Applications of NMR spectroscopy in understanding the gold biochemistry

Gold-based drugs have been successfully used for the treatment of rheumatoid arthritis. When administered, they undergo ligand exchange reactions in the body with biofluids, cells and proteins. NMR spectroscopy is a very useful technique for probing these ligand exchange reactions under physiological conditions. The strength of the binding ligands can be estimated by studying the chemical shift changes in 13C and 31P NMR. It is also a powerful method for investigating the kinetics and thermodynamics of the exchange reactions of gold drugs with biomolecules. The purpose of this review report is to highlight the importance of NMR spectroscopy in the study of gold biochemistry and to bridge the fairly large gap in the progress of this interesting area of bioinorganic chemistry.

Myocrisin and solganol are water soluble but insoluble in hydrophobic environments and thus have to be administered intramuscularly to prevent hydrolysis in acid gastric fluid.The lipophilic auranofin on the other hand can be administered orally [1,2].Auranofin possesses several potential advantages over the other gold drugs.These are oral administration, less kidney retention, equal distribution between the cellular components and serum proteins of the blood and inhibition of release of lysosomal enzymes, which are responsible for tissue damages [10].The crystal structure of auranofin shows that gold is linearly coordinated by the sulfur atom of thioglucose and the phosphorus of Et 3 P to give a discrete monomeric molecular species [11].Gold thiolates are polymeric in nature [12][13][14] (by sulfur bridging)  with a gold-gold interaction (Fig. 2).The structures of these polymers are very sensitive to the ionic strength and pH of the solutions [15].
The antiarthritic activity of gold(I) compounds may be related to the high affinity and selectivity of gold(I) for sulfhydryl sulfur as a biological ligand [16].Since gold(I) is extremely labile, these gold(I) complexes after their administration, undergo several ligand exchange reactions in the body with biofluids, cells and proteins [10,[17][18][19][20].The high affinity of gold(I) for sulfur and selenium ligands suggests that proteins including enzymes and transport proteins will be critical in vivo targets.Serum albumin, the principal extra cellular protein of blood, binds to about 90% of the gold in serum and functions as a defecto transport agent [21].With their chain structures, gold thiolates have a capacity, to react rapidly with a variety of ligands, like thiols, thiones, selenols and cyanide, while the reactions of auranofin are slower because of the strong binding of both ligands (triethylphosphine and thioglucose) to gold(I) [10,17,[22][23][24][25]. Gold(I) thiolates, (Gold(I), thiomalate and thioglcose) being polymers can not enter red blood cells (RBCs) [26,27], while auranofin being monomeric does enter into RBCs immediately after its absorption and binds to intracellular glutathione (GSH) and Cys-β-93's of hemoglobin (Hb) [25].The RBCs, which contain thiol and thione ligands, e.g., GSH, Hb and ergothionine (ErS), can form stable complexes with gold drugs [25,28].Gold has no known biological function in the body and hence there are no natural mechanisms for gold in the body.Therefore toxic effects may arise by the use of gold drugs [10].Toxic effects generally appear on skin, mucous membrane including gastrointestinal and renal problems.
NMR spectroscopy being a non-invasive method has found successful applications in the characterization of several species formed as a result of the drug action and thus allows studying several physiological consequences of the drugs [29].The exchange reactions of gold(I) drugs with various biological ligands have been studied in detail using NMR spectroscopy [17,[25][26][27][28][29][30].Proton, carbon-13 and nitrogen-15 NMR have proved to be very useful in the structural characterization of gold thiolates metabolites [17,[30][31][32].The sensitivity of 13 C and 15 N can be enhanced by using isotopically enriched samples or by polarization transfer techniques such as INEPT or DEPT.The phosphorus-31 nucleus with 100% natural abundance and high sensitivity makes 31 P NMR the most widely used technique for investigating the reactions of auranofin [29].The 31 P chemical shift is sensitive to the nature of the trans coordinated ligand and release of Et 3 P can be monitored by observation of Et 3 PO resonance [18,19,24,25,33].Changes in intensity of the resonances with time are helpful to follow the kinetics of the exchange reactions [34].This article describes a detailed review of the use of NMR spectroscopy in following the exchange reactions of anti-rheumatic gold(I) complexes with various biologically important ligands.The applications of NMR to study the structure-activity relationship are highlighted that would help in designing novel gold drugs and understanding their mechanism of action.

Interaction of gold drugs with thiols
Proton and carbon-13 NMR are successfully applied to monitor the exchange reactions of (Autm) n with thiols.The 1 H and 13 C NMR studies of the interaction of (Autm) n with thiomalate suggest the formation of new species with thiolate : Au ratio of 1.75 : 1.0 according to Eq. ( 1), which undergoes slow exchange with Htm [17].
The breakdown of (Autm) n polymer occurs by a similar route for other thiols such as cysteine [17].
The kinetic analysis of these reactions showed that they proceed through associative mechanism [17].
Generally it is established that reaction of thiols to [Au(SR)] n polymers results in the formation of bis(thiolate)aurate(I) species, which undergoes rapid exchange with additional ligand [35,36].Thiols with lower pK a values are in fast exchange with gold(I) and the resonances for free and bound thiomalate could be observed only for thiols with pK a greater than 9. 13 C NMR studies suggest that thiols with the lowest pK a values bind most strongly to gold(I).Thus binding of gold drugs with cysteine (pK a = 8.5) will be thermodynamically more favored over glutathione (pK a = 8.9).Due to ease of thiol exchange reactions, gold distribution in the body after drug treatment is very wide spread [17].
The interaction of (Autm) n with glutathione and the red blood cells has been studied by spin echo 1 H NMR [27,30].As (Autm) n is titrated with glutathione, a specific binding of gold to cysteine of glutathione is observed together with the release of thiomalate.The CH 2 resonance of cysteinyl residue changes from asymmetric quartet into a multiplet by the proximity of the metal center.The two resonances from the glutamyl residue of glutathione are also affected but to a smaller extent.The spectra of gold titration into red cell lysate were identical to the above pattern.The addition of myocrisin to a suspension of intact erythrocytes produced a different pattern of reactivity.In the 1 H NMR, the cysteinyl CH 2 resonances responded to the presence of the gold compounds but the glutamyl resonances do not.This shows that a gold-glutathione complex is not forming in the cytosol in an analogous manner to the lysate and the model system.A diminution of cysteinyl CH 2 resonance in the 1 H NMR independent of glutamyl resonances is normally regarded as characteristic of glutathione oxidation.These observations suggested that myocrisin stimulates oxidation of intracellular glutathione.The rate of transport of myocrisin across the membrane is slow [37].The reaction of myocrisin with the membrane bound thiol is represented as: Membrane-SH + Au 8 tm 9 −→ membrane-S-Au 8 tm 8 + tmH. ( It has been assumed that polymeric aurothiomalate coats the surface of red cells and prevents further entry of gold into cells.Consequently, the kinetics of the reaction with glutathione is expected to be slow.However, the response of glutathione to myocrisin in extracellular fluid is rapid suggesting that transport is not required [27].In similar experiments with the orally active auranofin the cysteinyl CH 2 disappears reflecting the ease of entry of this lipophilic drug into the cell and the increased glutathione exchange rate of gold(I) in the presence of the phosphine ligand [27,30].The exchange reactions of auranofin have been studied extensively using 13 C and 31 P NMR spectroscopy. 31P NMR is especially invaluable for investigating the behavior of Et 3 P released from auranofin.The most extensively studied metabolic reactions of auranofin (Et 3 PAuSATg) are those of serum albumin (AlbSH), the predominant carrier protein for serum gold.Auranofin reacts at Cys-34 of albumin via a ligand exchange reaction that displaces the sulfhydryl group with the formation of AlbS-Au-PEt 3 .Formation of AlbS-Au-PEt 3 species was identified by the appearance of a resonance at 38.8 ppm in the 31 P NMR spectra.The similarity of the chemical shift of AlbS-Au-PEt 3 to those of other thiolate adducts, RS-Au-PEt 3 suggests that auranofin reacts at Cys-34 with the displacement of ATgS − .The free acetylthioglucose liberated from auranofin reacts further with the AlbS-Au-PEt 3 and displaces PEt 3 , which is oxidized to Et 3 PO.Oxygen-17 NMR and GC-MS evidence that the principal oxidants are albumin disulfide bonds of albumin, where water is the major source of oxygen, which participates in the reaction [38].The sequence of reactions can be represented by Scheme 1 [18,39].
The 31 P NMR further confirms that Et 3 P is released from albumin bound complex rather from auranofin.It also gives indication about the gold binding at weak binding sites such as histidine and methionine residues [18].It has also been demonstrated by 31 P NMR that Et 3 PAu + bound to the weak binding of albumin can be transferred to the stronger binding site AlbS-AuPEt 3 to form the bis complex, AlbS(AuPEt 3 ) + 2 [40,41].The Et 3 PAu + entity is also liable to exchange in the presence of Me 3 PAuX and i− Pr 3 PAuX [41].
Phosphorus-31 NMR studies of the interaction of various R 3 P-Au-SATg complexes with albumin reveal that the rate of phosphine oxidation increases as the affinity of the thiol for gold(I) increases, while the rate decreases as the affinity of phosphine in R 3 P-Au-SR increases.In the reaction of Me 3 P-Au-SATg with albumin the smaller and less basic Me 3 P is displaced to form the corresponding Me 3 PO more readily than is Et 3 P in Et 3 P-Au-SATg [42].In the reaction of isopropyl analogue of auranofin, the i Pr 3 PO is formed only in small quantity, while the rest of the phosphine is oxidized to i Pr 3 PS.The experiment further demonstrates that in contrast the displacement of Et 3 P from Et 3 P-Au-SAlb by thiols, cyanide is required to displace i Pr 3 P from i Pr 3 P-Au-SAlb [43].
The values of 31 P chemical shifts of various species are given in Table 1.In Et 3 P-Au-SR complexes, the 31 P chemical shifts are correlated directly with the affinity of thiols for gold(I) and inversely with their pK SH values.The ability of various thiols to cause Et 3 PO formation from AlbS-Au-PEt 3 is; AT-gSH > TgSH > GSH, as expected from their basicity [33].The correlation of δ( 31 P) and pK a values helps to estimate the binding strength of other thiolate ligands.For example the δ( 31 P) for the adduct of hemoglobin with auranofin was observed at 34.0 ppm, which suggests the low affinity of hemoglobin towards gold compared to other thiols listed in Table 1.As a result of its low affinity, inter protein gold transfer from Hb(SAuPEt 3 ) 2 to mercaptalbumin was observed using 31 P NMR.As indicated by the 31 P NMR chemical shift values (Table 1), glutathione (GSH) is a more favorable binding site in RBC than hemoglobin.GSH does not however compete effectively with serum albumin for gold(I).The high affinity of albumin towards gold compared to hemoglobin and glutathione suggests that the accumulation/transfer of gold metabolites of auranofin in RBC is thermodynamically not favored.The 31 P NMR correlation predicted the direction of the inter protein (Hb to Alb) gold transfer reaction and thus it is helpful in predicting the position of equilibria [25].The transfer of Et 3 PAu + between protein species provide support to the sulfhydral shuttle mechanism for Et 3 PAu + transport across the cell membrane.
The 31 P NMR spectroscopy was successfully applied to study the mechanism of the reaction of AlbS with auranofin and its analogue, i Pr 3 P-Au-SATg.The kinetic analysis of the reaction between auranofin and serum albumin shows that the reaction is first order in albumin and zero order with respect to auranofin, with a rate constant, k 1 = 3.4 ± 0.3 × 10 −2 sec −1 for Eq.(2).However, under physiological conditions, where auranofin concentration is low, 10-25 µM ([albumin] = ∼400 µM) the reaction is suggested to follow the second order kinetics with a rate constant of 8 ± 2 × 10 2 M −1 s −1 [34].This high value of rate constant corresponding to a rapid ligand exchange reaction of auranofin with albumin explains the very strong affinity of Cys-34 for gold(I).The 1 H NMR study of the reaction of albumin with gold drugs also suggest that Cys-34 is the major binding site of albumin for gold(I) [45].A multistep mechanism was proposed [34] which can be explained as follow; AlbSH where * AlbSH and * AlbSAuPR 3 are conformationally altered albumin molecules in which Cys-34 is accessible to other solute molecules.In the crystal structure of albumin, Cys-34 is protected in a crevice, stabilized by in the ionized Cys − form possibly via hydrogen bonding to histidine 39 [46].A structural change, where it moves to a more solvent exposed position is required when it binds to gold(I) or a disulfide [47].The conformationally altered albumin species could not be identified by chromatography and the chromatographic methods suggested that the reaction is first order with respect to i Pr 3 P-Au-SATg.Thus the mechanism of this reaction could not be explained by chromatography and only NMR results describe the most probable mechanism.The rate law corresponding to this mechanism is; where, Saturation transfer experiments on i Pr 3 P-Au-SATg under equilibrium conditions yielded 2nd order rate constants for both the forward (1.2 × 10 2 M −1 S −1 ) and the reverse (3.9 × 10 M −1 S −1 ) directions.
Phosphorus-31 NMR spectroscopy can be used to probe the specific target sites for gold drugs.For example the 31 P NMR studies of the Et 3 PAuCl and (Et 3 P) 2 AuCl with human blood demonstrated that Et 3 PAuCl reacts with red blood cells binding to thiol sites of glutathione and hemoglobin, whereas (Et 3 P) 2 AuCl reductively attacks S-S linkage of plasma albumin (eq.) The bis complex accelerates rather than inhibits the SH-SS exchange reactions and therefore are not suitable as antiarthritic agent [48].

Exchange reactions with thiones
In the interaction of aurothiomalate with red cells, it has been observed that when all glutathione is complexed gold thiomalate reacts with ergothionine, which predominantly occurs in thione form [27,30].This observation suggested that Au(I) reacts with thiones less strongly than does to thiols.The reaction of thiones such as ergothionine [28], Imt (imidazolidine-2-thione) [49], Diaz (1,3-diazinane-2-thione) [50] and mercaptopurines [51] with (Autm) n usually leads to a ternary complex, [>C=S-Au-tm] without ejecting thiomalate.The excess ligands are in rapid exchange with the bound ligand.A comparison of the 13 C chemical shifts of the carbon atom attached to the coordinating sulfur atom of various thione ligands at the 1 : 1 ratio of thione :(Autm) n is given in Table 2.It is observed that the thiolated nucleosides bind more strongly than do other thiones with gold(I). 13C and 15 N NMR studies of the interaction of (Autg) n with thiones demonstrate that thiones bind to (Autg) n weakly and (Autg) n polymer does not undergo exchange reactions with thione ligands completely [54].This reflects that although gold thiolates are polymeric, they have different chemical reactivities.It seems that the Au-S bond in (Autg) n is stronger compared to that in (Autm) n .On the other hand, the reactions of thiol [18,33] and CN − [31,55]  Recently, the reactions of auranofin (Et 3 PAuSATg) with various thiones have been monitored by 13 C and 31 P NMR [56,57].These studies revealed that both Et 3 P and SATg − ligands were replaced by thiones simultaneously from gold(I) in auranofin forming [>C=S-Au-SATg] and [Et 3 P-Au-S=C<] type complexes.These ligands are consequently oxidized to Et 3 PO and (SATg) 2 respectively.Some of the liberated E 3 P also reacts with thiones forming Et 3 P-S=C< species.Thus the Et 3 P released from auranofin  can react with the cellular species in addition to its oxidation during exchange reactions in vivo.The Et 3 PO resonance in 31 P NMR is observed after several days, which confirms the slower rate of these exchange reactions.The reactions of thiols are comparatively faster.The resonances for [>C=S-Au-SATg] and (SATg) 2 species also appeared in 13 C NMR after several days of addition of thiones to auranofin showing that these exchange reactions are very slow.The rate of reaction of auranofin towards thiones at 298 K is found to be approximately 1 × 10 −9 M/S.A comparison of the rate of formation of Et 3 PO from auranofin with various thiones is shown in Fig. 3. Thus among thiones the order of reactivity towards auranofin is Tu > Diaz > ErS > Imt.Since thiones can replace both ligands from auranofin, there is a possibility that they can undergo exchange reactions with the adducts formed between gold drugs and cellular thiols like glutathione.

Exchange reactions with disulfides
With its chain structure (Autm) n may have a capacity, to react with disulfides and diselenides in addition to thiols and selenols.Since on binding to (Autm) n , the disulfide (S-S) linkage should be reduced to the thiolate form, therefore redox, instead of simple exchange reactions are expected to occur in these interactions.In the exchange reactions of (Autm) n with disulfides, different kinds of exchange processes were observed for two different disulfides [58].In the case of Ellmans reagent, the proton NMR revealed two sets of signals for the ES moiety, indicating formation of two new products.These resonances were assigned to a mixed disulfide (ES-tm) and a polymer capped by ES moieties.The formation of new species was confirmed by Raman spectroscopy.This was explained by the following equation; 2ESSE Tm-(Au 8 tm 7 )-Tm −→ ES-Au 8 tm 7 -ES + 2EStm.(10) The overall exchange process can be represented as: In the case of lipoic acid, a slow direct reaction was observed, producing dithiomalate (tm) 2 as end product and no mixed disulfide was detected.The (tm) 2 resonances were observed after one week in 1 H NMR showing that the exchange process was very slow.
Myocrisin is also known to react with the disulfide sites on exofacial surface of the membrane proteins.The reaction occurs primarily by exchange at the terminal (Autm) n moieties.
This exchange process does not entail any redox behavior on the part of the sulfhydryl functions (glutathione) in the membrane proteins [27].
Auranofin is also known to bind with the disulfide sites of albumin as described by Eq. ( 14), but there is no systematic study describing the behavior of auranofin towards disulfides.
The AlbSSR-Et 3 PAuSATg complex is weakly bound and readily dissociates during separation by conventional gel chromatography [34].Phosphine gold(I) thiolate complexes react with disulfides to undergo a thiolate/disulfide exchange, as described by Eqs ( 15) and ( 16) for mononuclear and a dinuclear complex respectively [59].16) Proton NMR confirms that the reaction proceeds in a stepwise fashion in which the unsymmetrical disulfide forms first followed by the formation of the symmetrical disulfide.Variable temperature 31 P NMR experiments suggest that Au-Au bonding in gold(I) thiolate clusters influences the reactivity towards disulfides [60].The cluster complexes undergo thiolate/disulfide exchange reactions more rapidly than monomeric complexes [59].

Gold-cyanide metabolites
The reaction of cyanide with gold are of therapeutic interest because the physiological distribution of gold(I) is believed to be affected by the cyanide present in the blood.Cyanide is produced naturally in the body by oxidation of SCN − by the enzyme myeloperoxidase in polymorphonuclear leukocytes (PMN) [61,62].[Au(CN) 2 ] − has been identified as a common metabolite of the gold drugs in the blood and urine of chrysotherapy patients [63].The reactions of gold drugs and their metabolites with cyanide lead to the formation of intermediates, [RSAuCN] − and [Et 3 PAuCN], which undergo disproportionation generating [Au(CN) 2 ] − that is readily taken up by red blood cells according to Eq. ( 1) [24,31,61,62,64].Graham et al. investigated the effect of cyanide on (Autm) n activation.It was observed that the cellular uptake of gold from (Autm) n was slow in the absence of cyanide.Cyanide markedly increased the uptake of gold by red blood cells, 21.1% after incubation for 24 hours [65].Tobacco smoking is known to increase the concentrations of gold in red blood cells in patients treated with (Autm) n [66,67].Cyanide acts as a shuttle to carry gold into red blood cells, because of inhalation of HCN from tobacco smoke [64].
The very large formation constant of [Au(CN) 2 ] − (log β = 36.6)[75] is believed to drive the reaction in the forward direction generating [Au(CN) 2 ] − .These reactions can easily be monitored by 13 C NMR.However, for R 3 PAuCN complexes 31 P NMR has been successfully applied to study these reactions [70][71][72].In the 13 C NMR spectra of such complexes two resonances are observed for CN carbon, corresponding to LAuCN and [Au(CN) 2 ] − species.When 13 C and 15 N labeled CN was used the [Au(CN) 2 ] − resonance appeared as triplet and the resonance for LAuCN was a doublet.A similar observation was recorded in the 15 N NMR [69,[72][73][74].
For cyano(phosphine)gold(I) complexes the 31 P NMR spectra revealed two signals, one each from R 3 PAuCN and [Au(R 3 P) 2 ] + species.The 13 C and 15 N NMR spectra of R 3 PAuCN complexes also showed two resonances in the low field region corresponding to Eq. ( 13).The 13 C and 15 N NMR chemical shifts were found to have opposite dependence on the basicity of phosphines in R 3 PAuCN complexes.The 15 N resonances shifted upfield, while the 13 C resonances shifted downfield with increase in the basicity of the phosphine.This opposite behavior can be explained on the basis of back bonding between gold(I) and cyanide.The donation of electron density from electron rich metal center to cyanide increases the double bond character of the Au-C bond resulting in a deshielding effect on carbon.Similarly, the reduction in the C-N bond order in the π species is accompanied by an upfield shift in 15 N NMR [72].
Similar to R 3 PAuCN complexes, the ligand scrambling reactions were also investigated in [thioneAuCN] and [selenoneAuCN] complexes.Both types of the complexes yielded two resonances for the CN group in 13 C and 15 N NMR, consistent with the scrambling equilibria given in Eq. ( 20).The equilibrium constants were calculated using relative intensities of CN resonances in LAuCN and [Au(CN) 2 ] − complexes in 13 C NMR, which gave the relative concentration of all three species [73,74].
Aurocyanide ([Au(CN) 2 ] − ) is rapidly taken up by polymorphonuclear leukocytes, and red blood cells and could affect the function of these cells [76].In RBCs, hemoglobin and glutathione are the main protein target for gold.The experiments showed that most of the gold in RBCs is bound to glutathione.Although, the concentration of cyanide is very low in RBCs (0.3-1 µM) [77,78], there is a high formation constant between cyanide and gold(I), ∼10 39  [79] and therefore bis(glutathione)gold(I) and dicyanogold(I) are likely to be found in RBCs.The mechanism by which [Au(CN) 2 ] − is transported through the cell membrane is still a subject of current research [62,64,80].However, the most convincing mechanism is the one in which gold(I) complexes undergo ligand exchange reactions to bind to sulfhydryl groups immobilized on or in the membrane.A series of such exchange reactions then passes the gold across the membrane and into the cells, where it may react with molecules containing sulfhydryl groups such as glutathione [81].The formation of [RSAuCN] − has also been reported as partially displacing CN − from [Au(CN) 2 ] − with thiols such as cysteine and glutathione in aqueous solution, although the formation constant of [RSAuCN] − is less than [Au(CN) 2 ] − [82].
[Au(CN) 2 ] − may undergo oxidation by OCl − generating gold(III) metabolites [80].The reactions of [Au(CN) 2 ] − with OCl − have been studied by 13 C NMR.The reversible, pH-dependent changes occurred by the oxidation of [Au(CN) 2 ] − can be described by Eqs (23) and ( 24): On addition of hypochlorite, the initial 156 ppm of [Au(CN) 2 ] − was replaced by resonances at 121.0 {[Au(CN) 2 (OH)Cl] − } ppm and then by 122.7 ppm {[Au(CN) 2 (OH) 2 ] − }, as increasing amounts of OCl − were added to the solution.These chemical shift changes towards the chemical shift of [Au(CN) 4 ] − (δ C = 106 ppm) are consistent with the direction of the gold(I) to gold(III).The transformation of the species resonating at 121 ppm, to the one resonating at 122.7 ppm is associated with a ligand exchange reaction (Eq.( 24)).Additional hydroxide is also generated according to the half reaction for reduction of the hypochlorite ion, Eq. ( 25): When a solution exhibiting only the 122.26): This oxidation and reduction process demonstrates that there is a potential redox cycle involving dicyanoaurate(I) and di-or tetra-cyanoaurate(III) species that could be established in vivo during chrysotherapy when macrophages are stimulated to undergo the oxidative burst [80].

NMR studies of antitumor gold complexes
Both gold(I) and gold(III) coordination compounds exhibit cytotoxic properties towards several tumor cells and both types of complexes are also effective on cells resistant to cisplatin [7][8][9].NMR spectroscopy is also a useful tool to study the interaction of antitumor phosphine gold(I) complexes such as [Au(dppe) 2 ]Cl (dppe = 1,2-bis(diphenyphosphino)ethane).The effect of [Au(dppe) 2 ]Cl on the metabolism of L1210 leukemia cells extracts was investigated by 1 H and 31 P NMR.It has been observed that [Au(dppe) 2 ]Cl causes an increase in the lactate levels and accumulation of amino acids, suggesting a drug induced increase in the rate of glycolysis and inhibition of protein synthesis [84].The reactions of [Au(dppe) 2 ]Cl with human blood plasma, red blood cells have been monitored by 1 H and 31 P NMR [85]. 31P NMR studies suggest that some [Au(dppe) 2 ] + is transferred from plasma to red blood cells with a half life of about 2 hrs at 25 • C. The rate of uptake of [Au(dppe) 2 ]Cl from plasma to red cells was evaluated by monitoring the change in intensity of the [Au(dppe) 2 ] + -phospholipid 31 P resonances with time.NMR results suggested that nearly half of the [Au(dppe) 2 ] + added to plasma was transferred to red cells.The complex binds within the cell membrane and consequently its mobility is reduced resulting in broadening of 31 P resonances.Although thiols exchange reactions play a major role in the metabolism of auranofin both in blood plasma and in cells, the complex [Au(dppe) 2 ] + did not affect the 1 H NMR resonances of intracellular GSH consistent with its low reactivity towards thiols in model systems [85]. 31P NMR studies have also shown that [Au(dppe) 2 ] + (unlike auranofin) remains essentially intact in human plasma and does not react significantly with glutathione or albumin [86].This may allow it to reach intracellular target sites, which are inaccessible to auranofin and contribute to its broader spectrum of antitumor activity.Being structurally different, the linear two coordinate and tetrahedral gold(I) phosphine complexes may follow different mechanisms.Reactions with sulfhydryl groups are likely to be involved for auranofin and Et 3 PAuCl, whereas lipophilic cationic properties of [Au(dppe) 2 ]Cl and [Au(dpype) 2 ]Cl may be important [9]. 31P NMR studies of tetrahedral bisphosphine gold(I) complexes with pyridyl substituents (R = 2-pyridyl/4-pyridyl in Fig. 1) have shown that these are stable for at least 30 hrs when incubated in blood plasma at 37 • C. Whereas the 2-pyridyl complex partitions between plasma and the RBCs, the 4-pyridyl complex (which has higher solubility) is retained in the plasma as evidenced by 31 P NMR [87].
For linear digold bis phosphine complexes XAu(dppe)AuX (which are cytotoxic against a wide range of tumors [88]), 31 P NMR studies show that thiols induce conversion of the linear complexes in to tetrahedral complex, [Au(dppe) 2 ] + and this reaction also occurs in blood plasma.The spectrum of antitumor activity of GS-Au(dppe)Au-SG (GS = glutathione) is similar to that of [Au(dppe) 2 ] + , which is the ring closed product being the major metabolite of the bridged digold complex in vivo [89].The complex (Ph 3 P)Au(dppp)Cl is also known to undergo disproportionation giving the [Au(dppp) 2 ] + complex [90].

Conclusions
NMR spectroscopy is found to be a useful technique to observe the exchange reactions of gold drugs even in the complex biological media and allows simultaneous detection of many metabolites involved in different metabolic pathways.For gold(I) phosphine drugs, 31 P NMR is useful for probing the ligand exchange reactions and the possible cellular target sites.The kinetics of ligand exchange reactions still requires further details.Cyanide plays a key role in the metabolism of gold drugs and [Au(CN) 2 ] − has been identified as common metabolite.The reactions of cyanide are successfully monitored by 13 C NMR because of the appearances of sharp CN resonances in the region of 140-160 ppm.The possible role of thiocyanate in the transport of gold(I) needs to be explored.Ligand exchange reactions of [Au(CN) 2 ] − with sulfhydryl groups provide a possible mechanism by which [Au(CN) 2 ] − is transported across the cell membrane.Similar to the oxidation of OCl − , oxidative addition of [Au(CN) 2 ] − with other oxidizing agents like disulfides and H 2 O 2 can provide a good comparison.Further studies are required to explain the formation of gold(III) metabolites.For antitumor gold complexes, there is a need to study the interactions between gold complexes and mitochondrial enzymes and other cellular targets that could lead to cell death.The present study would enhance our understanding of the reaction between gold(I) drugs and cellular species, which in turn helps to understand the mechanism of action of these drugs.

Fig. 3 .
Fig. 3. Rate of formation of Et 3 PO from auranofin with various thiones (the percentage of Et 3 PO was determined by integration of 31 P NMR spectra).

Table 1
a Two resonances due to diastereotopic Et 3 PAu + moieties.