Interactions of organotin(IV) halides with reduced glutathione in aqueous solution

Interactions of Organotin(IV) Halides with Reduced Glutathione in Aqueous Solution

E. Capolongo, A. M. Giuliani, M. Giomini, and U. Russo

FC. Department of Pharmacology, University of Padova, Padova.-AMG. Department of Inorganic Chemistry, University of Palemw, . Palermo.--MG. Department of Chemistry, University of Rome “La Sapienza, ” Rome.-UR. Department of Inorganic, Metallorganic, and Analytical Chemistry, University of Padova, Padova, Italy

ABSTRACT

Glutathione (GSH) is a compound extremely common among many living organisms in which it plays a fundamental role in the processes of detoxification. Also, organotin(IV) derivatives are more and more commonly used in technological processes or as antitumor drugs. So it seemed interesting to investigate the possible interactions between GSH and organotin compounds in water. Particularly, it has been studied because of its role in the organic radicals linked to the tin center on the stoichiometry and the structure of the adducts. Information’was obtained following the reaction between Me,SnCl,, (n = 1 to 3) and GSH by Miissbauer and NMR spectroscopies on the assumption that changes of the characteristic parameters such as the quadrupole splitting and the chemical shift and coupling constants, respectively, are closely related to modifications in the coordination of the tin atom.

In the case of addition of GSH to a solution of CH,SnCl, in HEPES, complex equilibria are evidenced, while in the case of Me,SnCl, and of Me,SnCl on1 a single species is present. Mijssbauer effect data together with point charge calculations and Y H NMR results point to a 21 and a 1:l complex, respectively; GSH is coordinated by the Sthiolic atom and the coordination geometry around the tin center appears to be trigonal bipyramidal.

INTRODUCTION

, Reduced glutathione (GSH) is a simple molecule extremely common in all living organisms. In fact, it is present in cells of both vegetables and animals, mainly in the liver, kidneys, e-rocytes, central nervous system, and skin [l]. Even if it is readily oxidized in the presence of oxygen [2], inside the living cells it is mainly

Address reprint requests and correspondence to: Professor U. Russo, Department of Inorganic, Metallorganic, and Analytical Chemistry, University of Padova, Via Loredan 4, I-35131 Padova, Italy.

Joumal of Inorganic Biochemhy, 49,275-293 (1993) 275 0 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/93/$6.00

,

276 F. Capolongo et al.

present in the reduced form (about 95%) [31 at very high concentration, between 0.1 and 10 mM [ll. GSH can act as a reducing agent oxidizing itself to the dimeric S-bridged form or as a ligand with three binding groups, the thiolic sulfur and the amidic and acidic oxygen atoms. Thanks to its wide distribution and reactivity, GSH is involved in a large variety of physiological and metabolic processes, as for instance, protection of cell membranes and proteic macro- molecules from peroxides, free radicals, and xenobiotic metal compounds, and the assemblage and degradation of proteins (41. In this way, GSH forms stable complexes with a large number of metals, mainly toxic at least at high concentration, which may either enter the organisms because they are present in the environment as pollutants or are purposely introduced as drugs.

On the other hand, tin derivatives are used in many technological processes and are present in a large number of synthetic materials [51; moreover they form numerous classes of inorganic and organometallic compounds under study as potential antitumor drugs [6, 71. In this way, their interaction with living organisms and, finally, with man will surely increase within the next years giving rise to pollution and toxicological problems [5]. The solution of these problems will require a deep knowledge of the mechanisms of cell detoxification, a process that probably involves a reaction with GSH. These facts prompted us to study the interaction of organotin(IV) derivatives with GSH in aqueous solution at physiological pH, to investigate the possible formation of stable complexes, and, if this is the case, to determine their stoichiometry and coordination geometry.

To the best of our knowledge, the only compounds reported in the literature are those formed by reaction of GSH with tributyl and triphenyl tin derivatives [g-lo]. At least in the solid state these compounds present a 2:l metal-to-ligand ratio with coordination geometries around the tin centers which seem to be different for the two tin compounds. Miissbauer spectroscopy shows the presence of two different tin sites in each compound with different coordination geometries. While much work has been performed on the interactions of organotin(IV) derivatives with amino acids, DNA, and related compounds in solution [ll-151, only a potentiometric titration has been reported on the Me,SnCl/GSH system [HI. The coordination of GSH appears to take place almost exclusively through the Sthiol atom to form a 1:l complex. It seemed worth to study the effects of the metal-to-ligand ratio, of the environment, and of the acidity on the formation of adducts between mono-, di-, and tri-methyl tin and reduced GSH.

EXPERIMENTAL

Methyltin chlorides were purchased from Alfa Ventron and purified by sublimation; reduced and oxidized glutathione and N-ethylmaleimide (NEM) were obtained from Janssen, and used without any further purification; HEPES and TRIS were used as received from Carlo Erba.

The solutions used in the Miissbauer experiments were 200 mM in the buffer and 10e2 M in the tin salt; the appropriate amount of GSH was added and the pH quickly readjusted to 7.4 by means of NaOH. When different pH values were required, HCl or NaOH were added to the solutions of the tin derivative and GSH in bidistilled water to obtain the right value. The final volume in every case was 2.0 ml.

INTERACTIONS OF ORGANOTIN WITH GLUTATHION 277

Mossbauer spectra were recorded on a Laben Master 4000 instrument at 80 K using a Harwell cryostat. The Ca ‘I9 SnO, source (15 mCi, New England Nuclear) at room temperature was moved at constant acceleration and with a triangular wave form. Suitable computer programs have been employed in the fitting procedure of the experimental spectra to Lorentxian line-shapes. The solution samples in Teflon holders, containing 2.0 ml of the tin solution, were quickly frozen in liquid nitrogen before mounting in the cryostat for the measurements. The MWbauer spectrum recorded for one of the frozen solutions is reported in Figure 1 as an example of the quality.

The quadrupole splitting values have been calculated under the assumptions of the point charge model using the partial quadrupole values reported in the literature 1121. They were compared with the experimental values to obtain structural information on the coordination geometry around the tin center. The agreement between the calculated and the experimental AE, values is accepted when the difference is within f0.4 mm/set.

The NMR spectra were recorded either on an AMX-600 or on an AM-400 Bruker spectrometer, operating at 600 and 400 MI-Ix on protons, respectively. The following stock solutions were prepared for the measurements:

i. A TRW2HCl 200 miU solution in 2H20. The exchangeable hydrogens of the compound had previously been substituted with deuterium, by repeated lyophilizations from 2H20 solutions. The pH of the buffer was 7.41. The solution was deaerated and kept under nitrogen.

ii. A reduced glutathione 80 mM solution in deuterated 200 mM TRIS buffer. The pH was adjusted to 7.10 by addition of small aliquots of NaO’H. The solution was kept under nitrogen and at ca. 4°C.

iii. 20 mM solutions of Me,SnCl,_, (n = 2,3) in a!eutemted 200 mM TRIS buffer. The pH was adjusted at a value around 6.3-6.5 by addition of NaO’H. This pH was chosen because at the tin concentration used, the hydrolysis products which form at a higher pH 1161 may give rise to precipitates.

1.000

al UO.996

II-J lTJo.sa-4

:

I.‘.’ ’ ’ ’ ’ ’ ’ ’ 1 ’ ’ ’ ’ ’ ’

Source Velocity (mm/set) FIGURE 1. The ‘19Sn Miksbauer spectrum of a frozen solution in HEPES of the GSH/Me,SnCI, system in 2.21 ratio recorded at 80.0 K.

278 F. Cupolongo et rtl

iv. 20 mM HEPES or ,TRIS solutions at pH ca. 7.3 in 2H20. I-IEPES and TRIS were first lyophilized several times from ‘H,O to substitute the exchangeable hydrogens with deuterium and then deaerated and kept under nitrogen.

v. 80 mM solution of GSH in deutemted 20 mh4 HEPES or TRIS solution. The pH was ca. 3.3. The solutions were kept under nitrogen at ca. 4°C.

vi 20 mM, solutions of MeJnCI,, (n = 1,2,3) @I 20 mM deutemted HEPES or TN.9 buffer solution. The -pH, acidity, were not adjusted by addition of NaO* H.

All pH values are uncorrected for the isotope effect. The sampels were prepared immediately before the measurements by mixing the appropriate amounts of solutions i, ii; and iii (or iv, v, and ‘vi), to obtain the desired tin-to-glutathione ratios and sealed in the NMR tube under nitrogen to avoid oxidation of the GSH; The pH of .each sample was checked and adjusted at ca 7.3 by addition of NaO*H. The solvent deuterium resonance was’ used for field-frequency lock; all chemical shifts reported are relative to TMS.

The TRIS buffer was used in preference of HEPES because it is more favorable for NMR studies exhibiting only one resonance, which was suppressed by presaturation.

RESULTS AND DISCUSSION

The study of the interactions in solution is based on i19Sn MSssbauer and ‘H NMR spectroscopies, whose parameters are sensitive to the coordination geometry of the tin atom. SMoreover, within the limits of the point charge approxima- tion and the hypothesis of ideal geometries around the tin center, it is bpossible, making use of the MGssbauer parameters, to propose a structure for each compound eventually formed in the reactions.

The Aqueous Model Systdm: Species Present in Aqueous Solutionb of MeSnClj and GSH

The dissolution of MeSnCl., in water produces a strongly acidic solution, pH = 1.72, as a consequence of the loss of the chloride ion, the coordination of water molecules, and their subsequent hydrolysis. This is clearly shown in the Miissbauer spectrum of the solution; the hyperfine parameters (see Table 1) are in the range typical for solid state [AlkSn(OlOHl, 1171 while point charge calculations indicate [MeSn(H,O),(OH),l+, for which the calculated AE, is 2.11 mm/s, as the most probable compound present in the solution. On increasing the pH to physiological values by addition of NaOH, a small decrease of both the isomer shift and the quadrupole splitting indicates the loss of protons from the coordinated water molecules. According to point charge calculations, one proton is lost per complex molecule with the consequent formation of MeSn(H,OXOH), for which a AE, value of 1.58 mm/s is calculated in good agreement with the experimental one of 1.75 mm/s (see Table 1). If the pH is lixed to 7.4 by means of HEPES, one molecule of the buffer substitutes the water in the coordination sphere of the tin and the point charge model indicates that the aminic nitrogen is involved in the coordination. In fact, the calculated AE, is now 1.92 mm/s and compares favorably with the experimental value of 1.83 mm/s.

The addition of GSH to this solution causes evident effects in the Miissbauer spectrum: a second species is formed in an amount that is related to the


TABLE 1. Miissbauer Effect Parameters for the GSH/MeSnCl, System in Aqueous HEPES Buffer at pH 7.4 Recorded at 80.0 K

GSH/MeSnCl, Sa AEQ PI P.2 Area Ratio (mm/xc) (mm/& (mm/see) (mm/set) (A%)

0 5.0

8.0

12.0

23.0

0, pH = 1.72’ 0, pH = 7.45’

0.51 1.83 0.94 0.83 100 1.04 1.69 l.OOb l.OOb 31 0.45 1.82 0.77b 0.77b 69 1.28 1.81 1.02b 1.02b 41 0.48 1.88 0.79b 0.79b 59 1.25 1.84 1.06b l&P 48 0.46 1.87 0.82b 0.82b 52 1.33 1.79 0.83b 0.83b 48 0.51 1.85 0.81b 0.81b 52 0.82 2.00 0.80 0.82 100 0.51 1.75 0.94 0.86 100

’ Referred to room temperature CaSnO,. b Parameters constrained to a fixed ratio. ’ Reactions in absence of HEPES.

ligand-to-metal ratio R (see Fig. 2). The amount of this second component compared to the starting material increases until R is about 12. From this point on, the two tin compounds are present in the solution approximately in the same amount. While the hyperfine parameters of the first species are virtually identical with those of the starting material, the new component presents values that are rather difficult to rationalize. In fact, while the quadrupole splitting is the same of the HEPES-tin derivative, and this seems to indicate that large variations in the coordination geometry have not taken place, the isomer shift is

~‘-_~-s’,dj.....“.“,.‘....““““““’,......” ..i. .b’ 10.00 15.00 20.00 25.00

R PLGURB 2. Graphic correlation between the relative amount of the two species formed in the GSH/MeSnCI, system obtained from the Miissbauer spectra as a function of the metal-to-ligand ratios R.

280 F. Capohgo et al.

AE C.IC =-I.91 AE _,==+ 1.62

MC4

OH\ (./ql-Q

Nmnid

AE &=+2_ 14 AE _,&=c 1.92

FIGURE 3. Some possible tin coordination spheres in the HEPES buffer solution for the 1:l GSH/MeSnCl, system.

now much higher (see Table 1). Thus a new ligand, less electronegative, has entered the coordination sphere substituting probably a hydroxide ion, but point charge calculations are not able to distinguish among the many different possibilities (see Fig. 3).

This system has been studied by ’ H NMR only in 20 mM deuterated HEPES buffer solution (tin was 10 or 5 mM): the pH of the stock 20 mM solution of the organotin in 20 mm HEPES was 1.51 and its value increased gradually to 2.70 when the samples at increasing GSH concentrations (R values increasing to 8) were prepared according to the procedures described.

Even in these acidic conditions more than one species exists in solution: simple solvated cations have been evidenced at pH < 2 and polycations in the pH region 2 to 4 by polarographic measurements 08) while Miissbauer data indicate the presence of [MeSn(H,O),(OH),l+.

In the absence of glutathione several proton resonances are observed for the methyltin moiety (see Fig. 4a1, confirming the presence of several hydrolyzed species in solution, some of which seem to be polymeric (broad lines). Upon the gradual addition of GSH, a new resonance grows at lower fields (S = 1.19 ppm), while the others progressively decrease in intensity. At GSH to tin ratio 8:l only this new resonance is observed together with a weak, narrow one at 0.89 ppm, that is probably due to a monomeric species (see Fig. 4b).

The *J&r, ’ H) value for the resonance at 1.19 ppm (the chemical shift remains virtually the same at all R values) is constant and equal to 81 f 1 Hz. This corresponds, according to Holmes and Kaesz [19], to an apparent s-char- acter of 38% in the tin atomic orbital involved in the Sn-C bond, suggesting a considerable rehybridization of the tin atomic orbitals.

The Aqueous Model System: Species Present in Aqueous Solutions of Me,SnCl, and GSH

The behavior of Me,SnCl, in water as a function of pH and in HEPES solution has already been described in detail [12]. The addition of GSH to a HEPES

INTERACTIONS OF ORGANOTIN WITH GLUTATI-IION 281

(b)

1 I I I

1.25 1.00 0.75 0.50 6 (ppm)

FIGURE 4. Partial ‘H NMR spectra of the GSH/MeSnCI, system in 20 mM deuterated HEPES solution (B, = 14.1 T, T = 300 K); (a) GSH/MeSnCl, = O/l; (b) GSH/MeSnCl, = 8/l.

solution of Me,SnCl, gives rise to a continuous decrease of the quadrupole splitting and a simultaneous increase of the isomer shift (see Table 2). The plot of these values against the ligand-to-metal ratio R (see Fig. 5) is linear up to a value of two, after which both the quadrupole splitting and the isomer shift remain virtually constant. This may be interpreted as good evidence for the formation of a well defined complex with a 2:l stoichiometry, in agreement with the findings with related ligands [12-14, 201. So, for example, the reaction with cysteine produces a 2:l adduct with an isomer shift of 1.28 mm/s and a quadrupole splitting of 2.32 mm/s: for this compound a trigonal bipyramidal geometry (tbp) and a C,SnS,N environment for the tin atom have been proposed on the base of Miissbauer data [12, 131. The similarity of these hyperhne parameters with those obtained in the reaction with GSH supports the hypothesis that the two compounds present a similar coordination geometry around the tin atom. The three equatorial positions should be occupied by the

282 F. Capohgo et al.

TABLE 2. Miissbauer Effect Parametesr for the GSH/Me,SnCl, System in Aqueous HEPES Buffer at pH 7.4 Recorded at 80.0 K

‘GSH/Me,SnCI, lYa Ratio (mm/set)

AEQ (mm/set)

r, (mm/=)

r, (mm/set)

0 1.09 3.13 0.87 0.89 0.5 1.10 2.97 1.06 0.99 1.0 1.17 2.75 1.17 0.98 1.4 1.28 2.47 1.10 0.91 1.8 1.33 2.36 1.05 0.99 2.0 1.31 2.31 0.97 0.97 2.2 1.31 2.21 1.05 1.02 2.6 1.37 2.24 1.01 0.95 3.0 1.33 2.25 0.87 0.91 4.0 1.38 2.27 0.86 0.86 6.0 1.35 2.26 0.80 0.81 8.0 1.33 2.25 0.96 0.93

a Referred to room temperature CaSnO,.

two methyl and an Sthiol groups, while a second Sthiol and an Namino should occupy the two axial positions. In this way a good agreement between the experimental and the quadrupole splitting calculated by the point charge model is achieved (see Fig. 6). It is clear, anyway, that AE, does not change whether the amino nitrogen comes from a chelating GSH or from HEPES. The involvement of the thiolic group of GSH in the bonds to the tin atom is confirmed by the absence of any reaction of Me,SnCl, with the oxidized form of GSH. The thiolic group is no longer available for bonding and the amidic or the acidic groups do not seem to be able to enter the coordination sphere of the tin atom as there is no variation in the Mossbauer parameters with respect to those of

3.50 3

3.00

G

Q 2.50 h I

I - - *

2.00

1.50 :

Lo I. l * 1

1.00

FIGURE 5. Miissbauer hyperflne parameters for the GSH/Me,SnCl, system in 200 mM HEPE!3 as a function of the different ligand-to-metal ratios R.


N”

b\ y14”i-”

SUW

M8 89”- NW

OH

AEcalc=+2.73

N”

&\ &l-S,

41 Me

OH

AE _=-2.78

N” Ml \

an-su* k4l FIGURE 6. Some possible tin

SUIbl coordination spheres for the 2/l

hEa,==- 1.92 GSH/Me,SnCl, system at pH = 7.4.

the starting HEPES solution. Similarly, N-ethylmaleimide, known to block sulfurization reactions [21], prevents the formation of any complex of GSH with Me,SnCl,, at least under our experimental conditions. In this way the formation of an adduct between GSH and Me,SnCl, seems to be clearly established, together with a 2:l stoichiometry and a tbp structure.

In order to obtain further information about the details of the reaction between GSH and Me,SnCl,, a Miissbauer study of the reaction in water at increasing pH values has been carried out at the constant ligand-to-metal molar ratio of 2:l. For pH values lower than 2.0, no reaction takes place as the quadrupole splitting value remains identical with that of the pure aqueous solution due to the hydrated Me,Sn*+ ion (see Table 3). In fact in strongly acidic solutrons GSH is fully protonated and positively charged 1231. Electro- static repulsion may be efficient enough to prevent any interaction bewteen the two ions. As the pH is slowly increased (see Fig. 71, the Miissbauer spectra of

TABLE 3. Mijssbauer Effect Parameters for the GSH/Me,SnCl, System in 2:l Ratio in Water as a Function of pH Recorded at 80.0 K

Sa AEQ i

PH (mm/xc) (mm/xc)

1.31 1.52 4.15 2.00 1.51 4.16 2.55 1.33 3.31 3.50 1.32 2.95 4.50 1.32 2.62 5.66 1.33 2.36 6.66 1.35 2.19 8.02 1.34 2.25 9.37 1.30 2.26

10.20 1.19 2.40 12.12 0.91 2.30 ‘,’

’ Referred to room temperature CaSnO,.

r, (mm/se4

0.90 0.88 0.98 1.14 1.21 0.97 0.85 0.87 0.90 0.99 0.87

crrur$ecl

0.90 0.87 1.01 1.15 1.09 0.85 0.89 0.87 0.91 0.80 0.82


Lo 1.00 --------\ 1 0.00 m

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

PH

FIGURE 7. Mijssbauer hyperfine parameters for the GSH/Me,SnCI, system in a 2/l ratio in aqueous solution as a function of pH.

the solutions change, approaching the one obtained in HEPES solution at a pH value of about 6.0. At this pH, GSH loses the two carboxylic protons and becomes negatively charged. In this form the interaction with the still positively charged organotin ion leads to the formation of the bis adduct. As the hyperiine parameters are virtually identical with those obtained in the HEPES solution, the tin environment may be considered to be the same in the two solutions. The complexation takes place when the thiolic sulfur is still protonated and the aminic nitrogen belongs to a chelating GSH rather than to a HEPES molecule. By further increasing the pH, that is in a basic water solution, AE, seems to slightly increase while S decreases, probably as a consequence of a hydrolysis reaction of the complex.

NMR measurements on samples with GSH to organotin ratios from 0:l to 8:1, at 10 or 5 mM tin concentrations, in 200 mM deuterated TRIS buffer, have been performed. A typical proton spectrum is reported in Figure 8, while chemical shifts and proton-tin coupling constraints for the Me,Sn(IV) moiety are collected in Table 4 and a plot of S vs the GSH to tin molar ratio, R, is shown in Figure 9. The behavior is strictly comparable to that found for frozen solutions of Me,Sn(IV)-GSH in HEPES buffer by l19Sn Miissbauer spectroscopy. Thus, the NMR data support the proposed metal/ligand 1:2 stoichiometry proposed for the complex.

Attempts to fit the S vs R curve by the MINSQ program [23] for nonlinear curve fitting assuming a model with a 1:2 complex were unsuccessful. A good fitting is instead obtained with a two-complex model Cl:1 and 1:2), although standard deviations are exceedingly high; moreover the model requires also, as an input datum, the chemical shift of the 1:2 complex, which is not available. One can only safely say that the K1,, value is of the order of magnitude of lo2 and K,:, of 104. From the 2J(Sn,‘H) coupling constants obtained from the


-.;o 4.700----- -iYr-x-- z- I I I I 4.25 3.75 3.50 2.50 2.25 2.00 1.75 1.50 1.25 1.00 .75

WY

FIGURE 8. ‘H NMR spectrum of a GSH/Me,SnCl, system in deuterated TRIS buffer (B, = 9.4 T, T = 300 K, [TRIS] = 200 mM, [Me,SnCl,l = 5 mM, GSH/Me,SnCl, = 8/l, pH = 7.53).

TABLE 4. ‘H NMR Parameters for the GSH/Me,SnCl, System in 200 mM Deuterated TRIS Buffer (B, = 9.4 T, T = 300 K, [Sn] = 10 or 5 mM, pH = 7.3 f 0.2)

GSH/Me,SnCl, ,. 6 CH,Sn Ratio (ppm from TMS)

2J(“9Sn, ‘H) 0-k)

0 0.7608 83.3 129 0.2 0.734 79.6b 125 0.5 0.751 77.P 123 0.8 0.775 74.4b 121 1.0 0.788 74.0b 121 1.5 0.829 70.7b 118 2.0 0.856 69.9 118 3.0 0.876 68.2 117 4.0 0.881 67.9 117 5.3 0.882 67.7 116 8.0 0.880 67.9 117

a pH = 6.53. bAverage of ‘19Sn and “‘Sn couplings.


.70 h I I I I

0 1 2 3 4 5 6 7 6 R

FIGURE 9. Proton chemical shift of the Me,Sn(IV) moiety in 200 mM deuterated TRIS buffer solution of the GSH/Me,SnCl, system as a function of the different ligand-to-metal ratios (B, = 9.4 T, T = 300 K; pH = 7.3 f 0.3).

satellites, it is possible to calculate a value for the C-Sn-C angle, 8, making use of the Lockhart equation [24]:

8 = O.O105l?l* - 0.79912JI + 122.4 (1)

and the calculated values are collected in Table 4. The *J value changes from 83.3 Hz for the diorganotin(IV) alone in the buffer to 67.8 Hz at high R values. Me,Sn(IV) in water at pH near 7 has a tetrahedral geometry around tin [121; however, from Eq. (l), a 8 value of 129” for the compound in TRIS buffer is obtained. This result suggests that the TRIS molecule may occupy a coordination position around the tin, giving rise to a trigonal bipyramidal geometry, with equatorial methyl groups and distorted toward a tram-octahedron, as has been found in HEPES buffer [12].

The addition of glutathione shifts the geometry towards a regular trigonal bypiramidal structure. The suggestion, based on Miissbauer results, that the tin environment is C,SnS,N, with GSH coordinated through the thiolic S atom, is further supported by the substantial R-dependent upfield shift observed for the p cysteine protons resonance, and, to a lesser extent, for the (Y cysteiene proton resonance (data not reported).

Similar series of measurements taken in the presence of a low concentration of either TRIS or HEPES deuterated buffer (tin was 10 or 5 mM and buffer 20 mM) and without adjustment of the pH, yield the values of ‘J reported in Table 5. The pH in both series varied between 2.7 and 3.3 in the whole R range.

Making use of Eq. 1 one observes a decrease of the 8 angle from 150 to 125” in the case of the TRIS buffer, and from 142 to 123” for HEPES containing solutions. These results indicate that the average geometry around the metal atom goes from distorted tnzns-octahedral to trigonal bipyramidal in both cases.

The initial (R = 0) 2J value is in agreement with what is known from the literature [16] and at high R values the geometry is the same found in buffered solution at pH ca. 7.3. These experimental findings suggest that GSH may coordinate to dimethyltin(IV) moieties even in acidic solutions, but a considerable excess of ligand is necessary.

TABLE 5.


Tbo-Bond Tin-Proton Coupling Constants in Hz for the GSH/Me$nCl, System in 20 mM Deuterated TRIS or HEPES Solution ([Sn] = 10 or 5 mM, B0=14.1T,T=300K)

GSH/Me,SnCl, Ratio TRIS HEPES

0 102.1 95.7 0.2 loo.1 90.7 0.5 97.4 88.6 0.8 95.8 85.5 1.0 94.2 84.3 1.5 90.5 82.2 2.0 88.5 80.7 3.0 85.9 80.0 4.0 82.5 78.2 5.3 80.6 77.2 8.0 79.3 76.3

It should be considered, that in this pH range, GSH is present in the form of the xwitterionic monoanion, with ca. 30% of the negatively charged dianion, with the protonated terminal amino group [22]; the interaction with the formally positively charged organotin moiety may thus be rather inefficient, while compe- tition with the buffer species, particularly HEPES, could be significant.

The Aqueous Model System: Species Present in Aqueous Solutions of Me,SnCI and GSH

As already pointed out, triorganotin(IV) derivatives are known to form stable compounds with GSH: nBu$n+ and Ph,Sn+ give 2:l complexes at least in the solid state [8,9], while no solid compound has been obtained with Me,SnCl due to solubility problems, even if some interaction has been proposed to take place in solution [lo]. Miissbauer spectra of frozen water solutions of Me&Cl and GSH support this hypothesis.

The pH of a water solution of 10 mM Me,SnCl is slightly acidic, 4.1, and is indicative of the formation of hydrolyzed species. The Miissbauer spectrum of this solution can well be fitted to a single doublet with AEo value of 3.80 mm/set. Point charge calculations exclude a tetrahedral coordination geometry around the tin center, while they are compatible with a tbp. Under this hypothesis, the three methyl groups lie in the equatorial plane while the two apical positions are occupied either by two water molecules, or, more likely, by a water molecule and a hydroxide ion. By increasing the pH to physiological values by adding NaOH, the quadrupole splitting decreases to a value of 2.87 mm/set (see Table 61, that is in perfect agreement with that calculated for the fruns-Me,Sn(OH),- species. The situation is rather different if the pH is fixed to 7.4 by means of HEPES. The Miissbauer spectrum can still be fitted to a quadrupole doublet, but the AE, value is now 3.31 mm/set; this increase can be due to the substitution of an OH ion with a HEPES molecule bonded to the tin center by an aminic nitrogen (see Fig. 10). If GSH is now added, the quadrupole splitting decreases almost linearly with the GSH/Me,SnCl ratio down to 2.4 mm/set when the ratio is 1:l (see Table 5). From this point on, the AEo values remain virtually constant indicating that a stable 1:l adduct has been formed in solution (see Fig. 11). Again, point charge calculations have


TABLE 6. Miissbauer Effect Parameters for the GSH/Me,SnCI System in Aqueous HEPES Buffer at pH 7.4 Recorded at 80.0 K

GSH/Me,SnCI S* Ratio (mm/see)

AEQ

(mm/set) r1

(mm/set) r2

(mm/set)

0 1.28 3.31 0.81 0.85 0.1 1.27 3.27 0.87 0.93 0.2 1.31 3.12 0.83 0.85 0.35 1.27 2.97 0.97 0.99 0.50 1.33 2.80 0.99 0.96 0.60 1.28 2.79 0.96 1.02 0.80 1.31 2.69 0.95 0.94 1.00 1.34 2.41 0.84 0.79 2.00 1.31 2.37 0.82 0.93 3.50 1.37 2.42 0.80 0.84 5.00 1.34 2.38 0.81 0.82

’ Referred to room temperature CaSn03.

proven useful to indicate the possible structure of this compound: an octahedral structure can be excluded and only tetrahedral or trigonal bipyramidal geometries are possible (see Fig. 11). As it seems rather unlikely to be a reduction of the coordination number, the tbp structure is proposed for this adduct. The organic radicals occupy the equatorial plane, a thiolic sulfur from a GSH molecule one of the apical positions, while the other is occupied by a water molecule or by an aminic nitrogen of either a GSH or a HEPES molecule. Much more intriguing is the situation when a 1:l GSH:Me,SnCI mixture is allowed to react in water at different pH values. For pH values lower than about 4.0, as in the case of the mono- and di-methyltin( no reaction seems to take place as the Mossbauer hyperfine parameters do not show any change in respect to the

3.20

3.00

2.60

2.40 l *

*

2.00 m 0.00 1 .oo 2.00 3.00 4.00 5.00

R

FIGURE 10. Mijssbauer hyperfine parameters for the GSH/Me,SnCl system in 200 mM HEPES solution as a function of the different ligand-to-metal ratios R.


AE talc

=-3.15

AE AE CdC =- 1.94 talc =-2.22

Me. I ‘Sn

Mb/ \

S

AE _,==+2.45

OH

Y \

Sk--Ma

k4/ s

FIGURE 11. Some possible tin coordination spheres in the HEPES buffer solution for the 1:l GSH/Me,SnCl system.

pure water solution (see Table 7). For pH higher than 7.5, a new species is obtained; its MSssbauer parameters prove that it is different from what was obtained in HEPES probably because the water molecule or the aminic nitrogen in the second apical position has been substituted by an OH- ion. In the pH range between 4.0 and 7.5 the situation is very complex as two different species are present at the same time in a roughly constant 1:l ratio. These two compounds present approximately the same 6 value, which is constant through- out the investigated pH interval, and quite different AE, values (see Table 7).

TABLE 7. MGssbauer Effect Parameters for the GSH/Me,SnCl System in 1:l Ratio in Water as a Function of pH Recorded at 80.0 K

S” *EC2 PH (mm/set) (mm/w)

2.87 1.33 3.73 3.87 1.36 3.63 5.17 1.37 3.81

1.39 2.43 5.45 1.34 3.59

1.30 2.15 6.10 1.41 3.39

1.30 2.04 6.62 1.36 2.82

1.32 1.90 7.26 1.33 2.64

1.29 1.83 7.51 1.33 2.11 9.24 1.33 2.15

a Referred to room temperature CaSnO,. b Parameters constrained to a tixed ratio.

l-1 (mm/se4

1.05 0.80 0.73b 0.87b 0.75b 0.87b 1.02b 0.72b 0.87b 0.7P 0.97b o.sP 0.86 0.87

l-2 (mm/set)

0.75 0.81 0.73b 0.87b 0.75b 0.87b 1.02b 0.72b 0.87b 0.7P 0.97b 0.81b 0.90 O.%

100 100 52 48 52 48 49 51 47 53 49 51

100 100

Capohgo et al.

This parameter decreases for both the species on increasing the pH until, for pH greater than 7.5, the two doublets collapse and a single compound is again obtained. No useful indication can be obtained from the point charge model for the two compounds formed in neutral solution.

This system has been studied by ‘H NMR in 200 mM deuterated TRIS buffer solution at glutathione to tin ratios ranging from OA to 8:l; a typical spectrum is shown in Figure 12 and the NMR data (chemical shifts and coupling constants) for the SnMe group of the whole series are collected in Table 8.

The plot of 6 vs R (see Fig. 13) suggests a 1:l complexation of GSH and Me,Sn(IV) in agreement with the Miissbauer findings. Nonlinear curve fitting [23] of the 6 vs R data yields a value of (1.6 If: 0.3) X lo3 for the formation constant of the complex. The 2J(Sn, ’ H) values obtained from the satellites show only minor variations when R is changed from 0 to 8, indicating that the geometry around the tin atom remains practically unchanged.

The 8 value of ca. 113” calculated from Eq. 2 1241 (Table 7)

0 = 0.0161~J)2 - 1.32l?( + 133.4 (2)

corresponds to the geometry of a highly distorted trigonal bipyramid, which has been obtained from ‘i9Sn Miissbauer measurements for Me,Sn(IV) in water at alkaline pH [ll or coordinated to the thiol group of cysteine [271.

In this case also, ‘H NMR measurements have been carried out either in deuterated TRIS or in deuterated HEPES solution at low concentration of the

_...~.._ ._._-___ _ __7 ._..T___-.._.- _ ._ _~_.. _-__.. _

4.2s 4.00 3.75 3.50 3.25 3.00 2.75 2.50 a.25 TL -T-- 3.75 3.50 i.25 I.00 .75 .50 WY

FIGURE 12. ‘H NMR spectrum of the a GSH/Me,Sn(VI) system in deuterated TRIS buffer (B, = 9.4 T, T = 300 K, [TRISI = 200 mM, [Me,SnCll = 5 mM, GSH/Me,SnCI = 8/l, pH = 7.46).

INTEIWCI’IONS OF ORGANOTIN WITH GLUTATHION 291

TABLE 8. ‘H- NMR Parameters for the GSH/Me,SnCl System in 200 mM Deuterated TRIS Buffer (B, = 9.4 T, T = 300 K, [Sn] = 10 or 5 mM, pH = 7.4 f 0.1)

GSH/Me,SnCI s CH,Sn

Ratio (ppm from TMS)

0 0.427 0.2 0.444 0.5 0.465 0.8 0.482 1.0 0.493 1.5 0.504 2.0 0.510 3.0 0.517 4.0 0.515 5.3 0.511 8.0 0.511

zJ(“9Sn, ’ H) @Ix)

65.2 115.8 63.2’ 114.3 62.3’ 113.7 62.8 114.0 62.3 113.7 62.0 113.4 61.7 113.2 61.5 113.1 61.5 113.1 61.4 112.7 61.5 113.1

‘Average of ‘19Sn and “‘Sn couplings; the two satellites could not be resolved.

buffer (tin was 10 or 5 mM and the buffer 20 mM) and no pH adjustment. In either case the initial (R = 0) pH value was ca. 5.6, while by increasing the amount of added GSH it decreased to ca. 3.5. The *J(Sn, ’ H) value is indepen- dent from the GSH to tin ratio (*J = 68.4 f 0.2 and 69.1 f 0.1 I-Ix in HEPES and ‘IRIS buffer, respectively) and the 8 value calculated from Eq. 2 [24] is about 119”, corresponding to a trigonal bipyramidal geometry, with equatorial methyl groups. The finding that the geometry does not change by addition of the ligand suggests the coordination of the solvent and/or buffer molecules in the absence of GSH. The R-dependence of the SnMe chemical shift in HEPES solution (Fig. 14) suggests again the formation of a complex with a 1:l stoichiometry even in acidic solution, while in the presence of IRIS the data are completely uninfor- mative.

.4Q . I I I t I I I I

0 1 2 3 4 5 6 7 6 R

FIGURE 13. Proton chemical shift of the Me,Sn(IV) moiety in 200 mM deuterated TRIS buffer solution of the GSH/Me,SnCl. system as a function of the different ligand-to-metal ratios R (B, = 9.4 T, T = 300 K, pH = 7.3 f 0.3).


.56 t

1.0 R

FIGURE 14. Proton chemical shift of the Me,Sn(IV) moiety in 20 mM deuterated HEPES buffer solution of the GSH/Me,SnCl system as a function of the different ligand- to-metal ratios R (B, = 14.1 T, T = 300 K; pH, see text).

CONCLUSIONS

The present Miissbauer study on frozen aqueous solutions of GSH and mono-, di-, and tri-organotin(IV) derivatives, together with the results of the point charge model applied to the AE, values and of the ‘H NMR measurements on the same systems in solution at room temperature, allowed us to obtain useful information on the interactions of organotins with an important biological compound. It seems, to be proven that, at least in HEPES or TRIS at physiological pH, there are strong interactions between the reduced form of GSH and all the organotins that have been tested. In every case, an involvement of the thiolic sulfur is very likely, even at pH where it is still protonated, with the formation of a pentacoordinate complex with a tbp geometry around. the tin center. In the case of the mono methyl derivative an equilibrium between the starting material and another not identified species is obtained, while with the two other organotin derivatives stable and well defined complexes are formed. In these last cases, the stoichiometric formulas are different from each other, presenting a 2:l and a 1:l ligand-to-metal ratio, respectively, and are different also from what is expected from the solid state work B-101. However, these results may give some clue for the study of the mechanism of defense of the cells against xenobiotic pollutants and of the activity of metal based drugs.

77~ financial support of the CNR (Rome), contribution No. %.002%.CToo3 and of the Minister0 dell’Universitci e della Ricerca Scientifica, Progetti di Interesse Nazionale, is gmte- filly acknowledge. We wish to thank the NMR Laboratory of the lJniversi@ of Florence for the use of the AMX-600 spectrometer and Mr. Massimo Lucci for recording the spectm. Thanks are aLFo due to Professor Maurizio Paci, University “Tor Vergata ” Rome, for giving access to the AM-400 spectrometer.

REFERENCES

1. S. N. Kosower and M. E. Kosower, Int. Rev. of Citologv 54, 109 (1978). 2. L. Floh& and W. A. Giinzler, in Glutathione: Metabolism and Function, I. M. Arias

and W. B. Jakoby, Eds., Kroc Found. Ser., Vol. 6, Raven Press, New York, 1976.


3. L. Floh& and W. A. Giinzler, in Glututhione, L. Flohi? et al., Eds., Thieme, Stuttgart, 1974, p. 132.

4. N. S. Kosower, K. R. Song, and E. M. Kosower, B&him. Biophys. Actu, 192, 1 (1969).

5. S. Nicklin and M. W. Robson, Appf. 0~. Chem. 2,487 (1988). 6. A. Crowe, in Tin-BasedAntitumor Drugs, M. Gielen, Ed., Springer-Verlag, 1989, p. 69. 7. N. R. Lomax and V. L. Narayanan, Chemical Structures of Interest to the Divkion of

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Sot., Dalton Trans. 519, 3903 (1989).

Received June 7, 1992; accepted Juiy 31, 1992

Interactions of organotin(IV) halides with reduced glutathione in aqueous solution

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