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The constitution of platinum blues
J ¢nom nacl Chem 19~7, Vol 39, P0. 437-439. Pergamon Press.
Printed in Great Britain
THE CONSTITUTION OF PLATINUM BLUES CHARLES M. FLYNN, JR., T. S. VISWANATHAN and R. BRUCE MARTIN Chemistry Department, University of Virginia,Charlottesville, VA 22901. U.S.A.
(Received 17June I976) h,bstract--A wide variety of ligands were tested with several Pt(II) starting complexes in order to determine requirements for formation of platinum blues. Primary amides yield blues with K2PtCI,. 2,4-Dihydroxy-3unsubstituted pyrimidines (uracils) yield blues with cis (NH3)~Pt(H:O):~÷. Other nucleic acid bases and replacement of ammonia by substituted amines in the starting Pt(lI) complex failed to yield blue products. Spectrophotometric analysis in the visible region reveals that solutions of blues are complex and variable mixtures of several components The rate of appearance of Pt blues displays an initial induction period and is dependent upon the uracil to Pt(II) mole ratio, absolute concentration of Pt(II) and pH. Pt blues are formed slowly and are not equilibrium mixtures as the constitution depends upon prior history of the sample. It is suggested that in many Pt blue systems only a fraction of the Pt is involved in production of the blue color. Most of the features of the investigated Pt blues may be accounted for by supposing that they consist of short stacked polymers with Pt-Pt bonds. INTRODUCTION In contrast to the yellow or colorless nature of most platinum complexes, some ligands yield intense blue or purple complexes that are referred to as platinum blues. Though the reaction of PtCI42 and (RCN)2PtClz with primary amides to yield deep purple to dark blue products has been known for more than half a century, their structure is uncertain. In a combination physical method and X-ray study, the platinum blue of trimethylacetamide was formulated as a Pt(IV) complex[l], Interest in the structure of platinum blues has quickened as a result of their recently proved effectiveness as antitumor agents in mammals without the associated severe kidney damage produced by antitumor cis (NH3)2PtC12 [2]. The reaction of cis [(NH3)2Pt(H20)z] 3+ with uracils produces blues that are potent antitumor agents. Though we have investigated the reactions of a wide variety of ligands with several Pt(II) starting complexes, this report emphasizes those combinations which gave blue or otherwise visibly colored products. EXPERIMENTAL
Cis (NH3)2PtI2 was prepared by a procedure improved over that reported [3]. THe preparation was carried out in dim light, which seemed to minimize formation of dark iodoplatinum precipitates. A solution of 5.0g (12 mmole) K2PtCI4was treated with KI [12.0 g ~72mmole) in 18 ml water; vs previously used Pt:l = 1:8], heated quickly to 70°, allowed to cool (0.5 hr), filtered and treated with 12-13 ml 2.0 M NH3. After 0.5 hr the product was filtered, washed with cold water and dried in a desiccator. The analogs with methylamine and ethylenediamine[4] were prepared similarly. Frans(NH3)2Ptl2 was prepared by dissolving the cis complex in aqueous NH3 and evaporating. [(en)Pt(H:O)2](NO~): solutions were prepared as described ~reviously[4] and cis[(NHO~Pt(H20)2](NO~) was prepared analogously. During both preparation and storage the solutions were protected from light. Though the last compound has been reported to be photosensitive[5], no photoisomerization of the light protected solutions was detected by the thiourea test[6]. ~,olutions of cis[L2Pt(H~O)~](HSO,)z (L=NH3 or CH3NH2) were prepared similarly using a hot, nearly saturated 1:1 Ag2SO,:H~SO, solution. THe mixture was allowed to cool after addition of L:PtI2 and stirred overnight as for the nitrate salts. l(en)Pt(H20)2]SO, and trans[(NH3)2Pt(H20)2]SO,solutions were i3repared similarly. Other chemicals were high quality commercial !~roducts. 437
A large number of ligands were tested for formation of blues and their spectra monitored over several weeks. Due to the high absorbances, 1 mm path length cells and neutral density filters were often employed. In all cases the samples were stored away from bright light at room temperature, about 20°. Some solutions were buffered at pH 2 with HSO4- and at pH 5 with acetic acid buffer systems. '~C magnetic resonance spectra were recorded on a JEOL PS-100P/ECI00 spectrometer operating at 23.5 kG. RESULTS
With K2PtCI,, in a 3:1 ligand to metal molar ratio, oxamic acid, oxamide (warm) and orotic acid give dark gray-blue solutions. With a large (103) excess of ligand, acetamide yields a deep blue solution. N-substituted amides did not give blue products. K2PtCh did not yield blue products with any of the other compounds mentioned below. A solution 2 mM in PtCI42 and 4 mM in biuret at pH 5 does develop a purple-gray color with absorption maxima at 610, 485 and 400nm, all with E > 2000. With ten times greater concentrations the color is bluer. With cis [(NH3)2Pt(H20)2] 2+, either as sulfate or nitrate, purple, blue, or green products were obtained in a 1:3 molar ratio with uracil, l-methyluracil, uridine, the three corresponding 5-methyl (thymine) compounds, dihydrouracil, and barbituric acid (blue ppt). 3-substituted uracils did not give blue products. In agreement with a previous report[2], trans-[(NH3)2Pt(H20)z]S04, cis[(CH3NH2)2Pt(H20)2]S04 and [(en)Pt(H20)2]SO4 did not yield blue products even with 2,4-dihydroxy-3unsubstituted pyrimidines. Appearance of Pt blues occurs most readily below pH 5, perhaps due to formation of an inert dihydroxo bridged Pt(II) dimer near pH 6.5 [4]. Orange to red colors were obtained from cis [(NH3)2Pt(H20)2]SO4 and the primary amides of acetic, trimethylacetic, phenylacetic and benzoic acids at a 3:1 ligand to Pt molar ratio. These solutions displayed an obsorption band at 480 nm. Similar colors were also obtained with the same ligands and cis-[(CH3NH2)2Pt(H20)2]S04 or [(en)Pt(H20)2]SO4. These red-orange colors do not appear to have been reported previously. The rate of appearance of a blue color is usually slow. Figure 1 shows the slow growth of green-blue color in solutions originally containing thymidine and 0.040 M cis-[(NH3)zPt(H20)2] 2~ at several molar ratios. It is
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Fig. 1. Appearance of blue color in solutionsoriginallycontainingthymidineand 0.040M cis(NH,)2Pt(H20)22÷ buffered at pH 2 with HSO, . The molar absorptivity on the ordinate is based on the total original Pt(II) concentration.The one digitnumbernear the centerof each curveis the molarratio of thymidineto Pt(II).The three digit numbersat the beginningand end of each curve identifythe wavelengthin nm at the maximumof a relatively broad absorptionband. evident that there is an induction period for the appearance of a blue color suggestive of an autocatalytic reaction. The higher ligand to Pt molar ratios produce greater initial rates. Greater reaction rates are obtained at the same molar ratios with 0.10MPt(II) and the absorptivities become too great to measure. A peak about 113 as strong as that in the red develops at about 450 nm. Growth of the 450 nm peak is nearly identical for the 3 : 1 and 2:1 ratio solutions. Depending upon conditions with 1-substituted uracils, peaks or shoulders appear from 450-480, 560-580 and 680-720 nm. These solutions yield colors from purple to blue-green as the ligand to Pt mole ratio is varied from 0.5 to 3. The dominant peak may be in either of the last two ranges. This variability and that indicated in Fig. 1 suggests that these solutions contain a vareity of species that contribute to color in the visible region. Qualitatively we have found that production of a blue color with 1-substituted uracils is not simply proportional to the cis[(NH3)2Pt(H20)2]2÷ concentration; solutions less than 1 mM in Pt do not yield blue colors even after long standing. This lack of production of a blue color may be due to failure to achieve equilibrium or to a prolonged induction period. In experiments with 1-methyluracil or thymidine with cis[(NH3)2Pt(H20):]2+ blue colors were allowed to develop at 25-100 mM Pt for 30-40 days. The course of decrease in visible absorbance of diluted solutions of these aged samples was compared with the increase in absorption of fresh solutions of identical overall composition. It is evident that a common equilibrium position was not achieved. Thus the species present in pyrimidine blue solutions are not in equilibrium and are dependent upon prior history of the solution. Less of an induction period is evident in the growth of a blue color in solutions containing 2 mM PtCL2- and a 500-1000 fold excess of acetamide. At pH 2 or 5 the absorbance reaches a plateau after about 10 days with maxima from 580-600nm (e2300-2600) and at about 515 nm (~ 1400-1800). Dilution of a solution at pH 2 yields an insignificant decrease in absorptivity, suggesting the importance of kinetic factors in determining solution composition. Despite the apparent similarities in absorption spectra of the pH 2 and pH 5 solutions, extractions with nitromethane of an aged pH 5 solution yield only a
minor proportion of blue material, while extraction at pH 2 gives a substantial portion of royal blue material with an absorption maximum at 585 nm in the nitromethane phase leaving a blue-violet aqueous phase. Formation of a blue color in the 1-methyluracil system at pH 2 was found to be independent of atmosphere: air, 02 stream, N2 stream and vacuum sealed samples all behaved similarly. Either the system is insenstitive to oxygen or so sensitive that our precautions were insufficient. Attempts to prepare crystalline materials in the 1-methyluracil system were unsuccessful. Some experiments pertaining to the oxidation state of Pt were performed on a green-blue solution containinga 2:1 ratio of 1-methyluracil and cis[(NH3)2Pt(H20)2] 2÷. Addition of KI gave a dark brown precipitate, while addition of KI to a solution of cis[(NH3)2Pt(H20)2](HS04): alone yields yellow cis (NH3)2PtI2. Addition of Fe(II), Fe(III), or H202 did not produce a visible effect. Addition of a proportion of V(II) corresponding to < 1/5 of the Pt in the solution immediately discharged the green-blue color. It is difficult to take nuclear magnetic resonance spectra of pyrimidine blues, a result which suggests the presence of paramagnetic materials. Proton magnetic resonance spectra yield broad peaks. The 13C magnetic resonance spectrum of a 3 month old deep blue solution (E600) originally containing0. I M cis (NH3)2Pt(H20)22÷ and 0.3 M thymidine at pH < 2 shows only free ligand peaks. A blue 1:1 solution of cis[(NH3)2Pt(H:O)2]2+ and uridine gave three sets of 13Cmagnetic resonance peaks. One set is that of free ligand, L, and a second set corresponds well to that found for enPdL2[7]. The third set is most reasonably assigned to a 1:1 complex with uridine. In both the thymidine and uridine containingsolutions, peaks ascribable to the blue material are simply not evident, even with low intensities. DISCUSSION
In the case of pyrimidines, 2,4-dihydroxo-3unsubstituted pyrimidines yield Pt blues. Substitution at the 1, 5 and 6 positions does not prevent formation of these uracil blues, while substitution at the 3-position does. Substitution of the proton at 3N in uracils is a feature of the reaction of Pd(II) and Pt(II) complexes [7, 8]. [dienPt(OH2)]2+and [(en)Pt(OH2)2]2+and
-139
The constitutionof platinumblues the corresponding Pd(II) complexes displace the 3N proton upon complexation without forming blues[8]. Though a displaceable proton at 3N appears essential for forming blues, its substitution by Pt(II) is insufficient to produce a blue. Uracil blues are most easily formed with cis [(NH3)2Pt(H20)2]~' . The trans complex and substituted ammonia molecules including en do not yield blues. The cis NH, feature may be critical in not producing steric hindrance or in providing a covalent linkage with Pt or another ligand molecule. Due to the slowness of the reactions producing blues, ammonia, even when not present originally, might be produced from promoted hydrolysis of coordinated ligands such as nitriles and primary amines. A multiplicity of species exist in solutions of Pt blues. Evidence for this conclusion is presented in Fig. 1 where the shifting of the wavelength of maximum absorbance is a common feature in the formation of blues. These shifts in the absorbance maximum reflect only a piece of the underlying changes occurring in the absorption spectra as blue colors develop. Under different conditions even the absorption maximum in the visible spectrum occurs at widely different wavelengths, e.g. from 560-580 and 680-720nm. The ratio of absorbances in these two wavelength regions is highly variable. Evidence such as that shown in Fig. 1 indicates a maximum of two substituted uracils per Pt in a blue, with a suggestion that even one might suffice. The two NH3 molecules may then remain bound to Pt in a blue. The complexity of Pt blue systems has been noted by most other investigators [1,2]. Several lines of evidence suggest that only a fraction {< 10%) of the Pt and ligand in solution contribute to the color of uracil blues. Evidence for an unusual multiplicity of species has just been mentioned. The apparent molar absorptivity near 600 in the visible spectra based on total Pt concentration is at least five times lower than expected for a fully allowed transition. Deep blue solutions of two different uracils gave no lines in the t3C magnetic resonance spectra assignable to a new species. Finally, the blue color is immediately discharged by only a fractional equivalent of V(II). That only a fraction of the Pt present is involved in formation of blues is likely to be the case in many preparations described in the literature. One advantage of absorption spectroscopy in studying solutions that are mixtures is that we know by definition that the blues are being investigated while other physical methods may be measuring properties of other molecules ir~ the solution. A variety of arguments suggest that the oxidation state of some Pt in the blue solutions is fractionally greater than 2+. Though some formulations of blues have been as Pt(IV), substantiating evidence in terms of redox properties has not been given. Discharge of a uracil blue color was achieved with only a fractional equivalent of V{ll). THe reaction with I to give a brown rather than a yellow product also suggests an oxidation state greater than 2-~. Neither Pt(II) nor Pt(IV) species are paramagnetic yet the nuclear magnetic resonance spectra of the blue solutions suggest the presence of some paramagnetism. Even in the absence of O2 small amounts of oxidized Pt(II) species may be produced by disproportionation and bv reaction with an oxidizing uracil molecule.
Most of the points presented above may be accomodated by supposing that Pt blues are small polymers of variable chain length. Attempts by several groups of investigators to obtain crystals have been unsuccessful. Monomeric complexes of both Pt(II) and Pt(IV) are usually yellow or colorless. The failure of solutions with low Pt concentration to yield blue colors and the induction period for appearance of a visible absorption as illustrated in Fig. 1 also suggest polymer formation. The wide variation in absorption maxima and other spectral characteristics is accounted for by chains of varying length and different Pt-Pt spacings in the different chains. Viewed in this way platinum blues are simply a generic term for the the whole gamut of species that absorb in the yellow to near-IR region. Known stacked polymeric structures with short Pt-Pt bonds support this type of structure for Pt blues. Blues have not been observed with Pd and the tendency of this metal to form stacks with Pd-Pd bonds is rarer than with Pt[9]. The bis(oxalato)platinate complex forms polymers which vary in color from orange through brown and violet to blue as the chain length increases[10], The mean oxidation state of Pt in this and other stacked polymers ranges from just greater than 2+ to 2.4+ consistent with the conclusions of this paper on the presence of some paramagnetism and the oxidation state in solutions containing Pt blues. To form deeply colored Pt-Pt bonded polyomers the Pt monomers must approach to about 3.2A[11]. Allowing for equatorial steric hindrance, the approximatte axial thicknesses dueotO ligands coordinated to Pt are as follow: NH3, 3.4A; uracil ring, 3.4A; CH3NH2, 4.6,~; and en, 4.2A. Thus the failure of substituted ammonia ligands to form blues is explained by their greater thickness which prohibits formation of the short Pt-Pt contacts reo~uired to generate a blue color. The similarity of the 3.4 A base stacking distance in the B form of DNA with the 3.2 A Pt-Pt distance in blue polymers points up the compatibility of complexed uracil:s meeting the steric requirements for stacking in Pt blues. The exact nature of the bonding involved and the reasons for failure of other nucleic acid bases to yield Pt blues are matters for further inquiry. Acknowledgement--This research was partially supported by :a research grant from the National Cancer Institute. REFERENCES
1. D. B. Brown, R. D. Burbank and M. B. Robin, J. Am. Chem. Soc. 91, 2895 (1969). 2. J. P. Davidson, P. J. Faber, R. G. Fischer. Jr., S. Mansy. H. J. Peresie, B. Rosenberg and L. Van Camp, (am'er Chemotherapy Reports, Part 1 15, 287 (1975) 3. S. C. Dhara, Ind. J. Chem. 8, 193 (1970). 4. M. C. Lira and R. B. Martin, Z lnorg. Nucl. Chem. 38. 1911 (1976). 5. J. R. Perumareddiand A W. Adamson.J. Phys. ('hem. 72.414 (1968). 6. A. A. Grinberg, M. Serator and M. 1. Gelfman. Zh Neorv. Khim. 13, 3289 (i968). 7. D. J. Nelson, P. L. Yeagle, T. L Miller and R B. Martin, Bioinorganic Chem. 5, 353 (1976). 8. M. C. Lim and R. B. Martin, J. lnorg. Nucl. Chem. 38. 1915 (1976). 9. J. S. Millerand A. J. Epstein, Prog. Inorg. Chem. 20, 1 (1976). 10. K. Krogman and P. Dodel, Chem. Ber. 99, 3408 (1966). 11. K. Krogman, Angew. Chem. Int. Ed. Engl. 8. 35 (1969!.
Printed in Great Britain
THE CONSTITUTION OF PLATINUM BLUES CHARLES M. FLYNN, JR., T. S. VISWANATHAN and R. BRUCE MARTIN Chemistry Department, University of Virginia,Charlottesville, VA 22901. U.S.A.
(Received 17June I976) h,bstract--A wide variety of ligands were tested with several Pt(II) starting complexes in order to determine requirements for formation of platinum blues. Primary amides yield blues with K2PtCI,. 2,4-Dihydroxy-3unsubstituted pyrimidines (uracils) yield blues with cis (NH3)~Pt(H:O):~÷. Other nucleic acid bases and replacement of ammonia by substituted amines in the starting Pt(lI) complex failed to yield blue products. Spectrophotometric analysis in the visible region reveals that solutions of blues are complex and variable mixtures of several components The rate of appearance of Pt blues displays an initial induction period and is dependent upon the uracil to Pt(II) mole ratio, absolute concentration of Pt(II) and pH. Pt blues are formed slowly and are not equilibrium mixtures as the constitution depends upon prior history of the sample. It is suggested that in many Pt blue systems only a fraction of the Pt is involved in production of the blue color. Most of the features of the investigated Pt blues may be accounted for by supposing that they consist of short stacked polymers with Pt-Pt bonds. INTRODUCTION In contrast to the yellow or colorless nature of most platinum complexes, some ligands yield intense blue or purple complexes that are referred to as platinum blues. Though the reaction of PtCI42 and (RCN)2PtClz with primary amides to yield deep purple to dark blue products has been known for more than half a century, their structure is uncertain. In a combination physical method and X-ray study, the platinum blue of trimethylacetamide was formulated as a Pt(IV) complex[l], Interest in the structure of platinum blues has quickened as a result of their recently proved effectiveness as antitumor agents in mammals without the associated severe kidney damage produced by antitumor cis (NH3)2PtC12 [2]. The reaction of cis [(NH3)2Pt(H20)z] 3+ with uracils produces blues that are potent antitumor agents. Though we have investigated the reactions of a wide variety of ligands with several Pt(II) starting complexes, this report emphasizes those combinations which gave blue or otherwise visibly colored products. EXPERIMENTAL
Cis (NH3)2PtI2 was prepared by a procedure improved over that reported [3]. THe preparation was carried out in dim light, which seemed to minimize formation of dark iodoplatinum precipitates. A solution of 5.0g (12 mmole) K2PtCI4was treated with KI [12.0 g ~72mmole) in 18 ml water; vs previously used Pt:l = 1:8], heated quickly to 70°, allowed to cool (0.5 hr), filtered and treated with 12-13 ml 2.0 M NH3. After 0.5 hr the product was filtered, washed with cold water and dried in a desiccator. The analogs with methylamine and ethylenediamine[4] were prepared similarly. Frans(NH3)2Ptl2 was prepared by dissolving the cis complex in aqueous NH3 and evaporating. [(en)Pt(H:O)2](NO~): solutions were prepared as described ~reviously[4] and cis[(NHO~Pt(H20)2](NO~) was prepared analogously. During both preparation and storage the solutions were protected from light. Though the last compound has been reported to be photosensitive[5], no photoisomerization of the light protected solutions was detected by the thiourea test[6]. ~,olutions of cis[L2Pt(H~O)~](HSO,)z (L=NH3 or CH3NH2) were prepared similarly using a hot, nearly saturated 1:1 Ag2SO,:H~SO, solution. THe mixture was allowed to cool after addition of L:PtI2 and stirred overnight as for the nitrate salts. l(en)Pt(H20)2]SO, and trans[(NH3)2Pt(H20)2]SO,solutions were i3repared similarly. Other chemicals were high quality commercial !~roducts. 437
A large number of ligands were tested for formation of blues and their spectra monitored over several weeks. Due to the high absorbances, 1 mm path length cells and neutral density filters were often employed. In all cases the samples were stored away from bright light at room temperature, about 20°. Some solutions were buffered at pH 2 with HSO4- and at pH 5 with acetic acid buffer systems. '~C magnetic resonance spectra were recorded on a JEOL PS-100P/ECI00 spectrometer operating at 23.5 kG. RESULTS
With K2PtCI,, in a 3:1 ligand to metal molar ratio, oxamic acid, oxamide (warm) and orotic acid give dark gray-blue solutions. With a large (103) excess of ligand, acetamide yields a deep blue solution. N-substituted amides did not give blue products. K2PtCh did not yield blue products with any of the other compounds mentioned below. A solution 2 mM in PtCI42 and 4 mM in biuret at pH 5 does develop a purple-gray color with absorption maxima at 610, 485 and 400nm, all with E > 2000. With ten times greater concentrations the color is bluer. With cis [(NH3)2Pt(H20)2] 2+, either as sulfate or nitrate, purple, blue, or green products were obtained in a 1:3 molar ratio with uracil, l-methyluracil, uridine, the three corresponding 5-methyl (thymine) compounds, dihydrouracil, and barbituric acid (blue ppt). 3-substituted uracils did not give blue products. In agreement with a previous report[2], trans-[(NH3)2Pt(H20)z]S04, cis[(CH3NH2)2Pt(H20)2]S04 and [(en)Pt(H20)2]SO4 did not yield blue products even with 2,4-dihydroxy-3unsubstituted pyrimidines. Appearance of Pt blues occurs most readily below pH 5, perhaps due to formation of an inert dihydroxo bridged Pt(II) dimer near pH 6.5 [4]. Orange to red colors were obtained from cis [(NH3)2Pt(H20)2]SO4 and the primary amides of acetic, trimethylacetic, phenylacetic and benzoic acids at a 3:1 ligand to Pt molar ratio. These solutions displayed an obsorption band at 480 nm. Similar colors were also obtained with the same ligands and cis-[(CH3NH2)2Pt(H20)2]S04 or [(en)Pt(H20)2]SO4. These red-orange colors do not appear to have been reported previously. The rate of appearance of a blue color is usually slow. Figure 1 shows the slow growth of green-blue color in solutions originally containing thymidine and 0.040 M cis-[(NH3)zPt(H20)2] 2~ at several molar ratios. It is
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Fig. 1. Appearance of blue color in solutionsoriginallycontainingthymidineand 0.040M cis(NH,)2Pt(H20)22÷ buffered at pH 2 with HSO, . The molar absorptivity on the ordinate is based on the total original Pt(II) concentration.The one digitnumbernear the centerof each curveis the molarratio of thymidineto Pt(II).The three digit numbersat the beginningand end of each curve identifythe wavelengthin nm at the maximumof a relatively broad absorptionband. evident that there is an induction period for the appearance of a blue color suggestive of an autocatalytic reaction. The higher ligand to Pt molar ratios produce greater initial rates. Greater reaction rates are obtained at the same molar ratios with 0.10MPt(II) and the absorptivities become too great to measure. A peak about 113 as strong as that in the red develops at about 450 nm. Growth of the 450 nm peak is nearly identical for the 3 : 1 and 2:1 ratio solutions. Depending upon conditions with 1-substituted uracils, peaks or shoulders appear from 450-480, 560-580 and 680-720 nm. These solutions yield colors from purple to blue-green as the ligand to Pt mole ratio is varied from 0.5 to 3. The dominant peak may be in either of the last two ranges. This variability and that indicated in Fig. 1 suggests that these solutions contain a vareity of species that contribute to color in the visible region. Qualitatively we have found that production of a blue color with 1-substituted uracils is not simply proportional to the cis[(NH3)2Pt(H20)2]2÷ concentration; solutions less than 1 mM in Pt do not yield blue colors even after long standing. This lack of production of a blue color may be due to failure to achieve equilibrium or to a prolonged induction period. In experiments with 1-methyluracil or thymidine with cis[(NH3)2Pt(H20):]2+ blue colors were allowed to develop at 25-100 mM Pt for 30-40 days. The course of decrease in visible absorbance of diluted solutions of these aged samples was compared with the increase in absorption of fresh solutions of identical overall composition. It is evident that a common equilibrium position was not achieved. Thus the species present in pyrimidine blue solutions are not in equilibrium and are dependent upon prior history of the solution. Less of an induction period is evident in the growth of a blue color in solutions containing 2 mM PtCL2- and a 500-1000 fold excess of acetamide. At pH 2 or 5 the absorbance reaches a plateau after about 10 days with maxima from 580-600nm (e2300-2600) and at about 515 nm (~ 1400-1800). Dilution of a solution at pH 2 yields an insignificant decrease in absorptivity, suggesting the importance of kinetic factors in determining solution composition. Despite the apparent similarities in absorption spectra of the pH 2 and pH 5 solutions, extractions with nitromethane of an aged pH 5 solution yield only a
minor proportion of blue material, while extraction at pH 2 gives a substantial portion of royal blue material with an absorption maximum at 585 nm in the nitromethane phase leaving a blue-violet aqueous phase. Formation of a blue color in the 1-methyluracil system at pH 2 was found to be independent of atmosphere: air, 02 stream, N2 stream and vacuum sealed samples all behaved similarly. Either the system is insenstitive to oxygen or so sensitive that our precautions were insufficient. Attempts to prepare crystalline materials in the 1-methyluracil system were unsuccessful. Some experiments pertaining to the oxidation state of Pt were performed on a green-blue solution containinga 2:1 ratio of 1-methyluracil and cis[(NH3)2Pt(H20)2] 2÷. Addition of KI gave a dark brown precipitate, while addition of KI to a solution of cis[(NH3)2Pt(H20)2](HS04): alone yields yellow cis (NH3)2PtI2. Addition of Fe(II), Fe(III), or H202 did not produce a visible effect. Addition of a proportion of V(II) corresponding to < 1/5 of the Pt in the solution immediately discharged the green-blue color. It is difficult to take nuclear magnetic resonance spectra of pyrimidine blues, a result which suggests the presence of paramagnetic materials. Proton magnetic resonance spectra yield broad peaks. The 13C magnetic resonance spectrum of a 3 month old deep blue solution (E600) originally containing0. I M cis (NH3)2Pt(H20)22÷ and 0.3 M thymidine at pH < 2 shows only free ligand peaks. A blue 1:1 solution of cis[(NH3)2Pt(H:O)2]2+ and uridine gave three sets of 13Cmagnetic resonance peaks. One set is that of free ligand, L, and a second set corresponds well to that found for enPdL2[7]. The third set is most reasonably assigned to a 1:1 complex with uridine. In both the thymidine and uridine containingsolutions, peaks ascribable to the blue material are simply not evident, even with low intensities. DISCUSSION
In the case of pyrimidines, 2,4-dihydroxo-3unsubstituted pyrimidines yield Pt blues. Substitution at the 1, 5 and 6 positions does not prevent formation of these uracil blues, while substitution at the 3-position does. Substitution of the proton at 3N in uracils is a feature of the reaction of Pd(II) and Pt(II) complexes [7, 8]. [dienPt(OH2)]2+and [(en)Pt(OH2)2]2+and
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The constitutionof platinumblues the corresponding Pd(II) complexes displace the 3N proton upon complexation without forming blues[8]. Though a displaceable proton at 3N appears essential for forming blues, its substitution by Pt(II) is insufficient to produce a blue. Uracil blues are most easily formed with cis [(NH3)2Pt(H20)2]~' . The trans complex and substituted ammonia molecules including en do not yield blues. The cis NH, feature may be critical in not producing steric hindrance or in providing a covalent linkage with Pt or another ligand molecule. Due to the slowness of the reactions producing blues, ammonia, even when not present originally, might be produced from promoted hydrolysis of coordinated ligands such as nitriles and primary amines. A multiplicity of species exist in solutions of Pt blues. Evidence for this conclusion is presented in Fig. 1 where the shifting of the wavelength of maximum absorbance is a common feature in the formation of blues. These shifts in the absorbance maximum reflect only a piece of the underlying changes occurring in the absorption spectra as blue colors develop. Under different conditions even the absorption maximum in the visible spectrum occurs at widely different wavelengths, e.g. from 560-580 and 680-720nm. The ratio of absorbances in these two wavelength regions is highly variable. Evidence such as that shown in Fig. 1 indicates a maximum of two substituted uracils per Pt in a blue, with a suggestion that even one might suffice. The two NH3 molecules may then remain bound to Pt in a blue. The complexity of Pt blue systems has been noted by most other investigators [1,2]. Several lines of evidence suggest that only a fraction {< 10%) of the Pt and ligand in solution contribute to the color of uracil blues. Evidence for an unusual multiplicity of species has just been mentioned. The apparent molar absorptivity near 600 in the visible spectra based on total Pt concentration is at least five times lower than expected for a fully allowed transition. Deep blue solutions of two different uracils gave no lines in the t3C magnetic resonance spectra assignable to a new species. Finally, the blue color is immediately discharged by only a fractional equivalent of V(II). That only a fraction of the Pt present is involved in formation of blues is likely to be the case in many preparations described in the literature. One advantage of absorption spectroscopy in studying solutions that are mixtures is that we know by definition that the blues are being investigated while other physical methods may be measuring properties of other molecules ir~ the solution. A variety of arguments suggest that the oxidation state of some Pt in the blue solutions is fractionally greater than 2+. Though some formulations of blues have been as Pt(IV), substantiating evidence in terms of redox properties has not been given. Discharge of a uracil blue color was achieved with only a fractional equivalent of V{ll). THe reaction with I to give a brown rather than a yellow product also suggests an oxidation state greater than 2-~. Neither Pt(II) nor Pt(IV) species are paramagnetic yet the nuclear magnetic resonance spectra of the blue solutions suggest the presence of some paramagnetism. Even in the absence of O2 small amounts of oxidized Pt(II) species may be produced by disproportionation and bv reaction with an oxidizing uracil molecule.
Most of the points presented above may be accomodated by supposing that Pt blues are small polymers of variable chain length. Attempts by several groups of investigators to obtain crystals have been unsuccessful. Monomeric complexes of both Pt(II) and Pt(IV) are usually yellow or colorless. The failure of solutions with low Pt concentration to yield blue colors and the induction period for appearance of a visible absorption as illustrated in Fig. 1 also suggest polymer formation. The wide variation in absorption maxima and other spectral characteristics is accounted for by chains of varying length and different Pt-Pt spacings in the different chains. Viewed in this way platinum blues are simply a generic term for the the whole gamut of species that absorb in the yellow to near-IR region. Known stacked polymeric structures with short Pt-Pt bonds support this type of structure for Pt blues. Blues have not been observed with Pd and the tendency of this metal to form stacks with Pd-Pd bonds is rarer than with Pt[9]. The bis(oxalato)platinate complex forms polymers which vary in color from orange through brown and violet to blue as the chain length increases[10], The mean oxidation state of Pt in this and other stacked polymers ranges from just greater than 2+ to 2.4+ consistent with the conclusions of this paper on the presence of some paramagnetism and the oxidation state in solutions containing Pt blues. To form deeply colored Pt-Pt bonded polyomers the Pt monomers must approach to about 3.2A[11]. Allowing for equatorial steric hindrance, the approximatte axial thicknesses dueotO ligands coordinated to Pt are as follow: NH3, 3.4A; uracil ring, 3.4A; CH3NH2, 4.6,~; and en, 4.2A. Thus the failure of substituted ammonia ligands to form blues is explained by their greater thickness which prohibits formation of the short Pt-Pt contacts reo~uired to generate a blue color. The similarity of the 3.4 A base stacking distance in the B form of DNA with the 3.2 A Pt-Pt distance in blue polymers points up the compatibility of complexed uracil:s meeting the steric requirements for stacking in Pt blues. The exact nature of the bonding involved and the reasons for failure of other nucleic acid bases to yield Pt blues are matters for further inquiry. Acknowledgement--This research was partially supported by :a research grant from the National Cancer Institute. REFERENCES
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