSH in 50 mM phosphate buffer at a) pH 7.4 and b) pH 5.3 and at
298 K. Peak assignments: a, 2; f, 7+; k, 12+; i, 13+; l, 14+; m, 15+ and n, 16.
Mass spectra for all fractions in this figure are shown in Fig. S10 and S11,
ESI.†
6
bridged diruthenium product [((h -cym)Ru)2(GS)3]+ (13, ap-
pearing as peak i in Fig. 7b) and a hetero-thiolato-bridged
6
diruthenium complex [((h -cym)Ru)2(GS)2(RS)]+ (14, peak l in
Fig. 7b, R = (C6H4)CONH(C6H5)) as indicated by the doubly-
6
charged ion peaks at m/z 695.64 (calc. m/z 695.64 for{[((h -
Discussion
2+
6
cym)Ru)2(GS)3] + H} ) and 656.89 (calc. m/z 656.89 for {[((h -
2+
6
cym)Ru)2(GS)2(RS)] + H} ) (Fig. S11, ESI†), respectively, due
Ruthenium arene anticancer complexes [(h -arene)Ru(en)Cl]PF6
to the dissociation of the en ligand and the substitution of
the mercaptobenzanilide ligand by GSH accompanied by the
release of the mercaptobenzanilide ligand (peak a in Fig. 7b).
Six hours later, two other diruthenium products appeared and
increased in concentration for 72 h. The MS results (Fig.
S11, ESI,† Table 3) indicate that these two species are the
(arene = p-cymene (1) or biphenyl) can form strong adducts with
the thiolate groups of cysteine,21 glutathione22,45 and albumin.23
Intriguingly, Ru-coordination can induce the oxidation of thiolato
to sulfenates or sulfinates22,23,45 in the presence of molecular oxy-
gen. Ruthenium coordination stabilizes the sulfenato ligands.22,25
The free sulfenic acids are highly reactive and readily oxidized
further to sulfinic or sulfonic acids.26,27 Such oxidation of Ru-
SR bonds probably accompanied by protonation46 leads to the
ready displacement of the sulfur ligand by guanine N725 and DNA
binding.22 However, the fate of the released sulfenic acid ligands is
unknown.
Cysteine-215 in the catalytic site of the enzyme PTP1B under-
goes redox regulation involving the formation of a sulfenyl-amide
intermediate on oxidation by H2O2 and subsequent reduction
with glutathione back to an active thiol.37,40 The formation of
the sulfenyl-amide intermediate is thought to protect the thiol
from further oxidation to sulfinate or/and sulfonate which can
irreversibly deactivate it.37,40 This unusual reaction prompted us
to consider the possibility that ruthenium coordination to the
6
hetero-thiolato-bridged adducts [((h -cym)Ru)2(GS)(RS)2]+ (15)
6
and [((h -cym)Ru)2(RS)2(CHCO2)(PO3)] (16; the formate and
phosphate ligands come from the HPLC solvents and the phos-
phate buffer, respectively). On the basis of HPLC peak areas, only
ca. 13% of complex 7 remained intact after 72 h reaction with
10-fold excess GSH. Thus, the pathway for reaction of complex 7
with GSH at pH 5.3 shown in Scheme 5 can be proposed.
We also treated the fully characterized di-ruthenium thiolato
adduct 8 with H2O2 and/or GSH by following the same procedures
described above. No oxidized products with H2O2 or ligand-
exchanged adducts with GSH were detectable after a 72 h reaction
(data not shown), suggesting that this diruthenium thiolato
complex is inert towards both GSH and H2O2.
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catalytic cysteine of PTP1B might stabilize the sulfenate/sulfinate
intermediate, prevent reactivation of the catalytic cysteine and
thereby inhibit the enzyme. Indeed, in a preliminary study,
ruthenium arene complex 1 was found to inhibit PTP1B with
an IC50 of 19 mM.41 In the present work, therefore, we have
studied reactions of 2-mercaptobenzanilide (2), a chemical model
for Cys215 of PTP1B, with ruthenium anticancer complex 1,
including the formation and redox behavior of the resulting
thiolato products.
Although compound 2 is well recognized as a chemical model
of PTP1B,38,39 its X-ray crystal structure has not been previously
reported. The crystallographic data obtained herein (Fig. 1a, Table
2) show that the distance between the sulfur atom (S1) of the thiol
and the nitrogen atom (N1) of the amide in compound 2 is similar
to that between the sulfur atom in Cys215 and the nitrogen atom of
the amide of Ser216 in PTP1B.37,40,43 This is evidence that the ortho-
substituted benzene scaffold of 2 indeed provides a good model
for the proximity of the amide and the cysteine thiol group at the
active site of PTP1B,39 although the aromatic thiol in 2 may have a
different reactivity towards ruthenium arene complexes compared
to the aliphatic thiol47 of the cysteine residue in the catalytic site
of PTP1B.
between the NH of the en ligand and the carbonyl oxygen, which
may account for the higher stability of the ruthenium adduct
with compound 2 compared to GSH. The thiolato adduct with 2
(complex 7) is soluble and stable in aqueous solution (pH 7.4);
only 6% of 7 hydrolyzes after 60 h incubation at 298 K (Fig. S5,
ESI†). However, at pH 5.3, nearly 15% of 7 hydrolyzed after 60 h
incubation. Interestingly, the hydrolysis involved the displacement
of the en ligand instead of the thiolate ligand, affording the
di-aqua adduct 9 (Scheme 3, Fig. S5, ESI†). Sulfur-containing
ligands such as cysteine and methionine are known to exhibit
high trans effects in Pd(II),48 Pt(II)49 and Ru(II)21 coordination
chemistry, labilizing metal-N bonds in the trans position. On the
other hand, at low pH the ethylenediamine (en) ligand is readily
protonated, making it a better leaving group, accounting for the
dissociation of en from complex 7 in weakly acidic solutions. This
trans effect imposed by S coordination to Ru was also observed in
the reaction of complex 1 with the thiol compound 2, and complex
7 with H2O2 or with GSH at pH 5.3. At pH 5.3, the majority of 2 is
present in the RSH form (pKa 5.7). When RSH attacks ruthenium
in 1, therefore, the proton can be transferred to the outgoing
en,46 leading to the formation of the final triply-thiolate-bridged
6
diruthenium product [((h -cym)Ru)2(RS)3]+. For the reaction of
Oxidation of compound 2 with H2O2 was fast, almost complete
within 0.5 h. However, during this time, the main product was the
disulfide compound 3 instead of the sulfenyl-amide compound 4
which formed after 0.5 h of reaction. This was accompanied by the
formation of the sulfonic acid 5 and the sulfinyl-amide compound
6 (Fig. 2a, Scheme 1). The sulfenyl-amide product 4 was separated
from the reaction mixture and crystallographically characterized.
Compound 4 is usually considered to be a good model for studying
the GSH-mediated reactivation of the sulfenyl-amide intermediate
of PTP1B. The X-ray structure of 4 (Fig. 1b) shows the presence of
the sulfenyl-amide (S1–N1) bond, with a bond length (1.7186(16)
complex 7 with H2O2 or GSH, as a consequence of the labilization
of the en ligand in 7 by this trans effect, a significant amount of the
6
diaqua adduct [(h -cym)Ru(RS)(H2O)2]+, the thiolato-bridged
6
diruthenium product [((h -cym)Ru)2(GS)3]+ and the hetero-
6
thiolato-bridged diruthenium complex [((h -cym)Ru)2(GS)2(RS)]+
(R
=
(C6H4)CONH(C6H5))
were
formed
(Fig.
6
and 7b).
To mimic H2O2-mediated oxidation and the subsequent GSH-
mediated reduction of the oxidized derivative of the catalytic thiol
group coordinated to ruthenium of PTP1B, the redox behavior of
the thiolato complex 7 was investigated and compared with that
of the model compound 2. During the early stages (<0.5 h) of
the reaction of 7 with H2O2 at pH 7.4, only a minor amount of
the sulfenyl-amide compound 4 was observed. This is the oxidized
derivative of the dissociated thiolato ligand which most probably
resulted from the hydrolysis of complex 7 (vide supra). At a later
stage (>7.5 h), the sulfinyl-amide compound 6 (2-phenyl-1,2-
benzisothiazol-3(2H)-one 1-oxide, Scheme 1) which also resulted
from the oxidation of the dissociated thiolato ligand in 7 became
detectable, accompanied by the formation of the Ru(II) sulfinato
˚
A) similar to that of the sulfenyl-amide bond in oxidized PTP1B
40
˚
(1.7 A). Unlike the puckered five-membered ring containing
the sulfenyl-amide bond in the crowded catalytic site of PTP1B,
the five-membered sulfenyl-amide ring in compound 4 is almost
coplanar with the benzene ring (Fig. 1b). However, this structural
difference seemed to have little effect on the reduction of this
sulfenyl-amide compound by GSH.39 In the presence of a 10-fold
excess of GSH, compound 4 was completely reduced back to its
initial thiol form, i.e. compound 2, within 5 min, directly analogous
to the reactivation of the sulfenyl-amide intermediate during the
redox regulation of PTP1B.37,40
6
complex [(h -cym)RuII(en)(RS(O)2)]+ (10) and a possible Ru(III)
6
thiolato adduct [(h -cym)RuIII(en)(RS)]2+ (11). The Ru(II) sulfinato
Having fully characterized the model compound 2 as well as
its oxidation by H2O2 and subsequent reduction of the oxidized
sulfenyl-amide product with GSH, we then studied reactions
between compound 2 and the ruthenium complex 1. At a 1 : 1
molar ratio, the reaction reached equilibrium within 4 h, affording
complex 10 resulted from the oxidation of the thiolate ligand in 7,
and the Ru(III) thiolato complex 11 may be formed by oxidation
of the Ru(II) in 7 (Fig. S6, ESI,† Table 3). Although there seems
to be little precedent for such an oxidation, there is a report of
6
the oxidation of the Ru(II) in the organoruthenium complex [(h -
6
the mono-ruthenium thiolato complex [(h -cym)Ru(en)(RS)]+ (7+,
C6Me6)Ru(tpdt)] (tpdt = 3-thiapentane-1,5-dithiolate) on reaction
with Ph2MCl2 (M = Ge or Sn).50 However, more than 50% of
complex 7 remained intact after 72 h of reaction. At pH 5.3, due
to the increase of the oxidation capacity of H2O2, more sulfinato
and sulfenato Ru(II) complexes formed. The protonation of the
sulfenate ligand in 10 in turn labilizes the Ru–S(OH) bond,46
probably accounting for the formation of more sulfenyl-amide
compound 4 (Fig. 6). However, there was still ca. 12% of 7
detected intact after reaction with excess H2O2 for 72 h. These
data imply that the coordinated thiolate ligand in 7 is much less
R = (C6H4)CONH(C6H5)). Interestingly, in the presence of a 10-
fold excess of GSH, this mono-ruthenium thiolato complex was
still the main product, indicating that complex 1 has a higher
affinity for the thiol in 2 than for GSH. This might imply that
PTP1B would form stronger adducts than GSH with ruthenium
complex 1 in cells.
The crystallographic data (Table 4, Fig. 3) show that the
coordination of Ru with the sulfur atom in 2 allows the formation
˚
of a strong H-bond (NH ◊ ◊ ◊ OC length: 2.02 A, Fig. S4, ESI†)
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reactive towards H2O2 than the free thiol in 2 which was completely
oxidized by 1.4 mol equiv. H2O2 within 0.5 h.
towards both H2O2 and GSH. Such reactions may be implicated
in the mechanism of action of this class of ruthenium arene
anticancer complexes. With appropriate chemical modification
to reduce their cytotoxicity, organometallic ruthenium complexes
may have potential for treatment of diabetes as a family of novel
PTP1B inhibitors.
When the mono-ruthenium thiolato complex 7 was first reacted
with H2O2 at pH 7.4 for 24 h and then with an excess of GSH
(after removal of excess H2O2 by catalase), the small amount of
sulfenyl-amide compound 4 resulting from the oxidation of the
dissociated thiolate group from 7 was quickly reduced back to
its initial form, compound 2, accompanied by the formation of
small amounts of triply-glutathione-bridged diruthenium complex
13 and mixed disulfide compound GS-S(C6H4)CONH(C6H5)
(17). The diruthenium complex 13 formed through the direct
substitution of the thiolato ligand in 7 by GSH, leading to the
further release of the thiolato ligand in 7 (Fig. 5b) such that only
ca. 10% of thiolato complex 7 was present in its initial form after
72 h reaction (Fig. 7b). These data imply that under physiological
conditions, intracellular GSH may activate the thiolate in 7 by
displacing the thiolato ligand, though the ligand exchange is very
slow (Fig. 5).
Experimental
Materials and methods
6
[(h -p-cym)Ru(en)Cl]PF6 (1) was synthesized as described
previously.11 AgPF6 and trifluoroacetic acid (TFA) were purchased
from Alfa, acetonitrile (HPLC grade) from Merck, glutathione
(GSH, reduced), hydrogen peroxide, 2-mercaptobenzoic acid, ani-
line, sodium dihydrogen phosphate and NH4PF6 from Sinopharm
Chemical Reagent Beijing Co., Ltd, p-nitrophenyl phosphate (p-
NPP) from Sigma. The recombinant protein tyrosine phosphatase
1B (PTP1B) was a gift from Dr Maolin Guo at University of
Massachusetts Dartmouth.
At pH 5.3, the dissociation of the en ligand from 7 allowed
formation of more glutathione-bridged diruthenium adduct 13
and mixed-thiolate-bridged adducts 14 and 15. However, the
amount of the free thiol compound 2 released from 7 did not
increase compared with that formed at pH 7.4 (Fig. 7).
Synthesis
2-Mercaptobenzanilide (2). Model compound 2 was synthe-
sized following a reported procedure.42 A suspension of o-
mercaptobenzoic acid and aniline at a molar ratio of 1 : 1 in
chlorobenzene was heated to 343 K on an oil bath under argon,
and small of amount of PC13 was gradually added over half an
hour. After the addition was complete, the mixture was heated
at reflux with stirring under Ar for 3 h. The resulting reaction
mixture was filtered while hot, and the solvent evaporated to yield
a white solid which was purified by flash column chromatography
(10 : 1 EtOAc/hexane). Recrystallization from methanol gave
white needle crystals suitable for X-ray diffraction studies. Yield:
0.87 g (23.7%). MS: m/z 230.02 [M+].
Conclusions
The mercaptobenzanilide compound 2 and its oxidized derivative
the sulfenyl-amide compound 4 are models for species which form
in the active site of the enzyme PTP1B. Compounds 2 and 4 are
characterized here for the first time by X-ray crystallography. The
X-ray structures clearly indicate that the ortho-substituted benzene
scaffold of 2 indeed provides a good model for the proximity
of the amide and the cysteine thiol group at the active site of
PTP1B. This is the key structural feature of the catalytic site
leading to the formation of the sulfenyl-amide intermediate 4
which in turn protects the catalytic thiol from further oxidization
into sulfinate, sulfonate, and can be reactivated by GSH-reduction
back to thiol form. Obviously, a simple aliphatic thiol compound
cannot duplicate this characteristic redox behavior.
2-Phenyl-1,2-benzisothiazol-3(2H)-one (4). Compound 4 was
prepared according to a reported procedure.51 To a well stirred
mixture of benzamide (25 mg, 0.0975 mmol) and TFA (0.0225 mL,
0.29 mmol) in 3 mL of CH2Cl2, a solution of phenyliodine(III)bis-
(trifluoroacetate) (PIFA) (62.5 mg, 0.145 mmol) in 4.5 mL of
CH2Cl2 was added. The resulting mixture was stirred at 273 K
for 1 h, then filtered, and the solvent removed by evaporation.
The crude mixture was purified by flash column chromatography
(using dichloromethane) to afford the product as a white solid,
which was further purified by recrystallization from ethanol to
give colorless block crystals suitable for X-ray diffraction studies.
Yield: 8 mg (36%). MS: m/z 228.00 [M+].
6
Reaction of the ruthenium arene anticancer complex [(h -p-
cym)Ru(en)Cl]PF6 (1) with 2 at pH 7.4 produced only the thiolato
mono-ruthenium arene complex [(h -cymene)Ru(en)(RS)]+ (7, R =
6
(C6H4)CONH(C6H5)) even in the presence of a 10-fold excess of
glutathione, implying that the ortho-substituted benzene enhances
the reactivity of the thiol in 2 due to the formation of the strong
H-bond between the NH of the en ligand and the carbonyl oxygen.
In contrast, at pH 5.3 the reaction of 1 with 2 gave rise to only a
triply-S-bridged thiolato adduct [((h -cymene)Ru)2(RS)3]+ (8) due
6
to the labilization of the en chelating ligand in 1.
HPLC-ESI-MS
Further studies indicated that the coordination of ruthenium to
the thiol sulfur of the model compound 2 largely retarded the oxi-
dation (deactivation) by H2O2 of this sulfur to the sulfenyl-amide
intermediate 4. As a consequence, the reduction (reactivation) of
the sulfenyl-amide intermediates by glutathione was significantly
inhibited. The thiolate ligand –S(C6H4)CONH(C6H5) in 7 is only
slowly displaced by excess GSH and reactivated towards H2O2
oxidation. The activity of the thiolate in 7 towards H2O2 is much
lower than that of the thiol in 2. In addition the thiolate ligands
in the triply-S-bridged diruthenium complex 8 are relatively inert
Positive-ion electrospray ionisation mass spectra were obtained
on a Micromass Q-TOF mass spectrometer (Waters) coupled to
an Agilent 1200 HPLC system using a XDB-C18 column (4.6 ¥
150 mm, 80 A, 5 mm, Agilent) with a flow rate of 1.0 mL min-1 and a
˚
splitting ratio of 1 : 10. Mobile phases were A, water (purified using
a MilliQ Reagent Water System) containing 0.1% TFA, and B,
acetonitrile containing 0.1% TFA. Gradient elution was achieved
as follows (solvent B): 10% within 0–2 min; 10%–42% over 2–3
min; 42% within 3–8 min; 42%–68% over 8–9 min; 68% within
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9–15 min; 68%–90% over 15–16 min; 90% within 16–19 min and
reset to 10% at 20 min. The spray voltage was 3.0 kV, and the
cone voltage 35 V. The desolvation temperature was 413 K and
source temperature 373 K. Nitrogen was used as both cone gas
and desolvation gas with a flow rate of 40 L h-1 and 400 L h-1,
respectively. The collision energy was set up to 9.9 V. All spectra
were acquired in the range of 100–2000 m/z. Data were collected
and analyzed on a MassLynx (version 4.0).
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X-ray crystallography
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Single-crystal X-ray diffraction studies for 2-mercaptobenzanilide
(2) and 2-phenyl-1,2-benzisothiazol-3(2H)-one (4) were carried
out using graphite monochromated Mo-Ka radiation (l =
˚
0.71073 A) on a Rigaku Saturn 724 CCD area detector. The
6
X-ray diffraction data of [(h -cym)Ru(en)(RS)]PF6 (7, R =
(C6H4)CONH(C6H5)) were obtained on a Rigaku RA-Micro7HF
˚
diffractometer with Confocal Mo-Ka radiation (l = 0.71073 A)
6
and [((h -cym)Ru)2(RS)3]PF6 (8) on a Rigaku R-AXIS RAPID
diffractometer with graphite monochromated Mo-Ka radiation
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˚
(l = 0.71073 A) on a Rigaku raxis Rapid IP Area Detector. All
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data were collected at 173 K, and structure solution and refinement
were performed using SHELXL-97.
2001, 1396.
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PTP1B inhibition assay
Following a similar procedure to that described in the literuture,52
the PTP1B activity was determined at 298 K in a reaction mixture
(pH 7.2) containing 10 mM pNPP as substrate, 20 mM MOPS
and 2 mM DTT. Inhibition assays were performed in the same
buffer on a 96-well plate. Firstly, 10 mL of ruthenium complex 1 at
various concentrations was mixed with 70 mL of enzyme in MOPS
(20 mM) buffer (the final concentration of PTP1B was 250 nM),
and the resulting mixture was incubated at 298 K for 30 min.
Then 5 mL DTT was added and the mixture was incubated at 298
K for another 30 min to restore enzyme activity, and finally 10
mL of pNPP (0.02 M) substrate was added. After incubation for
30 min at 298 K, the enzymatic reactions were stopped by addition
of 5 mL of 1 M NaOH, and the absorption was determined at
405 nm. Nonenzymatic hydrolysis of the substrate was corrected
by measuring the control samples without addition of enzyme.
IC50 values were generated by fitting the concentration-dependent
inhibition curves using the program Origin (version 7.5). All data
points were obtained in sextuplicate.
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Acknowledgements
We thank the NSFC (grant Nos: 90713020, 20975103, 21005081
and 21020102039), the 973 Program of MOST (2007CB935601),
the Chinese Academy of Sciences (Hundred Talent Program),
and the European Research Council (grant no. 247450) for
support, and Dr Maolin Guo of the University of Massachusetts
Dartmouth for PTP1B.
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