Inorganic Biochemistry

Journal of Inorganic Biochemistry 99 (2005) 690697 www.elsevier.com/locate/jinorgbio

JOURNAL OF

Oxidation of Zn(N2S2) complexes to disulfonates: relevance to zinc-nger oxidation under oxidative stress
Rodolphe Alves de Sousa a, Erwan Galardon a, Mathieu Rat a, Michel Giorgi b, Isabelle Artaud a,*
a

Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite Rene Descartes, ` 45 rue des Sts Peres, 75270 Paris Cedex 06, France b Service Commun de Cristallochimie, Faculte des Sciences et Techniques, Universite Paul Cezanne (Aix-Marseille 3), avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France Received 10 September 2004; received in revised form 12 November 2004; accepted 19 November 2004

Abstract A series of Zn(N2S2) complexes has been prepared, and characterized. They have dierent nitrogen donors such as, either two amidates, two amines, two imines or one amidate and one imine. A bis-amidato dithiolato complex has been structurally characterized by single crystal X-ray diraction analysis, and exhibits a distorted tetrahedral structure. Oxidation of all these complexes with dioxirane or anhydrous H2O2 results in the formation of a unique product, the disulfonate species. Most often, zinc was found to be released during the course of the oxidation. The bis-imine/bis-sulfonate species is the only one to retain zinc. This complex was crystallized with two pyridine molecules. Its crystal structure reveals a distorted octahedral environment around the zinc cation. 2004 Elsevier Inc. All rights reserved.
Keywords: Zinc-nger; Thiolate; Oxidation; Sulfonate; Oxidative stress

1. Introduction Zinc is following iron the most important metal in biology. The best-known role associated to its Lewis acidity is its function in the active site of hydrolytic enzymes or oxidoreductases such as alcohol dehydrogenase [1]. In this catalytic sites zinc binds a water molecule along to three protein residues such as histidine, glutamate, aspartate and/or cysteine promoting a zinc core of nitrogen, oxygen and sulfur donors. Beside this catalytic motif, zinc is also found in structural motif assisting in the folding of proteins. The characteristics of these zinc sites are that there are no bound water molecules and four protein ligands. Zinc-nger motifs are a

Corresponding author. Tel.: +33 1 42862189; fax: +33 1 42868387. E-mail address: isabelle.artaud@univ-paris5.fr (I. Artaud).

particular case of structural zinc sites which are found in a large number of eukaryotic transcription factors [2]. They are involved in the formation of a polypeptide loop which recognizes and binds to specic DNA sequences. In this case, zinc is typically coordinated to two histidines and two cysteines. The presence of cysteines makes the catalytic [3] and structural [4] zinc sites, as well as ZnS clusters [5] found in metallothioneins, sensitive to reactive oxygen species produced in the situation of oxidative stress. This cysteine oxidation results in the disruption of zinc thiolate bonds and the release of zinc ion [47]. When the sulfhydryl groups are oxidized beyond the disulde state, zinc decoordination is not reversible with reductants, resulting in enzyme inactivation and disruption of gene transcription. The nal oxidation state of cysteine has not been determined and is supposed to be the sulnic or the sulfonic acid which cannot be converted back to thiol [4]. While a

0162-0134/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2004.11.019

R. Alves de Sousa et al. / Journal of Inorganic Biochemistry 99 (2005) 690697

691

large body of synthetic [8,9] and theoretical [9] work has been undertaken these last years to understand the high nucleophilicity of zinc-thiolate in biological systems, such a work aimed at understanding the reactivity of zinc-ngers toward strong oxidants was not conducted so far. To address the question of the nal oxidation state of cysteines under strong oxidizing conditions, we have prepared several Zn(II) complexes of tetradentate N2S2 ligands and submitted them to strong oxidants such as H2O2 or dioxirane. In these complexes zinc is coordinated to two thiolates and two nitrogen donors such as two deprotonated amides, two amines, two imines or one imine and one amidate. In every case thiolates are oxidized to sulfonates, most often with subsequent release of zinc from the tetradentate ligands. The bis-imine/disulfonate species is the sole product to retain zinc.

2. Results and discussion The ligands or proligands used in this study are shown in Scheme 1. Three of them have been previously synthesised, namely N,N 0 -(2-thioacetyl-isobu tyryl)-o-phenylenediamine (1a) [10], N,N 0 -(3-mercapto3-methylbutyryl)-o-phenylenediamine (1b) [11], and 2,2 0 -dimethyl-2,3,2 0 ,3 0 -tetrahydro-[2,2 0 ]-bibenzothiazolyl (2) [12,13]. The third compound (3) was prepared in this work and its synthesis is detailed in supplementary data. Ligands (1a), (1b) and (3) were metallated in dimethylformamide (DMF) with ZnCl2. Two equivalents of NaOMe were rst added to deprotonate the thiolates for (1b) or to open the thiazolidinyl ring for (2) and (3), and/or deprotect the thiolate(s) for (1a) and (3). These experiments were performed under strictly anaer-

obic conditions due to the high sensitivity of thiolates toward dioxygen. This uncontrolled oxidation results in a mixture of oxidized species including disuldes, and sulfur oxidized species which have not been further characterized. After metallation, one or two additional base equivalents were added to deprotonate the amides. The Zn(N2S2) complexes, (4a), (4b) and (7b), shown in Scheme 1, were isolated as the tetraethylammonium salts after exchange of Na+ into Et4N+ with Et4NCl. The bis-imine dithiolate complex (5), and the bis-amine dithiolate complex (6) were obtained according to the procedures described by Corbin and Work [13]. Treatment of the thiazolidine (2) with Zn(OAc)2 aorded (5) which could be further reduced with NaBH4 to yield (6). All the complexes were characterized by 1H NMR, IR, and by elemental analysis for (4a) and (4b). Complex (7) was more fragile, and a complete elimination of residual salts either from (7a) (sodium salt) or (7b) (Et4N salt) was very dicult. Thus, the exact masses of (7a) and (7b) were determined by high resolution mass spectrometry (HRMS). As there were no examples of Zn(N2S2) complexes in which the zinc cation was bound to two carboxamido nitrogens and two thiolates, crystals of (4b) suitable for X-ray analysis were grown in a mixture of acetonitrile-diethylether. The crystal structure of (4b) shown in Fig. 1, reveals as for complex (6) [14] a pseudo-tetrahedral environment around the Zn(II) ion. The ZnN and ZnS bond distances (see legend of Fig. 1) compare well in both complexes. However, the bit angles NZnS are greater in (4b) than in (6) as a result of the geometric constraints imposed in a six-membered N,S chelate relative to a ve-membered one. All the zinc-thiolate complexes were stable toward dioxygen. They were submitted to oxidation with

O NH HN (CH2)n SR RS

H N O (CH2)n S 2

H N O S HN S 3 HN O S

1a n = 0, R = COCH 3 1b n = 1, R = H N 2O N N (CH2)n Zn S S O (CH2)n 2 Et 4N+ HN NH Zn S S 6

Scheme 1.

N Zn S S 5 N Zn S S N O

4a n = 0 4b n = 1

7a X = Na 7b X = Et4N

692

R. Alves de Sousa et al. / Journal of Inorganic Biochemistry 99 (2005) 690697

Fig. 1. Thermal ellipsoid plot (50% probability level) of the anion of (4b); H atoms, counter-ions and water molecules are omitted for clarity. Only one cationic monomer is represented. Selected bond distances (A): Zn1N1, 2.035(5); Zn1N2, 2.050(5); Zn1S2, 2.292(2); Zn1S1, 2.3210(19); O1C7, 1.268(8); O2C12, 1.255(8); N1C7, 1.334(8); N2C12, 1.324(9). Selected bond angles (): N1Zn1N2, 81.8(2); N1Zn1S2, 129.02(17); N2Zn1S2, 103.47(19); N1Zn1S1, 97.75(17); N2Zn1S1, 126.35(18); S1Zn1S2, 116.40(8); C7C8C9, 116.5(6); C12C13C14, 115.0(7).

complexes (8a) and (8b) display mNH and mCO vibrations around 3200 and 1670 cm1 as expected for free amide. Using H2O2 instead of dioxirane leads to identical results except that the complete conversion of thiolates to sulfonates requires ten equivalents of this oxidant. With a lower amount of oxidant, residual starting products were always observed along with the disulfonate species. With the amidate-imine dithiolate zinc complex (7b), dierent oxidation products were isolated whether H2O2 or dioxirane were used as oxidants. H2O2 oxidation results in the formation of the corresponding free amide-imine disulfonate salt (11) (Scheme 3) whereas dioxirane aords the aldehyde (12) due to the possible cleavage of the imine bond of 11 with the residual water retained in the acetone solution of dioxirane. Oxidation of the neutral bis-amine dithiolate complex (6) with dioxirane yields the zinc disulfonate salt (10a) (data not shown). Zinc was then removed upon treatment with two equivalents of NaOH promoting the formation of the sodium disulfonate salt (10b) which was characterized by elemental analysis. Decoordination of Zn2+ was evidenced by IR spectroscopy. While the mNH vibration of (6) appears as an intense and sharp band located at 3154 cm1, this band is broadened and shifted to higher frequencies centered at 3365 and

dimethydioxirane (DMD) or H2O2 used as the anhydrous H2O2/urea complex. Oxidation of complexes (4a) and (4b) with six equivalents of dioxirane leads to the formation of the corresponding tetraethylammonium disulfonate salts (8a) and (8b) shown in Scheme 2. Interestingly, addition of a substoichiometric amount of dioxirane to (4a) or (4b) did not lead to the intermediate formation of S- or O-bonded sulnate or free disulnate salt, but only to a mixture of starting product and free disulfonate salt. The release of Zn(II) following the oxidation of (4a) and (4b) was suggested by IR spectroscopy and conrmed by mass spectrometry and elemental analysis of (8a) and (8b). In contrast to complexes (4a) and (4b), the IR spectra of which show carbonyl stretching frequencies mCO around 1540 cm1 typical of coordinated carboxamido nitrogens, those of

H O HN DMD 12 7b SO3 - Et 4N+ O

urea, H2 O2

N HN SO3 -

2 Et4N+

SO3 11

Scheme 3.

O NH HN (CH2)n SO3 -

O (CH2)n

SO3 -

N Zn O O S O O S O O

HN SO3 -

NH SO3 -

2 Et4N+ 8a n = 0 8b n = 1 9

Zn2+ or 2 Na+ 10a, Zn2+ 10b, 2Na+

Scheme 2.

R. Alves de Sousa et al. / Journal of Inorganic Biochemistry 99 (2005) 690697

693

3370 cm1 for the zinc (10a) and sodium (10b) disulfonate salts, respectively, as expected for free amine. This shift shows that the zinc center only interacts as a counter-cation like Na+ with the sulfonate groups. Very recently, a similar result has been described for the H2O2 oxidation of a diamino dithiolato zinc derivative of N,N 0 -bis-2-methyl-mercaptopropyl-N,N 0 -dimethylethylenediamine [15]. Finally for the last compound (5), in contrast to the previous complexes for which thiolate oxidation is associated to the cleavage of the zinc sulfur bonds and release of zinc, dioxirane oxidation of this imine-thiolate complex gives the corresponding bis-imine bis-sulfonate complex (9) (Scheme 2). Compound (9) has been characterized by elemental analysis, as well as 1H NMR and IR spectroscopies. Its IR spectrum exhibits a mCN vibration at 1651 cm1, a stretching frequency value very close to that observed for the starting bis-imine dithiolate complex (5) (mCN = 1643 cm1). Crystals suitable for X-ray analysis were grown by diusion of diethylether in an acetonitrile solution of (9) with two equivalents of pyridine. While (9) was only sparingly soluble in acetonitrile, it readily solubilized after adding two pyridine equivalents. The molecular structure shown in Fig. 2 reveals that the coordination around the zinc is octahedral. Selected bond angles and distances are listed in the legend of Fig. 2. The ligand occupies four coordination sites with the two imine nitrogens in cis position and the two sulfonate oxygens in trans position. Two pyridine

molecules complete the octahedral coordination set. Among the few metal sulfonate complexes reported in the literature [1622], all are six-coordinate, with two additional water molecules for tetradentate ligands. Zinc coordination is limited to pyridine-2-sulfonates [20,21] or tris-(3-tert-butylpyrazolyl)-methanesulfonate [22] which contain strong pyridine or pyrazolyl nitrogen donors and form stable ve- or six-membered N,O chelate ring, respectively. Compound (9) contains such two ve-membered N,O chelate rings with two strong imine nitrogen donors. Formation of disulfonate complexes by oxidation of Zn(N2S2) precursors can be explained by a three-step reaction involving rst the oxidation of thiolates to S-bonded sulnates, then isomerization of S-bonded to O-bonded sulnates and further oxidation to O-bonded sulfonates. The absence of any detection of S-bonded sulnate intermediates suggests that these species are unstable and rearrange very rapidly into O-bonded sulfinates. The formation of free sulfonates from (4), (6) and (7) suggests that neither carboxamidate nor amine are strong enough nitrogen donors to retain zinc during this interconversion. In that regard, all attempts to insert zinc into the tetradentate bis-amide bis-sulfonate ligands (8a) or (8b) or bis-amine bis-sulfonate one (10b) have failed whether the zinc salt was ZnCl2 or the more reactive Zn(CH3CN)n(ClO4)2. To be tightly bound to a metal centre, amides have to be deprotonated. This deprotonation proceeds readily after binding of the

Fig. 2. Thermal ellipsoid plot (50% probability level) of complex (9) associated to two pyridine molecules. H atoms are omitted for clarity. Selected bond distances (A): Zn1N1, 2.1833(16); Zn1N2, 2.1926(16); ZnN3, 2.0924(17); Zn1N4, 2.1017(18); Zn1O3, 2.1812(15); ZnO6, 2.1774(15); S1 O2, 1.4355(18); S1O1, 1.4444(18); S1O3, 1.4700(16); S2O4, 1.4431(17); S2O5, 1.4433(16); S2O6, 1.4690(15). Selected bond angles (): N1Zn1 N2, 74.05(6); N4ZnN2, 93.39(6); N4Zn1N1, 157.56(7); N3Zn1N1, 94.77(7); O6Zn1O3, 170.82(6); N4Zn1O3, 84.67(6); N3Zn1O3, 90.4.(7); N4Zn1O6, 89.46(7); N3Zn1O6, 83.98(6); N3Zn1N4, 102.58(7).

694

R. Alves de Sousa et al. / Journal of Inorganic Biochemistry 99 (2005) 690697

amide under its conjugated iminol form which causes the amides pKa to drop. Amine nitrogens are also not strong ligands and for these two nitrogen donors, insertion of zinc in N2X2 chelates rst requires the binding of zinc to two strong X donors. In that regard thiolates have a high anity for zinc, but sulfonates are obviously poor oxygen donors for zinc and do not allow the insertion of this cation in the corresponding tetradentate N2(SO3)2 chelates.

Single Reection Horizontal ATR unit (Zirconium Selenium crystal) (attenuated total reection method: ATR method). Electrospray ionisation (ESI) mass spectra were performed on a Thermo Finnigan LCD Advantage spectrometer and fast atom bombardment (FAB) ones were recorded at ENS in Paris. Elemental analyses were carried out by the microanalysis service at Paris VI University or at Gif sur Yvette CNRS. All experiments were run under an argon atmosphere in schlenk tubes. 4.2. N,N 0 -(2-thioacetyl-isobutyryl)-o-phenylenediamine (1a), N,N 0 -(3-mercapto-3-methylbutyryl)-ophenylenediamine (1b) Compounds (1a) [10] and (1b) [11] were prepared as previously described. 4.3. 2,2 0 -dimethyl-2,3,2 0 ,3 0 -tetrahydro-[2,2 0 ]bibenzothiazolyl (2) Compound (2) as well as the corresponding bis-imine and bis-amine zinc complexes (5) and (6), respectively, were synthesised according to Corbin and Works procedures [13]. 4.4. Complex (4a) and general procedure for the synthesis of the dithiolate zinc complexes Two equivalent of 1 M NaOMe in MeOH (1.5 mL) were added to a DMF solution (5 mL) of the proligand (1a) (300 mg, 0.76 mmoL), then the reaction mixture was stirred at 50 C for 1 h 30 min. After cooling to room temperature, the solution was transferred over solid ZnCl2 (103 mg, 0.76 mmoL) and heated for 2 h at 50 C. Then, two other base equivalents were added. One hour later the solution was transferred over solid Et4NCl (250 mg, 1.5 mmoL) and stirred at room temperature for 1 h. The solvents were then removed in vacuo and the oily residue dissolved in CH3CN (20 mL). The insoluble salts were ltered o. After removal of half of the solvent, the solution was poured into anhydrous diethylether (150 mL) aording a white powder which was collected by ltration and dried in vacuo. Yield: 330 mg, 65%. (4a) C30H56N4O2S2Zn 2H2O (670.34): Calc. C, 53.75; H, 9.02; N, 8.36. Found: C, 53.72; H, 9.02; N, 8.16%. IR (neat): m = 1534 (mCO) cm1. 1H NMR (250 MHz, [D6] DMSO): d = 1.20 (t, 3 J = 7.5, 24H, CH3, Et4N), 1.48 (s, 12H, CH3), 3.24 (q, 3J = 7.5, 16H, CH2, Et4N), 6.51 (dd, 3J = 6.5, 4 J = 3.5, 2H, Har), 8.36 (dd, 3J = 6.5, 4J = 3.5, 2H, Har). 4.5. Complex (4b) The formation of the thiolates needs no heating and was carried out at room temperature with two equivalents of base. Yield from 100 mg of (1b) (0.3 mmoL):

3. Conclusion In this work, we have prepared several Zn(N2S2) complexes related to zinc-nger which contain in addition to two thiolates two amines, two carboxamidates, two imines or one imine and one carboxamidate. All are oxidized with H2O2 or dioxirane to disulfonate derivatives. Apart from the bis-imine bis-thiolate ligand for which zinc coordination is retained during thiolate to sulfonate oxidation, for the other compounds, this oxidation is associated to disruption of ZnS bonds and subsequent zinc release. This result might be relevant to zinc-nger oxidation under oxidative stress conditions. In zinc-ngers, zinc is bound to two histidine/cysteine pairs which are located at a distance of several amino-acid residues and thereby, eventhough histidine nitrogens are strong donors, there is no strong N,N 0 chelate eect to prevent zinc decoordination during the conversion of S-bonded to O-bonded sulnates. In a paper published a few years ago [23], it has been shown that the cysteines of a peptide corresponding to a transcription factor were oxidized with H2O2 in the presence of Zn(II) into thiosulnates which are, as sulfonates, reactive sulfur oxidized species supposed to be generated under conditions of oxidative stress [24,25] Finally, we describe a very simple method to cleanly oxidize dithiolates into disulfonates under mild conditions. As a unique requirement, the thiolates have to belong to a N2S2 tetradentate ligand suitable for the formation of Zn(N2S2) complex.

4. Experimental section 4.1. General All solvents and chemicals were purchased from SDS and Aldrich, respectively. Dimethylformamide (DMF) and CH3CN were dried using standard procedures and stored over 4 A molecular sieves under an argon atmosphere. 1H NMR spectra were recorded on a Bruker ARX-250 spectrometer and chemical shifts were reported in ppm downeld from trimethylsilane (TMS). IR spectra were obtained with a PerkinElmer Spectrum One FT-IR spectrometer equipped with a MIRaclee

R. Alves de Sousa et al. / Journal of Inorganic Biochemistry 99 (2005) 690697

695

95 mg, 45%. Anal. Calc. for C32H60N4O2S2Zn H2O (698.4): C, 55.03; H, 9.24; N, 8.02. Found: C, 55.40; H, 9.07; N, 8.04%. IR (KBr pellet) (m cm1): 1545 (mCO). 1H NMR in [D6]DMSO (d ppm): 1.20 (t, 3J = 7, 5, 24H, CH3, Et4N), 1.22 (s, 12H, CH3), 2.15 (s, 4H, CH2), 3.25 (q, 3J = 7.5, 16H, CH2, Et4N), 6.52 (dd, 3 J = 6.5, 4J = 3.5, 2H, Har), 8.39 (dd, 3J = 6.5, 4J = 3.5, 2H, Har). 4.6. Complex (7a) The same procedure was followed, but only one base equivalent was required after transferring the solution over solid ZnCl2 since there was only one amide function to deprotonate. The sodium salt (7a) was isolated by precipitation from an CH3CN solution into Et2O. Yield from (IV) (110 mg, 0.31 mmoL): 84 mg, 68% (based on a molecular weight of 572.42 for (7a) corresponding to the formula C15H19N2NaOS2Zn DMF NaCl 2.5H2O determined by elemental analysis. IR (neat) (m cm1): 1660 (mCN), 1524 (mCO). 1H NMR in [D6]DMSO (d ppm): 1.19 (s, 6H, CH3), 1.48 (s, 6H, CH3), 3.37 (s, CH2), 6.95 (t, 3J = 7.5, 1H, Har), 7.32 (t, 3 J = 7.5, 1H, Har), 7.48 (d, 3J = 7.5, 1H, Har), 7.79 (d, 3 J = 7.5, 1H, Har), 8.24 (s, 1H, CH). FAB-MS: m/ z = 371 [M-Na+]. HRMS: Calc. C15H19N2OS2Zn [MNa+]: m/z = 371.0230; found 371.0239. 4.7. Complex (7b) The tetraethylammonium salt (7b) was puried by several precipitations from an acetone solution into Et2O. Yield from (3) (100 mg, 0.28 mmoL): 87 mg, 61%. C23H39N3OS2Zn (503.08). IR (neat) (m cm1): 1636 (mCN), 1549 (mCO). 1H NMR in CD3CN) (d ppm): 1.22 (t, 3J = 7.2, 12H, CH3, Et4N), 1.5 (s, 6H, CH3), 3.18 (q, 3J = 7.2, 8H, CH2, Et4N), 3.38 (s, 2H, CH2), 6.95 (t, 3J = 7.4, 1H, Har), 7.32 (t, 3J = 7.4, 1H, Har), 7.39 (d, 3J = 7.4, 1H, Har), 7.75 (d, 3J = 7.4, 1H, Har), 8.14 (s, 1H, CH). FAB-MS: m/z = 371 [M-Et4N+]. HRMS: Calc. C15H19N2OS2Zn [M-Et4N+]: m/ z = 371.0230; found 371.0239. 4.8. Typical procedure for oxidation of the dithiolate zinc complexes with dimethyldioxirane Stock solutions of dimethyldioxirane (DMD) in acetone were prepared and titrated by oxidation of methylphenysulde to the corresponding sulfoxide as previously reported [26]. Six equivalents of DMD in acetone were added to a DMF solution of the zinc complex (100 mg in 5 mL DMF) in three portions at 0 C and stirred at this temperature for 1 h. Then, the reaction mixture was allowed to warm to room temperature and stirred for 1 h. Then the solution was concentrated in vacuo, ltered to eliminate insoluble salts and the

disulfonate species isolated by precipitation in anhydrous Et2O at 0 C. 4.9. 2-[2-(2-methyl-2-sulfo-propylamino)-phenylcarbamoyl]-propane-2-sulfonic acid bis-tetraethylammonium salt (8a) Yield from (4a) (105 mg, 0.16 mmoL): 83 mg, 75%. Anal. Calc. for C30H58N4O8S2 0.5H2O (675.01): C, 53.31; H, 8.80; N, 8.29. Found: C, 53.43; H, 8.50; N, 8.08%. IR (neat) (m cm1): 3250 (mNH), 1677 (mCO), 1220 and 1173 (mas SO3), 1021 msSO3 . 1H NMR in [D6]DMSO (d ppm): 1.21 (t, 3J = 7, 24H, CH3, Et4N), 1.47 (s, 12H, CH3), 3.25 (q, 3J = 7, 16H, CH2, Et4N), 7.12 (dd, 3J = 5.5, 4J = 4, 2H, Har), 7.74 (dd, 3J = 5.5, 4 J = 4, 2H, Har), 9.90 (s, 2H, NH). 4.10. 2-methyl-1-[2-(3-methyl-3-sulfo-butyrylamino)phenylcarbamoyl]-propane-2-sulfonic acid bis-tetraethylammonium salt (8b) Yield from (4b) (99 mg, 0.15 mmoL): 60 mg, 58%. Anal. Calc. for C32H62N4O8S2 3H2O (716) : C, 51.31; H, 9.15; N, 7.18. Found: C, 51.87; H, 9.45; N, 7.72%. IR (KBr pellet) (m cm1): 3270 (mNH), 1670 (mCO), 1210 and 1180 mSO3 , 1035 msSO3 . 1H NMR in [D6]DMSO (d ppm): 1.21 (t, 3J = 7, 24H, CH3, Et4N), 1.29 (s, 12H, CH3), 2.84 (s, 4H, CH2), 3.25 (q, 3J = 7, 16H, CH2, Et4N), 7.15 (dd, 3J = 5.5, 4J = 4, 2H, Har), 7.56 (dd, 3J = 5.5, 4J = 4, 2H, Har), 9.45 (s, 2H, NH). 4.11. Complex (9) Yield from (5) (163 mg, 0.45 mmoL): 200 mg, 97%. Anal. Calc. for C16H14N2O6S2Zn H2O 0.3DMF (499.76): C, 40.61; H, 3.65; N, 6.44. Found: C, 40.71; H, 3.61; N, 6.26%. IR (neat) (m cm1): 1651 (mCN), 1274, 1261, 1171, 1158, 1140, 1083, 1015 and 995 mSO3 . 1H NMR in [D6]DMSO (d ppm): 2.56 (s, 6H, CH3), 7.18 (d, 3J = 7.5, 1H, Har), 7.47 (t, 3J = 7.5, 1H, Har), 7.67 (t, 3J = 7.5, 1H, Har), 7.84 (d, 3J = 7.5, 1H, Har). 4.12. 2,2 0 -(butan-2,3-diylidene-diamino)di(benzenesulfonic acid sodium salt) (10b) After oxidation of (6) as above-described and evaporation of the solvents to dryness, Zn2+ was exchanged with 2 Na+ by adding two equivalents of 2 N aqueous NaOH to a DMF solution of the zinc disulfonate salts. Zn(OH)2 was eliminated by ltration and a yellow powder was isolated by precipitation in anhydrous Et2O. Then the solid was dissolved in water, and insoluble products were eliminated by ltration. After lyophilisation, compound (10b) was obtained as a yellow powder. Yield from (6) (250 mg, 0.68 mmoL): 283 mg, 94%.

696

R. Alves de Sousa et al. / Journal of Inorganic Biochemistry 99 (2005) 690697

Anal. Calc. for C16H18N2NaO6S2 0.4DMF (473.67): C, 43.61; H, 4.43; N, 7.10. Found: C, 43.71; H, 4.73; N, 6.98%. Ir (neat) (m cm1):3370 (mNH), 1175 and 1135 masSO3 , 1018 msSO3 . 1H NMR in methanol d4 (d ppm) (the product is a mixture of two diastereoisomers. Only the signals of the major isomer could be completely assigned and are described): 1.26 (d, 3J = 7.5, 6H, CH3), 3.93 (m, 2H, CH), 6.60 (t, 3J = 7.5, 1H, Har), 6.80 (d, 3 J = 7.5, 1H, Har), 7.24 (t, 3J = 7.5, 1H, Har), 7.38 (d, 3 J = 7.5, 1H, Har). 4.13. 2(2-Formyl-phenylcarbamoyl)-propane-2-sulfonic acid tetraethylammonium salt (12) Oxidation of (7b) with DMD yielded (12) in quantitative yield. C20H35N2O4S (399.58). IR (neat) (m cm1): 3260 (mNH), 1674 (mCO), 1215 and 1172 masSO3 , 1026 msSO3 . 1H NMR in CD3CN) (d ppm): 1.22 (t, 3 J = 7.2, 12H, CH3, Et4N), 1.56 (s, 6H, CH3), 3.18 (q, 3 J = 7.2, 8H, CH2, Et4N), 7.25 (t, 3J = 7.4, 1H, Har), 7.61 (t, 3J = 7.4, 1H, Har), 7.79 (d, 3J = 7.4, 1H, Har), 8.47 (d, 3J = 7.4, 1H, Har), 10 (s, 1H, CHO), 11,45 (s, 1H, NH). FAB-MS: m/z = 270 [M-Et4N+]. HRMS C11H12NO5S [M-Et4N+]: Calc. 270.0436; found 270.0429. 4.14. 2-Methyl-1-{[2-(2-methyl-2-sulfo-propionylamino) -benzylidene]-amino}-propane-2-sulfonic acid bis-tetratethylammonium salt (11) One millilitre of a 0.5 M DMF solution of H2O2/urea was added to 1 mL of a DMF solution of (7b) (25 mg, 0.05 mmoL). After stirring for one day, the solvents were removed in vacuo. The residue was dissolved in CH3CN (1 mL) and transferred into anhydrous Et2O at 0 C. The resulting yellow precipate was collected by ltration and dried in vacuo to give (11). Yield from (7b) (15 mg, 0.037 mmoL): 16 mg, 64%. C31H60N4O7S2 (664.97). IR (neat) (m cm1): 3359 (mNH), 1662 (mCO), 1586 (mCN), 1180 masSO3 , 1033 msSO3 . 1H NMR in CD3CN) (d ppm): 1.22 (t, 3J = 7.5, 24H, CH3, Et4N), 1.58 (s, 6H, CH3), 3.18 (q, 3J = 7.5, 16H, CH2, Et4N), 3.87 (s, 2H, CH2), 7.12 (t, 3J = 7.5, 1H, Har), 7.38 (t, 3 J = 7.5, 1H, Har), 7.52 (d, 3J = 7.5, 1H, Har), 8.42 (s, 1H, CH), 8.58 (d, 3J = 7.5, 1H, Har), 12.01 (s, 1H, NH). FAB-MS: m/z = 534.2 [M-Et4N+]. HRMS: Calc. C23H40N3O7S2 [M-Et4N+]: m/z = 534.2308; found 534.2297. 4.15. X-ray crystallography Diraction data were measured on a Bruker-Nonius Kappa CCD diractometer. The structures were solved by direct methods and rened using the program SHELXL97 [27]. Crystal data for (4b): Mw = 1360.69, triclinic, colorless crystal (0.3 0.3 0.2 mm3), a =

11.8980(5) A, b = 16.604(1) A, c = 20.304(1) A, a = 97.007(3), b = 90.104(4), c = 108.475(4), V = 1, 3772.5(3) A3, space group P  Z = 2, q = 1.198 g cm3, l(Mo Ka) = 7.96 cm1, 26,053 reections measured at 173 K in the 026.36 h range, 13,796 unique, 797 parameters rened on F2 using 13,796 reections to nal indices R[F2 > 4rF2] = 0.1181, wRw 1r2 F 2 0:1841P 2 o 3:5878P 0:3062 where P F 2 2F 2 =3. The last o c residual fourier positive and negative peaks were equal to 2.295 and 0.803 respectively: strong residual positive fourier peaks were located at about 1 A from the zinc cations. Several empirical absorption corrections were tested but did not solve the problem. All the crystals obtained were twins, including the measured one, however the reciprocal lattices could be partially separated during the integration process, which can explain the renement problems and the moderate quality of the resolution. The complex crystallizes as two monomers with four tetraethylammonium cations (two of them being disordered and rened on two sites of occupancies equal to 0.5) and two water molecules. Crystal data for (9 + 2 py): Mw = 617.98, monoclinic, colorless crystal (0.3 0.3 0.3 mm3), a = 11.7590(3) A, b = 22.9320(8) A, c = , b = 115.398(2), V = 2719.01(17) A3, space 11.1620(5) A group P21/c, Z = 4, q = 1.51 g cm3, l(Mo Ka) = 11.05 cm1, 5284 unique reections measured at 223 K in the 0.8926.31 h range, 352 parameters rened on F2 using 5284 reections to nal indices 2 R[F2 > 4rF2] = 0.0315, wRw 1=r2 F 2 0:0279P o 2 2 2:239P 0:0755 where P F o 2F c =3. The last residual fourier positive and negative peaks were equal to 0.246 and 0.304 respectively. CCDC 237073 and CCDC 249789 contain the supplementary crystallographic data for (4b) and (9 + 2py) respectively. These data are available free of charge at www.ccdc.cam. ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 IEZ, UK; Fax: (internat.) + 44-1223-336-033; E-mail: deposit@ccdc.cam.ac.uk.

Appendix A. Supplementary data Synthetic route as well as characteristics of compound (3). Complete crystallographic data for N2S2Zn complex (4b) and N2(SO3)2(Py)2Zn complex (9 + 2Py) (CIF). Supplementary data associated with this article can be found, in the online version at doi:10.1016/ j.jinorgbio.2004.11.019.

References
[1] W.N. Lipscomb, N. Strater, Chem. Rev. 26 (1996) 23752433. [2] E.H. Cox, G.L. McLendon, Curr. Opin. Chem. Biol. 4 (2000) 162165.

R. Alves de Sousa et al. / Journal of Inorganic Biochemistry 99 (2005) 690697 [3] J.P. Crow, J.S. Beckman, J.M. McCord, Biochemistry 34 (1995) 35443552. [4] M. Casadevall, B. Sarkar, J. Inorg. Biochem. 71 (1998) 147152. [5] W. Maret, B. Vallee, Proc. Natl. Acad. Sci. USA 95 (1998) 3478 3482. [6] R.A. Quesada, R.W. Byrnes, S.O. Krezoski, D.H. Petering, Arch. Biochem. Biophys. 334 (1996) 242250. [7] J.F. Collet, J.C. DSouza, U. Jakob, J. Biol. Chem. 278 (2003) 4532545332. [8] G. Parkin, Chem. Rev. 104 (2004) 699767. [9] D.C. Fox, A.T. Fiedler, H.L. Halfen, T.C. Brunold, J.A. Halfen, J. Am. Chem. Soc. 126 (2004) 76277638. [10] S. Chatel, A.S. Chauvin, J.P. Tuchagues, P. Leduc, E. Bill, J.C. Chottard, D. Mansuy, I. Artaud, Inorg. Chim. Acta 336 (2002) 1928. [11] M. Rat, R. Alves de Sousa, J. Vaissermann, P. Leduc, D. Mansuy, I. Artaud, J. Inorg. Biochem. 84 (2001) 207213. [12] E. Bayer, E. Breitmaier, Chem. Ber. 101 (1968) 15791593. [13] J.L. Corbin, D.E. Work, Can. J. Chem. 52 (1974) 10541058. [14] W. Tsaglalidis, D. Rehder, J. Biol. Inorg. Chem. 1 (1996) 507 514. [15] C.A. Grapperhaus, C.S. Mullins, P.M. Kozlowski, M.S. Mashuta, Inorg. Chem. 43 (2004) 28592866.

697

[16] V.E. Kaasjager, E. Bowman, S. Gorter, J. Reedijk, C.A. Grapperhaus, J.H. Reibenspies, J.J. Smee, M.Y. Darensbourg, A. Derecskei-Kovacs, L.M. Thomson, Inorg. Chem. 41 (2002) 1837 1844. [17] T.M. Cocker, R.E. Bachman, Chem. Commun. (1999) 975976. [18] R.K. Henderson, E. Buwman, A.L. Spek, J. Reedijk, Inorg. Chem. 36 (1997) 46164617. [19] M. Murata, M. Kojima, A. Hioki, M. Miyagawa, M. Hirotsu, K. Nakajima, M. Kita, S. Kashino, Y. Yoshikawa, Coord. Chem. Rev. 174 (1998) 109131. [20] T.S. Lobana, I. Kinoshita, K. Kimura, T. Nisshioka, D. Shiomi, K. Isobe, Eur. J. Inorg. Chem. (2004) 356367. [21] K. Kimura, T. Kimura, I. Kinoshita, N. Nakashima, K.I. Kitano, T. Nishioka, K. Isobe, Chem. Commun. (1999) 497498. [22] W. Klaui, M. Berghahn, W. Frank, G.J. Reiss, T. Schonherr, G. Rheinwald, H. Lang, Eur. J. Inorg. Chem. (2003) 20592070. [23] Y. Xu, D.E. Wilcox, J. Am. Chem. Soc. 120 (1998) 73757376. [24] G.I. Gilles, K.M. Tasker, C. Jacob, Free Rad. Biol. Med. 31 (2001) 12791283. [25] G. Gilles, C. Jacob, Biol. Chem. 383 (2002) 375388. [26] W. Adam, J. Bialas, L. Hadjiarapojlou, Chem. Ber. (1991) 2377. [27] G.M. Sheldrick, SHELXL-97, Program for the renement of crystal structures, University of Gottingen, Germany, 1997.