A 13 C NMR study of the interactions of Ag 13 CN and Ag ( CN ) − 2 with thiomalic acid , L-methionine and DL-selenomethionine

Complexation of Ag as AgNO3, solid AgCN and Ag(CN) − 2 by labeled and unlabeled L-methionine, DLselenomethionine and d,l-thiomalate were studied by nuclear magnetic resonance methods. The 13C NMR indicates that only Ag react with the both L-methionine, DL-selenomethionine at neutral and higher pH via CO2 and S or Se atom forming a chelate. The Ag(CN)2 and AgCN do not bind to either of these two ligands at any pH. The Ag 13CN, which is an insoluble polymer, can react with thiomalate to form chelate complexes at neutral pH. Various structures for the chelate formations are proposed.


Introduction
There is considerable interest devoted to the reactions of the dicyanoaurate (I) ion, Au(CN) −  2 , because it exhibits anti-HIV activity [1].It is also an important metabolite of chrysotherapy agents (anti-arthritic gold drugs) [2,3].
Although, the formation constant for Au(CN) − 2 is known to be about 10 36 [4], this important metabolite is known to react with thiols where one of the cyanide can be exchanged with addition of thiomalate (tmS − ) [5] and with glutathione [6].However, little is known about the reactivity of Ag(CN) −  2 complex, although the coordination chemistry of the thiocyanate group has been exploited extensively, and the crystal structures of a large number of thiocynate compounds are known [7][8][9].Recently, Stocker et al. [10] studied the complexation of thiourea and its derivatives with AgCN where monomer and dimer complexes are formed which are separated and studied by X-ray spectroscopy.

NMR measurements
All NMR measurements were carried out on a Jeol JNM-LA 500 NMR spectrophotometer at 297 K.The 13 C NMR spectra were obtained at 125.65 MHz with 1 H broadband decoupling.The spectral conditions were: 32k data points, 0.967 s acquisition time, 1.00 s pulse delay and 45 • pulse angle.

Solution preparations
Experiment 1: 0.10 M L-Met was prepared in 1.0 cm 3 D 2 O.The pH* was adjusted to neutral, acidic and basic as shown in Table 1. 1 equivalent of solid (0.0168 g) AgNO 3 (or and with 1 equivalent of KAg(CN) 2 ) was added to the above solution and pH* was adjusted as indicated in the same table.There were no precipitates observed at neutral pH* and acidic medium but some ppts were observed at the basic medium.Similar experiments were repeated with SeMet.The results are summarized in Table 1.
Experiment 2: 0.20 M L-methionine (0.0250 g unlabel Met + 0.0050 g 13 C-CH 3 -Label Met) was prepared in 1.0 cm 3 D 2 O.The pH* was adjusted to neutral, acidic and basic as shown in Table 2. 1 eq. of (0.0336 g) AgNO 3 was added to the above solution and pH* was adjusted as indicated in the same table.There were no precipitates observed at neutral pH* and acidic medium but some ppts were observed at the basic medium.Similarly 0.0250 g SeMet was mixed with 0.042 g 13 C-CH 3 -Label SeMet which is equivalent to 0.20 M 1.0 cm 3 solution.Followed by 1 equivalent addition of AgNO 3 as a solid to each solution.The results are summarized in Table 2.In this experiment, the KAg(CN) 2 interactions with Met a Met-AgNO 3 at pH* 7 gave a ppts.therefore, solution was decanted and ran 13 C NMR spectrum.b ppts of Met-AgNO 3 were dissolved in DMSO-d 6 and ran spectrum.c Still at such a high pH* the ppts.were not dissolved, so the solution was decanted and ran the NMR.The ppts were attempted to dissolve in DMSO-d 6 but the ppts turned immediately black and therefore no attempt was made to run the spectrum.and SeMet were not studied because in the previous experiment it was found that it does not react with either of these ligands.
The pH* was adjusted to neutral, acidic and basic as shown in Table 3. 0.0084 g AgNO 3 was added to the above solution and pH* was adjusted as indicated in the same table.At such a low concentration after addition of AgNO 3 still some ppts were observed.Similarly 0.0075 g SeMet 13 C-CH 3 -Label SeMet was prepared in 1.0 cm 3 solution and 1 equivalent of solid AgNO 3 was added again some ppts were observed.
The results are summarized in Table 3.We used labeled ligands to see if we can identify 1 J( 77 Se-13 C) for the SeMet or 1 J( 109 Ag-13 C) for the Met which will give information about the nature of the bonding between Ag(I) and Met or SeMet.
Experiment 4: 0.30 M thiomalate (tmS − ) was prepared in D 2 O and added 1 eq of Ag 13 CN as a solid.
Ag 13 CN itself is insoluble in water [10]; however, in the presence of tmS − it dissolved completely.The pH* titration was carried out as indicated in Fig. 1.Same experiment was repeated with unlabel AgCN to see the reproducibility of the experiment as indicated in Fig. 1B.Fresh new sample of 0.30 M thiomalate (tmS − ) was prepared in D 2 O and added 1 equivalent of KAg(CN) 2 as a solid, the spectrum of the experiment is shown in Fig. 1C.

Results and discussion
The AgCN is an insoluble polymer and does not react with thioether and selenoether type ligands under study [10].There is no change in the chemical shifts of Met and SeMet in the presence of Ag(CN) − 2 , therefore it can be concluded that Ag(CN) − 2 does not interact with either of these two ligands as noted in Table 1.However, AgNO 3 does react with these two ligands at neutral and at higher pH*.The possible structures of the Ag complexes formed with Met and SeMet are shown in scheme 1.As noted in Table 1, the chemical shift difference between free and bound CO − 2 carbon is 2.86 ppm at neutral pH*, however, only 0.20 ppm difference was observed in the acidic medium.Similarly, a chemical shift difference of 3.22 ppm for the CO − 2 carbon was observed for SeMet and its Ag + complex at neutral pH* and 1.00 ppm difference was observed in the acidic medium.Since both CO − 2 and γ-CH 2 carbon resonances are affected on complexation, we may infer that both Se (and S) and CO − 2 groups are involved in bonding to Ag(I) ion.Therefore, we can rule out structures I and II.
The structures III and IV would require only α-CH and Se attached groups (i.e.γ-CH 2 and -CH 3 ) would be affected.Since we do not observe any substantial changes in the shifts of these carbons upon complexation, we can rule out structures III and IV.Therefore, the only possibilities of binding are proposed in the following structures V and VI, where CO − 2 and γ-CH 2 resonances are shifted the most.We have earlier reported the interactions of Met and SeMet with Hg(II) [11] and with CH 3 Hg(II) [12]  in aqueous solution.The chemical shift difference between free and bound -CH 3 of SeMet show of about 9.22 ppm, however for Met the difference is around 4 ppm.Our recent study of solution and solid state 13 C NMR between thiourea (TU) and selenourea (SeU) with Hg(II) show that for Hg(TU) 2 Cl 2 and Hg(SeU) 2 Cl 2 , it is 6.9 and 9.7 ppm respectively indicating the softer nature of Se which binds more strongly to Hg compared to S atom [13].
Figure 1A shows the 13 C resonances of thiomalate (tmS − ) (0.30 M) solution in D 2 O and after adding 1 eq of Ag 13 CN as a solid.Ag 13 CN itself is insoluble in water [10], however, in the presence of tmS − it dissolved completely.The pH* was below 4 which was adjusted to 6.90.The free tmS −13 C NMR resonances at pH* 7 appeared at f 1 = 42.17,f 2 = 45.42,f 3 = 181.07and f 4 = 180.36ppm.At 1 : 1 ratio of tmS − : Ag 13 CN they appeared at 47.66 ppm (with broadening), 43.52, 183.36 and 180.26 ppm respectively.The 13 CN − resonance is very broad at 148.88 ppm and a small sharp resonance appeared at 120.04 ppm.Six new resonances in the high-field region appeared at 72.54, 70.92, 63.39, 61.28, 61.24 and 60.21 ppm.
Figure 1B    2 but the intensities of these resonances remained the same indicating the equilibrium concentration of the product does not increase appreciably when the concentrations of the reactants was increased.This perhaps indicates the equilibrium constant for these reactions are very small and lie towards the reactants.
Although AgCN is known to be a polymer [10] and insoluble in water at any pH, the addition of thiomalate was able to dissolve it even in the acidic pH.Stocker et al. [10] have prepared series of complexes of thiourea and its derivatives with AgCN.AgCN was able to form complexes with various stoichiometries bonding to AgCN via sulphur atom of the ligands.
The above three experiments which were repeated twice to check reproducibility shows interesting features, e.g. in all three experiments the CN − region is very broad except in Fig. 2A because here labeled Ag 13 CN was used.This broadening is due to the formation of various species like RS-Ag- 13 CN − .The 13 CN NMR chemical shift difference between Au( 13 CN) − 2 and RS-Au-13 CN − is very small only 1.13 to 1.88 ppm [14,15].Therefore one would expect the similar difference between Ag 13 CN and RS-Ag-13 CN − species.The bonding to Ag with oxygen is well established in PPh 3 -Ag-NO 3 complex where two oxygen of nitrate is bonded to Ag [16] via oxygen atom forming a dimer which showed some antimicrobial activities.Therefore it can be concluded in the proposed study that oxygen bonding to the Ag(I) is possible and it may increase the activities of the complexes.

Conclusions
We have demonstrated that insoluble AgCN polymer can be dissolved in the presence of thiomalate which forms various chelating species.The KAg(CN) 2 also reacts with thiomalate but it does not react with Met or SeMet at any pH.
shows the 1 : 1 equivalent of unlabel AgCN: tmS − , where the pH was maintained at 7.0.The CN − resonance was very broad.Two carboxylate resonances appeared at 183.28 ppm and 180.25 ppm, and a new resonance appeared at 171.85 ppm.In the high field region b1 and b2 resonances appeared at 47.68 and 43.57ppm respectively.Again five new resonances appeared at 72.54, 70.92, 63.39, 61.24 and 61.21 ppm.All these five resonances are of very low intensity.

Figure
1C shows the spectrum of 1 : 1 ratio of Ag(CN) − 2 : tmS − at pH* 6.90.Again the CN − resonance was very broad.Two carboxylate resonances appeared at 182.69 ppm and 180.39 ppm respectively.In the high field region b1 and b2 resonances appeared at 46.72 and 43.07 ppm respectively.Again four new resonances appeared at 72.54, 70.92, 63.39 and 61.24 ppm.All these four resonances of with very low intensity.Figure 2A,B,C is the expansion of the low field region.Several attempts were made

Fig. 2 .
Fig. 2. A, B and C are the same as in Figs 1A, 1B and 1C but corresponding to high-field region (130 to 190 ppm).

Table 2 13
C NMR measurements of a pH* titration of L-Met, SeMet itself and in the presence of AgNO 3 at a 1 : 1 (0.20 M) ratio for both Met and SeMet each reactions

Table 3 13
C NMR measurements of a pH* titration of13C-CH 3 -L-Met,13C-CH 3 -SeMet itself and in the presence of AgNO 3 at a 1 : 1 (0.05 M each) ratio each separately