Cisplatin--DNA cross-link models with an unusual type of chirality-neutral chelate amine carrier ligand, N,N dimethylpiperazine (Me2ppz): Me2ppzPt(guanosine monophosphate)2 adducts that exhibit novel properties

Most simple cis-PtA2G2 complexes that model the G-G cross-link DNA lesions caused by the clinically used anticancer drug cis-PtCl2(NH3)2 undergo large fluxional motions at a rapid rate (A2 = two amines or a diamine; G = guanine derivative). The carrier amine ligands in active compounds have NH groups, but the fundamental role of the NH groups has been obscured by the dynamic motion. To assess carrier ligand effects, we examine retro models, cis-PtA2G2 complexes, in which dynamic motion has been reduced by the incorporation of steric bulk into the carrier ligands. In this study we introduce a new approach employing the chirality-neutral chelate (CNC) ligand, Me2ppz (N,N'-dimethylpiperazine). Because they lie in the Pt coordination plane, the methyl groups of Me2ppz do not clash with the 06 of the base of G ligands in the ground state, but such clashes sterically hinder dynamic motion. NMR spectroscopy provided conclusive evidence that Me2ppzPt(GMP)2 complexes (GMP = 5'- and 3'-GMP) exist as a slowly interconverting mixture of two dominant head-to-tail (HT) conformers and a head-to-head (HH) conformer. Since the absence of carrier ligand chirality precluded using NMR methods to determine the absolute conformation of the two HT conformers, we used our recently developed CD pH jump method to establish chirality. The most abundant HT Me2ppzPt(5'-GMP)2 form had A chirality. Previously this chirality was shown to be favored by phosphate-cis G NIH hydrogen-bonding interligand interactions; such interactions also favor the HT conformers over the HH conformer. For typical carrier ligands, G O6 and phosphate interactions with the carrier ligand NH groups also favor the HT forms. These latter interactions are absent in Me2ppzPt(GMP)2 complexes, but the HT forms are still dominant. Nevertheless, we do find the first evidence for an HH form of a simple cis-PtA2G2 model with A2 lacking any NH groups. In previous studies, the absence of the HH conformer in cis-PtA2G2 complexes lacking carrier NH groups may be due to the presence of out-of-plane carrier ligand bulk. Such bulk forces both G O6-G O6 and G O6-carrier ligand clashes, thereby disfavoring the HH form. The major DNA cross-link adduct has the HH conformation. Thus, for anticancer activity, the small bulk of the NH group may be more important than the H-bonding interaction.     

Modification of second-sphere communication, leading to an unusually high abundance of the head-to-head conformer of cisplatin cross-link retro models

Rapid atropisomerization of cisplatin-DNA cross-link models, cis-PtA(2)G(2) (A(2) = two amines or a diamine, G = guanine derivative, bold font indicating a guanine not linked to another guanine), makes their NMR spectra uninformative. The conformers [two head-to-tail (DeltaHT and LambdaHT) conformers, one head-to-head (HH) conformer] are detected in (CCC)PtG(2) retro models (CCC = chirality-controlling chelates designed to reduce rotation around the G N7 to Pt bond by destabilizing the transition state). Clear trends are found with four CCC ligands, 2,2'-bipiperidine (Bip) and N,N'-dimethyl-2,3-diaminobutane (each with S,R,R,S and R,S,S,R configurations at the chelate ring N, C, C, and N atoms, respectively). S,R,R,S ligands favor left-handed G base canting and the LambdaHT form; R,S,S,R ligands favor right-handed canting and the DeltaHT form. The HH conformer is normally negligible unless G = 5'-GMP. However, understanding this 5'-phosphate effect is complicated by possible interligand interactions of the 5'-phosphate with the N1H of the cis-5'-GMP and a CCC NH; these interactions are referred to as second-sphere (SSC) and first-to-second-sphere (FSC) communication, respectively. We now investigate the four (CCC)PtG(2) models with 1-Me-5'-GMP, a G lacking the N1H needed for SSC. The phosphate location makes FSC possible in the major but not the minor HT form. The major form should increase from pH 3 to pH 7 because the phosphate is deprotonated at pH 7. However, the major DeltaHT form for the R,S,S,R pair did not change in abundance, and the major LambdaHT form for the S,R,R,S pair actually decreased. Thus, FSC is weak. At pH approximately 7 the HH conformer of the S,R,R,S pair had an abundance (40-44%) higher than that in any reported cis-PtA(2)G(2) adduct. FSC involving one 1-Me-5'-GMP could play a role. The high HH abundance and use of a pH jump experiment with (S,R,R,S)-BipPt(1-Me-5'-GMP)(2) allowed us to obtain the first deconvoluted CD spectrum for a cis-PtA(2)G(2) HH conformer. The CD features for the HH conformer are much weaker than for the HT conformers. Our findings are best interpreted to indicate that FSC is not important in aqueous solution, especially for the HT form. Weak FSC is consistent with recent models of the cross-link in duplexes. In contrast, crystals of fluxional models often reveal FSC, but not the more important SSC. SSC was unrecognized until our retro model studies, and the new results reinforce the value of studying retro models for identifying interactions in solution.     

Factors influencing conformer equilibria in retro models of cisplatin-DNA adducts as revealed by moderately dynamic (N,N'-dimethyl-2,3-diaminobutane)PtG(2) retro models (G = a guanine derivative)

Typical cis-PtA(2)G(2) models of key DNA lesions formed by cis-type Pt anticancer drugs are very dynamic and difficult to characterize (A(2) = diamine or two amines; G = guanine derivative). Retro models have A(2) carrier ligands designed to decrease dynamic motion without eliminating any of three possible conformers with bases oriented head-to-tail (two: DeltaHT and LambdaHT) or head-to-head (one: HH). All three were found in NMR studies of eight Me(2)DABPtG(2) retro models (Me(2)DAB = N,N'-dimethyl-2,3-diaminobutane with S,R,R,S and R,S,S,R configurations at the chelate ring N, C, C, and N atoms, respectively; G = 5'-GMP, 3'-GMP, 5'-IMP, and 3'-IMP). The bases cant to the left (L) in (S,R,R,S)-Me(2)DABPtG(2) adducts and to the right (R) in (R,S,S,R)-Me(2)DABPtG(2) adducts. Relative to the case in which the bases are both not canted, canting will move the six-membered rings closer in to each other ("6-in" form) or farther out from each other ("6-out" form). Interligand interactions between ligand components near to Pt (first-first sphere communication = FFC) or far from Pt (second-sphere communication = SSC) influence stability. In typical cases at pH < 8, the "6-in" form is favored, although the larger six-membered rings of the bases are close. In minor "6-out" HT forms, the proximity of the smaller five-membered rings could be sterically favorable. Also, G O6 is closer to the sterically less demanding NH part of the Me(2)DAB ligand, possibly allowing G O6-NH hydrogen bonding. These favorable FFC effects do not fully compensate for possibly stronger FFC dipole effects in the "6-in" form. SSC, phosphate-N1H cis G interactions favor LambdaHT forms in 5'-GMP and 5'-IMP complexes and DeltaHT forms in 3'-GMP and 3'-IMP complexes. When SSC and FFC favor the same HT conformer, it is present at >90% abundance. In six adducts [four (S,R,R,S)-Me(2)DABPtG(2) and (R,S,S,R)-Me(2)DABPtG(2) (G = 3'-GMP and 3'-IMP)], the minor "6-out" HT form at pH approximately 7 becomes the major form at pH approximately 10, where G N1H is deprotonated, because the large distance between the negatively charged N1 atoms minimizes electrostatic repulsion and probably because the G O6-(NH)Me(2)DAB H-bond (FFC) is strengthened by N1H deprotonation. At pH approximately 10, phosphate-negative N1 repulsion is an unfavorable SSC term. This factor disfavors the LambdaHT R form of two (R,S,S,R)-Me(2)DABPtG(2) (G = 5'-GMP and 5'-IMP) adducts to such an extent that the "6-in" DeltaHT R form remains the dominant form even at pH approximately 10.     

A rare example of three abundant conformers in one retro model of the cisplatin-DNA d(GpG) intrastrand cross link. Unambiguous evidence that guanine O6 to carrier amine ligand hydrogen bonding is not important. possible effect of the Lippard base pair step adjacent to the lesion on carrier ligand hydrogen bonding in DNA adducts

Guanine O6 to carrier ligand hydrogen bonding is a central feature of many hypotheses advanced to explain the anticancer activity of cis-type anticancer drugs, cis-PtA(2)X(2) (A(2) = diamine or two amines). Early structural evidence suggested that cis-Pt(NH(3))(2)(d(GpG)) (the cross-link model for the key cisplatin-DNA adduct) and other cis-PtA(2)(d(GpG)) adducts exist exclusively or mainly as the HH1 conformer with head-to-head (HH) bases. The dynamic motion of the d(GpG) in these adducts is too rapid to permit definitive characterization of both the conformation and the H-bonding. Hence, we use retro models having A(2) ligands designed to slow the motion. Here, we employ Me(2)ppz (N,N'-dimethylpiperazine), which lacks NH groups. Me(2)ppz is unique in having sp(3) N-methyl groups directly in the coordination plane, allowing the coexistence of multiple conformers but hindering dynamic motion in Me(2)ppzPt(d(GpG)) and Me(2)ppzPt(GpG) retro models. Dynamic processes are decreased enough in Me(2)ppzPt(d(GpG)) to permit HPLC separation of three abundant forms. After HPLC separation, the three re-equilibrate, proving that the three forms must be conformers and that Me(2)ppz has little influence on conformer distribution. This marks the first reported characterization of three abundant conformers for one cis-PtA(2)(d(GpG)) adduct. From NMR evidence, the Me(2)ppzPt(d(GpG)) HH1 conformer has uncanted bases. Another conformer, one of two recently discovered conformer types, has head-to-tail (HT) bases with Delta chirality. For this Delta HT1 form, several lines of evidence establish that the dinucleotide moieties have essentially identical structures in d(GpG) (and GpG) adducts of Me(2)ppzPt and other cis-PtA(2) complexes. For example, the shifts of the highly structure-sensitive G H8 NMR signals are almost identical for the Delta HT1 form of all adducts. In previous models, the stabilization of the Delta HT1 form could be attributed to G O6 H-bonding to A(2) NH groups. Such H-bonds are not possible for Me(2)ppz. The unambiguous conclusions are that G O6 H-bonding is weak and that neither canting nor H-bonding is essential in HH forms. These two features are present in almost all other small models but are essentially absent in the cross-link base pair (bp) step in duplexes. We conclude from our work that the forces favoring canting and H-bonding are weak, and we hypothesize that steric effects within the Lippard bp step adjacent to this cross-link bp step easily overcome these forces.     

Neglected bidentate sp2 N-donor carrier ligands with triazine nitrogen lone pairs: platinum complexes retromodeling cisplatin guanine nucleobase adducts

Rapid rotation of guanine base derivatives about Pt-N7 bonds results in fluxional behavior of models of the key DNA intrastrand G-G cross-link leading to anticancer activity of Pt(II) drugs (G = deoxyguanosine). This behavior impedes the characterization of LPtG2 models (L = one bidentate or two cis-unidentate carrier ligands; G = guanine derivative not linked by a phosphodiester group). We have examined the formation of LPtG2 adducts with G = 5'- and 3'-GMP and L = sp(2) N-donor bidentate carrier ligands [5,5'-dimethyl-2,2'-bipyridine (5,5'-Me2bipy), 3-(4'-methylpyridin-2'-yl)-5,6-dimethyl-1,2,4-triazine) (MepyMe2t), and bis-3,3'-(5,6-dialkyl-1,2,4-triazine) (R4dt)]. NMR spectroscopy provided conclusive evidence that these LPt(5'-GMP)2 complexes exist as interconverting mixtures of head-to-tail (HT) and head-to-head (HH) conformers. For a given G, the rates of G base rotation about the Pt-N7 bonds of LPtG2 models decrease in the order Me4dt > Et4dt > MepyMe2t > 5,5'-Me2bipy. This order reveals that the pyridyl ring C6 atom + H atom grouping is large enough to impede the rotation, but the equivalently placed triazine ring N atom + N lone pair grouping is sterically less impeding. For the first time, the two possible HH conformers (HHa and HHb) in the case of an unsymmetrical L have been identified in our study of (MepyMe2t)Pt(5'-GMP)2. Although O6-O6 clashes involving the two cis G bases favor the HT over the HH arrangement for most LPtG2-type complexes, the HH conformer of (R4dt)Pt(5'-GMP)2 adducts has a high abundance (approximately 50%). We attribute this high abundance to a reduction in O6-O6 steric clashes permitted by the overall low steric effects of R4dt ligands. Under the reaction conditions used, 3'-GMP forms a higher abundance of the LPt(GMP)2 adduct than does 5'-GMP, a result attributable to more favorable second-sphere communication in the LPt(3'-GMP)2 adduct than in the LPt(5'-GMP)2 adduct.     

Investigation relevant to the conformation of the 17-membered Pt(d(GpG)) macrocyclic ring formed by Pt anticancer drugs with DNA: Pt complexes with a Goldilocks carrier ligand

Platinum anticancer drug DNA intrastrand cross-link models, LPt(d(G*pG*)) (G* = N7-platinated G residue, L = R(4)dt = bis-3,3'-(5,6-dialkyl)-1,2,4-triazine), and R = Me or Et), undergo slow Pt-N7 bond rotation. NMR evidence indicated four conformers (HH1, HH2, ΔHT1, and ΛHT2); these have different combinations of guanine base orientation (head-to-head, HH, or head-to-tail, HT) and sugar-phosphodiester backbone propagation relative to the 5'-G* (the same, 1, or opposite, 2, to the direction in B DNA). In previous work on LPt(d(G*pG*)) adducts, Pt-N7 rotation was too rapid to resolve conformers (small L with bulk similar to that in active drugs) or L was too bulky, allowing formation of only two or three conformers; ΛHT2 was not observed under normal conditions. The (R(4)dt)Pt(d(G*pG*)) results support our initial hypothesis that R(4)dt ligands have Goldilocks bulk, sufficient to slow G* rotation but insufficient to prevent formation of the ΛHT2 conformer. Unlike the (R(4)dt)Pt(5'-GMP)(2) adducts, ROESY spectra of (R(4)dt)Pt(d(G*pG*)) adducts showed no EXSY peaks, a result providing clear evidence that the sugar-phosphodiester backbone slows conformer interchange. Indeed, the ΛHT2 conformer formed and converted to other conformers slowly. Bulkier L (Et(4)dt versus Me(4)dt) decreased the abundance of the ΛHT2 conformer, supporting our initial hypothesis that steric crowding disfavors this conformer. The (R(4)dt)Pt(d(G*pG*)) adducts have a low abundance of the ΔHT1 conformer, consistent with the proposal that the ΔHT1 conformer has an energetically unfavorable phosphodiester backbone conformation; its high abundance when L is bulky is attributed to a small d(G*pG*) spatial footprint for the ΔHT1 conformer. Despite the Goldilocks size of the R(4)dt ligands, the bases in the (R(4)dt)Pt(d(G*pG*)) adducts have a low degree of canting, suggesting that the ligand NH groups characteristic of active drugs may facilitate canting, an important aspect of DNA distortions induced by active drugs.     

Exploring the universality of unusual conformations of the 17-membered Pt(d(G*pG*)) macrochelate ring. Dependence of conformer formation on a change in bidentate carrier ligand from an sp3 to an sp2 nitrogen donor

Early studies on cis-PtA2(d(G*pG*)) (A2 = diamine or two amines, G = N7-platinated G) and cis-Pt(NH3)2(d(G*pG*)) models for the key cisplatin-DNA cross-link suggested that they exist exclusively or mainly as the HH1 conformer (HH1 = head-to-head G bases, with 1 denoting the normal direction of backbone propagation). These dynamic models are difficult to characterize. Employing carrier A2 ligands designed to slow dynamic interchange of conformers, we found two new conformers, DeltaHT (head-to-tail G* bases with a Delta chirality) and HH2 (with 2 denoting the backbone propagation direction opposite to normal). However, establishing that the non-HH1 conformations exist as an intrinsic feature of the 17-membered Pt(d(G*pG*)) ring requires exploring a range of different carrier ligands. Here we employ the planar aromatic sp(2) N-donor 5,5'-Me(2)bipy (5,5'-dimethyl-2,2'-bipyridine) ligand, having a shape very different from those of previously used nonplanar sp(3) N-donor bidentate carrier ligands, which often bear NH groups. The 5,5'-Me(2)bipy H6 and H6' protons project toward the d(G*pG*) moiety and hinder the dynamic motion of 5,5'-Me(2)bipyPt(d(G*pG*)). We again found HH1, HH2, and DeltaHT conformers with typical properties, supporting the conclusions that the new DeltaHT and HH2 conformers exist universally in dynamic cis-PtA2(d(G*pG*)) adducts, including cis-Pt(NH3)2(d(G*pG*)), and that the carrier ligand typically has little influence on the overall structure of the Pt(d(G*pG*)) macrocyclic ring of a given conformer. The sizes of the G H8 to H6/H6' NOE cross-peaks indicate little base canting in all 5,5'-Me(2)bipyPt(d(G*pG*)) conformers, suggesting that carrier-ligand NH groups favor the canting of one G base in the HH1 and HH2 conformers of typical cis-PtA2(d(G*pG*)) adducts.     

NMR studies of models having the Pt(d(GpG)) 17-membered macrocyclic ring formed in DNA by platinum anticancer drugs: Pt complexes with bulky chiral diamine ligands

The highly distorted Pt(d(G*pG*)) (G* = N7-platinated G) 17-membered macrocyclic ring formed by cisplatin anticancer drug binding to DNA alters the structure of the G*G* base pair steps, canting one base, and increases dynamic motion, complicating solution structural studies. However, the ring appears to favor the HH1 conformation (HH1 denotes head-to-head guanine bases, 1 denotes the normal direction of backbone propagation). Compared to cisplatin, analogues with NH groups in the carrier ligand replaced by bulky N-alkyl groups are more toxic and less active and form less dynamic adducts. To examine the molecular origins for the biological effects of steric bulk, we evaluate Me(4)DABPt(d(G*pG*)) models; the bulk and chirality of Me(4)DAB (N,N,N',N'-tetramethyl-2,3-diaminobutane with S,S or R,R configurations at the chelate ring carbons) impede dynamic motion and enhance the utility of NMR methods for identifying and characterizing conformers. Unlike past studies of adducts with such bulky carrier ligands, in which no HH conformer was found, the Me(4)DABPt(d(G*pG*)) adducts did form the HH1 conformer, providing compelling evidence that the sugar-phosphate backbone can impose constraints sufficient to overcome the alkyl-group steric effects. The HH1 conformer exhibits no significant canting. The (S,S)-Me(4)DABPt(d(G*pG*)) adduct has the least amount of the "normal" HH1 conformer and the greatest amount of the ΔHT1 conformer (ΔHT1 = head-to-tail G* bases with Δ chirality) ever observed (88% under some conditions). Thus, our results lead us to hypothesize that the low activity and high toxicity of analogues of cisplatin having carrier ligands with N-alkyl groups arise from the low abundance and minimal canting of the HH1 conformer and possibly from the adverse effects of an abundant ΔHT1 conformer. The new findings advance our understanding of the chemistry of the Pt(d(G*pG*)) macrocyclic ring and of the effects of carrier-ligand steric bulk on the properties of the ring.     

Imprinting structural information from a GpG ligand into the configuration of a chiral diamine ligand through second-sphere communication in platinum(II) complexes

Cisplatin forms the cis-Pt(NH3)2(d(GpG)) cross-link with DNA. We have recently created novel d(GpG) conformations by using "retro models" (complexes having bulky carrier ligands designed to slow d(GpG) dynamic motion). Our results define four conformer classes: HH1, HH2, delta HT1, and delta HT2, with a head-to-head or head-to-tail base orientation and a phosphodiester backbone with a normal (1) or opposite (2) propagation direction. Moreover, each G residue can be syn or anti, and the base canting can be left-handed (L) or right-handed (R). Thus, 32 variants of cis-Pt(NH3)2(d(GpG)) are conceivable, but the adduct is too dynamic to study. Thus far, by using retro models, we have obtained evidence for five variants with d(GpG) but only four with GpG. We therefore selected Me2DAPPt(GpG) complexes for study by 1H and 31P NMR spectroscopy, CD spectroscopy, and molecular mechanics and dynamics (MMD) calculations. Coordinated Me2DAP (N,N'-dimethyl-2,4-diaminopentane) has N, C, C, N chiral centers designated, for example, as R,R,R,R. This ligand has greater flexibility and more readily inverted N centers than ligands used previously in GpG retro models. One goal was to determine whether the GpG ligand can control the configuration of a carrier ligand. (R,R,R,R)-Me2DAPPt(GpG) forms the anti, anti HH1 R variant almost exclusively. Equal populations of the two possible linkage isomers of (S,R,R,R)-Me2DAPPt(GpG) are formed, both favoring the anti, anti HH1 R, variant; however, the isomer with the 5'-G cis to the S nitrogen has sharper signals, suggesting that interligand interactions are more favorable. Indeed, this linkage isomer was the major product of isomerization when (R,R,R,R)-Me2DAPPt(GpG) was kept at pH approximately 9.5 to allow N center equilibration. Steric clashes between the Me2DAP C-Me groups and the G O6 atoms found by MMD calculations appear to disfavor the HH1 conformer of (S,S,S,S)-Me2DAPPt(GpG) and (S,S,S,R)-Me2DAPPt(GpG) complexes. These two complexes have a significant population of the anti, syn delta HT1 conformer, as indicated by broad 1H NMR signals and by 31P NMR and CD data. Equilibration of (S,S,S,R)-Me2DAPPt(GpG) at pH 9.5 leads to a mixture of (S,S,S,S)-Me2DAPPt(GpG) and at least one isomer of (S,S,S,R)-Me2DAPPt(GpG). Thus, second-sphere communication (hydrogen bonding and steric interligand interactions) influences both GpG conformation and Me2DAP configuration.     

The neglected Pt-N(sulfonamido) bond in Pt chemistry. New fluorophore-containing Pt(II) complexes useful for assessing Pt(II) interactions with biomolecules

Treatment of cis-Pt(Me2SO)2Cl2 with DNSH-tren afforded [Pt(DNSH-tren)Cl]Cl and with DNSH-dienH, under increasingly more basic conditions, led to Pt(DNSH-dienH)Cl(2), Pt(DNSH-dien)Cl, and Pt(DNS-dien). (DNSH = 5-(dimethylamino)naphthalene-1-sulfonyl, linked via a sulfonamide group to tris(2-aminoethyl)amine (DNSH-tren) and diethylenetriamine (DNSH-dienH); the H's in DNSH-dienH designate protons sometimes lost upon Pt binding, i.e., sulfonamide NH for the dienH moiety and H8 for the DNSH moiety). Respectively, the three neutral DNSH-dienH-derived complexes are difunctional, monofunctional, and nonfunctional and exhibit decreasing fluorescence in this order as the dansyl group distance to Pt decreases. 2D NMR data establish that Pt(DNS-dien) has a Pt-C8 bond and a Pt-N(sulfonamido) bond. Pt(DNSH-dien)Cl and [Pt(DNSH-tren)Cl]Cl bind to N7 of 6-oxopurines (e.g., 5'-GMP, 3'-IMP, and 9-ethylguanine) and sulfur of methionine (met). Competition and challenge reactions for Pt(II) with met and 5'-GMP typically reveal that met binding is favored kinetically but that 5'-GMP binding is favored thermodynamically. This common type of behavior was found for [Pt(DNSH-tren)Cl]Cl. In contrast, Pt(DNSH-dien)Cl had reduced kinetic selectivity for met. This unusual behavior undoubtedly arises as a consequence of the bound Pt-N(sulfonamido) group, which donates strongly to Pt (as indicated by relatively upfield dien NH signals) and which places the bulky DNSH moiety close to the monofunctional reaction site. The decrease in the relatively upfield shifts of the DNSH group signals indicates that this group stacks with the purine. This stacking could explain the unprecedented, relatively low reactivity of a Pt complex bearing a dien-type ligand toward met vs 5'-GMP.     

Pyridine-carboxylate complexes of platinum. Effect of N,O-chelate formation on model bifunctional DNA-DNA and DNA-protein interactions

This paper reports on the chemistry of platinum complexes containing bidentate pyridine-carboxylate (pyAc = pyridin-2-yl-acetate and picEt = pyridine-2-ethylcarboxylate, ethylpicolinate) (N,O) ligands. The pyridine-2-acetate and ethylpicolinate ligands form six- and five-membered chelates, respectively, upon formation of the Pt-carboxylate bond. In all reactions with picEt with various platinum complex starting materials, spontaneous de-esterification of the pendant carboxylate ester occurs to give directly the chelates K[PtCl(2)(pic-N,O)]-trans-[Pt(pic-N,O)(2)] and SP-4,2-[PtCl(pic-N,O)(NH(3))] without any evidence of intermediates. The de-esterification is solvent dependent, and molecular modeling was used to explain this reaction. The reactions of the geometric isomers of [PtCl(pyAc-N,O)(NH(3))] with 5'-guanosine monophosphate, 5'-GMP, and N-acetyl-l-methionine, AcMet, were investigated by NMR spectroscopy. The objective was to ascertain by model chemistry the feasibility of formation of ternary DNA-Pt-protein adducts in biology. Model nucleotide and peptide compounds were formed in situ by chloride displacement giving [PtL(pyAc-N,O)(NH(3))](+) (L = 5'-GMP or AcMet). Competitive reactions were then examined by addition of the complementary ligand L. Sulfur displacement of coordinated 5'-GMP was slow. For SP-4,3-[Pt(AcMet)(NH(3))(PyAc-N,O)](+), a rapid displacement of the sulfur ligand by 5'-GMP was observed, giving SP-4,2-[Pt(5'-GMP-N7)(pyAc-N,O)(NH(3))](+).     

Novel binding interactions of the DNA fragment d(pGpG) cross-linked by the antitumor active compound tetrakis(mu-carboxylato)dirhodium(II,II)

Insight into the N7/O6 equatorial binding interactions of the antitumor active complex Rh(2)(OAc)(4)(H(2)O)(2) (OAc(-) = CH(3)CO(2)(-)) with the nucleotide 5'-GMP and the DNA fragment d(pGpG) has been obtained by one- (1D) and two-dimensional (2D) NMR spectroscopy. The lack of N7 protonation at low pH values and the significant increase in the acidity of N1-H (pK(a) approximately 5.6 as compared to 8.5 for N7 only bound platinum adducts), indicated by the pH dependence study of the H8 (1)H NMR resonance for the HT (head-to-tail) isomer of Rh(2)(OAc)(2)(5'-GMP)(2), are consistent with bidentate N7/O6 binding of the guanine. The H8 (1)H NMR resonance of the HH (head-to-head) Rh(2)(OAc)(2)(5'-GMP)(2) isomer, as well as the 5'-G and 3'-G H8 resonances of the Rh(2)(OAc)(2) [d(pGpG)] adduct exhibit pH-independent titration curves, attributable to the added effect of the 5'-phosphate group deprotonation at a pH value similar to that of the N1 site. The enhancement in the acidity of N1-H, with respect to N7 only bound metal adducts, afforded by the O6 binding of the bases to the rhodium centers, has been corroborated by monitoring the pH dependence of the purine C6 and C2 (13)C NMR resonances for Rh(2)(OAc)(2)(5'-GMP)(2) and Rh(2)(OAc)(2) [d(pGpG)]. The latter studies resulted in pK(a) values in good agreement with those derived from the pH-dependent (1)H NMR titrations of the H8 resonances. Comparison of the (13)C NMR resonances of C6 and C2 for the dirhodium adducts Rh(2)(OAc)(2)(5'-GMP)(2) and Rh(2)(OAc)(2) [d(pGpG)] with the corresponding resonances of the unbound ligands at pH 8.0, showed substantial downfield shifts of Deltadelta approximately 11.0 and 6.0 ppm, respectively. The HH arrangement of the bases in the Rh(2)(OAc)(2) [d(pGpG)] adduct is evidenced by intense H8/H8 ROE cross-peaks in the 2D ROESY NMR spectrum. The presence of the terminal 5'-phosphate group in d(pGpG) results in stabilization of one left-handed Rh(2)(OAc)(2) [d(pGpG)] HH1 L conformer, due to the steric effect of the 5'-group, favoring left canting in cisplatin-DNA adducts. Complete characterization of the Rh(2)(OAc)(2[d(pGpG)] adduct revealed notable structural features that resemble those of cis-[Pt(NH(3))(2) [d(pGpG)]]; the latter involve repuckering of the 5'-G sugar ring to C3'-endo (N-type) conformation, retention of C2'-endo (S-type) 3'-G sugar ring conformation, and anti orientation with respect to the glycosyl bonds. The superposition of the low energy Rh(2)(OAc)(2) [d(pGpG)] conformers, generated by simulated annealing calculations, with the crystal structure of cis-[Pt(NH(3))(2) [d(pGpG)]], reveals remarkable similarities between the adducts; not only are the bases almost completely destacked upon coordination to the metal in both cases, but they are favorably poised to accommodate the bidentate N7/O6 binding to the dirhodium unit. Unexpectedly, the two metal-metal bonded rhodium centers are capable of engaging in cis binding to GG intrastrand sites by establishing N7/O6 bridges that span the Rh-Rh bond.     

Platinum complexes with NH groups on the carrier ligand and with only one guanine or hypoxanthine derivative. Informative models for assessing relative nucleobase and nucleotide hydrogen-bond interactions with amine ligands in solution

Complexes of the type syn-(R,S)-Me(3)dienPtL (Me(3)dien = N,N',N' '-trimethyldiethylenetriamine; L = guanine or hypoxanthine derivative) have two rotamers, a feature useful for assessing hydrogen-bond interactions between a Me(3)dien NH group and either the O6 or the phosphate group of the coordinated L. The two rotamers are defined as endo and exo for the rotamer with the six-membered ring of the purine on the same side and on the opposite side, respectively, of the coordination plane as the N-Me's. For L = 5'-GMP and 5'-IMP the endo rotamer is the exclusive form (at neutral and basic pH) or is present at 90% and more (low pH where 5'-phosphate group is protonated). A 5'-phosphate group can be positioned to form a direct H-bond with a Me(3)dien NH group only in the endo form; such an H-bond explains this high endo preference. Such a direct phosphate-NH H-bond is not possible for other complexes used in this study because either L has no phosphate group (9-EtG, Guo) or the phosphate is at the 3'-position (3'-GMP and 3'-IMP), too far for H-bonding. Nevertheless, a preference for the endo rotamer was observed for these L also. This result is opposite to that expected both from potential steric repulsion of the L O6 with the N-Me groups and also from the lack of a potential favorable H-bond interaction between L O6 and a Me(3)dien NH. For the 9-EtG adduct, the temperature dependence of the endo/exo equilibrium and the activation parameters for endo/exo interconversion suggest that the preference for the endo rotamer arises from the hydration of the Me(3)dien NH groups; such hydration is favorable in the endo rotamer. At basic pH, N1H deprotonation increases the H-bond capacity of O6, and the exo rotamer increases in stability, becoming the dominant rotamer for the 9-EtG and Guo adducts. For L = 3'-GMP and 3'-IMP, stabilization of the endo form upon phosphate deprotonation at neutral pH was observed. This result is attributed to an H-bonding network involving water, the 3'-phosphate, and the Me(3)dien NH groups.     

ortho-Metallated complexes of platinum(II) and diplatinum(I) containing the carbanions (2-diphenylphosphino)phenyl and (2-diphenylphosphino)-n-tolyl (n = 5, 6)

When the ortho-metallated complexes cis-[Pt(kappa(2)-C6H3-5-R-2-PPh2)2] (R = H 1, Me 2) are either heated in toluene or treated with CO at room temperature, one of the four-membered chelate rings is opened irreversibly to give dinuclear isomers [Pt2(kappa(2)-C6H3-5-R-2-PPh2)2(mu-C6H3-5-R-2-PPh2)2] (R = H 10, Me 11). A single-crystal X-ray diffraction study shows the Pt...Pt separation in 10 to be 3.3875(4) A. By-products of the reactions of 1 and 2 with CO are polymeric isomers (R = H 13, Me 14) in which one of the P-C ligands is believed to bridge adjacent platinum atoms intermolecularly. In contrast to the behaviour of 1 and 2, when cis-[Pt(kappa(2)-C6H3-6-Me-2-PPh2)2] (cis-3) is heated in toluene, the main product is trans-3, and reaction of cis-3 with CO gives a carbonyl complex [Pt(CO)(kappa(1)-C-C6H3-6-Me-2-PPh2)(2-C6H3-6-Me-2-PPh2)] 15, in which one of the carbanions is coordinated only through the carbon. Formation of a dimer analogous to 10 or 11 is sterically hindered by the 6-methyl substituent. Comproportionation of 1 or 2 with [Pt(PPh3)2L] (L = PPh3, C2H4) gives diplatinum(I) complexes [Pt2(mu-C6H3-5-R-2-PPh2)2(PPh3)2] (R = H 16, Me 17). An X-ray diffraction study shows that 17 contains a pair of planar-coordinated metal atoms separated by 2.61762(16) A. There is no evidence for the formation of an analogue containing mu-C6H3-6-Me-2-PPh2. The axial PPh3 ligands of 16 are readily replaced by ButNC giving [Pt2(mu-2-C6H4PPh2)2(CNBut)2] 18, which is protonated by HBF4 to form a mu-hydridodiplatinum(II) salt [Pt2(mu-H)(mu-2-C6H4PPh2)2(CNBut)2]BF4 [21]BF4. The J(PtPt) values in [21]BF4 and 18, 2700 Hz and 4421 Hz, respectively, reflect the weakening of the Pt-Pt interaction caused by protonation. Similarly, 16 and 17 react with the electrophiles iodine and strong acids to give salts of general formula [Pt2(mu-Z)(mu-C6H3-5-R-2-PPh2)2(PPh3)2]Y (Y = Z = I, R = H 19+, Me 20+; Z = H, Y = BF4, PF6, OTf, R = H 22+; Z = H, Y = PF6, R = Me 23+). A single-crystal X-ray diffraction study of [23]PF6 shows that the cation has an approximately A-frame geometry, with a Pt-Pt separation of 2.7888(3) A and a Pt-H bond length of 1.62(1) A, and that the 5-methyl substituents have undergone partial exchange with the 4-hydrogen atoms of the PPh2 groups of the bridging carbanion. The latter observation indicates that the added proton of [23]+ undergoes a reversible reductive elimination-oxidative addition sequence with the Pt-C(aryl) bonds.     

Application of capillary electrophoresis-mass spectrometry for the investigation of the binding behavior of oxaliplatin to 5'-GMP in the presence of the sulfur-containing amino acid L-methionine

Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) has been used for investigating the influence of the sulfur containing amino acid L-methionine (L-Met) on the binding behavior of oxaliplatin (trans-R,R-diaminocyclohexane-(oxalato)platinum(II)) to 5'-GMP. L-Methionine competes with 5'-GMP for the platinum binding site and forms as well as 5'-GMP adducts with oxaliplatin. The formation of the prognosed complexes [Pt(DACH)(L-Met-S,N)]+ and [Pt(DACH)(5'-GMP)2]2- (DACH = 1,2-diaminocyclohexane) could be proved directly by using CE-ESI-MS. Furthermore, we could now bring forward proofs, that the coordination of 5'-GMP with oxaliplatin is inhibited by L-methionine and could show, that the 5'-GMP ligands of the [Pt(DACH) (5'-GMP)2]2- complex can be replaced slowly by L-methionine whereas methionine can not be replaced by GMP.     

7-Methylguanine: protonation, formation of linkage isomers with trans-(NH3)2Pt(II), and base pairing properties

Three protonated forms of 7-methylguanine (7-MeGH, 1) with different counter ions, [7-MeGH(2)]X (X = NO(3), 1a; ClO(4), 1b; BF(4), 1c) and two Pt(II) complexes, trans-[Pt(NH(3))(2)(7-MeGH-N9)(2)](ClO(4))(2) (4) and trans-[Pt(NH(3))(2)(7-MeGH-N9)(7-MeGH-N3)](ClO(4))(2)·3H(2)O (5) are described and their X-ray crystal structures are reported. 1a-1c form infinite ribbons via pairs of intermolecular hydrogen bonds between N1H···O6 and N3···N2H(2) sites, with anions connecting individual ribbons, thereby generating extended sheets. 4 and 5 do not display unusual features, except that 5 represents a rare case of a bis(nucleobase) complex of Pt(II) in which linkage isomers occur. Unlike in a previously reported compound, [Pt(dien)(7-MeGH-N9)](NO(3))(ClO(4)), the Pt coordination planes and the 7-MeGH planes are not coplanar in 4 and 5. The hydrogen bonding behaviour of 7-MeGH, free and when platinated at N9 (complex 4), was studied in Me(2)SO-d(6). It revealed the following: (i) there is no detectable self-association of 1 in Me(2)SO solution. (ii) 1 and 1-methylcytosine (1-MeC) form Watson-Crick pairs. (iii) 4 does not self-associate. (iv) 4 associates with 1-MeC in the Watson-Crick fashion. (v) 4 and 1 interact in solution, but no model can be proposed at present. (vi) Remarkable interaction shifts between 4 and 1 occur when NH(3) is liberated from trans-(NH(3))(2)Pt(II) to give NH(4)(+) in Me(2)SO-d(6). Feasible models, which imply the presence of deprotonated 7-MeG(-) species are proposed. Finally, DFT calculations were carried out to qualitatively estimate the effect of 7-MeGH acidity in [Pt(dien)(7-MeGH-N9)](2+) in dependence of the dihedral angle between the Pt coordination plane and the nucleobase.     

HPIC-UV-ICP-SFMS study of the interaction of cisplatin with guanosine monophosphate

Interaction of cis-[Pt(NH3)2Cl2] (cisplatin) with 5'-guanosine monophosphate (5'-GMP) has been investigated for the first time by on-line coupling of high performance ion chromatography (HPIC) to inductively coupled plasma sector field mass spectrometry (ICP-SFMS). The time-dependent reaction course of the cisplatin-5'-GMP system was followed after incubation under simulated physiological conditions by monitoring the decrease in the concentration of 5'-GMP and the increase in the concentration of formed adducts, on the basis of speciation analysis. Because of the two-step mechanism an intermediate mono adduct was observed together with the major product, the bis adduct cis-[Pt(NH3)2(GMP)2]2-. The data obtained correlated well with those from earlier studies employing orthogonal techniques such as capillary electrophoresis (CE). Furthermore, HPIC-ICP-SFMS provided unambiguous stoichiometric information about the major GMP-adduct. For this purpose the platinum-to-phosphorus ratio was determined by simultaneously measuring 31P and 195Pt. To separate significant interferences from 15N16O+, 14N16O1H+, 12C18O1H+, and 13C17O1H+ on 31P, high-mass resolution (m/deltam = 4,500) proved to be mandatory. The P/Pt signal ratio of 2/1 obtained corresponds to the molar ratio in the bis adduct cis-[Pt(NH3)2(GMP)2]2-.     

Aryl, methyl-diplatinum complexes each with a metal-metal donor-acceptor bond and bridging 2-diphenylphosphinopyridine (PN) ligands: general synthetic approach and mechanism of isomerization

A general synthetic method has been designed to prepare a series of unsymmetrical cationic organo-diplatinum complexes each containing two bridging 2-diphenylphosphinopyridine (PN), PPh(2)py, ligands and a platinum-platinum donor-acceptor bond. Thus, reaction of cis-[PtR(2)(SMe(2))2] (R = Ph, p-MeC(6)H(4) or p-FC(6)H(4)), 1, or cis,cis-[R(2)Pt(micro-SMe(2))(2)PtR(2)](R = Me) with 2 equiv. or 4 equiv., respectively, of PN in CH(2)Cl(2) gave cis-[PtR(2)(PN-kappa(1)P)(2)], 2. When complex 2 was reacted with 1 equiv. of HX (X = CF(3)COO) in CH(2)Cl(2), an approximately 2 : 1 mixture of trans-[PtRX(PN-kappa(1)P)(2)], 3, and [PtR(eta(2)-PN)(PN-kappa(1)P)]X, 4, was obtained. The reaction of one equiv. of the latter monomeric mixture with 0.5 equiv. of cis,cis-[R'(2)Pt(micro-SMe(2))(2)PtR'(2)] (R' = Me) or one equiv. of cis-[PtR'(2)(SMe(2))(2)] (R' = p-MeC(6)H(4)) in CH(2)Cl(2) immediately gave the head-to-head (HH) stereoisomer of the diplatinum complex hh-[RPt(micro-PN)(2)PtR'(2)]X, 6. However, the same reaction in benzene gave the corresponding head-to-tail (HT) stereoisomer ht-[RPt(micro-PN)(micro-NP)PtR'(2)]X, 9, in pure form after a few hours. The conversion of the HH isomer 6 to the HT isomer 9 in CH(2)Cl(2) took place very slowly during 10 d, while the conversion in C(6)H(6) was much faster and took place over 5 h. Based on the observations, a mechanism for the conversion of the kinetic HH stereoisomer to the thermodynamic HT stereoisomer is suggested which involves association of X- with the N(2)PtR'(2) center following by one-arm dissociation of one of the PN bridging ligands from the nitrogen terminal in the HH isomer, and subsequent exchange of the ligating atom and reformation of the HT arrangement. The methyl-di p-tolyl dimer ht-[MePt(micro-PN)(micro-NP)Pt(p-MeC(6)H(4))(2)]X, 9e, in solution gradually isomerizes to ht-[(p-MeC(6)H(4))Pt(micro-PN)(micro-NP)PtMe(p-MeC(6)H(4))]X, 11, by an aryl ligand transfer. All the complexes were fully characterized using multinuclear (1H, 31P and 195Pt) NMR spectroscopy and the complexes ht-[PhPt(micro-PN)(micro-NP)PtMe(2)]X, 9a, and ht-[(p-MeC(6)H(4))Pt(micro-PN)(micro-NP)PtMe(p-MeC(6)H(4))]X, 11, were further characterized by single crystal X-ray crystallography.     

Influence of carrier ligand NH hydrogen bonding to the O6 and phosphate group of guanine nucleotides in platinum complexes with a single guanine ligand

Coordinated N,N',N"-trimethyldiethylenetriamine (Me3dien) has several possible configurations: two have mirror symmetry (R,S configurations at the terminal nitrogens) and the terminal N-Me's anti or syn with respect to the central N-Me (anti-(R,S) and syn-(R,S) isomers, respectively), and two are nonsymmetrical (R,R and S,S configurations at terminal nitrogens, rac denotes a 1:1 mixture of the two isomers). For each configuration, two Me3dienPtG atropisomers can be formed (anti or syn orientation of central N-Me and G 06, G = guanine derivative), and these can be observed since the terminal N-Me's decrease the rate of G rotation about the Pt-N7 bond. In symmetrical syn-(R,S)-Me3dienPtG derivatives with G = 9-EtG and 3'-GMP, the anti rotamer, which can form O6-NH H-bonds, was slightly favored over the syn rotamer but never more than 2:1. This anti rotamer is also favored by lower steric repulsion between the terminal N-Me's and G O6; thus, the contribution of O6-NH H-bonding to the stability of the anti rotamer could be rather small. With G = 5'-GMP, an O6-NH H-bond in the anti rotamer and a phosphate-NH H-bond in the syn rotamer can form. Only the syn rotamer was detected in solution, indicating that NH H-bonds to 5'-phosphate are far more important than to O6, particularly since steric factors favor the anti rotamer. Interconversion between rotamers was faster for syn-(R,S)- than for rac-Me3dien derivatives. This appears to be determined by a smaller steric impediment to G rotation of two "quasi equatorial" N-Me's, both on one side of the platinum coordination plane (syn-(R,S) isomer), than one "quasi equatorial" and one "quasi axial" N-Me on either side of the coordination plane (rac isomer).