Fachbereich Chemie, Universität Dortmund, Germany Metal Coordination to Nucleobases and its Consequences: Group Acidifications, Migration Processes and Multiple Metalation Marta Garijo Añorbe Ph.D. Thesis submitted to the Fachbereich Chemie der Universität Dortmund, Germany for obtaining the degree of a Doktor der Naturwissenschaften Ph.D. Advisor: Prof. Dr. Bernhard Lippert Referee: PD Dr. Andrea Erxleben This work was carried out between August 2002 and January 2006 under the supervision of Prof. Dr. B. Lippert at the Chair of Bioinorganic Chemistry, Fachbereich Chemie, Universität Dortmund, Germany. My special thanks go to my Ph. D. Supervisor Prof. Dr. Bernhard Lippert for the opportunity to carry out research in his group, the interesting topic, the chemistry that I learnt from him, the kindness with which he has always treated me and for all his help. I would also like to thank Dr. Jens Müller and Dr. Gabi Trötscher-Kaus for carefully reading my thesis and correcting my English; Fabian (“El Genio”) for helping me to translate my English summary into German; Dr. Andrea Erxleben for kindly acting as referee; my colleagues in the laboratory and the office, Patrick Lax, Dr. Michael Roitzsch, Dr. Michael Willermann, Dr. Ralf Nowak, Marta Morell, Dr. Pablo Sanz, Dr. Deepali Gupta, Dr. Kathrin Schmidt, Dr. Clodagh Mulcahy, Dr. David Amantia, Pilar Brandi and especially to Myriam Gil and Weizheng Shen for the good atmosphere in the office and the great time we spent together; Marta Morell, Dr. Thorsten Oldag, Dr. Pablo Sanz and Dr. Uwe Zachwieja for their help with the X-ray structures and for teaching me all I know about crystallography; Dr. Jens Müller and Marta Morell for measuring my NMR samples after working hours; Dr. Pablo Sanz for recording 195Pt NMR spectra. Patrick Lax for the DFT calculations; Thorsten Grund for his help with technical support; Prof. Dr. Burkhard Costisella and Annette Danzmann for the innumerable NMR spectra that they carried out (too many pD dependences!), for the nice conversations and for the good work atmosphere; Markus Hüffner for the exact determinations of the elemental analysis and Wilga Buß for recording the Raman spectra; Michaela Market (and Lara), Dr. Dinah Dux, Dr. Andrea Erxleben, Dr. Jola Hermann, Dr. Irene Szymanski, Barbara Müller, Lars Holland, Fabian Polonius, Dominik Böhme, Oliver Gerbersmann, Simone Goritz, Dr. Elisa Barea, Christine Klimek, Sultan Cosar and Mahdi Hadjizadeh-Ziabari for the the nice atmosphere at work; Dr. Bülent Ceyhan for being there when I needed to talk to someone about chemistry and life; Javier Cuesta for his chemical advices. Prof. Dr. Helmut Sigel and his group for their cooperation with some of the pD dependences; Simon Albracht and Olga Karsten, my “Auszubildende”, for their help in preparing compounds; Lucia Staude for her contribution to the section 2.10; Birgit Thormann for her help from the beginning until the end and Burkhard Wellnitz for his “lustige” jokes; Prof. Dr. Luis Oro and Prof. Dr. Fernando Lahoz for the Erasmus Stipendium and the possibility of coming to Dortmund; Dr. Miguel Sanz, Pilar Flores, Inés López, Myriam Gil and Dr. Joaquín Gomis for their friendship and for being always there when I needed them; also for the good time we spent together in Dortmund; Magnus Nigmann for his patience with me and for helping me in a number of ways, especially with my German; Anastasia Bouziani and Rebecca Friese for supporting me during these years in our “WG” and for their friendship; the team from Limericks (Irish pub), where I had great times, especially to Sherie, Steve, Stan, Cherry Q. and Yusuf, for improving my English; all my Spanish friends, that they were always waiting for me in Spain. Do not worry, I will come back soon! my family, Ángel, Maribel, Beatriz, Angelines, Pepe, Raúl, Pilar, Jesús for their encouragement and support. I would like to thank in particular my parents for their help and support during these years. I dedicate this thesis to them. Dedicado a mis padres. Index 1 INTRODUCTION..........................................................................................1 2 RESULTS AND DISCUSSION.....................................................................7 2.1 1H NMR Method for Determination of pKa .............................................7 2.2 The pKa Values of Nucleobases.............................................................8 2.3 Electrostatic Effects and H bonds.......................................................10 2.4 Altered pKa Values of Nucleobases in Metal Complexes ..................14 2.5 9-MeA System .......................................................................................15 2.5.1 N6H2 Acidification of Adenine by a Single PtII ............................................................ 17 2.5.2 N6H2 Acidification of Adenine by Twofold PtII Binding through N1 and N7 ................ 19 2.5.2.1 Mixed Adenine/Cytosine Complexes of cis- and trans-a2Pt(II) .......................... 20 2.5.2.2 trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8) as Starting Compound.... 21 2.5.2.2.1 Crystal Structure of trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8)..... 21 2.5.2.2.2 NMR Spectroscopy of trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8) 26 2.5.2.2.3 pH Dependence of trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8) ..... 27 2.5.2.3 trans-[(NH3)2Pt(N7-9-MeA)(N7-9-MeA-N1)(dienPt)]4+ (9) .................................. 28 2.5.2.4 Comparison of trans-[(NH3)2Pt(N7-9-MeA-N1)2(dienPt)]4+ (9) with trans- [(NH3)2Pt(N7-9-MeA-N1)2(dienPt)2] 6+ (10) .......................................................... 32 2.5.3 Acidification of N6H2 in Trinuclear Bis(Adenine-N1,N7) Complexes with cis- and trans- Geometries.................................................................................................................. 33 2.5.3.1 Extent of Formation of H-Bonded Species......................................................... 36 2.5.3.2 Protonation of cis-[(NH3)2Pt{(N1-9-MeA-N7)Pt(NH3)3}2](NO3)6·2H2O (11) and cis- [(NH3)2Pt{(N7-9-MeA-N1)Pt(dien)}2](NO3)6 (12) ................................................. 37 2.5.3.3 Quantum-mechanical Calculations of cis-[(NH3)2Pt{(N1-9-MeA- N7)Pt(NH3)3}2](NO3)6·2H2O (11) ......................................................................... 37 2.5.3.4 Mixed Adenine/Guanine Complexes of cis- and trans-a2Pt(II)........................... 38 2.5.3.4.1 trans,trans,trans-[(NH3)2Pt(N7-9-MeA-N1)(dienPt)(N7-9-EtA-N1){(NH3)2Pt(9- MeGH-N7)}](ClO4)3(NO3)3 (13) ...................................................................... 39 2.5.3.4.2 Comparison of (13) with trans,trans,trans-{(NH3)2Pt(N7-9-MeA- N1)2[(NH3)2Pt(9-EtGH-N7)]2} 6+ (14) ............................................................... 44 2.5.4 PtII Migration from N1 to N6 in 9-Methyladenine ........................................................ 46 2.5.4.1 Migration in trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8) ..................... 46 2.5.4.2 trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N7,N6)](ClO4)3·3.5H2O (15).................... 50 2.5.4.3 pKa Value of N7,N6-diplatinated 9-MeA in (15).................................................. 56 Index 2.5.4.4 PtII Migration in Bis(9-MeA) Complexes............................................................. 56 2.5.4.4.1 [(9-MeA-N7)Pt(NH3)3]Cl2·2H2O as Starting Compound (16).......................... 57 2.5.4.5 Linkage Isomerization of cis-[(NH3)2Pt(N1-9-MeA-N7)2{Pt(NH3)3}2](NO3)6 (11). 61 2.5.4.6 cis-[(NH3)2Pt(N1-9-MeA-N7)(N6-9-MeA-N7){Pt(NH3)3}2]5+ (17) ......................... 63 2.5.4.7 cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2](NO3)4·6H2O (18) ............................... 66 2.5.5 Multiple Metalation of 9-Methyladenine ...................................................................... 71 2.5.5.1 Palladium Binding to 9-Methyladenine............................................................... 72 2.5.5.1.1 NMR Studies with dienPdII and 9-MeA .......................................................... 74 2.5.5.1.2 {[(dien)Pd]3(9-MeA─-N1,N7,N6)}Cl3.5(PF6)1.5·3H2O (19) ................................ 79 2.5.6 Tris(cytosine) Complexes ........................................................................................... 84 2.5.6.1 PtC3A.................................................................................................................. 84 2.5.6.2 Water Cluster: [Pt(1-MeC-N3)3(OH)](ClO4)0.5(OH)0.5·7H2O (21) ........................ 87 2.6 1,9-DimeAH+ System.............................................................................95 2.6.1 pKa values of trans-[(NH3)2Pt(1,9-DimeAH-N7)(1-MeC-N3)](NO3)3 (22) .................... 96 2.6.2 pKa values of trans-[(NH3)2Pt(1,9-DimeAH-N7)(9-MeGH-N7)](NO3)3 (23) ................. 97 2.6.3 Migration of CH3 in 1,9-DimeAH+ and Dimroth Rearrangement................................. 98 2.6.3.1 Possibility of Migration in trans-[(NH3)2Pt(1,9-DimeAH-N7)(1-MeC-N3)](NO3)3 (22)...................................................................................................................... 99 2.6.3.2 trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)](NO3)2 (24) .................................. 101 2.6.3.3 Possibility of Migration in trans-[(NH3)2Pt(1,9-DimeAH-N7)(9-MeGH-N7)](NO3)3 (23).................................................................................................................... 106 2.6.3.4 trans-[(NH3)2Pt(6,9-DimeA-N7)(9-MeGH-N7)](NO3)2·5H2O (25)...................... 107 2.7 1-MeC System .....................................................................................112 2.7.1 trans-[(NH3)2Pt(1-MeC-N3)2]2+ (26)........................................................................... 113 2.7.2 trans-[(NH3)2Pt(9-MeA-N7)(1-MeC-N3)]2+ (27) ......................................................... 114 2.8 Solvent Effects ....................................................................................117 2.8.1 Mixture of Acetone and D2O ..................................................................................... 119 2.8.1.1 20% Acetone / 80% D2O.................................................................................. 119 2.8.1.2 80% Acetone / 20% D2O.................................................................................. 121 2.8.2 Mixture of Methanol and D2O.................................................................................... 122 2.9 Acidity of Aqua Ligands in [(NH3)2Pt(nucleobase)(H2O)]n+..............124 2.9.1 Acidity Constants of trans-[Pt(NH3)2(H2O)2]2+ ........................................................... 125 2.9.2 Comparison with its cis-Isomer................................................................................. 126 2.9.3 Effect of Intramolecular H Bonding on the Basicity of a Metal-Bound Hydroxide..... 127 2.9.3.1 cis- and trans-[(NH3)2Pt(1-MeC-N3)(H2O)]2+ .................................................... 128 2.9.3.2 cis- and trans-[L2Pt(9-MeGH-N7)(H2O)]2+ ........................................................ 130 Index 2.10 Pyrazole System .................................................................................132 2.10.1 trans-[(NH3)2Pt(pzH)Cl](NO3) (28) as Starting Compound ................................... 135 2.10.1.1 NMR Studies .................................................................................................... 140 2.10.1.2 Reaction of trans-[(NH3)2Pt(pzH)2](NO3)2 with trans-[(NH3)2Pt(1-MeC-N3)(H2O)]2+ .......................................................................................................................... 141 2.10.1.3 Reaction of trans-[(NH3)2Pt(pzH)Cl]+ with AgNO3 ............................................ 143 2.10.1.4 Oxidation of Pt(II) to Pt(IV) in trans-[(NH3)2Pt(pzH)Cl]+.................................... 144 2.10.1.4.1 trans,trans,trans-[(NH3)2Pt(pzH)2Cl2](ClO4)2·H2O (32) ............................... 145 2.10.1.5 Reaction of trans-[(NH3)2Pt(pzH)Cl]+ with 9-MeGH ......................................... 149 2.10.1.6 trans,trans-[(NH3)2Pt(9-MeGH-N7)(N1-pz-N2)(NH3)2Pt(1-MeC-N3)](NO3)3 (34) ... .......................................................................................................................... 152 3 EXPERIMENTAL SECTION.....................................................................155 3.1 Instrumentation and Measurements .................................................155 3.1.1 Determination of pH- and pD-values ........................................................................ 155 3.1.2 NMR Spectroscopy ................................................................................................... 155 3.1.3 IR Spectroscopy........................................................................................................ 156 3.1.4 Elemental Analysis.................................................................................................... 156 3.1.5 X-Ray Crystallography .............................................................................................. 156 3.1.6 DFT Calculations ...................................................................................................... 157 3.2 Synthesis of Complexes ....................................................................157 3.2.1 Materials.................................................................................................................... 157 3.2.2 Preparation of Compounds....................................................................................... 158 3.2.2.1 cis-[(NH3)2Pt(1-MeC-N3)(N7-9-MeA-N1)(dienPt)](ClO4)4 (5) ........................... 158 3.2.2.2 trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8) ....................................... 159 3.2.2.3 trans-[(NH3)2Pt(N7-9-MeA)(N7-9-MeA-N1)(dienPt)]4+ (9) ................................ 159 3.2.2.4 trans-[(NH3)2Pt{(N7-9-MeA-N1)(dienPt)}2]6+ (10) ............................................. 160 3.2.2.5 cis-[(NH3)2Pt{(N1-9-MeA-N7)Pt(NH3)3}2](NO3)6·2H2O (11) .............................. 160 3.2.2.6 cis-[(NH3)2Pt{(N7-9-MeA-N1)(dienPt)}2](NO3)6 (12) ......................................... 161 3.2.2.7 trans,trans,trans-[(NH3)2Pt(N7-9-MeA-N1)(dienPt)(N7-9-EtA-N1){(NH3)2Pt(9- MeGH-N7)}]6+ (13) ............................................................................................ 161 3.2.2.8 trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N7,N6)](ClO4)3·3.5H2O (15).................. 162 3.2.2.9 [(9-MeA-N7){Pt(NH3)3}]Cl2·2H2O (16)............................................................... 162 3.2.2.10 cis-[(NH3)2Pt(N1-9-MeA-N7)(N6-9-MeA-N7){Pt(NH3)3}2]5+ (17) ....................... 163 3.2.2.11 cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2](NO3)4·6H2O (18) ............................. 163 3.2.2.12 [{(dien)Pd}3(9-MeA-N1,N7,N6)]Cl3.5(PF6)1.5·3H2O (19) .................................... 163 3.2.2.13 [Pt(1-MeC-N3)3(OH)](ClO4)0.5(OH)0.5·7H2O (21) .............................................. 163 Index 3.2.2.14 trans-[(NH3)2Pt(1,9-DimeAH-N7)(9-MeGH-N7)](NO3)3 (23)............................. 164 3.2.2.15 trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)](NO3)3 (24) .................................. 164 3.2.2.16 trans-[(NH3)2Pt(6,9-DimeA-N7)(9-MeGH-N7)](NO3)2·5H2O (25)...................... 165 3.2.2.17 trans,trans,trans-{(NH3)2Pt(N1-pz-N2)2[(NH3)2Pt(1-MeC-N3)]2}6+ (29)............. 166 3.2.2.18 trans-[(NH3)2Pt{(pzH)Cl}2](NO3)2·H2O (30) ....................................................... 166 3.2.2.19 trans,trans-[(NH3)2Pt(pzH)2Cl2][(NH3)2Pt(pzH)2] (ClO4)4·H2O (31) and trans- [(NH3)2Pt{(pzH)Cl}2](ClO4)2·H2O (32)................................................................ 167 3.2.2.20 trans-[(NH3)2Pt(pzH)(9-MeGH-N7)](NO3)2 (33)................................................ 167 3.2.2.21 trans,trans-[(NH3)2Pt(9-MeGH-N7)(N1-pz-N2)(NH3)2Pt(1-MeC-N3)](NO3)3 (34) ... .......................................................................................................................... 168 4 APPENDIX; X-RAY TABLES...................................................................169 5 SUMMARY...............................................................................................175 6 ZUSAMMENFASSUNG ...........................................................................179 7 RESUMEN ...............................................................................................183 8 REFERENCES.........................................................................................187 List of abbreviations......................................................................................197 List of compounds.........................................................................................199 Tables of the pD dependences.....................................................................201 1. Introduction 1 1 INTRODUCTION The heterocyclic rings of nucleobases are generally not involved in acid- base equilibria, since pKa values (pKa < 4 or pKa > 9) are outside the physiological pH range.[1] This picture is changed if nucleobases are modified, e. g. following alkylation or metal binding. For example, the N1H group in 7,9- dimethylguaninium has a pKa value of 7.22 ± 0.01,[2] compared to a value of 9.56 for 9-methylguanine,[3] and metal binding to N7 generally lowers the pKa value of this proton by one to two log units, depending on the metal, net charge, etc.[2-4] Another example, PtII bonded to N7 of a guanine model base reduces the pKa from 10.1 to ca. 8.8 (Figure 1).[2] HN N N N O H2N -H+ +H+ R CH3 N N N N O H2N R CH3 HN N N N O H2N R PtII -H+ +H+ N N N N O H2N R PtII pKa~7.2 pKa~8.8 1 2 3 4 5 6 9 8 7 71 7,9-dimethylguaninium Figure 1: Examples of modification of pKa values through alkylation or metal binding. It is known that in biological macromolecules the environment of an acidic proton can likewise modify its pKa value, in either direction. In special nucleic acid structures, for example, the “i motif” of hemiprotonated cytosine or DNA 1. Introduction 2 triplex structures containing cytosine⋅guanine⋅cytosinium triplets, this phenomenon is well established.[5] Shifts of the pKa values of nucleobase protons into the physiological pH range are currently of great interest in that they potentially permit acid-base catalysis.[6] In principle, systems with pKa values reasonably close to 7 could act as acid/base catalysts in biological reactions. It has been reported that an adenine residue in ribosomal RNA with the highly unusual pKa value of 7.6 ± 0.2 acts as a catalyst in the ribosomal peptidyl transferase center,[7] and nucleobase functions with near-neutral pKa values have been associated with ribozyme catalysis of the Hepatitis Delta Virus (HDV).[8] This ribozyme was the first RNA enzyme proposed to use a proton- transfer mechanism for catalysis (Figure 2). There is a possibility of hydrogen bonding between the oxygens of the phosphate and this contributes significantly to the ground-state stabilization of the protonated C75 (cytosine 75). The cis- cleavage reaction may be initiated by specific base deprotonation of the 2´-OH group of the first nucleobase (NB), followed by nucleophilic attack at the phosphate. A charge redistribution in the transition state involving a proton transfer from the N3 of protonated C75 to the 5´-oxyanion leaving group drives the reaction equilibrium in favour of the cleavage products. A scheme of the different bases which can attack the 2´-OH group and the different acids is shown in Figure 2. Similarly, an unusual pKa value of 6.5 for an adenine base close to the active site of a Pb-dependent ribozyme (“leadzyme”) has been described.[9] Other authors[10-12] and ourselves[3,4,13,14] have demonstrated in numerous instances that metal binding to a nucleobase acidifies protons of NH groups and, conversely, causes an apparent increase in nucleobase basicity upon metal coordination to a deprotonated, and hence anionic, nucleobase.[15,16] This is because the proton has a stronger polarizing effect than (most) divalent metal ions.[13a] The effect depends on a combination of factors such as the charge of the metal(s), the number of metal ions, the coligands, the distance between the metal center and the proton, and hence the site(s) of metal binding.[10c] 1. Introduction 3 O OHO PO O O O NB O OHO NB O OO NB N N NH2 O R N N N N NH2 R NH N NH2 O R O OO NB HO OH-H+ B N NH HN NH B H N H H H P O O P O O HO-Mn+ 5´ 3´ his basesacids n+M-OH2 O Figure 2: Present views of catalysis in the ribozymes using different bases and acids. In the HDV ribozime, Cytosine 75 with a shifted pKa value is essential for catalysis. In this work, one of the most interesting parts is the effect of twofold PtII binding to N1 and N7 of adenine model nucleobases. Specifically, the acidification of the exocyclic amino group at the 6-position was of interest. As has recently been demonstrated by Lüth et al.,[17] the acidity of this amino group is not only affected by electronic effects of coordinated metal ions (here: PtII at N1 and N7), but in addition by steric effects, viz. an efficient stabilization of the deprotonated amino group (Figure 3). For example, it has been found that while the acidity of this amino group (pKa ca. 16.7[18] of free base) is increased by 4 - 6 log units upon PtII binding at N1 and N7 (pKa ca. 10.8-12.6), it is further 1. Introduction 4 increased (to pKa~7.9) in the complex trans, trans, trans-[(NH3)2Pt(N7-9-MeA- N1)2{(CH3NH2)2Pt(1-MeU-N3)}2](ClO4)4. The drop in this pKa value was attributed to a synergy of electronic effects (Lewis acidification by two metal ions) and favourable geometrical conditions, that is, an efficient stabilization of the deprotonated adenine by intramolecular H-bond formation with the neutral adenine ligand (Figure 4). N N N N NH2 R N N N N N R N N N N N R PtII PtII -H+ +H+ H -H+ +H+ H H2N N N N N NH2 R PtII PtII H2N pKa = 16.7 pKa = 7.9 ~~ 71 9 6 1 6 7 9 Figure 3: Acid-base equilibria of N9-blocked adenine involving neutral and anionic species: the free nucleobase (top) and the N1,N7-diplatinated complex with a record-low pKa value (bottom). 1. Introduction 5 N N N N N N N N N Pt Pt Pt NH3 NH3 CH3H3C U Mea Mea U Mea Mea NH H H Figure 4: Intramolecular H-bond formation between the deprotonated adenine and the neutral adenine ligand.[17] In a sense, the situation is reminiscent of that found in dinuclear complexes of PtIV containing the amido-ammine bridging ligands H5N2-.[19] One of the scopes of this thesis is to examine in more detail the effects of metal coordination on the pKa values of a nucleobase, hence on its acidity and basicity, respectively, and consequences arising from perturbations. These physicochemical properties are of enormous significance not only for the charge of the nucleobase, but also for its tautomeric structure, and its ability to engage in hydrogen bonding and base recognition.[20] Another scope of this thesis is to study the migration of the platinum entity in Pt-complexes. The isomerization reactions of Pt–nucleobase adducts are expected to be rare owing to the inertness and thermodynamic stability of the Pt–N bond.[21] Unfortunately, data on thermodynamic stability constants for Pt–N complexes is very limited because of the inertness of the Pt-compounds. Considering the greater basicity of the N1 site over the N7 site in purine bases, the N7→N1 migration of Pt may be anticipated. In fact, this type of isomerization has been observed in (dien)PtII complexes of inosine[22] and adenosine[23]. Both isomerization reactions have been proposed to follow a similar mechanism, i.e. the change of PtII binding mode proceeds via N1,N7-diplatinated species. 1. Introduction 6 Although findings[22,23] suggest greater thermodynamic strength for the N1 platinated complexes over the N7 bound species, migration of Pt also seems to be possible in the opposite direction. In the case of (dien)PtII, this process only occurs in acidic solution. Under neutral and basic conditions no isomerization was observed. In addition, the N1,N7-diplatinated complex is perfectly stable at pH 2.8. Therefore, it was concluded that protonation of the unplatinated N7 site is necessary for the migration reaction. Very interestingly, this N1→N7 migration was found to occur intramolecularly, since addition of excess of Cl– (a good inactivator for PtII) caused no significant difference in the overall process.[24] It was suggested that PtII remains hydrogen bonded to the C(6)–N group during migration which could explain the rapid and efficient conversion to the N7 bound species, since after breaking the Pt–N1 bond very fast deprotonation (N7) and protonation (N1) take place. Platinum migration from endocyclic to exocyclic nitrogen has also been observed.[25] Migration of PtII from the N1 site to the exocyclic C(6) – NH2 group, proceeds in strongly basic solution without any detectable redox reaction. Two mechanistic explanations may be given for the adenine N1→N6 isomerization, both of which require deprotonation of the exocyclic amino group. First, migration of Pt may be analogous to the Dimroth rearrangement, in which an alkyl group formally migrates from heterocyclic nitrogen to an α-amino or α-imino group.[26] Some examples of this reaction will be explained in Section 2.6.3. 2.1. 1H NMR Method for Determination of pKa 7 2 RESULTS AND DISCUSSION 2.1 1H NMR Method for Determination of pKa There are numerous ways to determine pKa values of chemical compounds experimentally.[27] Potentiometric titration and ultraviolet (UV) spectroscopy are classical methods, for example. These techniques have been applied for nucleobases and also for metal-nucleobase complexes. For the determination of nucleobase pKa values, which are in the pH range of aqueous solutions (pH 0─14), NMR methods are widely used today. Acidity constants have been determined in this work by measuring the pD dependence of the chemical shifts in 1H NMR experiments in D2O. In case of D2O as solvent, 0.4 log units are added to the pH-meter readings (pH*) in D2O to give the corresponding pD values.[28,29] The pD was adjusted with DNO3 and NaOD solutions of different concentrations. The change of the chemical shift of one or several C-H protons in dependence on the pD values was evaluated by a Newton-Gauss non-linear least-squares curve-fitting procedure. Frequently the methyI resonances of the ligands proved to be more suitable for pKa determination than the aromatic protons because they do not undergo isotopic exchange with time. If the molecule has two deprotonation sites –and thus also two pKa values– the relationship between the observed chemical shift (δobs) and the varying pD value is described by equation (1), which was taken from the literature[30] and reformulated for the present situation: )pD2pp()pDp( )pD2pp( BH )pDp( BHB . BH22BHBH BH22BH 2 2 BH 10101 1010 ⋅−+− ⋅−+− +++ ++ + + + ++ ⋅+⋅+= KKK KKK obs δδδδ (1) 2.2. The pKa Values of Nucleobases 8 Equation (1) can be easily adjusted to the situation where only one deprotonation site is present, thus giving equation (2): )pDp( )pDp( BHB obs.. BH BH 101 10 − − + + + + ⋅+= K Kδδδ (2) In equations (1) and (2), δB, δBH+ and δBH22+ represent the chemical shifts of nucleobase species and complexes, which are not protonated (δB), protonated (δBH+) once or twice (δBH22+). The values pKBH+ and pKBH22+are the negative logarithms of the acidity constants of BH+ and BH22+ respectively. These acidity constants, which describe the situation in D2O can be transformed to aqueous solution (H2O) by application of equation (3).[31] 015.1 45.0p p O2D 2OH −= KK (3) Error limits given correspond to one time the standard deviation (1σ). 2.2 The pKa Values of Nucleobases The pKa values of the heterocyclic moieties of the five nucleobases of nucleic acids – guanine (G), adenine (A), cytosine (C), thymine (T), uracil (U) – are well outside the physiological pH range. At physiological pH, the heterocycles are therefore neutral (Fig. 5 and Fig.6). 2.2. The pKa Values of Nucleobases 9 HN N N N NH2 N N N N NH2 N N N N NH HN N N H N O H2N HN N N N O H2N N N N N O H2N HN N NH2 O N N NH2 O N N NH2 O HN N O O R' HN N O O R' N N O O R' H pKa pKa ~ 3.9 ~ 17 AH2 2+ ~ 3.6 ~ 10.0 GH2 2+ G2- ~ 4.6 ~ 17 ~10.1 ~10.5 UH2 2+/ TH2 2+ R R R R R R R R R R R R < -2 A G C UH/TH 1 6 3 7 9 1 2 3 6 7 9 1 2 3 6 1 2 3 6 Figure 5: Lewis formulas of nucleobases A, G, C, U(T) in different protonation states. Only single mesomeric and preferred tautomeric forms are given. Additionally, protonated forms of A, G, U(T) and twofold deprotonated forms of G are not shown. R = CH3, alkyl,....R’ =CH3 2.3. Electrostatic Effects and H bonds 10 A GGH + pH 1470 GH2 2+ AH + G - C - CCH + T - TH2 2+ /TH + Figure 6: Distribution of protonated states of nucleobases in dependence of pH. The pKa values of the nucleobases can be modified through alkylation or metal coordination. In this work, both modifications have been studied. 2.3 Electrostatic Effects and H bonds The understanding of alterations on pKa values and base pairing properties of nucleobases following metal coordination begins with an in-depth understanding of the forces determining the binding strength of metal- nucleobase interactions. Among others, ab initio quantum mechanical calculations have greatly contributed to achieve this goal. Our present understanding of metal-nucleobase bond formation may be summarized as follows. i) The electrostatic interaction between a metal ion and the nucleobase dipole appears to be the major contribution.[32] In the gas phase, this attraction is dominant, but in solution and in the solid state it can be minimized or even canceled as a consequence of countereffects of a polar solvent (e.g., water, and by anions). The large dipole moment of guanine (7.5 D) and its favorable orientation have been proposed to rationalize, at least in the case of the isolated base, the general high affinity of metal ions for the N7 position. In double-helical DNA, unlike in single-stranded DNA, the situation is more complex in that the affinity of metal ions for G-N7 is strongly modulated by the nature of the base on 2.3. Electrostatic Effects and H bonds 11 the 5´-side, hence depends on the molecular electrostatic potential and also on the accesibility of N7.[33] In contrast, the absolute values of the dipole moment of adenine (2.9 D) and its orientation renders it less favorable for N7 metal binding. The steric hindrance of the exocyclic amino group undoubtedly adds to this situation. ii) Hydrogen bonds between coligands of the metal (e.g., aqua or ammonia ligands) and the nucleobase add to complex stability.[34-36] An unexpected and surprising result of the computations was the finding that the exocyclic amino group of adenine in its N7 metal complexes can become substantially pyramidalized and can act as a weak hydrogen-bond acceptor for aqua ligands of the metal.[36] So the “neighbour group effects” on individual pKa values is very important. This term refers to the significance of favorable or unfavorable hydrogen-bonding interactions in stabilizing or destabilizing protonated or deprotonated nucleobase sites. If more than a single nucleobase is attached to the metal, the possibilities for stabilization or destabilization of protonated or deprotonated sites increases. In the system with N1,N7 diplatinated 9-MeA nucleobases, this aspect has been studied in more detail.[37] Depending on the nature of coligands, pKa values for deprotonation of the exocyclic amino group of 9-MeA have been found to vary between a high of ~ 12 and a minimum of 7.9. There are at least two possible interpretations for these findings: (1) Assuming that values of ~ 12 essentially reflect the acidifying effects of the two metal ions, then the two PtII ions were cause of an ~ 105-fold acidification of the exocyclic amino group, and the minimum value of 7.9 means an additional 104-fold acidification due to favorable neighbour group effects. The minimum pKa value observed in a trinuclear complex of trans-a2PtII containing a central (9-MeA-N7)Pt(N7-9-MeA) cross-link and two Pt(1-MeC-N3) units coordinated to the N1 positions of the two 9-MeA ligands,[17] has been attributed to the hydrogen-bond donor properties of the amino group of the neutral 9-MeA, which stabilizes the ─NH─ group of the second adenine base. The role of the amino group of the neutral 9-MeA is thus similat to that of amino groups in certain metalhydroxo species where it likewise 2.3. Electrostatic Effects and H bonds 12 stabilizes the anion and adds to the acidity of the metal aqua complex.[38] A survey of pKa values of a class of related complexes of cis- and trans-a2PtII as well as a3PtII with 9-methyladenine and 9-ethyladenine ligands and other nucleobases[37] seem to be in aggrement with this hypothesis. (2) An alternative interpretation would be to assume a priori additive effects of two metal entities at N1 and N7 in acidifying the exocyclic group, which are reduced by an insufficient stabilization of the deprotonated group due to unfavorable intramolecular hydrogen-bonding interactions. For example, the individual acidifications of the N(6)H2 group of 9-MeA in complexes of dienPtII with N1 coordination (≤ 6 log units) and with N7 coordination (≤ 4 log units) do not add up, as the pKa of the diplatinated complex (pKa ~ 11)[11] displays an acidification of only ~ 6 units. This situation is in contrast to the roughly additive effects of N1 methylation and N7 PtII coordination in trans-[(NH3)2Pt(1,9-DimeAH-N7)2]4+. It is obvious that details of fine tuning of pKa values still need to be settled. However, presently available pKa data in any case support the general view that an appropriately located hydrogen bond donor such as an exocyclic amino group of a nucleobase causes a lowering of the pKa value of N(6)H2 of adenine complexes, whereas a hydrogen bond acceptor such as an exocyclic carbonyl group of another nucleobase, increases the pKa (Figure 7). Considering the fact that in all these compounds am(m)ine coligands of the Pt(II) are present, one might argue that hydrogen bonding among these ligands and the deprotonated adenine site could have provided the stabilization required to reduce the pKa. In fact, DFT calculations are supportive of such a scenario (see Chapter 2.5). At the same time, they reveal an alternative to a direct ─NH─···H2N interaction, namely, via a solvent molecule.[37] We do not wish to exclude such a possibility, even though it does not readily rationalize the spread in pKa values experimentally observed in Pt am(m)ine complexes. If true (for all compounds) pKa values should display only slight variations. 2.3. Electrostatic Effects and H bonds 13 N N N N N Pt CH3 Pt N N N N N Pt CH3 Pt H H H2 N H C O ~~ ~~ 1 1 7 7 +H+ -H+ N N N N N Pt CH3 Pt H C O ~~ 1 7N N N N N Pt CH3 Pt H N ~~ 1 7 H H +H+ -H+ repulsion H bonding H bonding: low pKa repulsion: high pKa H Figure 7: Effects of neighbour groups on N(6)H2 acidity of N1,N7 diplatinated adenine: Low pKa by efficient hydrogen bonding between ─NH2 donor and ─NH─ versus high pKa due to repulsion between ─NH─ and O acceptor. iii) In addition to electrostatics and hydrogen-bond formation, polarization, and charge-transfer effects, also termed nonelectrostatic effects, are active.[39] These refer to changes associated with a redistribution of electrons in the bonds within the nucleobase of the metal complex. They are less influenced by solvent and counterions. Not unexpectedly, substitution of nucleobase protons (e.g., of N─H) by metal ions increases this contribution as compared to binding of the metal to an available lone pair of an endocyclic nitrogen or an exocyclic oxygen atom. The influence of the solvent will be discussed in chapter 2.8. 2.4. Altered pKa Values of Nucleobases in Metal Complexes 14 2.4 Altered pKa Values of Nucleobases in Metal Complexes Metal binding to exocyclic oxygen atoms of nucleobases such as O2 or O4 of T(U), O6 of G, and O2 of C [40, 41, 42-45] is expected to acidify endocyclic NH and exocyclic NH2 protons and reduce the basicity of endocyclic N as well as that of other carbonyl groups (T, U). The effect of metal coordination to any of the endocyclic N atoms can be also expressed in terms of a loss in basicity of the remaining endocyclic N atoms, hence in a reduced tendency to accept a proton and to become a metalated cation. Nucleid acids are composed of nucleosides which are joined through phosphodiester linkages. Nucleosides are composed of a nucleobase to a ribose ring. Nucleosides can be phosphorylated by specific kinases in the cell, producing nucleotides, which are the molecular building blocks of DNA and RNA. Each nucleotide consists of a cyclic sugar (ß-D-ribose in RNAs, ß-D-2´ deoxyribose in DNA), which is phosphorylated in the 5´position of the sugar and carries a heterocyclic ring at the C1´ position (ß-glycosyl C1´-N bond). The heterocycles are, in general, the purine bases guanine, adenine and the pyrimidine bases cytosine as well as thymine (DNA) or uracil (RNA). There are many more reports on pKa changes of nucleobase funcionalities in which neutral nucleobases provide endocyclic N atoms as bonding sites. These have been studied in this thesis. First of all, we are going to discuss the case of adenine. 2.5. 9-MeA System 15 2.5 9-MeA System The ligand 9-methyladenine (Figure 8) is used as a model compound for adenosine. The structural difference between adenosine and 9-MeA is the replacement of the sugar moiety by a methyl group in the latter. Adenine was methylated at N9 according to Krüger.[46] N N N N NH2 H 1 3 5 7 9 CH3 H 2 4 6 8 Figure 8: Amino form of 9-methyladenine. N-9 substituted adenines can exist theoretically in four tautomeric forms, with the first one, the amino form (I), being the preferred tautomer (Figure 9). pKa values of 9-MeA and its cationic forms are listed in Table 1. N N N N NH2 CH3 HN N N N NH CH3 N N H N N NH CH3 N N N H N NH CH3 (I) (II) (III) (IV) 1 3 7 9 6 Figure 9: The four possible tautomeric forms of 9-methyladenine: (I): amino tautomer, (II), (III) and (IV): imino tautomers. 2.5. 9-MeA System 16 Table 1: pKa values of 9-methyladenine determined potentiometrically and with UV-spectroscopy [for 9-MeA]. Position pKa values [47] N7 ( at 9-MeAH+-N1 ) -0.37 ± 0.06 N1 ( at 9-MeAH+-N7 ) 1.30 ± 0.31 N7 ( at 9-MeA ) 2.43 ± 0.30 N1 ( at 9-MeA ) 4.10 ± 0.01 Exocyclic amino group NH2 16.7 Adenine provides three unprotonated endocyclic nitrogen atoms (N1, N3, N7) at physiological pH, all of which are potential metal binding sites. The basicity order (affinity for H+) is N1>N7>N3.[48] The N9-substituted purine nucleobase adenine exhibits several potential binding sites for metal ions. These include the ring nitrogens N1, N3 and N7, and the exocyclic amino group. In native DNA, however, various Pt compounds seem to prefer the N7 site [22]. The N7 site of purine nucleobases is the major target for anticancer Pt drugs and related compounds in DNA. Because of the general inertness of Pt(II)/Pt(IV) and the high thermodynamic stability of the Pt-N bond, substitution of the nucleobases in the relevant Pt adducts is expected to be very difficult. In this respect, findings on relatively easy rearrangements of various Pt(II) bisadducts at the oligonucleotide level are of great interest, though the mechanism of the rearrangement is not known yet.[25] We have extended our studies of PtII-adenine complexes to compounds with different amine ligands (cis-[a2PtII], [a3PtII], trans-[ma2PtII], and [dienPtII], where a=NH3 and ma=NH2CH3). In addition, we have employed other nucleobases as coligands (Figure 10). We have been particularly interested in the effect of cytosine since this nucleobase also contains a suitably located exocyclic amino group for stabilization of an NH group of adenine. 2.5. 9-MeA System 17 A7 A7 1A7 1A7 1A7 C3 3C 7A1 1A7 1A7 1A7 T3 1A7 U3 A (NH3)3PtA L A L A 1A 7A A7 3C A7 7A 3C 7A 1 7GH 7GH 7A 1 3T 7A 1 3U 1 4 5 3 6 7 2 11 12 1098 14 13 15 16 or (dien)PtA cis-a2PtAL (a=NH3 or NH2CH3) trans-a2PtAL (a=NH3 or NH2CH3) 1A7 3CC3 GH7 3T 1A7 GH7 3U 1A7 1A7 7A1 Figure 10: Schematic representation of PtII complexes studied in this work. Coordination sites are indicated. 2.5.1 N6H2 Acidification of Adenine by a Single Pt II The acidification of adenine nucleobases by a coordinated metal ion can be expressed in terms of a loss in basicity for accepting a proton at one of the three available endocyclic nitrogen atoms, N1 (preferably), N7, or N3. PtII binding to N7 of a N9-substituted adenine, for example, 9-methyladenine, makes protonation of the preferred N1 position more difficult by approximately 2 log units, that is, the pKa value of N1H drops from 4.10 in free 9-methyladeninium (9-MeAH+) to approximately 2 in its PtII complexes.[13] If PtII is bonded to N1, protonation occurs at N7 with a pKa value of approximately 1.2,[49] and if the N3 site is carrying a PtII entity, the overall basicity of the adenine ring drops by 4 log units, with N7 then being more basic than N1.[13a,50] 2.5. 9-MeA System 18 Table 2: pKa values of the N6H2 group in 9-alkyladenine complexes of PtII.[39] For simplification, the N1 and N9 alkylnucleobases were named with the general symbols of the nucleobases (A, G, C, T, U). [a] Determinated by potentiometry. The remaining values were determinated by 1H NMR spectroscopy (n.d. = not determined). The compounds with numbers were studied in this thesis. Compound Cation Composition pKa1 pKa2 A [(dien)Pt(A-N7)]2+ > 13[11] - 1 [(dien)Pt(A-N1)]2+ > 11 - B [{(dien)Pt}2(A-N1,N7)] 4+ ca. 11[11] - 2 cis-[a2Pt(A-N7)(C-N3)] 2+ > 12.6 - 3 trans-[a2Pt(A-N7)2] 2+ > 12.8 n. d. 4 cis-[a2Pt(A-N7)2] 2+ > 13 n. d. 5 cis-[a2Pt(C-N3)(N7-A-N1)Pt(dien)] 4+ 10.79 6 cis-[{a2Pt(C-N3)}2(A-N1,N7)] 4+ 11.03 - 7 trans-[{a2Pt(C-N3)}2(A-N1,N7)] 4+ 10.00 - C trans,trans-[(ma)2Pt(C-N3)(N1-A-N7)Pta2(GH-N7)] 4+ 10.66[37] - D trans,trans-[(ma)2Pt(A-N7)(N1-A-N7)Pta2(GH-N7)] 4+ 10.08[37] - E trans,trans-[a2Pt(T-N3)(N7-A-N1)Pt(ma)2(GH-N7)] 3+ 12.06[51] - F trans,trans-[a2Pt(U-N3)(N7-A-N1)Pt(ma)2(GH-N7)] 3+ 12.62[51] - 11 cis-[a2Pt{(N1-A-N7)Pta3}2] 6+ 8.7, 9.10[a] 10.7, 10.9[a] 12 cis-[a2Pt{(N7-A-N1)Pt(dien)}2] 6+ 9.23[a] 10.56[a] G1 trans,trans,trans-[a2Pt(N7-A-N1)2{a2Pt(GH-N7)}2] 6+ 8.67[a] 10.96[a] G2 trans,trans,trans-[a2Pt(N7-A-N1)2{(ma)2Pt(GH-N7)}2] 6+ 8.57[a] 10.61[a] H trans,trans,trans-[a2Pt(N7-A-N1)2{a2Pt(T-N3)}2] 4+ 8.61[a] 11.31[a] I trans,trans,trans-[a2Pt(N7-A-N1)2{(ma)2Pt(U-N3)}2] 4+ 7.94[17] 11.66[17] The acidifying effect of metal coordination on any of the ring nitrogen atoms is also reflected by a drop in the pKa value of the exocyclic amino group (Table 2). Of course, this is numerically different from the ΔpKa values measured for the protonated endocyclic nitrogen atoms but it also depends on the site of metal binding. It appears that N1PtII binding to adenine nucleobases causes a larger acidification of the N6H2 group than N7 PtII binding: In the N1(dien)PtII complex of 9-MeA (1) deprotonation starts above pH* 11,[11] and with [(NH3)3Pt(adenosine-N1)]2+ a pKa value of 12.4 has been reported.[12] Our own 2.5. 9-MeA System 19 findings with cis-[(NH3)2Pt(9-MeA-N7)(1-MeC-N3)](ClO4)2⋅H2O (2) and with compounds trans-[(NH3)2Pt(9-MeA-N7)2](ClO4)2⋅2H2O and cis-[(NH3)2Pt(9-MeA- N7)2](NO3)2⋅2H2O (3 and 4, respectively) are also consistent with a relatively moderate effect of the metal ion at N7 (pKa values > 12.6), although it might be argued that the charge effect of the metal is reduced in the bis(nucleobase) complex relative to that in a mono(nucleobase) complex. Anyway, there is more stabilization of deprotonated adenine in case of the trans-isomer. In the 1H NMR spectra, the two deprotonation steps of the trans-[(NH3)2Pt(9-MeA-N7)2]2+ are well separated. For the cis complex, however, the second one occurs at high pD (Figure 11). 0 2 4 6 8 10 12 14 3,80 3,82 3,84 3,86 3,88 3,90 3,92 3,94 3,96 3,98 4,00 4,02 4,04 4,06 trans-[(NH 3 ) 2 Pt(9-MeA-N7) 2 ]2+ (3) cis-[(NH 3 ) 2 Pt(9-MeA-N7) 2 ]2+ (4) (δ) /p pm pD Figure 11: pD dependence of the N(9) CH3 resonance of (3) and (4) in the 1H NMR spectra. The first pKa corresponds to the protonation at N1 and the values are 1.00 ± 0.06 and 1.84 ± 0.04 respectively. 2.5.2 N6H2 Acidification of Adenine by Twofold Pt II Binding through N1 and N7 Coordination of a second PtII entity to an adenine nucleobase causes a more pronounced acidification of the exocyclic adenine amino group and permits ready detection in water. In early studies, pKa values of 11.0 - 11.3 have been 2.5. 9-MeA System 20 determined for [{(dien)Pt}2(9-MeA-N1,N7)]4+(B),[11] and 10.8 was the value reported for [{(NH3)3Pt}2(adenosine-N1,N7)]4+.[12] 2.5.2.1 Mixed Adenine/Cytosine Complexes of cis- and trans-a2Pt(II) The dinuclear mixed adenine/cytosine complexes (5) – (7) studied in this thesis (Figure 10) essentially confirm that coordination of a second PtII entity to an adenine causes a more pronounced acidification of the NH2 group. Still, there is an interesting detail to be noted: For the trans-[(NH3)2PtII] compound (7) a pKa value of 10.0 ± 0.1 is observed, which is significantly lower than the corresponding pKa value of 11.1 ± 0.1 for the cis isomer (6) (Table 2, pag. 18). In two previously described nucleobase triplets containing a central Pt(N1-adenine- N7)Pt unit,[28] trans,trans-[(NH3)2Pt(1-MeT-N3)(N7-9-MeA-N1)Pt(NH2CH3)2(9- EtGH-N7)](ClO4)3⋅5.2H2O (E) and trans,trans-[(NH3)2Pt(1-MeU-N3)(N7-9-EtA- N1)Pt(NH2CH3)2(9-EtGH-N7)]3+ (F), the pKa values for deprotonation of the exocyclic amino group of the adenine nucleobase in water were found to be substantially higher, around 12.1 and 12.6 in both cases. This difference of two log units from the value for (7) clearly suggests a substantial internucleobase effect (Figure 12). Although a charge influence is also likely to play a role, ((7) has a charge of +4; (E) and (F) have charges of +2 once the guanine ligands have undergone deprotonation), it is probably not dominant. For example, (D) has almost the same pKa value (10.08 ± 0.22) as (7), although it has a charge of only +3 once the guanine ligand is deprotonated. An internucleobase effect on the pKa value of adenine is further suggested by a comparison of (C) and (D): Both compounds have identical charges (and identical pKa values of the guanine ligands), yet the pKa values of the bridging adenine ligand are different (10.66 ± 0.03 and 10.08 ± 0.22, respectively). 2.5. 9-MeA System 21 N N N N O NH2 Pt R aa N N N N NH R Pt a a N N O O R Pt aa N N NH2O N N N N H N R Pt N N HN O R a a A U(T) C A C G 7 + repulsion 3+ possible H bond E,F R H Figure 12: Internucleobase effects on deprotonated adenine group N6H– in (E) and (F) (left) and (7) (right). 2.5.2.2 trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8) as Starting Compound In order to study the acidification of N6H2 in a trinuclear bis(adenine- N1,N7), this compound was synthesized. The starting compound trans- [(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8) (with dien = diethylenetriamine) was prepared by reaction of [(dienPt)(9-MeA-N1)]2+ and trans-DDP. As counterion perchlorate was applied. 2.5.2.2.1 Crystal Structure of trans-[(NH3)2ClPt(N7-9-MeA- N1)(dienPt)](ClO4)3 (8) Crystals of trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8) were isolated from an aqueous solution and characterized by X-ray crystallography. 2.5. 9-MeA System 22 The complex (8) crystallizes in the triclinic space system. In the refinement process of the X-ray data, all non-hydrogen atoms of the crystal were refined anisotropically. The hydrogen atoms were placed at geometrical idealized positions and refined isotropically. Crystal data, data collection and refinement parameters for (8) are summarized in Table A-1 (see Appendix). The solid state structure of (8) consists of an adenine nucleobase coordinated to two platinum atoms, which are binding to N1 and N7 of the 9- methyladenine as well as three perchlorate counter ions. The platinum binding through N1 (Pt1), is coordinated to the three nitrogen atoms of the dien ligand and the other one (Pt2), through N7, is in addition coordinated to a chloride and two ammonia ligands in trans-position. A view of the cation trans- [(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (11) with the labeling scheme is shown in Figure 13. The geometry of the (dien)Pt1 moiety is normal and compares well with published data.[52] Thus, the N1E-Pt1-N3E angle deviates markedly from 180° (167.5(4)°), unlike the N2E-Pt1-N1B angle, which is 177.9(4)°. The dien ring displays the characteristic sting ray structure, with C2E and C3E out of the Pt coordination plane by 0.69(2) and 0.44(2) Å, respectively. The other C atoms (C1E and C4E) are by 0.08(2) and -0.18(2) Å out of the platinum coordination plane. Pt-N distances about the Pt1 center range from 1.992(9) to 2.056(9) Å. A list of selected distances and angles between atoms of trans-[(NH3)2ClPt(N7-9- MeA-N1)(dienPt)](ClO4)3 is given in Table 3. The two Pt1-N-C angles are clearly different; in the case of the CH group, the Pt1-N1B-C2B angle is 118.5(8)° and on the side containing the exocyclic amino group, the Pt1-N1B-C6B angle is larger, 121.2(7)° 2.5. 9-MeA System 23 Pt2 Cl Pt1 N7B N1B N21 N22 N1E N3E N2E C1E C2E C3E C4E Figure 13: View of the cation trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)]3+ of (8). Table 3: Selected distances (Å) and angles (º) for non-hydrogen atoms in 8. Pt1-N2E 1.992(9) N2E-Pt1-N1B 177.9(4) Pt1-N1B 2.008(9) N2E-Pt1-N1E 85.3(4) Pt1-N1E 2.044(9) N1B-Pt1-N1E 92.5(4) Pt1-N3E 2.056(9) N2E-Pt1-N3E 84.3(4) Pt2-N7B 2.007(9) N1B-Pt1-N3E 97.8(4) Pt2-N22 2.034(9) N1E-Pt1-N3E 167.5(4) Pt2-N21 2.041(9) C2B-N1B-C6B 120.7(10) Pt2-Cl 2.294(3) N7B-Pt2-N22 92.0(4) N7B-Pt2-N21 89.0(4) N22-Pt2-N21 179.0(4) N7B-Pt2-Cl 179.1(3) N22-Pt2-Cl 88.0(3) N21-Pt2-Cl 91.1(3) The trans-(NH3)2ClPtII entity presents normal distances and angles between atoms. 2.5. 9-MeA System 24 The atoms of the 9-methyladenine base are coplanar, with a r.m.s. deviation of 0.014. The dihedral angle between the adenine ring and the platinum(1) coordination plane is 85.8(3)°. However, the dihedral angle with the other platinum(2) coordination plane is lower, 78.4(2)°. The atoms of this plane have a r.m.s deviation of 0.011. Pt-N1 and Pt-N7 vectors are approximately perpendicular to each other [87.7(2)°]. The unit cell of complex (8) has two molecules, which are identical by a center-inversion symmetry operation. For the discussion of the hydrogen bonding pattern, the different hydrogen bonds of both molecules are going to be explained separately. There are not intramolecular hydrogen bonds, because the distance between the exocyclic amino group of the adenine nucleobase and one of the nitrogen of the dien ligand is too long: N6B···N1E, 3.77(1) Å. There are no intermolecular hydrogen bonds interactions between ligands of neighbouring cations. The perchlorates are involved in intermolecular hydrogen bonds. The O14 of the perchlorate anion forms a hydrogen bond to the proton of the dien ligand (H3E), O14···N3E(H3E), 2.98(1)Å and N2E(H2E) of the same dien ligand, forms a hydrogen bond with a O41(a) of another perchlorate anion, O41(a) (- x+1, -y, -z+1)···N2E(H2E), 2.90(1) Å. One of the ammine ligands is also involved in hydrogen bonds with two different perchlorate anions: N21··· O31, 3.09(1) Å and N21···O41, 3.09(1) Å (Figure 14). 2.5. 9-MeA System 25 N21 O41 O31 O41a N2E N3E O14 Figure 14: View of intermolecular H bonding between symmetry-related pairs of trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8). In the other orientation there are also hydrogen bonds between the cations and the perchlorate anions. The distances are the following: O53···N31, 3.11(1) Å; O51···N31, 3.26(1) Å; N31···O21, 3.05(1); O23···N6b(H6b) (-1+x, y, z), 3.089(4) Å and O61···N3D(H3D), 2.94(1) Å (Figure 15). N31 O51 O53 O21 O23 O64 N3D N6b Figure 15: View of intermolecular H bonding between another symmetry-related pairs of trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8). 2.5. 9-MeA System 26 The arrangement of the trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)]3+ cations is shown in Figure 16. Perchlorate anions are located between the layers acting as connection. No π-stacking interactions are observed between aromatic nucleobases. (i) (ii) Figure 16: View of the crystal packing of the cations of (8).Symmetry operations: (i) –x+1, -y, -z+1; (ii) -1+x, y, z. 2.5.2.2.2 NMR Spectroscopy of trans-[(NH3)2ClPt(N7-9-MeA- N1)(dienPt)](ClO4)3 (8) The first characterization of (8) prepared in situ was done by means of NMR spectroscopy. The 1H NMR spectrum of a solution containing the cationic trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)]3+ entity displays two signals in the aromatic region. One of them is a sharp singlet, while the other one is a broad signal, corresponding to the H2 and H8 protons of the 9-methyladenine base. In addition, a sharp singlet (s) of the methyl group of 9-MeA and the CH2 resonances of the dien ligand are observed. When the solution is in the neutral pH, the signal corresponding to H8 as well as the CH3 singlet of 9-MeA have 2.5. 9-MeA System 27 chemical shifts of δ = 8.86 and 3.95 ppm, respectively; signals associated with the dien ligand range from 2.7 to 3.3 ppm (Figure 17). The assigment of H8 of 9- MeA was established by an NOE experiment. CH3 HDO H8 H2 dien 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 δ / ppm Figure 17: 1H NMR spectrum in D2O of the trans-[(NH3)2ClPt(N7-9-MeA- N1)(dienPt)]3+ at room temperature (pD = 4.9). 2.5.2.2.3 pH Dependence of trans-[(NH3)2ClPt(N7-9-MeA- N1)(dienPt)](ClO4)3 (8) The acidity constant of trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)]3+ has been determined by pD dependent 1H NMR measurements. In the course of this study, a complication of the spectra at high pH was observed. At pD > 8, two signals occured for the methyl group of the 9-MeA, which is a consequence of a migration of Pt, leading to the formation of one new compound. This will be explained in Chapter 2.5.4. 2.5. 9-MeA System 28 2.5.2.3 trans-[(NH3)2Pt(N7-9-MeA)(N7-9-MeA-N1)(dienPt)] 4+ (9) In order to compare the pKa value of compound (8) with that of a similar one, in which an additional adenine is binding to one of the platinum atoms, trans-[(NH3)2Pt(N7-9-MeA)(N7-9-MeA-N1)2(dienPt)]4+ was synthesized (9). The difference between compound (8) and (9) is a substitution of the chloro ligand by a 9-MeA. Two different ways for the synthesis of the complex with two adenine nucleobases have been worked out. The first one consists of the treatment of (8) with one equivalent of silver nitrate. After removal of AgCl, one equivalent of 9- MeA was added. The subsequent substitution reaction was carried out in 24 hours at 40 °C. The resulting 1H NMR spectra were very complicated because of the formation of other species, which could not be identified. Therefore, a different strategy was needed. The compound trans-[(NH3)2Pt(N7-9-MeA)(N7-9- MeA-N1)(dienPt)]4+ was prepared by reaction of trans-[(NH3)2Pt(9-MeA-N7)2]2+ (3) with [(dienPt)(H2O)2]2+. The resulting 1H NMR spectra recorded at different pD values are shown in Figure 18. The resonances A, B1, B2 and C correspond to three different compounds. The structure and the formulas of these complexes are summerized in Table 4. The resonance A corresponds to the methyl group of the starting material [trans-[(NH3)2Pt(9-MeA-N7)2]2+ (3)]. The resonance B1 corresponds to the methyl group of the adenine containing the dienPtII entity at N1 in the desired product trans-[(NH3)2Pt(N7-9-MeA-N1)2(dienPt)]4+ (9). The resonance B2 corresponds to the methyl group of the adenine with the N1 position free in trans-[(NH3)2Pt(N7-9-MeA-N1)2(dienPt)]4+ (9). Finally, the resonance C corresponds to the methyl group of the compound trans- [(NH3)2Pt(N7-9-MeA-N1)2(dienPt)2]6+ (10). 2.5. 9-MeA System 29 pD = 1.3 pD = 3.57 pD = 5.53 pD = 11.27 pD = 11.50 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 A C B2 B1 A A A A C C C C B1 B2 B2 B1 B2 B1 B2 B1 δ / ppm Figure 18: Stackplot of the aliphatic region of 1H NMR spectra at different pD values (from top to bottom: pD = 1.3, 3.57, 5.53, 11.27, 11.50) of the mixture of three compounds. The chemical shifts of B1 are strongly affected by basic pD. It can be well seen that it is easy to distinguish the different species coexisting in the solution. If we consider the methyl groups, we can observe four signals, which correspond to three different compounds. There is one singlet, which corresponds to the starting compound trans-[(NH3)2Pt(9-MeA-N7)2]2+(3). This complex has been studied before (see part 2.5.1; its behavior in solution 2.5. 9-MeA System 30 with different pD´s has been followed). There are two singlets, which can be assigned to both methyl groups of the 9-MeA nucleobases of the desired dinuclear compound trans-[(NH3)2Pt(N7-9-MeA)(N7-9-MeA-N1)(dienPt)]4+ (9). And finally, the last signal corresponds to the trinuclear complex trans- [(NH3)2Pt(N7-9-MeA-N1)2(dienPt)2]6+ (10). This will be discussed further in the next chapter. The pD dependence of compound (9) was easy to follow, because the determination of the pKa value of (3) has previously been shown (Figure 11) and the pD dependence of the compound (10) will be shown later. Table 4: Summary of the three different compounds observed in the reaction of trans-[(NH3)2Pt(9-MeA-N7)2]2+ (3) with [(dienPt)(H2O)2]2+. The resonance A corresponds to the complex (3); the resonances B1 and B2 to (9) and the resonance C to (10). Compound Formula CH3- Res. A7 7A 2+ trans-[(NH3)2Pt(9-MeA-N7)2]2+ (3) {starting material} A A7 7A 4+ 1 B2 B1 trans-[(NH3)2Pt(N7-9-MeA-N1)2(dienPt)]4+ {desired product (9)} B1 B2 A7 7A 6+ 11 trans-[(NH3)2Pt(N7-9-MeA-N1)2(dienPt)2]6+ (10) C It should be noted that resonance B1 is not very sensitive at acid conditions, whereas it is very sensitive at basic conditions. On the contrary, the resonance B2 is very sensitive at acid conditions. This is due to the presence of a dienPtII entity at N1 in the 9-MeA, which contains B1. The N1 position in the other 9-methyladenine is free, so the methyl resonance corresponding to this 2.5. 9-MeA System 31 nucleobase B2, will be more affected at acidic conditions than the methyl resonance of the 9-MeA, which has a dienPtII entity at N1 position B1. The change in chemical shift of the adenine methyl groups B1 and B2 depending on pD is shown in Figure 19. Looking at the course of the two methyl groups over the pD range one can easily attribute the deprotonation of the exocyclic amino group. On one hand, in the pD range from 1 to 3 the methyl group B2 undergoes a strong upfield shift (Δδ > 0.1 ppm), whereas the other CH3 group B1 is not so much affected (Δδ ≈ 0.04 ppm). The first pKa value corresponds to the protonation at N1 position and its value is estimated to be < 2. On the other hand, in the pD range from 8 to 10 the CH3 group B1 of the adenine ligand undergoes a strong upfield shift (Δδ ≈ 0.15 ppm), whereas the other adenine resonance is hardly affected (slight upfield shift of Δδ ≈ 0.04 ppm). This indicates, that deprotonation of the exocyclic amino group occurs first at the adenine moiety, carrying a dienPtII entity coordinated to N1. The second pKa value corresponding to this inflection point is 9.75 ± 0.1. This value is markedly decreased in comparison with the pKa value (~ 16.7)[18] of the free nucleobase. Thus, the presence of two platinum atoms coordinated to N1 and N7 positions influences the acid-base properties of the exocyclic amino group. In addition to this effect, there is an intramoleculary stabilization by a H-bond donor (Figure 20). 0 2 4 6 8 10 12 14 3,80 3,85 3,90 3,95 4,00 4,05 4,10 B1 B2 (δ) / pp m pD Figure 19: pD dependence of the CH3 proton chemical shifts of trans- [(NH3)2Pt(N7-9-MeA-N1)2(dienPt)]4+ (9). 2.5. 9-MeA System 32 The deprotonation of the second N6H2 group could not be determined. The estimated pKa3 value is >13. N N N N NH2 N N N N H2N H3C CH3 Pt Pt N N N N NH N N N N HN H3C CH3 Pt Pt H -H+ N N N N NH N N N N HN H3C CH3 Pt Pt+H + -H+ +H+ Figure 20: Assignment of the three deprotonation steps. First, deprotonation at N1 (not shown); second at NH2 of adenine with (dien)PtII at N1 and third, at NH2 of the other adenine. 2.5.2.4 Comparison of trans-[(NH3)2Pt(N7-9-MeA-N1)2(dienPt)] 4+ (9) with trans-[(NH3)2Pt(N7-9-MeA-N1)2(dienPt)2] 6+ (10) Treatment of trans-[(NH3)2Pt(N7-9-MeA-N1)2]2+ with two equivalents of trans-[(dienPt)(H2O)]2+ at 40°C during three days leads to the formation of (10). The NMR spectroscopic study of the deprotonation of the exocyclic group of both adenine nucleobases reveals that the first pKa value in water has a value of 8.3 ± 0.1 (corresponding to the deprotonation of NH2 of one adenine) and the pKa value corresponding to the deprotonation of the second adenine in water is 10.1 ± 0.1 (Figure 21). If we compare these values with the values obtained for (9), the pKa values of (10) are lower, because trans-[(NH3)2Pt(N7-9-MeA- N1)2(dienPt)2]6+ (10) has one platinum entity more than (9). N N N N NH2 N N N N H2N H3C CH3 Pt Pt Pt N N N N NH N N N N HN H3C CH3 Pt Pt Pt H N N N N NH N N N N HN H3C CH3 Pt Pt Pt+H + -H+ +H+ -H+ Figure 21: Deprotonation of the exocyclic amino groups in (10). 2.5. 9-MeA System 33 2.5.3 Acidification of N6H2 in Trinuclear Bis(Adenine- N1,N7) Complexes with cis- and trans- Geometries We have prepared several complexes of general composition Pt3A2 (A = 9-MeA or 9-EtA), with formally 1.5 Pt entities per adenine base and either a cis- or a trans-[a2PtII] or [ma2PtII] entity cross-linking two central adenine bases. Surprisingly, pKa values in the compounds studied are substantially lower than in the cases with two Pt entities per adenine (compare with the results in Table 2, pag. 18). This rules against the charges of the metal entities being the major determinants of ligand acidity. Several examples of trinuclear PtII complexes containing a single cis- [a2PtII] as well as two monofunctional a3PtII units have been studied. In cis- [(NH3)2Pt{(N1-9-MeA-N7)Pt(NH3)3}2](NO3)6·2H2O (11), the X-ray crystal structure of which has been reported before,[53] pD-dependent 1H NMR spectra in D2O and potentiometric titrations in H2O gave pKa1 values of approximately 8.7 (1H NMR spectroscopy, calculated for H2O) and 9.10 ± 0.03 (potentiometry, calculated for H2O) for deprotonation of the exocyclic amino group of the first adenine base and pKa2 values of approximately 10.7 (1H NMR spectroscopy) and 10.99 ± 0.10 (potentiometry) for deprotonation of the second adenine. In the NMR spectra, the two deprotonation steps of (11) are particularly well separated for the adenine H2 resonance (Figure 22). It is noted that above pD 7.5 and at ambient temperature only single sets of H2 and H8 resonances are observed; this is unlike the situation at lower pH values, where resonance doubling (with an intensity ratio of about 1:3) is observed due to slow nucleobase rotation. While the pKa2 value is in the range expected for a Pt2(9-MeA-N1,N7) species (see above), pKa1 is significantly shifted to lower values. We propose that the first proton loss from the exocyclic amino group is facilitated by an efficient stabilization of the deprotonated species involving donation of a proton from the N6H2 group of the second 9-MeA in a hydrogen bond (Figure 23). 2.5. 9-MeA System 34 5 6 7 8 9 10 11 12 13 14 8,2 8,4 8,6 8,8 9,0 9,2 H2 (δ) / pp m pD Figure 22: pD dependence of the H2 resonance of (11) in the 1H NMR spectra. Two distinct deprotonation processes for the two adenine ligands are indicated, with pKa values of 8.7 and 10.7 (converted to H2O). N N N N NH2 R N N N N H2N R Pt PtPt N N N N NH R N N N N NH R Pt PtPt H +H+ -H+ +H+ -H+ N N N N NH R N N N N HN R Pt PtPt N N N N NH2 R N N N N H2N R Pt Pt Pt N N N N NH R N N N N HN R Pt Pt Pt 4+ 1 1 3+ 1 2+ 1 1 1 1 1 2+ 1 1 4+ hh ht pKa1= 8.7 pKa2= 10.7 hh ht hh Figure 23: Proposed rotamer distribution of (11) depending on the pH of the solution. hh = head-head, ht = head-tail. 2.5. 9-MeA System 35 This scenario is supported by structural arguments: In the solid-state structure of (11)[53] the two adenine bases are in a head-head arrangement with the two exocyclic amino groups 3.35 Å apart (N6A···N6A’) and essentially perpendicular to each other. Following removal of a single proton only a slight tilting of the two bases would be required to lower the separation of the two exocyclic nitrogen atoms to well below 3 Ǻ and to permit stabilization of the deprotonated species by H-bond formation. Removal of a second proton (from the other adenine base) is expected to lead to mutual repulsion of the NH groups, to a larger separation of these groups, and probably, as a consequence, to base rotation into a head-tail orientation. With no extra stabilization of the deprotonated species possible, the pKa2 value is again in the “normal” range for diplatinated adenines, namely close to 11. When the positions of the cis-[a2PtII] and the [a3PtII] entities on the adenine bases are interchanged, that is, in cis-[(NH3)2Pt{(N7-9-MeA- N1)Pt(dien)}2](NO3)6 (12), differences between the pKa1 and pKa2 values are somewhat lower than in (11), but the values are still significantly apart (9.23 ± 0.08 and 10.56 ± 0.17, respectively; measured by potentiometry). The low pKa value of the first deprotonation step calls for a similar interpretation as in the case of (11). Although X-ray crystal structures are not available for (12) or for the cis-[(NH3)2Pt(9-MeA-N7)2]2+ fragment (4) with a head-head arrangement of the two bases, comparison with the positions of the O6 atoms in cis-[(NH3)2Pt(9- EtGH-N7)2]2+ (two guanines in the head-head orientation)[54] leaves no doubt that hydrogen bonding between the NH- and NH2 groups in (12) is feasible on steric grounds. Altogether, four compounds containing a central trans-[a2PtII] unit bridging two adenine nucleobases through their N7 positions were studied: trans,trans,trans-[(NH3)2Pt(N7-9-MeA-N1)2{(NH3)2Pt(9-EtGH-N7)}2](ClO4)6·6H2O (G1), trans,trans,trans-[(NH3)2Pt(N7-9-EtA-N1)2 {(CH3NH2)2Pt(9-MeGH-N7}2] (ClO4)6 (G2), trans,trans,trans-[(NH3)2Pt(N7-9-EtA-N1)2 {(NH3)2Pt(1-MeT-N3)}2] (ClO4)4·11H2O (H), and trans,trans,trans- [(NH3)2Pt(N7-9-EtA-N1)2 {(CH3NH2)2Pt(1-MeU-N3)}2] (ClO4)4·4H2O (I). As can be seen from Table 2 2.5. 9-MeA System 36 (Page 18), there is a further drop in the pKa1 value of 9-MeA for these compounds compared to the examples containing a central cis-[a2PtII] unit, with a minimum of 7.9 ± 0.3 (1H NMR spectroscopy) reached in the case of (I). With the mixed guanine/adenine complexes (G1) and (G2) deprotonation takes place at the two guanine ligands (N1 positions) prior to deprotonation at the bridging adenine ligands (N6H2). The pKa values for the guanine bases in (G1) and (G2) are between 7.1 and 7.6 and are thus also remarkably lower than in other cases of guanine model nucleobase complexes with PtII.[3,28] Interestingly, hemideprotonation and stabilization of the guanine anion through three hydrogen bonds with a neutral guanine ligand[55] could again be the reason for the observed low pKa values of the two bases, although the scenario of anion stabilization is radically different from that seen in the case of adenine bases. In principle, the argument of guaninate stabilization should hold up for any PtII complex containing guanine ligands. However, as an inspection of a model of deprotonated (G) reveals, there is the possibility of formation of a loop structure with intermolecular stacking of H-bonded guanines, which would make hemideprotonated (G) different from all mononuclear Pt complexes previously studied by us. Additional work is required to verify or disprove such a scenario. 2.5.3.1 Extent of Formation of H-Bonded Species If one accepts the idea that intramolecular hydrogen bonding, either directly or indirectly (through an H2O molecule, see below), is responsible for stabilization of the N6H– species and for the extra lowering of the pKa value, it is possible to estimate the degree of formation of the H-bonded structure. According to such an analysis[56] an extra acidification of 2 log units, say from 11 to 9 (the effect of two metals only, modified by charge considerations), corresponds to a degree of formation of the H-bonded species of more than 99%, and a ΔpKa value of 1.6 still requires a degree of formation of 97%. 2.5. 9-MeA System 37 2.5.3.2 Protonation of cis-[(NH3)2Pt{(N1-9-MeA-N7)Pt(NH3)3}2] (NO3)6·2H2O (11) and cis-[(NH3)2Pt{(N7-9-MeA- N1)Pt(dien)}2](NO3)6 (12) The relative ease of deprotonating the exocyclic amino group of the two 9-MeA nucleobases in (11) and (12) is contrasted by the superacidic conditions required to accomplish protonation of the 9-MeA ligands. According to results obtained from UV spectroscopy, protonation of (11) and (12) occurs with pKa values of –4.4 ± 0.3 and –4.3 ± 0.3, respectively (Ho scale). It is assumed that protonation takes place at the N3 position of 9-MeA. These values are somewhat lower than that of threefold protonated adenine, which also loses its first proton from N3.[2] 2.5.3.3 Quantum-mechanical Calculations of cis-[(NH3)2Pt{(N1-9- MeA-N7)Pt(NH3)3}2](NO3)6·2H2O (11) Geometry-optimized structures for the cation cis-[(NH3)2Pt{(N1-9-MeA- N7)Pt(NH3)3}2]6+ of (11) were calculated by Patrick Lax with the Gaussian 98 suite of programs.[57] The calculated structures are, however, different from the solid-state structure of (11) (nitrate salt, dihydrate)[53] in that one of the two adenine bases is strongly tilted with respect to the central cis-[(NH3)2PtII] plane. Optimizations converged toward geometries in which the N6H– group is stabilized by hydrogen bonding with an NH3 group, either from the (NH3)3PtII unit at N7 or from the cis-[(NH3)2PtII] unit at N1. This picture changed dramatically when a water molecule was inserted between the two N6 positions: Then, the two exocyclic amino groups were interconnected through hydrogen bonds extending from the water molecule (Figure 24). An additional hydrogen bond is formed between the exocyclic N6 amide group and the cis-oriented NH3 ligand (Nam). 2.5. 9-MeA System 38 OH2 N6 N6´ Nam Pt1 Figure 24: Geometry-optimized gas-phase structure of the cation of (11) with the H-bonding pattern involving N6H–, N6´H2, the water molecule, and one of the central NH3 ligands (Nam). 2.5.3.4 Mixed Adenine/Guanine Complexes of cis- and trans-a2Pt(II) The simple concept of replacement of a weakly acidic proton of a hydrogen bond between nucleobases by a metal entity of suitable geometry yields “metal-modified base pairs” or larger aggregates (Figure 25).[58,59] The metal complexes obtained in this way may be considered models of temporary or permanent interstrand cross-links of metal ions with nucleic acids.[60] If extended to metalated oligonucleotides, targeting single-stranded or double- stranded nucleic acids is relevant to antisense and antigene approaches for gene silencing.[61] Finally, regular metal cross-linking of duplex DNA (M – DNA) can lead to a situation in which DNA behaves as a molecular wire.[62] N H X N XM Figure 25: Concept of generating metal-modified base pairs. 2.5. 9-MeA System 39 In the course of studies on metal-modified nucleobase quartets[63] an example of an extreme acidification (109) of the 6-amino group of adenine in the complex trans,trans,trans-{(NH3)2Pt(N7-9-EtA-N1)2[(MeNH2)2Pt(1-MeU-N3)]2}4+ has been demonstrated.[17] In this chapter, there will be discussed a similar complex, with three platinum atoms, but only three nucleobases. 2.5.3.4.1 trans,trans,trans-[(NH3)2Pt(N7-9-MeA-N1)(dienPt)(N7-9- EtA-N1){(NH3)2Pt(9-MeGH-N7)}](ClO4)3(NO3)3 (13) Colourless crystals of trans,trans,trans-[(NH3)2Pt(N7-9-MeA- N1)(dienPt)(N7-9-EtA-N1){(NH3)2Pt(9-MeGH-N7)}](ClO4)3(NO3)3 (13) were obtained from a solution of trans-[(NH3)2(H2O)Pt(N7-9-MeA-N1)(dienPt)]4+ (aqua species of the cation (8)) and trans-[(NH3)2Pt(9-EtA-N1)(9-MeGH-N7)]2+ after several days at room temperature. Compound (13) crystallizes in the monoclinic C2/c space group. Unfortunately, these crystals were very small, and the observed reflections were not sufficiently strong to permit anisotropical refinement of all the atoms; for this reason in the refinement process of the X-ray data, only the platinum atoms were refined anisotropically. Not all the anions were properly refined. Complex (13) consists of two adenine nucleobases (9-MeA and 9-EtA), a 9-methylguanine model nucleobase, a (dien)PtII unit and two trans-a2PtII entities. trans,trans,trans-[(NH3)2Pt(N7-9-MeA-N1)(dienPt)(N7-9-EtA-N1){(NH3)2Pt(9- MeGH-N7)}](ClO4)3(NO3)3 (13) crystallizes in a characteristic Z-shape with the three bases and the (dien)PtII being nearly coplanar (Figure 26). As in related compounds,[63] Pt─N1 and Pt─N7 vectors are approximately perpendicular to each other [85.7(7)°]. Geometries of the square-planar coordination spheres of the three Pt atoms have normal values of angles and distances, taking into account the low quality of the crystal (Table 5). 2.5. 9-MeA System 40 Pt1 Pt2 Pt3 N7B N1B N7A N1A N7G N11 N12 N23 N24 Figure 26: View of the cation trans,trans,trans-[(NH3)2Pt(N7-9-MeA- N1)(dienPt)(N7-9-EtA-N1){(NH3)2Pt(9-MeGH-N7)}]6+ (13). Table 5: Selected distances (Å) and angles (º) for non-hydrogen atoms in 13. Pt1-N1A 1.98(2) N1A-Pt1-N7G 176(1) Pt1-N7G 2.07(2) N1A-Pt1-N12 94(1) Pt1-N12 2.12(2) N1A-Pt1-N11 84(1) Pt1-N11 2.17(3) N12-Pt1-N7G 89(1) N11-Pt1-N7G 93(1) N11-Pt1-N12 177(1) Pt2-N7A 2.02(3) N7A-Pt2-N7B 178(1) Pt2-N7B 2.04(3) N7A-Pt2-N24 90(1) Pt2-N24 2.04(4) N7A-Pt2-N23 87(1) Pt2-N23 2.06(3) N7B-Pt2-N24 92(1) N7B-Pt2-N23 91(1) N23-Pt2-N24 174(1) Pt3-N1B 2.14(3) N31-Pt3-N32 173(1) Pt3-N31 2.06(3) N31-Pt3-N33 86(1) Pt3-N32 2.05(4) N31-Pt3-N1B 93(1) 2.5. 9-MeA System 41 Pt3-N33 2.12(4) N32-Pt3-N33 88(1) N32-Pt3-N1B 93(1) N1B-Pt3-N33 176(1) The dihedral angles formed by the coordination planes of the platinum atoms and the different nucleobases are as follows: 77.4(6)° (Pt1-G), 81.3(6)°(Pt1-A), 89.9(7)° (Pt2-A), 87.4(7)° (Pt2-B) and 87.0(7)° (Pt3-B). This means, that with the exception of the first angle, all others are close to 90° and hence the vectors are close to perpendicular. A weak intramolecular contact was found between the exocyclic amino group of the guanine base and the exocyclic amino group of the 9-ethyladenine base: O6G···N6A, 3.37(4) Å. The formation of compound (13) was also studied by 1H NMR spectroscopy. The spectrum shown in Figure 27 was recorded after one week of reaction. As can be observed in this spectrum, resonances due to the starting compound have almost disappeared, while the new signals of (13) have appeared. The signals are easily assigned. The existence of different rotameric forms in D2O at ambient temperature is not observed. The corresponding H(A) signals show no doubling. Therefore, a rotation with a different activation energy about the central A-N7-Pt(2)-N7-A bonds is assumed, with the repulsive interaction of the two exocyclic A-NH2 groups favouring the Z-form, as found in the solid state (Figure 26). 2.5. 9-MeA System 42 H(A){ H8(G) CH (A1)3 CH (G)3 CH (Et-A2)3 CH (Et-A2)2 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 δ / ppm * * * * *** Figure 27: Resonances of the aromatic protons and the methyl groups in the 1H NRM spectrum of (13).The signals (*) correspond to the starting compound trans-[(NH3)2Pt(9-EtA-N1)(9-MeGH-N7)]2+. pD-dependent 1H NMR spectra (2 < pD < 14) reveal two deprotonation steps ocurring with pKa1 = 7.14 ± 0.07 and pKa2 = 8.32 ± 0.01 (calculated for H2O). The deprotonation of the second adenine occurs at pD > 10 (pKa3). The first pKa value is assigned to a single deprotonation step of NH2 group of adenine and the second one to the deprotonation of N1 of guanine. Comparison with literature data show that the amino group acidity (pKa ~ 16.7 in the free base[18]) increases upon PtII coordination to N7 or N1 by 2─3 log units and even more so if both positions are simultaneously platinated (pKa ~ 10.8─12.6[63,11,12]). The unusually low pKa1 of 7.14 can only be rationalized if in addition to the polarizing effect of the two metal ions a fundamental conformational change is taken into account. Here it is proposed that the transition of the Z-shaped cation to a U-shaped one takes place, which stabilizes the first deprotonated species {13-H+}5+ (Figure 28). On the basis of structural models as well as X-ray data of (13) it is obvious that (i) the three nucleobases and the dien entity can addopt a perfectly planar arrangement, and (ii) the exocyclic amino groups of the head- head arranged adenine bases are 3 Å or less apart. In fact, any reduction of the N7-Pt-N7 angle from 180° further reduces this separation. This short distance 2.5. 9-MeA System 43 permits an efficient stabilization of the NH─ group by the NH2 group of the second adenine through intramolecular H bond formation (Figure 28, bottom). The situation is reminiscent of that occurring in the N2H5─ ligand of the dinuclear PtIV complex [(NH3)3Pt(NH2)2(N2H5)Pt(NH3)3]5+.[19] This conclusion concerning the conformational change during the first deprotonation of (13) is in agreement with observations for linkage isomers, viz. complexes containing a central N1-Pt- N1 bond and additional PtII entities at the N7 positions.[63,64] There, mainly the electronic effects of the two metal ions are operative, hence pKa values are higher and close to the normal range (10─11). As the pH is raised further, the cation is expected to adopt again the Z-shape to avoid the mutual repulsion of the two amide groups. dien 6+ A A G 6+ A A G dien A A G dien 5+ dien 4+ A A G N N N N N Pt G Et H N N N N HN Pt dien Me H Pt -H+ +H+ -H+ +H+ rotation 1 1 7 7 Figure 28: Schematic presentation of the conformational pH switch (13). 2.5. 9-MeA System 44 The compound described here behaves as a pH switch,[16b] and hence changes its conformation from Z to U upon removal of a single proton and again from U to Z if a second proton is removed. The mutual orientation of the two adenine nucleobases and the close distance of the exocyclic amino groups make the compound a system with the potential of acting as an acid and a base in a reversible manner at physiological pH. The only requirement to achieve such a function would be to have the system confined in space to a situation in which the cation adopts a structure halfway between the two extremes (Figure 28, top). As a consequence of slight mechanical oscillations of the two halves a proton might be ejected or picked up from the environment. There is another interesting aspect of the cationic {13-H+}5+ in its U- shape, which refers to its possible interaction with (anionic) telomere sequences of DNA. The enzyme telomerase is a key player in the development of cancer and has been found to be inhibited by conjugated π-systems capable of stabilizing guanine quartet structures of the telomeres.[65] 2.5.3.4.2 Comparison of (13) with trans,trans,trans-{(NH3)2Pt(N7-9- MeA-N1)2[(NH3)2Pt(9-EtGH-N7)]2} 6+ (14) In order to compare the above mentioned compound (13) with a similar one, trans,trans,trans-{(NH3)2Pt(N7-9-MeA-N1)2[(NH3)2Pt(9-EtGH-N7)]2}6+ (14), synthetized by M. Lüth, was studied.[47] Four pKa values are expected, two corresponding to the deprotonation of the NH2 of adenine nucleobases and the other two corresponding to the deprotonation of N1 of the guanine nucleobases. The acidity constants were determined by potentiometry[47] and by 1H NMR spectroscopy. The values are summerized in Table 6. 2.5. 9-MeA System 45 Table 6: Different pKa values for (14) obtained with potentiometry. Position Potentiometry[47] pKa 9-MeA-NH2 7.13 ± 0.02 pKa1 11.0 ± 0.1 pKa4 9-EtGH-N1 7.59 ± 0.02 pKa2 8.67 ± 0.03 pKa3 The first deprotonation of the NH2 of one adenine presents a low value (7.13 ± 0.02). This phenomenon was also found before.[17] When this happens, an intramolecular hydrogen bond between the NH─ and the NH2 is possible (Figure 29). N N N N N N N N N NH H3C H3C H N N N N O NH2 Et N H N N N O NH2 Et Pt Pt Pt pKa2pKa1 pKa4 pKa3 5+ A G A G H H Figure 29: One of the possible arrangemenst of trans,trans,trans-{(NH3)2Pt(N7- 9-MeA-N1)2[(NH3)2Pt(9-EtGH-N7)]2}5+ (14). In this U conformation, there is a stabilization of the NH– group by the NH2 of the second adenine through an intramolecular H bond. Similar values were found applying 1H NMR spectroscopy. The 1H NMR studies reveal that the adenine deprotonates first, then two guanines and 2.5. 9-MeA System 46 eventually the second adenine. The signals are more affected in the case of the adenine (Figure 30). From pD 6.6 to pD 7.6, the H8 and H2 resonances become broad, however 9-EtGH resonances remain sharp. A tautomer equilibrium is feasible, given the relatively slight differences between pKa1 and pKa2. 2 4 6 8 10 12 14 8,1 8,2 8,3 8,4 8,5 8,6 8,7 8,8 8,9 9,0 9,1 9,2 9,3 H2(A) H8(A) H8(G) (δ) / pp m pD Figure 30: pD dependence of the resonances corresponding to the protons of adenine (H2 and H8) and the proton of guanine (H8). 2.5.4 PtII Migration from N1 to N6 in 9-Methyladenine In the course of our 1H NMR studies with platinated adenine nucleobases we noticed in many instances a complication in the spectra of samples kept at high pH conditions (pH*>10). We considered two scenarios, both of which are precedented in nucleobase chemistry, namely deamination of adenine and conversion into a hypoxanthine ligand, and/or migration of PtII from N1 or N7 to N6. The latter aspect has been studied in detail by Arpalahti and co- workers.[25,66,67] 2.5.4.1 Migration in trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8) In the course of the study of the pD dependence of complex (8), new 2.5. 9-MeA System 47 resonances were observed. During the determination of the acidity constant of (8), a new signal of a methyl group was observed at pD ~ 9. With time, the signal due to the starting compound disappeared and only the new singlet remained. The solution was brought to pD ~ 2.5 and the spectrum was compared with the spectrum of a solution of (8) at identical pD (Figure 31). There is a very strong difference in the chemical shifts of the resonances (particularly pronounced in the case of the heteroaromatic proton resonance). The resonances corresponding to the CH2 of the dien are also different in both spectra. The spectrum on the top in Figure 31 belongs to a solution of (8) in D2O at pD ~ 2.5. The spectrum on the bottom corresponds to a solution of (8), which was during one week at pD ~ 9 and then brought to pD ~ 2.5 with DNO3. Due to this difference, the spectrum on the bottom corresponds to another compound. It could be possible that the dienPt entity has migrated from N1 to N6. This could explain the doubling of the CH3-resonances after two days, probably due to the existence of rotamers (Figure 32). 9.00 8.90 8.80 8.70 8.60 8.50 8.40 8.30 8.20 8.10 4.00 3.80 3.60 3.40 3.20 3.00 2.80 ~ ~ ~ ~ ~ ~ H8 CH3 CH3H8 dien dien Figure 31: Possibility of migration in (8). Complex (8) in D2O at pD ~ 2.5 (top) and spectrum of (8) after being treated with NaOD and brought to pD ~ 2.5 (bottom) 2.5. 9-MeA System 48 ~ ~ ~ ~ 8.30 8.20 8.10 8.00 7.90 3.80 3.60 3.40 3.20 3.00 2.80 CH3}H8/2} δ / ppm N N N N NH Pt Pt H2Oa a CH3 N N N N HN Pt Pt H2Oa a CH3 3+ 3+ rotation Figure 32: Rotamers in trans-[(NH3)2(H2O)Pt(N7-9-MeA-N1)(dienPt)]3+ (8). 1H NMR Spectrum with two signals corresponding to the CH3 goups of both rotamers (D2O, pD ~ 9). In order to prove the existence of another species, trans-[(NH3)2ClPt(N7- 9-MeA-N1)(dienPt)](ClO4)3 (8) was dissolved in DMSO-d6 and the 1H NMR signals were followed with time (Figure 33). After ten minutes, a new signal B can be observed. This signal increases with time, while the signal corresponding to (8) A, decreases. After two hours, the starting compound (8) is gone, and only trans-[(NH3)2(DMSO)Pt(N7-9-MeA-N1)(dienPt)]3+ is present, in which the chloro atom is exchanged for one molecule of the deuterated solvent, DMSO (Figure 34). After one week, a new signal C appears. This signal was thought to correspond to [(dienPt)(9-MeA-N1)]2+ (1), but it was found that C does not agree with this assumption, since the chemical shifts of (1) in DMSO are not the same as the resonances of C. Therefore, we assume that C corresponds to the product of a migration of dienPtII entity from N1 to N6. 2.5. 9-MeA System 49 9.00 8.80 8.60 8.40 8.20 3.95 3.85 3.75 3.65 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~ ~ CH -A3 CH -B3 H8-A H2-A H8-B H2-B CH -B3H2-BH8-B CH -C3 H2-CH8-C t=10 min t=20 min t=45 min t=120 min t=1 week t=2 weeks δ / ppm Figure 33: 1H NMR spectra of (8) and related compounds in DMSO-d6 after different periods of time. 2.5. 9-MeA System 50 N N N N NH2 Pt Pt Cl a a CH3 3+ in DMSO-d6 N N N N NH2 Pt Pt DMSO a a CH3 3+ N N N N NH2 Pt CH3 2+ + Pt DMSO DMSO aa migration N N N N NH Pt Pt DMSO a a CH3 2+ A B C (1) Figure 34: Different species starting from trans-[(NH3)2(H2O)Pt(N7-9-MeA- N1)(dienPt)]3+ (8) with the course of time in DMSO-d6. 2.5.4.2 trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N7,N6)](ClO4)3·3.5H2O (15) We were successful in isolating a reaction product. By applying compound trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N1,N7)](ClO4)4 (7) and titrating it with NaOH to pH 11.1, we aimed to obtain crystals of the deprotonated form (7’). The isolated crystals (15) proved, however, to be a linkage isomer of (7’) (Figure 35) in which the Pt entity, which originally resided at N1, had moved to N6. (15) can be reprotonated to give (15’), which is formally the twofold- platinated rare imino tautomer of adenine.[13b,25,68,69] 2.5. 9-MeA System 51 N N N N NH2 Pt R C 1 7 N N N N NH Pt R C 1 7-H + +H+ N N N N NH R 1 7 -H+ +H+ 4+ Pt C 6 HN N N N NH R Pt 1 7 Pt C 6 3+ 3+4+ pKa= 10.0 pKa= 5.0 7 7´ 1515´ C Pt C Pt C Pt C Figure 35: Linkage isomerization of (7) to (15´) and relevant pKa values of the protonated forms, (7´) and (15). Note the large difference of 5 log units. The crystals were characterized by X-ray crystallography. Complex (15) crystallizes in the monoclinic space group. During the refinement of the structure, all non-hydrogen atoms in the strucuture were refined anisotropically and all hydrogen atoms were included in geometrically calculated positions. Details concerning the crystal, X-ray measurement, and the refinement of data are listed in Table A-2 (see Appendix). The angles between the platinum atom (Pt1) and (Pt2) and the four coordination sites are approaching 180°: N3B-Pt1-N7A, 174.6(3)° and N3C-Pt2- N6A, 174.7(2)°. Pt-N distances about the Pt center range from 1.988(6) to 2.046(5) Å. Selected distances and angles are provided in Table 7. The cation trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N7,N6)]3+ of (15) and the atom numering scheme are depicted in Figure 36. 2.5. 9-MeA System 52 Table 7: Selected distances (Å) and angles (º) for non-hydrogen atoms in 15. Pt1-N3B 2.018(7) N3B-Pt1-N7A 174.6(3) Pt1-N7A 1.988(6) Pt1-N1L 2.043(6) Pt1-N2L 2.034(7) Pt2-N3C 2.046(5) N3C-Pt2-N6A 174.7(2) Pt2-N6A 1.993(6) Pt2-N3L 2.025(6) Pt2-N4L 2.033(6) N6A-C6A 1.316(9) C2A-N1A-C6A 119.1(6) O2B···N4L 2.944(9) C2A-N3A-C4A 109.2(7) O2B···N6A 3.126(9) N3L···N1A 3.064(9) Pt1 Pt2 N3C N6A N3B N7A N3L N1A O2B N2L N4L Figure 36: X-ray structure of the cation of trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA- N7,N6)](ClO4)3·3.5H2O (15). As can be seen, the trans-[(NH3)2Pt(1-MeC-N3)]2+ residue has migrated from N1 to N6 and adopts a syn conformation with respect to N1 of the adenine nucleobase. While the cytosine ring opposite to N7 of the adenine ring is close 2.5. 9-MeA System 53 to coplanar with adenine (dihedral angle of 12.3°) and is involved in weak H- bond formation (O2B···N6A 3.13(1) Ǻ; O2B···N4L 2.944(9) Å; numbering as given in Figure 36), the cytosine opposite to N6 is at a substantial angle (45.5°) with the adenine plane. The N1 position of adenine is deprotonated in (15) (internal ring angle of 119.1(6)°, very similar to the value of 118.8(1)° in neutral 9-MeA[70]) but is involved in weak H-bond formation (3.06(1) Ǻ) with the NH3 ligand of Pt2 (N3L in Figure 36). Pairs of cations of (15) are arranged in such a way as to permit stacking of the adenine ring with the cytosine ring B of an adjacent cation (3.5–3.7 Ǻ). Additional contacts between cations of (15) are mediated by numerous hydrogen bonding interactions, which involve ClO4– anions, NH3 groups, and water molecules. None of these contacts is unusually short. With a single exception, direct contacts between bases of adjacent cations are not seen. The exception is a short contact between the oxygen atom of the cytosine ring coordinated to the N6-bonded Pt and the H8 atom of an adjacent adenine (2.53 Å; symmetry operation –x, –1/2 + y, –1/2–z). The syn orientation of the N6-bonded Pt (Pt2) is also seen in [(dien)Pt(9- MeA-N6)]2+, in which the 9-MeA ligand is neutral and carries a proton at N1.[25] From modeling studies, it appears that an anti orientation of Pt2 in (15) is unfavourable because of the presence of the co-ligands of Pt1 at N7. Of course, in the absence of a metal at N7, anti orientations of N6-bonded metal ions are possible,[66,69] sometimes in equilibrium between both forms,[71] and an anti orientation is realized if dinuclear, metal–metal bonded units (Rh2,[72] Mo2[73]) are attached to N7 and N6 simultaneously. A study of the hydrogen bonding pattern reveals the existence of a water oligomer. There are two types of hydrogen bonds involving water molecules: the first one, between water molecules interacting only with water molecules (formation of water polymer); and the second one, between water molecules interacting with the ones which form the water cluster and at the same time with the N3 position of the adenine (O4w and its symmetric one) or with the ammine 2.5. 9-MeA System 54 ligand of the Pt2, N2L. Each cation of (15) and its corresponding water molecules of crystallization forms intermolecular hydrogen bonds between the water molecules with the cation at 1-x, 1/2+y, 1/2-z, thus leading to a pair of related cations (Figure 37). O5w O2w N3A(ii) O4w(i) N2L(i) O3w(i) O3w O5w(i) O2w(i)N2L N3 O4w Figure 37: View of the water cluster of (15). The symmetry operator for the description of hydrogen bonds is (i) 1-x, 1/2+y, 1/2-z. Oxygen atoms of the water molecules that are involved in hydrogen bond formation exclusively between water molecules are : O5w···O3w, 2.730 Ǻ; O5w···O3w(i), 2.857 Ǻ. Oxygen atoms of the water cluster with other water molecules: O5w···O2w, 2.903 Ǻ and O5w···O4w(i), 2.011 Ǻ. The latter distance is artificially short because O5w and O4w have occupancy factors of 0.5 only. O3w is also only partially (50%) occupied. Oxygen atoms of the water which do not form the cluster, also join the nucleobase oligomer: O2w···N3A(ii) (x, 1/2– y,1/2+z), 2.811 Ǻ; O4w···N2L, 2.746 Ǻ. The principal motif of the water polymer is a cyclic water tetramer, in which the atoms O5w, O3w and its symmetric ones are joined via hydrogen bonds. The internal angles of the tetramer are 96.8° (O3w-O5w-O3w(i)) and 91.8° (O5w-O3w-O5w(i)). Figure 38 shows in detail the distances between atoms of the cyclic tetramer. 2.5. 9-MeA System 55 O5w O3w(i) O3w O5w(i) 2.730 2.857 Å Å Figure 38: View of the cyclic water tetramer present in (15). The 1H NMR spectrum of a freshly dissolved sample of (15) in D2O (pD 7.8, ambient temperature) indicates the presence of two different rotamer forms, but given the various possibilities (rotation about the Pt1–N7A bond, the Pt2– N6A bond, or the C6A–N6A bond; numbering as given in Figure 36), a straightforward interpretation is difficult. Aromatic adenine proton resonances are observed at δ = 8.36, 8.14, and 8.07 ppm with relative intensities of approximately 0.2:1:0.2, and two methyl resonances of 9-MeA– occur at δ = 3.86 and 3.81 ppm (ca. 3:0.6). As to cytosine resonances, two H6 and two H5 doublets (ca. 1:1) are clearly discernable (H6: δ = 7.69 and 7.65 ppm; H5: δ = 6.10 and 6.09 ppm), as are two CH3 singlets at δ = 3.51 and 3.47 ppm (ca. 1:1). There are indications for two additional weak doublets at approximately δ = 7.63 and 6.12 ppm, which are, however, superimposed with the other doublets. Partial isotopic exchange appears to be responsible for the weak intensities of two of the three aromatic protons of 9-MeA–. On the basis of a 2D NOESY spectrum we can assign the intense singlet at δ = 8.14 ppm to the H2 proton of 9-MeA–, as it does not exhibit a cross-peak with the methyl group at N9. This finding tentatively suggests that there is hindered rotation about the Pt1–N7A bond. 2.5. 9-MeA System 56 2.5.4.3 pKa Value of N7,N6-diplatinated 9-MeA in (15) The acidity of the proton at N1 of (15´) was determined by 1H NMR spectroscopy (pD dependence of CH3 of adenine and H2 of adenine) and found to be 5.0 ± 0.1(calculated for H2O). This value is lower by 2.6 log units than that of [(dien)Pt(9-MeA-N6)]2+, which is 7.65 ± 0.05,[25] and is a consequence of the second PtII at N7. The difference is reasonably close to the ΔpKa values for N1- protonated residues carrying a PtII at N7 (2.2 ± 0.1).[13] This suggests that the acidifying effect of multiple metal ion binding is roughly additive. Comparison with other metal ions reveals that the acidification brought about by PtII at N6 is moderate: For an RHgII complex the pKa value for N1H has been found to be 4.5,[69] for an RuII chelate (N7,N6) the value was 6.5,[74] and for [(NH3)5RuIII] values of 2.5 and 4.9 have been estimated,[10a,c] depending on the rotamer state (metal syn or anti with respect to N1H).[75] 2.5.4.4 PtII Migration in Bis(9-MeA) Complexes Due to the general inertness of PtII and PtIV and the high thermodynamic stability of Pt–N bonds, the factors affecting the initial binding step of Pt to DNA are considered crucial for understanding the biological activity of anticarcinogenic Pt drugs.[48,76,77] In this respect, findings on relatively facile Pt– N bond rearrangements at the oligonucleotide level are of prime interest[78,79] since they may occur as soon as the platinated oligonucleotides are hybridised with their complementary ribo- or deoxyribonucleotide strands.[78] But the exact mechanism for this type of rearrangement is unknown, rendering model studies in this field highly desirable. In some cases, Pt–N bond rearrangements have been reported in simple complexes within the nucleobase moiety[25,66] or within the auxiliary ligand.[80] A clear picture though, is not apparent regarding the factors controlling these migrations and they may even require the cooperation of other nucleophiles[79a] or involve PtIV as an intermediate.[81,82] 2.5. 9-MeA System 57 It has been shown that in isomeric bis(9-methyladenine) complexes under basic conditions, coordinated PtII undergoes an intramolecular N1→N6 or N7→N6 migration upon displacement of a NH2 proton.[83] Subsequently, the product, cis-[(NH3)2Pt(9-MeA-N6)(9-MeA-N7)]n+, undergoes a slow deamination reaction of the N7-bound 9-MeA to provide the corresponding hypoxanthine complex instead of a second migration step. Herein, we discuss an intramolecular migration of coordinated platinum(II) from the N1 site to the exocyclic amino group in 9-MeA. Although the exact migration mechanism has not yet been unequivocally determined, the platinum N1→N6 migration in adenine proceeds without any detectable redox reaction, thus differentiating it from the Pt migration from the ring nitrogen to the exocyclic amino group in platinated pyrimidine complexes, which involve PtIV as an intermediate.[81,82,84] 2.5.4.4.1 [(9-MeA-N7)Pt(NH3)3]Cl2·2H2O as Starting Compound (16) In order to study platinum migration in the complex cis-[(NH3)2Pt(N1-9- MeA-N7)2{Pt(NH3)3}2](NO3)6 (11), compound [(9-MeA-N7){Pt(NH3)3}]Cl2·2H2O (16) was prepared by reaction of [(9-MeA-N7)PtCl3] with NH3. After keeping the reaction in the refrigerator for a while, colourless crystals were obtained. These crystals were isolated and charaterized by X-ray crystallography. Complex (16) crystallizes in the monoclinic space system. In the refinement process of the X- ray data, all non-hydrogen atoms of the crystal were refined anisotropically. All the hydrogen atoms were found in the difference Fourier map and refined without restraints. A summary of crystallographic data, data collection parameters and refinement parameters of (16) data is given in Table A-3 (see Appendix). Figure 39 gives an illustration of [(9-MeA-N7){Pt(NH3)3}]2+ with the atom numbering scheme. 2.5. 9-MeA System 58 Pt N1L N2LN3L Figure 39: View of [(9-MeA-N7){Pt(NH3)3}]2+(16) with the atom numbering scheme. The geometry of the 9-methyladenine ligand does not differ very much from other reported adenine nucleobases coordinated via N7 to a platinum.[85] The most important effect is due to the coordination of the N7 site of the ring, which leads to a significant enlargement of 3° of the C5-N7-C8 angle. Significant differences of the bond distances compared with the 9-MeA[70] are a lengthening of the N7-C5 bond (∆ = 0.02 Å) and C4-C5 (∆ = 0.02 Å), and shortenings of the N9-C8 (∆ = -0.01 Å) bonds. Protons bonded to the corresponding atoms have normal bond distances, ranging from 0.74(7) to 1.13(8) Å. The atoms in the adenine nucleobase are almost coplanar, within a maximum deviation from the least-squares plane of less than 0.007 Å. However, the square-planar coordination of the platinum atoms presents a r.m.s. deviation of 0.041. The N1L and N3L atoms are situated below (-0.047(2) and -0.049(2) Å, respectively) the platinum coordination plane. The dihedral angle between the adenine ring and the platinum coordination plane is 80.6(1)°. The bond distances and angles of [(9-MeA-N7){Pt(NH3)3}]2+ cation are compiled in Table 8. 2.5. 9-MeA System 59 Table 8: Selected distances (Å) and angles (º) for non-hydrogen atoms in 16. Pt-N7 2.015(3) N7-Pt-N2L 177.02(16) Pt-N2L 2.039(4) N7-Pt-N1L 90.93(15) Pt-N1L 2.043(4) N2L-Pt-N1L 91.34(19) Pt-N3L 2.048(4) N7-Pt-N3L 89.28(16) N1-C2 1.340(5) N2L-Pt-N3L 88.6(2) N1-C6 1.350(5) N1L-Pt-N3L 176.82(16) N3-C2 1.329(6) C2-N1-C6 119.1(3) N3-C4 1.344(5) C2-N3-C4 111.0(3) N6-C6 1.335(5) C8-N7-C5 106.0(3) N7-C8 1.315(6) C8-N7-Pt 125.3(3) N7-C5 1.391(5) C5-N7-Pt 128.2(3) N9-C8 1.342(6) N9-C4 1.369(5) N9-C9 1.464(5) C4-C5 1.390(5) C5-C6 1.411(5) The hydrogen pattern is relatively uncomplicated; it is defined by interactions between the water molecules and the Cl2 anion. Water molecule O2w interacts with the ammonia ligand (N1L) and with the N3 position of the adenine nucleobase of another molecule: O2w···N1L, 2.907(6) Å and O2w···N3a(-x+2, -y-1, -z+1), 2.947(5) Å. The chloride ion (Cl2) interacts with a water molecule and with the ammino ligand (N2L) of two different neighbour molecules: Cl2···O1wa(-x+2, y-1, -z+3/2), 3.226(4) Å and Cl2···N2La(x+1/2, -y- 3/2, z+1/2), 3.224(5) Å (Figure 40). 2.5. 9-MeA System 60 O2w O1w Cl2 N2L N1L N3 Figure 40: View of the hydrogen bond pattern of (16). Figure 41 gives a view of the arrangement of the cations in the layers of the crystal, in which the unit motifs are repeated in all directions, but the cations in every line are rotated 180° respect to the other one. The separation distance between two consecutives layers is 3.33 Å. This distance means the π-stacking of the aromatic rings of the nucleobases is present, but only between pairs. Figure 41: Details of the packing of (16). The second step to obtain the complex (11) is the reaction of [(9-MeA- N7){Pt(NH3)3}]2+(16) with cis-(NH3)2PtII in a 2:1 ratio. 2.5. 9-MeA System 61 Treatment of cis-[(NH3)2Pt(N1-9-MeA-N7)2{Pt(NH3)3}2](NO3)6 (11) with base resulted in the migration of the coordinated PtII from the endocyclic N1 site to the exocyclic NH2 group as shown by a crystal structure determination of the reaction product. So, in aqueous NaOH, the compound (11) first undergoes an intramolecular migration to give the N1,N6-bound species cis-[(NH3)2Pt(N1-9- MeA-N7)(N6-9-MeA-N7){Pt(NH3)3}2]5+ (17), which can then be slowly transformed into the doubly-migrated species cis-[(NH3)2Pt(N6-9-MeA- N7)2{Pt(NH3)3}2]4+ (18). 2.5.4.5 Linkage Isomerization of cis-[(NH3)2Pt(N1-9-MeA- N7)2{Pt(NH3)3}2](NO3)6 (11) Complex (17) was obtained by addition of 1M NaOH to a solution of cis- [(NH3)2Pt(N1-9-MeA-N7)2{Pt(NH3)3}2](NO3)6 (11). This compound exists in solution as a mixture of two rotamers (head-head; head-tail). After several days, it was observed that the H8 resonance splits (pD ~ 9.7, δ = 8.6 ppm) in two sets occurring at approximately 9.0 and 8.2 ppm. The intensity of these new resonaces increases with time. At the same time, two new A(N-CH3) resonances in 1:1 ratio appear. It is suspected that there are two differently bonded 9-MeA ligands, presumably through linkage isomerization (Figure 42). 2.5. 9-MeA System 62 N NN N NH2 H3C Pt N N N N NH2 CH3 Pta3 N NN N NH2 H3C Pt N N N N NH CH3 Pta3 Pt aa +H+ -H+ Pt aa 7 1 71 7 1 71 (1) (2) 6+ 5+ N NN N NH2 H3C Pta3 N N N N NH CH3 Pta3 Pt aa N NN N NH2 H3C Pt N N N N HN CH3 Pta3 (17) Pt aa (B) (1) (2) Linkage isomerization Linkage isomerization 7 1 71 7 1 71 6 6 5+ 5+ +H+ -H+ N NN N NH H3C Pt N N N N NH CH3 Pta3 Pt aa (A1) 7 1 71 6 4+ N NN N HN H3C Pt N N N N NH CH3 Pta3 Pt aa 7 1 71 6 4+ (18) N NN N NH H3C Pt N N N N NH CH3 Pta3 Pt aa (D) 7 1 71 6 4+ +H+ -H+ N NN N NH H3C Pt N N N N HN CH3 Pta3 Pt aa (B1) 7 1 71 6 4+ N NN N NH H3C Pt N N N N HN CH3 Pta3 Pt aa (E) 7 1 71 6 4+ Linkage isomerization Linkage isomerization Linkage isomerization a3 a3 a3 a3 a3 a3 a3 a3 (11) Figure 42: PtII migration following N(6)H2 deprotonation. Different pathways for the formation of the possible linkage isomers of cis-[(NH3)2Pt(N1-9-MeA- N7)2{Pt(NH3)3}2]6+ (11). 2.5. 9-MeA System 63 Under basic conditions, the exocyclic amino group of compound (11) loses a proton. This deprotonation of the N6 amino group facilitates Pt migration from N1 to N6 to give cis-[(NH3)2Pt(N1-9-MeA-N7)(N6-9-MeA-N7){Pt(NH3)3}2]5+ (17); or from N7 to N6 to obtain the compound cis-[(NH3)2Pt(N1-9-MeA- N6)Pt(NH3)3(N1-9-MeA-N7){Pt(NH3)3}]5+ (B). In both cases, a second deprotonation is possible, so the compounds (A1) and (B1) would be obtained. Thus, a second migration step can take place, giving the compounds (18), (D) and (E), as shown in Figure 42. In order to elucidate which routes of migration take place, we tried to obtain crystals of the compounds under alkaline conditions. In one case a compound was successfully isolated and it was shown by X-ray crystallography to be cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2]4+ (18). Characterization of compound (17) was carried out by applying 1H NMR spectroscopy. There is no indication for N7→N6 migration; consequently one has to conclude that the Pt migration takes place from N1 to N6. 2.5.4.6 cis-[(NH3)2Pt(N1-9-MeA-N7)(N6-9-MeA-N7){Pt(NH3)3}2] 5+ (17) Treatment of cis-[(NH3)2Pt(N1-9-MeA-N7)2{Pt(NH3)3}2]6+ (11) with base produces (17), for which the 1H NMR spectrum displays two pairs of interconverting sets of nucleobase signals in the ratio of 1:1 for each of the converting sets (Figure 43). The resonances of the other H8/2 cannot be seen, because these signals disappear very rapidly due to isotopic exchange of hydrogen for deuterium from the deuterated solvent. 2.5. 9-MeA System 64 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 δ / ppm H8/2 H8/2 CH3 CH3 N N N N NH2 HN N N N NH Pt a a H3C CH3 Pta3 Pta3 H H H H 2 2 8 8 1 6 7 H8/2H8/2 Figure 43: 1H NMR spectrum of (17) in D2O (pD ~9.5) of the compound cis- [(NH3)2Pt(N1-9-MeA-N7)(N6-9-MeA-N7){Pt(NH3)3}2]5+ (17). The spectrum displays two sets of methyl resonances, one corresponding to the adenine nucleobase with cis-a2PtII at N1 (CH3) and the other one to cis-a2PtII at N6 position (CH3). The exchange processes likely to be in effect are the often encountered restricted rotation about the Pt-Nnucleob. bonds due to internal hydrogen bonding involving the non-complexed nitrogens of the nucleobases (e. g. N1, N6, N7) and suitable donor sources (e. g. NH3), and/or the syn/anti interconversion about the N6–C6 bond in the adenine moiety.[67,83] Therefore, either two different bases were present in (17), or a single base type was present but coordinated at different sites. But given the expectation based on previous observations[25,83] and the crystal obtained for a double metal migration product (see next chapter), 2.5. 9-MeA System 65 it was ascertained that the bis-complex, where one 9-MeA ligand is coordinated at the N1 site and the other at the N6 site, was formed. Unfortunately, attempts to crystallize this complex were not successful. The experiment for the determination of the acidity constant of N1H and NH2 of (17) was performed in relatively dilute solution (c = 2 mM). In Figure 44, the chemicals shifts of one methyl group and the adenine H8/2 hydrogen atoms are shown in dependence on pD. 2 4 6 8 10 12 14 3.78 3.80 3.82 3.84 3.86 3.88 3.90 3.92 pp m pD p (D O)Ka1 2 p (D O)Ka2 2 2 4 6 8 10 12 14 8,10 8,15 8,20 8,25 8,30 8,35 pp m pD p (D O)Ka2 2 p (D O)Ka1 2 Figure 44: 1H NMR pD-dependence (δ, ppm) of H8/2 and CH3 resonances in D2O of the cation cis-[(NH3)2Pt(N1-9-MeA-N7)(N6-9-MeA-N7){Pt(NH3)3}2]5+ (17). The values for calculating the first pKa value in the left diagramm correponds to the methyl group (CH3) and the diagramm in the right to one of the H8I2. In both diagrams, the NMR data were evaluated by a curve-fitting procedure based on equation (3) (see Section 2.1) which considers two deprotonation steps. The final results for deprotonation of the N1 position and NH2 were obtained by calculating the weighted mean of the pKa* values, giving pKa1(D2O) = 5.46 ± 0.03 and pKa2(D2O) = 11.52 ± 0.07. Finally, the pKa values determinated in D2O were transformed to aqueous solution by applying equation (4) resulting in pKa1(H2O) = 4.94 ± 0.03 pKa2(H2O) = 10.91 ± 0.07 2.5. 9-MeA System 66 In agreement with other N6-platinated compounds, the pKa value corresponding to the deprotonation of the N1 position is the expected range. 2.5.4.7 cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2](NO3)4·6H2O (18) Compound (11) was dissolved in water and the pH value was raised from 5.5 to 11 by adding 1M NaOH. The solution was kept in a closed vial until crystals of (18) appeared after several weeks. The structure of (18) was determinated at room temperature by X-ray crystallographic analysis (Figure 45). Compound (18) crystallizes in the monoclinic space group C2/c. All the hydrogen atoms were found in the difference Fourier map and refined without restraints. A summary of crystallographic data, data collection parameters and refinement parameters of (18) is given in Table A-4 (see Appendix). Pt2 N6N6a Pt1a Pt1 N7 N7a N11 N13 N12 Figure 45: View of the cation cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2]4+ of (18). The anions and water molecules are omitted for clarity. The cation of (18) consists of two 9-MeA model nucleobases coordinated to a cis-(NH3)2PtII entity via N6 and two (NH3)3PtII entities bound at N7. The cation (18) is centrosymmetric, with Pt2 sitting in the inversion center. 2.5. 9-MeA System 67 The angles between the platinum atom (Pt1) and the four coordination sites are nearly 180°: N7-Pt-N11, 178.63(19)° and N13-Pt1-N12, 177.8(2)°. The Pt1, N11, N7 atoms are situated below (-0.007(2), -0.027(2), -0.027(2) Å, respectively) and the N12 and N13 above (0.030(2) and 0.030(2) Å, respectively) the platinum coordination plane, with a r.m.s. deviation of 0.026. Pt1-N distances about the Pt1 center range from 2.010(4) to 2.045(5) Å. Similar Pt-N bond lengths are observed for the platinum atom (Pt2). Bond distances and angles between atoms of cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2]4+ (18) are listed in Table 9. Non-hydrogen atoms of each 9-MeA are essentially planar, with a r.m.s. deviation of 0.02. Distances and angles between atoms of the 9-MeA bases are not unusual in comparison to other reported adenine compounds. The N1 position of adenine is deprotonated in (18) (internal ring angle of 119.7(4)°, very similar to the value of 118.8(1)° in neutral 9-MeA[70]). Table 9: Selected distances (Å) and angles (º) for non-hydrogen atoms in 18. Pt1-N7 2.010(4) N7-Pt1-N13 91.04(19) Pt1-N11 2.045(5) N7-Pt1-N12 89.59(18) Pt1-N12 2.045(4) N13-Pt1-N12 177.8(2) Pt1-N13 2.029(4) N7A-Pt1-N11 178.63(19) N13-Pt1-N11 89.8(2) N12-Pt1-N11 89.6(2) Pt2-N6 2.017(4) N6-Pt2-N21 87.84(17) Pt2-N21 2.053(4) N6-Pt2-N6a 92.16(15) Pt2-N21a 2.053(4) N6-Pt2-N21a 176.79(18) Pt2-N6a 2.017(4) C6-N6 1.302(6) C2-N1-C6 119.7(4) The dihedral angle between the adenine ring and the platinum (Pt1) coordination plane is nearly perpendicular, 84.6(1)°. However, the angle between the adenine base and the other platinum atom (Pt2) is 63.4(1)°. The 2.5. 9-MeA System 68 dihedral angle formed between the adenine nucleobase and its symmetric one is 59.7(1)°. No intramolecular or intermolecular hydrogen bonds are observed within the cationic entity. The arrangement of the cis-[(NH3)2Pt(N6-9-MeA- N7)2{Pt(NH3)3}2]4+ cations in the solid state is shown in Figure 46. Figure 46: View of the arrangement of the cis-[(NH3)2Pt(N6-9-MeA- N7)2{Pt(NH3)3}2]4+ cations of (18). However, there are intermolecular hydrogen bond contacts between the cations of (18), the water molecules and the nitrate anions. The most important ones are: O1w···O23(nitrate), 2.82(2) Å,; O1w···N13a (–x+1, y, -z+1/2), 2.925(6) Å,; O2w···N21, 3.034(7) Å; O2w···N21a (–x+1, y, -z+1/2), 3.034(7) Å; O3w···N13, 2.933(7) Å; O11(nitrate)···N11, 3.072(8) Å; O11···N13, 2.933(7) Å; O13(nitrate)···N13, 2.933(7) Å; O22(nitrate)···N11a (–x+1, y, -z+1/2), 3.14(2) Å (Figure 47). 2.5. 9-MeA System 69 O2w O1w O22 N11a N13a O23 N21 O3w N11 O11 O13 Figure 47: Intermolecular hydrogen bonds in (18). Therefore, the crystal packing of (18) is based on interactions between cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2]4+ cation, water molecules and nitrate counter anions. As shown in Figure 48, the arrangement of the cations, the water molecules and the nitrate anions in the crystal does not allow π-stacking between the aromatic rings of the adenines. The layers are separated by the nitrate counter anions. Figure 48: Detail of packing of cis-[(NH3)2Pt(N6-9-MeA- N7)2{Pt(NH3)3}2](NO3)4·6H2O (18). 2.5. 9-MeA System 70 In the 1H NMR spectrum of (18) (Figure 49), only a single set of nucleobase signals is present, consistent with the fact that both 9-MeA bases are equivalent and hence have identical metal binding patterns. Due to the extreme conditions of the reaction (high pH values and long time), an isotopic exchange (H→D) is observed for one of the two aromatic protons of the adenine base. H8/2 CH3 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 δ / ppm Figure 49: 1H NMR spectrum in D2O (pD ~ 7.80) of the compound cis- [(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2]4+ (18). The spectrum displays one singlet for the CH3 and another singlet in the aromatic region, which corresponds to H8 or H2. The other proton has disappeared due to isotopic exchange. The individual acidity constants pKa1 (for the deprotonation of N1H of one adenine) and pKa2 (for deprotonation of N1H of the other adenine), calculated with a non-linear least squares fit after Newton-Gauss with equation (3) for each 2.5. 9-MeA System 71 proton, are given in Table 10. The values for the situation in water were then calculated by applying equation (4). Table 10: Negative logarithms of the acidity constants (pKa) of cis-[(NH3)2Pt(N6- 9-MeA-N7)2{Pt(NH3)3}2]4+ (18) in D2O and in H2O. Values for pKa of the proton H8/2 and the methyl group are given with one standard deviation (1σ). D2O H2O pKa1 pKa2 pKa1 pKa2 A–H8/2 5.48 ± 0.14 7.11 ± 0.12 4.95 ± 0.14 6.56 ± 0.12 A–CH3 5.34 ± 0.12 7.05 ± 0.11 4.82 ± 0.12 6.50 ± 0.11 Average 5.41 ± 0.13 7.08 ± 0.11 4.89 ± 0.13 6.53 ± 0.11 2.5.5 Multiple Metalation of 9-Methyladenine Twofold metal binding, to N1 and N7 is likewise quite common. Here it has been discussed some examples. N1,N7 binding gives a rise to two mutually perpendicular M–N vectors which permit construction of molecular rectangles and meanders with 90°-angles.[86] Threefold metal binding to a neutral, 9- blocked adenine via N1, N3 and N7 has recently been observed in a polymeric, helical complex.[87] Deprotonation of the exocyclic amino group prior to metal complexation does, however, not necessarily imply that the nucleobase becomes anionic. Rather, metal binding to this group can be accompanied by a shift of an amino proton to another site, e.g. N1, therefore generating a metalated form of a rare adenine tautomer.[25,69,88] As to the formation of N6 metalated species, initial metal coordination to N1 or N7 is likely to take place, followed by metal migration. This has been demonstrated in this work. In one case, the cis- (NH3)2PtII migrates from N1 to N6 (see Section 2.5.4.5). Once the metal resides at N6, it may adopt two different orientations, either syn to N1 or anti. Examples 2.5. 9-MeA System 72 exists for both cases.[69,88a] As far as consequences for the H bonding ability of a N6 metalated adenine nucleobase are concerned, the relevance to metal mutagenicity is an obvious one.[89] There are several reports on binding mode A (Figure 50) with simultaneous metal binding to N1, N6, N7,[90,91] leading to trinuclear, cyclic metal complexes with remarkable receptor properties. A record fourfold metalation of a 9-methyladenine dianion has been observed with M = (trpy)PdII (B) with metal entities simultaneously bound to N1, N6, N7, and C8 (Figure 50).[92] N N N N N M M R H A 6 1 7 N N N N N M M R H B 6 1 7 M M8 Figure 50: Multiple metalation of 9-(R)adenine. Modes of metal binding. 2.5.5.1 Palladium Binding to 9-Methyladenine A threefold metalation of a 9-methyladenine has been observed with metal entities simultaneously bound to N1, N6 and N7 (Figure 51). In this case, the metal entity is (dien)PdII. Two equivalents of (dien)PdII were added to a solution containing 9-MeA and the mixture was brought to pH 5-6. The fast rate of the reaction allows instantaneous coordination of Pd either at the N1 site (C) of the free nucleobase, or at the N7 site of the 9-MeA (B) or both sites, N1 and N7 (A) (Figure 51). In order to deprotonate the exocyclic amino group, the pH was brought to 8-9 by addition of 1M NaOH. In this case, a metal migration can occur, from N1 to N6 (E) or from N7 to N6 (F). According to our studies, we suggest that the migration occurs from N1 to N6. The addition of a third (dien)PdII to this solution of the di-nuclear complex (E or F), leads to the formation of a tri-nuclear palladium complex of 9-methyladenine: {[(dien)Pd]3(9- MeA-N1,N7,N6)}5+ (19 = D). 2.5. 9-MeA System 73 The reaction was followed by 1H NMR spectroscopy. It was very difficult to assign the signals to the different complexes. In order to simplify this problem, different reactions were carried out. N N N N NH2 CH3 N N N N NH CH3 N N N N HN CH3 N N N N NH CH3 N N N N NH CH3 -H++H+ linkage isomerlinkage isomer +1eq. dienPdII+1eq. dienPdII N N N N NH2 CH3 N N N N NH2 CH3 Pd PdPd Pd Pd Pd Pd PdPd Pd Pd Pd Pd AB C E D = (19) 1 7 1 7 1 7 1 7 1 6 7 6 1 7 1 6 7 2+ 4+ 2+ 3+ 3+ 3+ 5+ F Figure 51: Deprotonation of the N(6)H2 of the adenine nucleobase in {[(dien)Pd]2(9-MeA-N1,N7)}3+ (A) and competing migration of PtII from N1 or N7 to N6. Formation of {[(dien)Pd]3(9-MeA-N1,N7,N6)}5+ (19). 2.5. 9-MeA System 74 2.5.5.1.1 NMR Studies with dienPdII and 9-MeA When the yellow crystals {[(dien)Pd]3(9-MeA-N1,N7,N6)}5+ (19) are dissolved in D2O (pD ~ 8.5), one can observe multiple sets of H2 and H8 resonances and three major ones (N-CH3) (spectrum (i) in Figure 52). There are also several minor signals. This multiplicity apparently arises from a rapid equilibrium of different dienPdII complexes. If an excess of dienPdII is added to the solution of the crystals, spectrum (ii) is obtained (Figure 52). The pD was decreased to pD ~ 6.7 and only one majority signal can be observed. This signal corresponds to the dinuclear complex (A: {[(dien)Pd]2(9-MeA-N1,N7)}4+). We know that A is the dinuclear species, because a reaction of 9-MeA with three equivalents of [dienPd(H2O)]2+ at pD ~ 2.9 leads to a main signal, and this signal has the same chemical shifts at pD ~ 6.7. The chemical shifts of the resonances of A are the following: 3.88 ppm (s, CH3), 8.75 ppm (s, H2) and 8.66 ppm (s, H8). If the reaction is carried out with 0.33 equivalents of dienPdII and one equivalent of 9-MeA, no dinuclear complex is expected, only mononuclear complexes (A-N1 and A-N7). This was observed at pD ~ 5.3. 2.602.803.003.203.403.604.00 8.00 8.208.408.608.809.009.00 8.80 8.60 8.40 8.20 8.00 4.00 3.80 3.60 3.40 3.20 3.00 2.80 2.60 ~ ~ ~ ~ ~ ~ A B C D A H2A H8A H2C H8B H2B H8C H8D H2D (i) (ii) Figure 52: 1H NMR spectrum of (19) at pD ~ 8.5 (i). Addition of an excess of [dienPd(H2O)]2+ leads to spectrum (ii) on the bottom (pD ~ 6.7). 2.5. 9-MeA System 75 In order to find out which compounds correspond to the rest of the signals, the following experiment was carried out. Two equivalents of [dienPd(H2O)]2+ were added to a solution of 9-methyladenine at pD ~ 5.82. There is no signal of free adenine. But there are three principal sets of signals A, B and C (spectrum (iii) Figure 53). The chemical shifts at pD ~ 5.82 of the species B are 3.88 ppm (s, CH3), 8.52 ppm (s, H8) and 8.35 ppm (s, H2). The resonances of C are at 3.82 ppm (s, CH3), 8.60 ppm (s, H2) and 8.14 ppm (s, H8). The protons at lowest field correspond to the dinuclear complex A. The assignment of the proton resonances was made by performing the same reaction with deutered 9-methyladenine (9-MeA-d8) (spectrum (iv), Figure 53). Both reactions, with 9-MeA and with 9-MeA-d8, were brought to pD ~ 8.8 and to pD ~ 10.25. 9.00 8.90 8.80 8.70 8.60 8.50 8.40 8.30 8.20 8.10 H8C H2BH8B H2C H8A H2A H2A H2C H2B 3.60 3.40 3.20 3.00 2.80 2.60 A C B A B C ~ ~ ~ ~ ~ ~ (iii) (iv) Figure 53: Reaction of 2 dienPdII with 9-MeA at pD ~ 5.82 (iii). Comparison with the same reaction using 9-MeA-d8 (iv). At pD ~ 8.8 there are two new signals (D and E), one of them, E, is of very intensity. Signal A decreases strongly. C is the main signal (Figure 54). 2.5. 9-MeA System 76 Spectrum (v) corresponds to the reaction with 9-MeA and spectrum (vi) to the reaction with 9-MeA-d8. 9.00 8.80 8.60 8.40 8.20 8.00 3.80 3.60 3.40 3.20 3.00 2.80 B C E D H2A H8A H2C H8B H2B H2D/H2E H8C H2D }H8E H2A H2C H2B H2D/H2E } A A B E D C ~ ~ ~ ~~ ~ (v) (vi) Figure 54: 1H NMR spectrum of the reaction between 2 [dienPd(H2O)]2+ with 1 equivalent 9-MeA at pD ~ 8.8 (v) and with 9-MeA-d8 (vi). At pD ~ 10.25 the signal corresponding to A has disappeared and the resonance E has increased in intensity. The main signals are now D and E (see Figure 55). The chemicals shifts corresponding to the compound D are: 3.75 ppm (s, CH3), 8.10 ppm (s, H2) and 8.01 ppm (s, H8) and the ones corresponding to E are: 3.80 ppm (s, CH3), 8.07 ppm (s, H2) and 8.22 ppm (s, H8). 8.70 8.408.60 8.30 8.20 8.10 8.008.50 3.80 3.60 3.40 3.20 3.00 2.80 2.60 B C E D H2C H8B H2B H8E H8C H8D H2E H2D ~ ~ ~ ~ Figure 55: 1H NMR spectrum of the reaction of 2 [dienPd(H2O)]2+ with 9-MeA at pD ~ 10.25. 2.5. 9-MeA System 77 If another equivalent of [dienPd(H2O)]2+ is added to the reaction mixture at pD ~ 9, signal A reappears in low intensity and E decreases strongly. Now, the main signals are C and D (Figure 56). A B C D E H2A H8A H8B H2B H2C H8C H8D H2D H8E H2E 8.80 8.70 8.60 8.50 8.40 8.30 8.20 8.10 8.00 7.90 4.00 3.80 3.60 3.40 3.20 3.00 2.80 2.60 ~ ~~ ~ Figure 56: 1H NMR spectrum of the reaction of 3 [dienPd(H2O)]2+ with 9-MeA at pD ~ 9. Instead of adding two equivalents of [dienPd(H2O)]2+, the reaction is carried out by adding one equivalent of [dienPd(H2O)]2+ to one equivalent of 9- MeA and keeping the mixture at pD ~ 5.3, 40°C for a day. There are three sets of signals, one of which is due to free 9-methyladenine (Figure 57). A pD dependence was measured, in order to find out which resonance corresponds to {[(dien)Pd](9-MeA-N1)}2+ (C) and which one to {[(dien)Pd](9-MeA-N7)}2+ (B). 8.70 8.60 8.50 8.40 8.30 8.20 8.10 3.80 3.60 3.40 3.20 3.00 2.80 B C Free 9-MeA H8C H2 H8H2BH8B H2C ~ ~~ ~ Figure 57: 1H NMR spectrum of the reaction of one equivalent of dienPdII and one equivalent of 9-MeA at pD ~ 5.30. 2.5. 9-MeA System 78 It was observed, that the more acidic the solution is, the more free 9- methyladenine is present. Resonance C is always more intensive than signal B. Looking at the values of the chemical shifts of B and C summerized in Table 11, it can be noted that the resonances corresponding to the compound {[(dien)Pd](9-MeA-N1)}2+ (C) are not very sensitive at acidic conditions, since the N1 position is metalated. However, the resonances of {[(dien)Pd](9-MeA-N7)}2+ (B) are very sensitive at acidic conditions. In the pD range from 1.7 to 5.3 the H8 resonance of compound B undergoes a stronger upfield shift (∆δ = 0.271) than the H8 resonance of C (∆δ = 0.125). So in this pD range, the resonances of B are more affected than those corresponding to C. Based on this observation B is tentatively assigned to {[(dien)Pd](9-MeA-N7)}2+ and C to {[(dien)Pd](9-MeA- N1)}2+. However, these results are different in the case of Pt(II). The chemical shifts are also different from the values found by Jorma Arpalahti[93] for dienPt(Ado) isomers. Table 11: Values of the pD dependence from 1.7 to 5.3 of the1:1 reaction of dienPdII and 9-MeA. pD δH2C δH2B δH8C δH8B δCH3C δCH3B ppm 1.70 8.63 8.54 8.27 8.82 3.84 3.96 2.55 8.60 8.15 3.82 3.33 8.60 8.37 8.14 8.55 3.82 3.89 4.10 8.60 8.36 8.14 8.54 3.82 3.88 5.30 8.60 8.35 8.13 8.53 3.82 3.88 After the assignment of the signals A, B and C, it is proposed that signal D corresponds to (19), in other words, to {[(dien)Pd]3(9-MeA-N1,N7,N6)}5+. E could be the intermediate {[(dien)Pd](9-MeA-N7,N6)}4+. All the obtained results are summarized in Table 12. 2.5. 9-MeA System 79 Table 12: Chemical shifts of the different compounds containing one, two or three entities of dienPdII and one 9-methyladenine bonding through N1,N7 and N6. Signal Compound δCH3 δH2 δH8 A {[(dien)Pd]2(9-MeA-N1,N7)}4+ 3.88 8.75 8.66 B {[(dien)Pd](9-MeA-N7)}2+ 3.88 8.35 8.52 C {[(dien)Pd](9-MeA-N1)}2+ 3.82 8.60 8.14 D {[(dien)Pd]3(9-MeA-N1,N7,N6)}5+ (19) 3.75 8.10 8.01 E {[(dien)Pd](9-MeA-N7,N6)}4+ 3.80 8.07 8.22 2.5.5.1.2 {[(dien)Pd]3(9-MeA ─-N1,N7,N6)}Cl3.5(PF6)1.5·3H2O (19) Yellow crystals of {[(dien)Pd]3(9-MeA-N1,N7,N6)}Cl3.5(PF6)1.5·3H2O (19) were obtained from an alkaline solution containing 9-methyladenine, [(dien)Pd(H2O)]2+, NaCl and KPF6. The yellow crystals are not stable at room temperature, so they were measured at low temperature. They crystallize in the monoclinic P2(1)/c space group. In the refinement process of the X-ray data, all non-hydrogen atoms of the crystal were refined anisotropically. Crystal data, data collection and refinement parameters for (19) are summarized in Table A-5 (see Appendix). The solid state structure of (19) consists of three (dien)PdII entities bound to the N7, N1 and N6 positions of the anionic 9-methyladenine nucleobase. A view of the cation {[(dien)Pd]3(9-MeA–-N1,N7,N6)}5+ with the labeling scheme is shown in Figure 58. 2.5. 9-MeA System 80 Pd1 Pd2 Pd3 N7 N6 N1 N13 N11 N12 N21 N23 N22 N33 N32 N31 Figure 58: View of the cation {[(dien)Pd]3(9-MeA─-N1,N7,N6)}Cl3.5(PF6)1.5·3H2O (19). The Cl– and PF6– anions and the three water molecules are omitted for clarity. The (dien)PdII entities at N1 and N6 are mutually syn oriented, leading to a contact of 3.23 Å between Pd3 and Pd2. Consequently, Pd1 at N7 and Pd2 at N6 are anti, displaying a large separation of 5.24 Å. Pd-N distances to the nucleobase are between 2.031(3) (Pd1-N7) and 2.063(3) Å (Pd3-N1). It is to be noted that the Pd2-N6 distance, hence the bond distance to the deprotonated exocyclic amino group, is in between these extremes (2.041(3) Å), and not shorter, as might have been suspected. Geometries of the dienPdII entities are normal. In the case of dienPd1II entity, the N13-Pd1-N11 angle deviates markedly from 180° (166.93(13)°), unlike the N12-Pd1-N7 angle, which is 175.61(13)°. The dien ring displays the characteristic sting ray structure, with C12 and C13 out of the Pd coordination plane by -0.707(5) and -0.568 (5) Å, respectively. The two central CH2 groups (at either site of N12) are oriented toward the C8 of 9-MeA─. 2.5. 9-MeA System 81 In the case of the palladium atom coordinated at N6 of the adenine (Pd2) a similar situation is observed. There is a deviation from linearity of the angle between Pd2 and the two coordinated sites of the dien ligand cis positioned to the adenine nucleobase: N23-Pd2-N21, 164.59(13)°. This contrasts with the almost linear orientation of N22-Pd2-N6, 175.92(13)°. The dien ring also displays the characteristic sting ray structure, with C22 and C23 out of the Pd coordination plane by -0.609(5) and -0.587(5) Å, respectively. In this case, the central CH2 groups (at N22) are oriented toward the C5 of 9-MeA─. The largest deviation from an ideal square-planar coordination of the palladium atoms is present in Pd3, which is coordinated to the N1 position of the adenine nucleobase. The N33-Pd-N31 angle deviates markedly from 180° (162.88(12)°), unlike the angle N32-Pd3-N1, which is 178.65(13)°. The dien ring displays again the characteristic sting ray structure, with C32 and C33 out of the Pd coordination plane by -0.543(6) and -0.486 (5) Å, respectively. The central CH2 groups (adjacent to N32) are pointing toward the C2 of 9-MeA─. Pd1-N, Pd2-N and Pd3-N distances about the Pd center range from 2.002(3) to 2.063(3) Ǻ. A list of selected distances and angles involving the Pd atoms of (19) is given in Table 13. Table 13: Selected distances (Å) and angles (º) for non-hydrogen atoms in 19. Pd1-N7 2.031(3) N12-Pd1-N13 85.00(13) Pd1-N11 20.46(3) N12-Pd1-N7 175.61(139 Pd1-N12 2.005(3) N13-Pd1-N7 95.65(129 Pd1-N13 2.020(3) N12-Pd1-N11 84.25(13) N13-Pd1-N11 166.93(13) N7-Pd1-N11 94.51(12) Pd2-N6 2.041(3) N22-Pd2-N6 175.92(13) Pd2-N21 2.046(3) N22-Pd2-N23 83.63(13) Pd2-N22 2.010(3) N6-Pd2-N23 95.84(12) Pd2-N23 2.043(3) N22-Pd2-N21 84.52(13) N6-Pd2-N21 95.27(12) 2.5. 9-MeA System 82 N23-Pd2-N21 164.59(13) Pd3-N1 2.063(3) N32-Pd3-N31 83.88(12) Pd3-N31 2.036(3) N32-Pd3-N33 83.45(12) Pd3-N32 2.002(3) N31-Pd3-N33 162.88(12) Pd3-N33 2.055(3) N32-Pd3-N1 178.65(13) N31-Pd3-N1 95.71(12) N33-Pd3-N1 96.69(12) C6-N6 1.314(4) C8-N7-C5 105.1(3) C6-N1 1.391(4) N1-C6-C5 113.9(3) C2-N1 1.363(5) C2-N1-C6 120.0(3) The adenine ligand in (19) has normal distances and angles between atoms. But if we compare the distance C6-N6 between complex (19) and 9- MeA,[70] a slight shortening is observed for (19): 1.314(4) in (19) and 1.348(9) Å in 9-MeA. The atoms of the 9-methyladenine base are coplanar, with a r.m.s. deviation of 0.019. The dihedral angle between the adenine ring and the PdN4 coordination sphere of Pd1 is 84.43(8)°; hence the two planes are almost perpendicular. The dihedral angles between the adenine ring and the two other metal coordination planes are 54.12(9)° and 50.0(1)°, respectively for Pd2 and Pd3. The crystal packing of (19) is dictated by interactions between the {[(dien)Pd]3(9-MeA-N1,N7,N6)}5+ cation, the chloro counter anions and the crystallization water molecules. Weak interactions between fluoro ligands of the hexafluorophosphate anions and the N11H2 of the dienPd1 entity were found, and between PF6─ and C34H2 of the dienPd3 entity. As shown in Figure 59, the Cl2 anion in the crystal structure is shared by another cation, and forms hydrogen bonds with the protons of the dien ligand: Cl2···N13(H13), 3.253(3) Å, Cl2···N22b(H22b)(x-1, y, z), 3.155(3) Ǻ and Cl2···N32b(H32b)(x-1, y , z), 3.126(4) Å (Figure 59). 2.5. 9-MeA System 83 Cl2 N22b N32b N13 Figure 59: View along the a axis of the packing of the cation of (19). A view along the b axis, shown in Figure 60, reveals that there are also intermolecular hydrogen bonds between Cl4 anions and the protons of the dien group. These hydrogen bond contacts are: Cl4···N23(H23), 3.225(3) Ǻ, Cl4···N33a(H33a)(-x+2, -y+1, -z+1), 3.205(3) Å. Two consecutive cations are related by a center of symmetry. No intramolecular hydrogen bonds are present in the structure of (19). Cl4 N23a N23 Cl4a N33a N33 Figure 60: View along the b axis of the packing of the cation of (19). 2.5. 9-MeA System 84 Packing of (19) does not allow π-stacking interactions between aromatic rings. The Cl4 is also involved in another hydrogen bond with a water molecule: Cl4···O2w, 3.089(4) Ǻ. Hexafluorophosphate anions are located between ribbons of cations of (19) (Figure 61). Figure 61: Top view of a layer in the solid state structure of (19). 2.5.6 Tris(cytosine) Complexes 2.5.6.1 PtC3A In order to study if there is a possibility of stabilization of the N(6)H─ by the exocyclic amino group of a cytosine nucleobase, a tetrakis (nucleobase) complex with three cytosine ligand and one adenine ligand was studied. The starting compound [Pt(1-MeC-N3)3Cl]NO3·1.5H2O was synthesized according to a published method.[94] The platinum is coordinated through the N3 positions of three cytosine nucleobases. Its respective aqua species [Pt(1-MeC-N3)3(H2O)]2+ has been studied in solution by means of 1H NMR spectroscopy.[95] 2.5. 9-MeA System 85 Reaction of [Pt(1-MeC-N3)3(H2O)]2+ with 9-MeA in water (1:1 ratio, 40°C, pD ~ 4) gave two products together with some unreacted ligand (ca. 15% after 16 days).[95] According to a pD dependence of the 1H NMR resonances, they are identified as [Pt(1-MeC-N3)3(9-MeA-N7)]2+ (ca. 70%) and [Pt(1-MeC-N3)3(9- MeA-N1)]2+ (ca. 15%). The N7 linkage isomer [Pt(1-MeC-N3)3(9-MeA-N7)]2+ (20) was isolated as NO3─ salt in crystalline form, but the quality was not good enough for an X-ray crystal structure analysis. However, the X-ray structure of the ClO4─ salt has been previously described[95] and is expected to be similar to the nitrate salt as far as the structure of the cation is concerned. Figure 62 shows the 1H NMR spectrum of complex (20). Signals associated with the methyl groups of the 1-methylcytosine ligands appear in a 2:1 ratio at 3.43 ppm and 3.34 ppm, which suggests that the one at 3.43 ppm corresponds to the methyl group of the cytosine trans to the adenine nucleobase. The signals associated with H5 and H6 display two sets of doublets in a 2:1 ratio, which is also attributed to the different types of 1-methylcytosine ligands. Resonances due to the 9-MeA can readily be assigned: H8 (8.80 ppm,s); H2 (8.26 ppm, s) and CH3 (3.85 ppm, s). 2.5. 9-MeA System 86 H8 H2 H6(C) H5(C) CH (A)3 CH (C2)3 CH (C1)3 7A (2)C 3C(2) 2+ 3 C(1) 3 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 δ / ppm Figure 62: 1H NMR spectrum of [Pt(1-MeC-N3)3(9-MeA-N7)]2+ (20) in D2O (pD =7.54) The Pt atom is coordinated to the respective N3 sites of the three 1- methylcytosine ligands and the N7 position of a 9-methyladenine, and it adopts a square-planar PtN4 coordination geometry. The orientation of the cytosine rings in the ClO4─ salt[95] is head-tail-head (h-t-h), meaning that each cis- arranged 1-MeC ligand is oriented head-tail with respect to its neighbour. A resonable explanation for this orientation is the stabilization of the structure by intramolecular hydrogen bonds. Intramolecular hydrogen bonding between the exocyclic groups of pyrimidine bases requires a cis arragement of the respective ligands.[96] In the ClO4─ salt of (20), four intramolecular hydrogen bonds between N4 and O2 of the cytosine ligands are found[95] and the adenine ligand is oriented in a way that permits H-bonding interactions with both the two cytosine ligands in cis position, one being significantly shorter than the other. The N7 coordinated complex undergoes protonation at the N1 position with a pKa of 1.97 ± 0.03.[97] In order to check if this intramolecular interactions affect the pKa 2.5. 9-MeA System 87 value of the exocyclic amino group of the adenine, a pD dependence of the complex [Pt(1-MeC-N3)3(9-MeA-N7)]2+ (20) was recorded (Figure 63). We observed that the 1-MeC resonances are more affected than the 9-MeA resonances. So the pKa value obtained (pKa(H2O) = 12.63 ± 0.04) corresponds to the deprotonation of the NH2 of the cytosine nucleobase. To conclude, there is no stabilization between the 9-methyladenine and the 1-MeC ligands. This value compares well with the pKa value of cis- and trans-[Pt(1-MeC-N3)(9-MeA- N7)]2+ (see Section 2.4.2.1). 2 4 6 8 10 12 14 7,1 7,2 7,3 7,4 7,5 7,6 (δ) /p pm pD H6(C2) Figure 63: pD dependence of [Pt(1-MeC-N3)3(9-MeA-N7)]2+ (20). 2.5.6.2 Water Cluster: [Pt(1-MeC-N3)3(OH)](ClO4)0.5(OH)0.5·7H2O (21) In an attempt to isolate a deprotonated derivate of [Pt(1-MeC-N3)3(9- MeA-X)]2+ (X = N1 or N7), the reaction mixture was brought to basic pH by addition of 1M NaOH. Eventually colourless crystals were isolated and charaterized by X-ray analysis. However, the crystals proved to be [Pt(1-MeC- N3)3(OH)](ClO4)0.5(OH)0.5·7H2O (21), hence a deprotonated form of the starting compound [Pt(1-MeC-N3)3(H2O)]2+, which had not reacted with 9-MeA. Compound (21) crystallizes in the monoclinic system. In the refinement process, all the hydrogen atoms, including the ones of the seven water molecules, were found in the difference Fourier map and refined without 2.5. 9-MeA System 88 restraints. Crystallographic data, data collection parameters and refinement parameters of (21) data are listed in Table A-6 (see Appendix). Complex (21) proved to be quite interesting in that it contained a well- structured mixed hydroxo, aqua oligomer. Discrete water clusters represent a topical research issue. Of particular interest are, among others, fundamental aspects such as the geometrie of water structure,[97] the mobility of protons in hydrogen bonds between water molecules,[98] structural changes of ice in confined space,[99] or the role of water stabilizing biopolymer aggregates.[100] The crystallographic characterization of clusters or chains of water molecules is usually aided by the presence of guest molecules (such as in water clathrates),[101] by supramolecular architectures,[102] or even by the presence of simple coordination compounds.[103] In the compound (21), the H bonding between the nucleobases display well-structured water cluster which also interact with the heterocyclic rings. The relevance of water clusters in biological systems and chemical processes has been intensively studied in recent years.[97,104-109] The H bonded water molecules of the clusters can display different geometries. Some examples of tetramers,[110,111] pentamers,[112] hexamers,[113-116] octamers,[117,118] and decamers[119] have been reported. These water rings as basic units can be considered as building blocks for the formation of supramolecular entities. There are many examples of 1D,[120,121-123] 2D,[124-127] and 3D polymers.[103a] The platinum atom is coordinated to the respective N3 sites of three 1- methylcytosine ligands and to a OH group, which is coordinated to four water molecules, forming a cyclic pentamer (Figure 64). The Pt atom adopts a square- planar PtN3O coordination geometry. The metal is coplanar with the four donors with a deviation of ± 0.023 Ǻ. Selected bond lengths and angles are given in Table 14. 2.5. 9-MeA System 89 O1p O1w O2w O3w O4w N3C N3B N3A Pt Figure 64: Views of the water cluster of (21) linked to the platinum atom via the OH─ligand. Table 14: Selected distances (Å) and angles (º) for non-hydrogen atoms in 21. Pt-N3A 2.030(6) O1P-Pt-N3B 178.4(18) Pt-N3B 2.022(6) O1P-Pt-N3C 88.3(2) Pt-N3C 2.027(6) N3B-Pt-N3C 92.5(2) Pt-O1P 2.001(5) O1P-Pt-N3A 86.4(2) N4A···O2B 2.951(9) N3B-Pt-N3A 92.9(2) N4C···O2B 3.009(8) N3C-Pt-N3A 174.4(2) N4B···O2A 2.954(9) N4B···O2C 3.036(8) O1P···O1w 2.821(8) O1w···O2w 2.706(9) O2w···O3w 2.63(1) O3w···O4w 2.69(1) O4w···O1P 2.879(9) There are no unusual bond distances or angles either in the Pt coordination sphere or in the heterocycles. The atoms of the heterocyclic rings are approximately coplanar within ±0.0251, ±0.0138 and ±0.0087 Å, respectively for 1-MeCa, 1-MeCb and 1-MeCc. Their mean planes form dihedral angles with the Pt coordination plane which do not deviate significantly from a perpendicular 2.5. 9-MeA System 90 arrangement (89.9(2)° and 86.0(2)° for the cytosines trans to each other, 87.2(2)° for the cytosine trans to the OH group). The orientation of the cytosine rings is head-tail-head (h-t-h), meaning that each cis-arranged 1-MeC ligand is oriented head-tail with respect to its neighbour, a feature also confirmed for [Pt(1-MeC-N3)3(9-EtGH-N7]2+, for the precursor compound of (21), [Pt(1-MeC)3Cl]+[94], as well as for other structurally characterized Pt(II)[128] and Pd(II) complexes[96] containing three cytosine ligands. A reasonable explanation for the preference of the h-t-h orientation is the stabilization of the structure by intramolecular hydrogen bonds. Intramolecular hydrogen bonding between the exocyclic groups of pyrimidine bases requires a cis arrangement of the respective ligands.[96] Therefore the h-t- h arrangement in tris(1-methylcytosine) compounds and the analogous h-t-h-t arrangement in a tetrakis(1-methylcytosine) Pt(II) complex,[94] which likewise has been confirmed by X-ray analysis, allow the formation of the maximum number of intramolecular hydrogen bonds. In (21), four intramolecular hydrogen bonds between N4 and O2 of the cytosine ligands are found, ranging from 2.951(9) to 3.036(8) Å (Figure 65). O2A O2C N4C N4A O2B N4B Figure 65: Molecular cation of [Pt(1-MeC-N3)3(OH)]+·4H2O with intramolecular H bonds between cytosine ligands. The hydroxo ligand (O1P) is incorporated in a cyclic structure comprised of four additional water molecules, O1w, O2w, O3w and O4w. Other water 2.5. 9-MeA System 91 molecules (O5w and O6w) are joined to the cyclic pentamer (via O1w and O4w, respectively). The atoms of the cyclic pentamer are arranged in an envelope conformation, in which the O1P atom lies slightly (0.016(3) Ǻ) above the plane defined by the remaining four atoms. Distances between oxygens atoms within the pentamer are: O1P···O1w, 2.821(8) Å; O1w···O2w, 2.706(9) Å; O2w···O3w, 2.63(1) Å; O3w···O4w, 2.69(1) Å; O4w···O1P, 2.879(9) Å. Internal angles of the pentamer range from 93.25(3)° (O1P···O1w···O2w) to 120.5(3)° (O4w···O1P···O1w). Figure 66 shows in detail the distances and angles between atoms of the cyclic pentamer. O1P O1w O2w O3w O4w 2.821Å 2.706 2.630 2.690 2.879 Å Å Å Å 93.2° 120.5° 115.7° 94.0° 113.3° Figure 66: View of the cyclic pentamer present in (21). A study of the hydrogen bonding pattern shows the formation of a dense water polymer. There are two types of hydrogen bonds involving water molecules: the first one, between water molecules interacting only with water molecules (formation of water polymer); and the second one, between water molecules of the water polymer interacting with the exocyclic oxygen atoms of the cytosine rings. Each cation of (21) and its corresponding water molecules of crystallization forms intermolecular hydrogen bonds between the water molecules with the cation at 1/2–x, 1/2–y, -z, thus leading to a pair of centrosymmetrically related cations (Figure 67). The hydrogen bonds and the water molecules in orange are closest to the viewer. The symmetry operator (i) for the description of hydrogen interactions is 1/2–x, 1/2–y, -z. Oxygen atoms of the water molecules that are involved in 2.5. 9-MeA System 92 hydrogen bond formation exclusively between water molecules are: O5w (H- bonds with O1w and one symmetrical O6w), O6w (H-bonds with O4w, O8w, O7w and one symmetrical O5w). Oxygen atoms of the water molecules of the water polymer which also join the nucleobase polymer are: O2w (H bond with one symmetrical O2A), O3w (H bond with one symmetrical O2C). A detailed list of the interactions including the symmetry operator is given in Table 15. O1P O2C O2A O1w O5w O6w(i) O7w(i) O8w(i) O4w O6w O8w O7w O5w(i) O2C(i) O1w(i) O2w(i) O1P(i) O3w(i) Pt(i)Pt O3w O2w O4w(i) O2A(i) Figure 67: View of the polymeric structure of crystallization water in (21). In the 3D building of the polymer, the side defined by the O4w···O2w atoms of two pentamers of adjacent unit cells approach each other very closely. As a consequence of this, a cyclic hexamer can be observed between two pentamers. The hexamer and the pentamer entities share the O4w and O2w oxygen atoms. Table 15: Hydrogen bond distances (Å) involving the water of crystallization in 21. O2C···O3w(i) 2.750(1) O6w···O8w 3.040(2) O2A···O2w(i) 3.104(9) O4w···O6w 2.890(1) 2.5. 9-MeA System 93 O1w···O5w 2.920(1) O4w···O2w(i) 2.900(9) O5w···O6w(i) 2.950(1) O6w···O7w 2.530(2) Atoms in the hexamer adopt a slight chair conformation. The value of the sum of the internal angles of the ring is 482.2°; this value is much closer to 360° (planar structure) than to 657° (perfect chair conformation). The atoms O4w and symmetrical related atom O4w(i) lie 1.76(1) Ǻ out (below and above, respectively) of the plane defined by the other four water molecules (O2w, O3w, O2w(i), O3w(i)). The distances and angles in (21) are in agreement with reported examples that include this type of water cyclic architectures.[97,103,120] A perspective of two basic units of the water polymer is depicted in Figure 68. O2w O4w(i) O3w O3w(i) O2w(i) O4w O4w(i)O3w(i) O2w(i) O4w O3w O2w Figure 68: Top view of the cyclic water hexamer structure in (21) (left). Side view of the cyclic water hexamer structure showing its chair conformation (right). Probably, one of these water molecules corresponds to the (OH–)0.5, but it is very difficult to distinguish between a water molecule and a hydroxyl group, because these molecules are continuously moving in the crystal structure. The 2.5. 9-MeA System 94 distance between O6w and O7w is very short. It could be possible, that one of these oxygen atoms in fact represents a hydroxide rather than a water molecule. The 1H NMR spectrum of [Pt(1-MeC-N3)3(OH)]+ (21) (Figure 69) bears no peculiarities. There are two sets of inequivalent cytosine ligands of 2:1 ratio. The chemical shifts of cytosine H6 and H5 doublets, in position trans to the hydroxo ligand, are 7.43 and 5.89 ppm. However, the chemical shifts of H6 and H5 of the cytosines cis to OH are 7.54 and 5.96 ppm respectively. Two different sets of methyl groups can be likewise observed, at 3.41 (for 1-MeC cis to OH group) and 3.35 ppm (for 1-MeC trans to OH group). H6 H5 CH3 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 δ / ppm Figure 69: 1H NMR spectrum of [Pt(1-MeC-N3)3(OH)](ClO4)0.5(OH)0.5·7H2O (21) (D2O, pD ~ 7.1) The pH dependence of (21) was measured (Figure 70). The first pKa value corresponds to the deprotonation of the aqua ligand and it was found to be in water pKa = 5.72 ± 0.06. This value agrees with the one calculated for the acidity of the aqua ligand in the complex cis-[(NH3)2Pt(1-MeC-N3)(H2O)]2+ 2.6. 1,9-DimeAH+ System 95 (Chapter 2.8). The second deprotonation step begins at pD 12 and corresponds to the loss of one proton of the NH2 of the cytosine nucleobase. 2 4 6 8 10 12 3,406 3,408 3,410 3,412 3,414 3,416 3,418 3,420 3,422 3,424 (δ) / pp m pD Figure 70: Representation of the chemical shifts of H5 of the 1-methylcytosine in (21) in dependence of pD. 2.6 1,9-DimeAH+ System Methylation of the N1 site of 9-methyladenine acidifies the protons of the exocyclic amino group considerably. Thus, the pKa of the cationic 1,9-DimeAH+ is 9.1[129] as compared to 16.7 for neutral 9-MeA[18] (Figure 71). As expected, metal coordination to N7 of 1,9-DimeAH+ acidifies the N(6)H2 group further. 1,9- DimeA is a model of the corresponding nucleoside, which occurs in its protonated form (1,9-DimeAH+) as a rare base in tRNAs[130] and occasionally in rRNAs.[131] It is known that there is a difference between deprotonation of the cationic 1,9-dimethyladeninium cations in the here described complexes and deprotonation of the neutral ligand 9-methyladenine. However, this difference is a formal one only, as both the methyl group at N1 and the metal binding to this site are causing an acidification of the exocyclic NH2 group. The effect is only larger in the case of methylation (ΔpKa = 16.7 – 9.1 = 7.6) as compared to platination (ΔpKa ~ 4[37]), thus leading to a shift of qualitatively identical processes from strongly alkaline medium (Pt(9-MeA-N1), pKa = 12 – 13) to less alkaline medium (1,9-DimeAH+, pKa = 9.1). In both instances, binding of 2.6. 1,9-DimeAH+ System 96 (additional) PtII at N7 reduces these pKa values further, by approximately identical 3 log units. Moreover, the steric situations (possibility of formation of an intramolecular hydrogen bond) in both cases are closely similar. As previously shown[132], the two pKa values of trans-[(NH3)2Pt(1,9-DimeAH- N7)2]4+ are 4.1 ± 0.2 and 6.4 ± 0.3. N N N N NH Me Me N N N N NH Me H -H+ +H+ pKa = 9.1 Me 1 6 3 7 9 Figure 71: pKa value of 1,9-DimeAH+. Two forms of the bis(1,9-DimeA) complex of trans-(NH3)2PtII containing PtII bonded to N7 with different protonation states of the adenine ligands have been prepared and characterized by 1H NMR spectroscopy: trans-[(NH3)2Pt(1,9- DimeAH-N7)(1-MeC-N3)](NO3)3 (22) and trans-[(NH3)2Pt(1,9-DimeAH-N7)(9- MeGH-N7)](NO3)3 (23). 2.6.1 pKa values of trans-[(NH3)2Pt(1,9-DimeAH-N7)(1-MeC- N3)](NO3)3 (22) pKa values of the mixed 1,9-dimethyladeninium, 1-methylcytosine complex (22) were determined by pD dependent 1H NMR spectroscopy. There were several problems associated with this method, as in part previously reported.[132] First, due to 1,9-DimeA(H) rotation, all proton resonances of this ligand are considerably broadened, in particular at pD values where deprotonation of the 1,9-DimeAH+ ligand takes place. Second, PtII migration was a serious problem even at pD ~ 5. Third, isotopic exchange of the 2.6. 1,9-DimeAH+ System 97 heteroaromatic protons took place, causing disappearance of the respective resonances. However, the presence of a second nucleobase (in this case 1- MeC) also proved advantageous, because (de)protonation reactions of 1,9- DimeA(H) were sensed by these ligands as well. Δδ values were considerably smaller though. Compound (22) was studied in the pD range 1.5 – 12.5. Determination of the acidity of the 1,9-DimeAH+ ligand was straightforward, giving an average value of 6.84 ± 0.05 (D2O), corresponding to 6.29 ± 0.05 in H2O for the four resonances of the 1,9-DimeA(H) ligand. An intramolecular hydrogen bonding between N(6)H2 of 1,9-DimeAH and the exocyclic amino group of the cytosine should be possible (Figure 72). No deprotonation of the exocyclic amino group of the 1-MeC ligand was observed up to pD 10. However, a major rearrangement took place when a sample of (22) was kept at this pD for some time. This process will be discuss in the next chapter. N HN N N NH2 H3C H3C N N H2N O CH3 Pt a a A C N HN N N H N H3C H3C N N HN O CH3 Pt a a A C H -H+ +H+ Figure 72: Intramolecular hydrogen bond between NH– of 1,9-DimeA and the exocyclic amino group of 1-methylcytosine in the deprotonated form of (22). 2.6.2 pKa values of trans-[(NH3)2Pt(1,9-DimeAH-N7)(9- MeGH-N7)](NO3)3 (23) Compound (23) has been prepared on a small scale. pD dependent 1H NMR spectra for the individual nucleobase resonances gave a pKa value of 7.16 ± 0.06 in H2O. This can be considered as an average of two 2.6. 1,9-DimeAH+ System 98 pKa values. Here two pKa values are expected, one of the exocyclic amino group of the adenine and the other one due to the deprotonation at N1 of the guanine ligand. Probably, the difference between both of them is so small, that more data points are needed to get individual pKa values. Even then the possibility of a tautomer equilibrium must not be overlooked. Due to the appearance of new resonances the spectra become very complicated, however. In comparison with the complex trans-[(NH3)2Pt(1,9-DimeAH-N7)(1-MeC- N3)](NO3)2 (22), here no stabilization of the 1,9-DimeA ligand by hydrogen bonding is expected in the deprotonation form of (23) (Figure 73). N HN N N NH H3C H3C Pt a a A N HN N N NH H3C H3C Pt a a A-H+ +H+ H N N N N O NH2G CH3 H N N N N O NH2G CH3 H Figure 73: No stabilization of deprotonated 1,9-DimeA ligand by intramolecular hydrogen bonding. 2.6.3 Migration of CH3 in 1,9-DimeAH + and Dimroth Rearrangement In the course of our studies with 1,9-DimeAH+ complexes, we frequently observed changes in the 1H NMR spectra when the solution was kept for some time. As X-ray crystallography shows, these new signals are due to species arising from a “migration” of the methyl group from the N1 position to the N6 position to give 1,9-dimethyladeninium. This process is called Dimroth- rearrangement.[133] The basis of the mechanism of this reaction is shown in 2.6. 1,9-DimeAH+ System 99 Figure 74. It concerns here a relocation, in which under ring opening the N1 and N6 atoms exchange their positions. The reaction can be already introduced in weakly alkaline solution by the attack of a hydroxyl ion on the C2 atom. It can be used to produce N6-alkylated derivates of adenine.[134] Details of this process applied to compounds (22) and (23) are reported below. N N N N NH2 H3C CH3 H O- N N N N NH2 H3C CH3 HO H2N N N N N CH3 HO H3C N N N N N CH3 H3C H -H2O N N N N NH CH3 H3C * + * * ** 1 2 6 Figure 74: Mechanism of the process Dimroth-rearrangement. This type of reaction involves hydrolytic cleavage of the N1–C2 bond, followed by rotation and recyclization. As, verified by 15N labelling, the endocyclic nitrogen N1 bearing the alkyl group becomes the exocyclic N6 atom.[135] 2.6.3.1 Possibility of Migration in trans-[(NH3)2Pt(1,9-DimeAH- N7)(1-MeC-N3)](NO3)3 (22) The determination of the acidity constant for the deprotonation of the NH2 position of 1,9-DimeA has been described in Section 2.6.1. In the course of the determination of this pKa value, we noticed new signals, which have been assigned to different migration processes. As an example for the change of the 2.6. 1,9-DimeAH+ System 100 chemical shifts of the methyl groups and the appearance of new signals, a stackplot of the resonances in the aliphatic region of the 1H NMR spectra of the trans-[(NH3)2Pt(1,9-DimeAH-N7)(1-MeC-N3)]3+ (22) system at different reaction times is shown in Figure 75. A A A A A B BB B B BC C C B B B A A A A A A C C C B A C C A B AC B S S pD = 2.10 pD = 7.83 After 2 days After 1 week After 3 weeks 4.40 4.20 4.00 3.80 3.60 3.40 δ / ppm (pD = 7.83) (pD = 7.83) (pD = 7.83) Figure 75: Stackplot of the methyl group signals of (22) in D2O at different pD values after different periods of time. A = trans-[(NH3)2Pt(1,9-DimeAH-N7)(1- MeC-N3)]2+ (23); B = trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)](NO3)2 (24); C = not determined; S = starting compound 1,9-DimeAH+. From pD = 2.10 to pD = 7.83 the signals corresponding to the methyl groups of the adenine, undergo upfield shifts with increasing pD. At pD = 2.10, the chemical shift difference between the CH3-N9 and CH3-N1 resonances is very small. However, at pD = 7.83 these resonances are more clearly separated. 2.6. 1,9-DimeAH+ System 101 It should be noted, that the resonance of the methyl group of the cytosine remains in the same region (~ 3.5 ppm). This is because the first deprotonation step corresponds to the deprotonation of the NH2 of the adenine. Another observation, which has been made after two days, is the appearance of new signals (C). This finding, which occurs at pD ~ 5 and higher, clearly indicates that new compounds are formed. Two different sets of signals are observed. We attribute these resonances to the migration of the trans- (NH3)PtII entity (compound C) and to the migration of the methyl group from N1 to N6 (compound B). Several NMR experiments have been carried out at different pD values and these indicate that the migration of the methyl group takes place first. After three weeks, the resonances of the starting compound have almost disappeared, whereas the resonances of the new compound (B) have increased in intensity. In oder to determine if this interpretation is correct, it was decided to react 6,9-DimeA with trans-[(NH3)2Pt(1-MeC-N3)Cl](NO3). 2.6.3.2 trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)](NO3)2 (24) One equivalent of AgNO3 was added to trans-[(NH3)2Pt(1-MeC- N3)Cl]+, in oder to obtain the aqua species. After removal of AgCl, one equivalent of 6,9-DimeA was added. The mixture was kept at 40°C for three days. Crystals of (24) suitable for X-ray crystal structure analysis were obtained by slow evaporation of the solvent at 4°C. Complex (24) crystallizes in the monoclinic P2/n space group (see X-ray Table A-7). X-ray crystallography reveals the presence of two nitrate ions. The solid state structure of (24) consists of a platinum atom coordinated to the N3 atom of the 1-methylcytosine and to the N7 atom of the 6,9- Dimethyladenine in a square-planar coordination geometry, with no observable distortions. A view of the trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)]2+ cation is 2.6. 1,9-DimeAH+ System 102 depicted in Figure 76. The angles between the platinum atom and the respectively trans-positioned ligands are nearly 180°: N7A-Pt-N3B, 176.5(5)° and N11-Pt-N12, 177.1(5)°. The Pt, N11 and N12 atoms are situated below (- 0.005(5), -0.052(6) and -0.052(6) Å, respectively) and the N7A and N3B above (0.055(6) and 0.053(6) Å, respectively) the platinum coordination plane, with a r.m.s. eviation of 0.048. Pt-N distances range from 1.985(12) to 2.060(12) Å. A list of selected distances and angles between atoms of trans-[(NH3)2Pt(6,9- DimeA-N7)(1-MeC-N3)](NO3)2 (24) is given in Table 16. Pt N3B N7A N11 N12 N6A C6M Figure 76: X-ray structure of the cation trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC- N3)]2+ (24). Table 16: Selected distances (Å) and angles (º) for non-hydrogen atoms in 24. Pt-N7A 1.985(12) N7A-Pt-N3B 176.5(5) Pt-N3B 2.014(13) N7A-Pt-N11 88.4(5) Pt-N11 2.050(12) N3B-Pt-N11 92.3(5) Pt-N12 2.060(12) N7A-Pt-N12 90.5(5) N6-C6 1.328(17) N3B-Pt-N12 89.0(5) N6-C6M 1.418(17) N11-Pt-N12 177.1(5) C2A-N1A-C6A 119.5(13) C8A-N7A-C5A 103.5(13) 2.6. 1,9-DimeAH+ System 103 C8A-N7A-Pt 130.2(11) C5A-N7A-Pt 126.2(10) C4B-N3B-C2B 120.5(15) The adenine base bonded via N7 to the platinum atom presents normal distances and angles between atoms. The atoms of the 9-methyladenine base are coplanar, with a r.m.s. deviation of 0.018. The dihedral angle between the adenine ring and the platinum coordination plane is 89.9(3)°.The cytosine ligand in (24) has normal distances and angles between atoms. The two external Pt-N- C angles are significantly different: the Pt-N3B-C2B angle is 114.7(11)° and the Pt-N3B-C4B angle is larger, 124.8(11)°. This is a common feature of many 1- MeC compounds of Pt.[136] The atoms of the 1-methylcytosine base are coplanar, with a r.m.s. deviation of 0.015. The dihedral angle between the cytosine ring and the platinum coordination plane is 89.5(4)°. So both nucleobases are nearly perpendicular to the plane of Pt. As a consequence of this fact, an intramolecular hydrogen bond is observed in the cationic entity: O2B···N6A, 3.11(2) Å (Figure 77). N6A O2B Figure 77: View of the intramolecular (3.11 Å) H bonding. 2.6. 1,9-DimeAH+ System 104 Complex (24) displays few intermolecular hydrogen bonds only. The exocyclic amino group N4B of the cytosine nucleobase of the cation of (24) forms hydrogen bonds only with the oxygen atom of one nitrate anion. The water molecule is bonded to another oxygen of this nitrate: N4B(H4B)···O23, 2.941 Å, and O22···O1w, 2.871 Å (Figure 78). O1w O22 O23 N4B Pt C6M Figure 78: Intermolecular hydrogen bonds in (24). 1H NMR spectra of (24) were recorded. In aqueous solution, the spectrum displays two sets of doublets in a 1:1 ratio in the aromatic region (H6 and H5 protons of 1-MeC) as well as a singlet corresponding to the methyl group of 1- MeC. The resonances corresponding to H8, H2, CH3-N1 and CH3-N9 are well separated (Figure 79). Chemical shifts of the signals are at 8.56 ppm (H8), 8.44 ppm (H2), 7.69 ppm (H6), 6.09 ppm (H5), 3.53 ppm (CH3-N6), 3.94 ppm (CH3- N9) and 3.35 ppm (CH3-C) over a wide pH range. If we compare these chemical shifts with the ones for compound (B) in Section 2.6.3.1., they are identical (B = 24). 2.6. 1,9-DimeAH+ System 105 H8 H2 H6 H5 CH (A-N9)3 CH (A-N6)3 CH (C)3 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 δ / ppm * Figure 79: 1H NMR spectrum of trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC- N3)](NO3)2 (24) in D2O (pD ~ 6.3)(* = solvent). As expected, removal of the proton at the N1 (6,9-DimeA) position of the complex trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)](NO3)2 (24) produces lowfield shifts of the proton and methyl groups signals at strongly acidic pH values. The pKa value of (24) (N1 position) was determined to be 2.07 ± 0.04 in D2O, corresponding to 1.60 ± 0.04 in water. This value agrees well with the pKa1 value of the deprotonation of N1 of N7 platinated 9-MeAH+. A graphical representation of the pH dependence is depicted in Figure 80. 2.6. 1,9-DimeAH+ System 106 0 1 2 3 4 5 6 7 8,40 8,45 8,50 8,55 8,60 8,65 (δ) /p pm pD H2(A) Figure 80: Representation of the chemical shifts of H2 of the adenine ligand in trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)](NO3)2 (24). 2.6.3.3 Possibility of Migration in trans-[(NH3)2Pt(1,9-DimeAH- N7)(9-MeGH-N7)](NO3)3 (23) Similar results were found for trans-[(NH3)2Pt(1,9-DimeAH-N7)(9-MeGH- N7)](NO3)2 (23). The determination of the acidity constant of (23) via pD dependent 1H NMR spectroscopy was discussed in Section 2.6.2. Figure 81 shows several selected 1H NMR spectra, which were taken at different pD values. In spectra with pD < 8 only two of the three resonances expected for the methyl groups are observed. At pD ~ 8.75 we observe the expected three resonances. With increasing pD, an increasing of number of new resonances can be observed. Formation of two novel compounds could account for this observation. On one hand, (B) could be a migration product of the methyl group from N1 to N6. On the other hand, (C) could be assigned to a migration product of the trans-(NH3)2PtII entity. 2.6. 1,9-DimeAH+ System 107 A A A A A A A A A B B B C B CH (A)3 CH (G)3 pD 8.75 9.48 12.08 3.90 3.80 3.70 3.60 3.50 3.40 3.30 δ / ppm } Figure 81: Stackplot of the methyl group region of 1H NMR spectra of (23) at different pD values. As can be observed, the resonances corresponding to the product, in which the methyl group has migrated, (B) appear before the (C) resonance. 2.6.3.4 trans-[(NH3)2Pt(6,9-DimeA-N7)(9-MeGH-N7)](NO3)2·5H2O (25) To prove that the signals of (B) correspond to the postulated product, 6,9- DimeA was reacted with trans-[(NH3)2Pt(9-MeGH-N7)(H2O)]2+.This reaction was successful as judged by the fact that the signals of product (25) corresponded to (B), consistent with methyl group migration from N1 to N6. 2.6. 1,9-DimeAH+ System 108 One equivalent of 6,9-DimeA and one equivalent of trans-[(NH3)2Pt(9- MeGH-N7)(H2O)]2+ were allowed to react at 40°C for three days. The solution was then kept in the refrigerator. Within one week, colourless crystals of trans- [(NH3)2Pt(6,9-DimeA-N7)(9-MeGH-N7)](NO3)2·5H2O (25) suitable for X-ray crystal structure analysis were obtained. The crystals were isolated and measured. The complex (25) crystallizes in the monoclinic P2(1)/c space group. Data for trans-[(NH3)2Pt(6,9-DimeA-N7)(9-MeGH-N7)](NO3)2·5H2O (25) were collected at room temperature. In the refinement process of the X-ray data, all non-hydrogen atoms of the crystal were refined anisotropically. The hydrogen atoms were placed at geometrical idealized positions and refined isotropically. Crystal data, data collection and refinement parameters for (25) are summarized in Table A-8 (see Appendix). The solid state structure of (25) consists of a platinum atom coordinated to the N7 atom of the 9-methylguanine and to the N7 atom of the 6,9- Dimethyladenine in a square-planar coordination geometry, with no observable distortions. A view of the trans-[(NH3)2Pt(6,9-DimeA-N7)(9-MeGH-N7)]2+ cation is depicted in Figure 82. The angles between the platinum atom and the four coordination sites are nearly 180°: N7G-Pt-N7A, 177.7(6)° and N11-Pt-N12, 177.4(5)°. The N11, N12 atoms are situated below (-0.041(7), -0.042(7) Å, respectively) and the Pt, N7A and N7G above (0.005(6), 0.039(7) and 0.039(7) Å, respectively) the platinum coordination plane, with a r.m.s. deviation of 0.036. Pt-N distances about the Pt center range from 1.990(14) to 2.061(14) Å. A list of selected distances and angles between atoms of trans-[(NH3)2Pt(6,9-DimeA- N7)(9-MeGH-N7)](NO3)2·5H2O (25) is given in Table 17. Table 17: Selected distances (Å) and angles (º) for non-hydrogen atoms in 25. Pt-N7G 1.990(14) N7G-Pt-N7A 177.7(6) Pt-N7A 1.999(15) N7G-Pt-N12 88.5(5) Pt-N11 2.061(14) N7A-Pt-N12 90.4(5) Pt-N12 2.034(14) N7G-Pt-N11 91.8(5) N6A-C6A 1.39(2) N12-Pt-N11 177.4(5) N6A-C6M 1.48(2) C8A-N7A-C5A 103.6(15) 2.6. 1,9-DimeAH+ System 109 C8a-N7A-Pt 125.5(13) C5A-N7A-Pt 130.8(13) C6A-N1A-C2A 119.2(17) C2G-N1G-C6G 126.3(16) The presence of two anions indicates that both nucleobases are neutral. Consequently, the N1 position of 6,9-DimeA is not protonated. The size of the internal ring angle C6-N1-C2 (119.2(17)°) is consistent with this view. Values for 6,9-DimeAH with protonated N1 positions have been previously published and were found to be between 122° [(AH)2](HPO4)·2H2O[137] and 124.3º of [AH](Br)·½H2O.[138] Pt N7G N7A N6A C6M N12 N11 Figure 82: View of the cation trans-[(NH3)2Pt(6,9-DimeA-N7)(9-MeGH-N7)]2+ of (25) The guanine base bonded via N7 to the platinum atom displays normal distances and angles between atoms. The atoms of the 9-methylguanine base are coplanar, with a r.m.s deviation of 0.020. The dihedral angle between the guanine ring and the platinum coordination plane is 65.0(3)°. The atoms of the adenine base are also practically coplanar (r.m.s. deviation, 0.011). The dihedral angle between the adenine ring and the plane containing the Pt is 73.1(3)°. There is one intramolecular hydrogen bond observed (Figure 83). This bond 2.6. 1,9-DimeAH+ System 110 takes place between the exocyclic O6 of 9-MeGH and the N6H of 6,9-DimeA. The distance between both atoms is 2.93(2) Å. There are also intermolecular hydrogen bonds, e.g. between guanine bases, giving rise to pairs of H bonds between N2G and N3G sites: The N2G(H2G)···N3Ga(-x, -y+1, -z+1), 3.02(2) Å and N3G···N2Ga(H2Ga)(-x, -y+1, -z+1), 3.02(2) Å (Figure 83). These hydrogen bonds have been also observed in other strucutres.[139] N2G N2Ga N3G N3Ga O6G N6A Figure 83: View along the b axis of the packing of the cation of (25). Intramolecular N6A(H)···O6G and intermolecular N2G(H)···N3G hydrogen bonds are observed. The crystal structure also contains five water molecules of crystallization and two nitrate anions. Crystal packing of (25) is based on interactions between the trans- [(NH3)2Pt(6,9-DimeA-N7)(9-MeGH-N7)]2+ cation and the nitrate counter anions. As shown in Figure 84, the oxygen atoms of NO3– in the crystal structure are implicated in hydrogen bonding with N1G and N2G of the guanine base, and one of the ammine ligands (N12) of the platinum. 2.6. 1,9-DimeAH+ System 111 Figure 84: Intermolecular hydrogen bonds in (25). A 1H NMR spectrum of (25) was recorded. In aqueous solution, the spectrum displays two singlets corresponding to the methyl group of guanine and its H8 proton as well as singlets corresponding to H8, H2, CH3-N1 and CH3- N9 of the adenine (Figure 85). Chemical shifts of the signals are at 8.43 ppm (H8-A), 8.33 ppm (H8-G), 8.67 ppm (H2), 3.78 ppm (CH3-N6), 3.94 ppm (CH3- N9) and 3.32 ppm (CH3-G) over a wide pH range. If we compare these chemical shifts with the ones for compound (B) in Section 2.6.3.3., they are identical, thereby confirming the interpretation made above. 2.7. 1-MeC System 112 H2(A) H8(G) H8(A) CH (A-N9)3 CH (A-N6)3 CH (G)3 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 δ / ppm Figure 85: 1H NMR spectrum of (25) in D2O (pD ~ 6.4). 2.7 1-MeC System As demonstrated before in the case of PtII adenine compounds [17,37,132], suitably positioned hydrogen bond donor groups can stabilize the deprotonated amino group of a nucleobase and thus lower the apparent pKa of the amino group. When comparing pKa values of a nucleobase in dependence of the other ligands around the metal centers, it is helpful to reduce the variables as much as possible. Thus, in compounds of type trans-(NH3)2Pt(nucleobase)X (with X = second nucleobase or other ligand) the influence of a NH3 ligand bound to PtII available to form a H bond, can be considered constant to a first approximation. Furthermore, keeping the charges of the compounds to be compared identical (e.g. +2) minimizes the effects of different charge. Finally, if the two trans- positioned ligands (nucleobase and X) do not differ too much in their trans influence, as should be the case with heterocyclic N-donors, we propose that any significant shifts in pKa values of the nucleobase are to be attributed primarily due to intramolecular interactions between the nucleobase and X. In the following, this idea will be examined for the types of complexes with the trans-(NH3)2PtII moiety, namely for trans-[(NH3)2Pt(1-MeC-N3)X]2+. 2.7. 1-MeC System 113 The pKa value of the exocyclic amino group of 1-methylcytosine has been reported to be 16.7.[18] Coordination of (NH3)3PtII to the N3 position acidifies the N(4)H2 group to pKa = 12.9 (H2O).[136] With all 1-MeC compounds studied here, 1H NMR spectroscopic complications occur at high pD (≥ 12) with time. These are due to a deamination reaction of the nucleobases, during which 1-methylcytosine is converted into 1- methyluracilate.[136,140] With the adenine containing complex trans-[(NH3)2Pt(1- MeC-N3)(9-MeA-N7)](ClO4)2 (27) a similar deamination to give hypoxanthine is feasible [141], in addition to the possibility of PtII migration.[25,66,67] 2.7.1 trans-[(NH3)2Pt(1-MeC-N3)2] 2+ (26) trans-[(NH3)2Pt(1-MeC-N3)2]2+ (26) exists in aqueous solution in an equilibrium of head-head (hh) and head-tail (ht) rotamers, with the latter predominating 3:1.[142,143] The first deprotonation step of (26) was followed by 1H NMR spectroscopy in D2O solution. trans-[(NH3)2Pt(1-MeC-N3)2] 2+ trans-[(NH3)2Pt(1-MeC¯-N3)(1-MeC-N3)] + + H+ 26 Separate H5 and H6 doublets for the two rotamers were detected throughout the pD range 7-13, whereas no separate signals were seen for the N(1)CH3 singlet. Agreement between pKa values obtained for the most intense signal sets N(1)CH3, H(5)(ht), H(6)(ht) was excellent, values being 13.04, 13.04, and 13.02 (D2O), giving an average of 13.03 in D2O and 12.39 in H2O, with a standard deviation of ± 0.07. For the minor rotamer agreement was less good, 13.01 (H5) and 12.90 (H6) in D2O.[144] Although, in principle, a lower pKa could have been expected for the hh rotamer, we do not consider this difference significant. The separation between the exocyclic N(4) sites in the hh form, 2.7. 1-MeC System 114 which is estimated to be around 4 Å, is certainly too large to expect any substantial interaction between these groups. Moreover, if important, the possibility of favorable interaction between -NH¯ and -NH2 probably would have caused a shift in the rotamer equilibrium toward the head-head form, a feature not seen in the experiments (Figure 86). N N NH2 OH3C N N H2N O CH3 Pt a a N N NH2 OH3C N N H2N O CH3 Pt a a rotation head-head head-tail Figure 86: Rotamers (hh = head-head; ht = head-tail) of (26). Although for [(dien)PtII(1-MeC-N3)]2+ we have reported also migration to N4 under conditions of high pH, with trans-[(NH3)2Pt(1-MeC-N3)2]2+ (26) such a process is not observed at room temperature. 2.7.2 trans-[(NH3)2Pt(9-MeA-N7)(1-MeC-N3)] 2+ (27) Compound (27) has previously been crystallized as its ClO4– salt.[49] pD dependent 1H NMR spectra for the individual nucleobase resonances gave pKa values of 12.7 each for N(1)CH3, H5, and H6 resonances of 1-MeC. Despite considerably smaller shifts, rather similar values were obtained for N(9)CH3 and H2 resonances of the 9-MeA ligand (12.6 and 12.7), but the H8 resonance of 9- MeA (δ 8.64 at pD 6[49]) could not be evaluated due to rapid isotopic exchange. The average pKa of (27), converted from D2O to H2O, is thus 12.0 ± 0.1 (Figure 87). 2.7. 1-MeC System 115 2 4 6 8 10 12 14 16 18 5,5 5,6 5,7 5,8 5,9 6,0 6,1 (δ) /p pm pD H5 1-MeC Figure 87: pD dependence of the H5 resonance of the 1-MeC of (27). The experimental values are represented by the solid line, and the dotted line corresponds to calculated values based on a fitting of the pD dependence. A comparison of the extent of upfield shifts of the individual proton resonances reveals that 1-MeC resonances are much more affected than those of 9-MeA: Δδ values (in ppm) in the pD range 7 - 12.8 are 0.38 (H6, 1-MeC), 0.24 (H5, 1- MeC) and 0.12 (CH3, 1-MeC), as compared to 0.02 (H2, 9-MeA) and 0.01 (CH3, 9-MeA). This finding strongly suggests that it is the cytosine nucleobase which undergoes deprotonation, rather than the adenine. We considered the existence of a tautomeric equilibrium between two species containing deprotonated cytosine and deprotonated adenine, with the former dominating, but were unable to prove such a possibility by applying Raman spectroscopy, for which characteristic differences between ring modes of the individual tautomers could have been expected. 1H NMR spectra of (27) recorded at different pD values are shown in Figure 89. As can be seen, at pD ~ pKa there is a definite broadening of all three 1-MeC resonances, whereas the 9-MeA resonances remain sharp. We propose that this feature is due to a dynamic process, viz. ligand rotation in order to accomplish intramolecular H bonding between –NH¯ of 1-MeC¯ and - NH2 of 9-MeA (Figure 88). 2.7. 1-MeC System 116 N N N N NH R Pt N N H2N O R a a A C H -H+ N N N N NH R Pt N N N O R a a A C H H rotation Figure 88: Ligand rotation in order to favour intramolecular hydrogen bond between the exocyclic amino group of the 9-MeA and the –NH¯ of 1-MeC¯ in (27). δ / ppm 12.77 10.91 6.90 3.75 pD C- CH3 A- CH3 C- H5 C- H6 A- H2 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ A-H8 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 4.2 4.0 3.8 3.6 3.4 } } Figure 89: 1H NMR spectra of (27) recorded at different pD values. 2.8. Solvent Effects 117 2.8 Solvent Effects It is well established that acid-base equilibria and consequently pKa values can change considerably with variations of the environment, for example, the polarity and dielectric constant of the solvent, solvation properties, and others. This aspect is of particular significance in biological processes that occur at the interface of water and biomolecule aggregates. For example, variations in pKa values of amino acid side chains in proteins as well as local conditions in the active site of enzymes have been intensely studied for this reason.[145] Differences in pKa values of identical amino acid side chains are observed in the denatured and unfolded state of proteins, which provide qualitative information concerning electrostatic interactions in this state.[146] Similarly, pKa values of acid-base indicators fixed via molecular imprinting techniques in solid matrixes can be shifted within surprinsingly large boundaries as a consequence of different local environments.[147] Regarding the reasons for variations in pKa values,[148] it is helpful to differentiate between cationic, neutral, and anionic acids. In Figure 90, cases relevant to nucleic acids are depicted. Variations in pKa values of acids can be attributed to the reason, which is going to be discussed in this chapter: polarity of the environment. A strongly hydrophobic environment inhibits solvation and hence stabilization of the ionic conjugate bases in (II) and (III). Ionization is thus suppressed in these cases, and the pKa values increase. On the other hand, the pKa of the cationic acid (I) is expected to decrease, hence the cationic acid becomes more acidic, since the resulting conjugate base is uncharged. 2.8. Solvent Effects 118 HN N NH2 O R N N NH2 O R HN N N N O H2N R N N N N O H2N R (I) (II) (III) R O P OH O O R O P O O O H+ H+ H+ + cationic neutral + acid conjugate base neutral anionic + anionic (di)anionic Figure 90: Schematic representation of feasible acid-base equilibria of nucleobase components. The pKa1 value of the 9-methyladenine nucleobase has been determined in different solvents, in order to compare the resulting values. The pKa1 has been studied in H2O as solvent, and the pKa correponding to the deprotonation at N1 position was found to be 4.3[149] or 4.5.[150] When the N1 position of 9-MeA is protonated, the 9-methyladeninium (A) is a cation, so it acts as an acid and when it loses a proton, its conjugate base is neutral (B) (Figure 91). 2.8. Solvent Effects 119 HN N N N NH2 N N N N NH2 CH3 CH3 H++ A B Figure 91: Acid-base equilibria of 9-methyladenine. 2.8.1 Mixture of Acetone and D2O To determine the pKa1 value of the 9-MeA in a mixture of acetone and deutered water, two different experiments were carried out. In the first one, the proportion acetone/D2O was 20 to 80 and in the other case it was 80 to 20. Water is a very polar solvent and it has a dielectric constant of 80.4.[151] Acetone is also a polar solvent, but less polar than water. Its dielectric constant is consequently lower than that of water and it has the value of 21.4.[151] As a result, when acetone is added to water, the dielectric constant of the medium decreases and the neutral 9-methyladenine is better solvated. As a consequence of the solvent mixture, the equilibrium is shifted to the right side, so the cationic specie (A) becomes more acidic, or in other words, the pKa value becomes lower. 2.8.1.1 20% Acetone / 80% D2O The first experiment was performed with a mixture of acetone and D2O in proportion 20 to 80 by volume. With protonated 9-methyladenine only one deprotonation step can be observed. This step at low pH refers to the deprotonation of the N1H site of the 9-methyladeninium ion. The second one, at higher pH, is due to the deprotonation of the exocyclic amino group. However, 2.8. Solvent Effects 120 the latter one cannot be observed as it occurs at pH>14. The 1H NMR resonances of the methyl group situated at N9, of the H8 and of the H2 were measured in dependence on pD. The NMR data for each proton were evaluated by a curve-fitting procedure based on equation (2) (see Section 2.1) by taking the deprotonation step into account as can be seen in Figure 92. The individual pKa value calculated from the different protons is given in Table 18. The final result for the deprotonation step is then obtained by calculating the mean of the mentioned individual results, giving eventually pKa/D2O,av. Table 18: Negative logarithmus of the acidity constant (pKa) of 9-MeA in a solution 80% D2O and 20% Acetone. pKa/D2O determined from the shift of pKa/D2O,av pKa/H2O,av CH3 H2 H8 N1H+ 4.27 ± .0.094.21 ± .0.064.23 ± .0.07 4.24 ± 0.07 3.73 ± 0.07 2 4 6 8 10 12 3,8 3,9 4,0 8,0 8,1 8,2 8,3 8,4 8,5 8,6 H2 H8 CH 3 (δ) / pp m pD Figure 92: pH dependence of the 1H chemical shifts of 9-MeA in the pD range from 2 to 11. 2.8. Solvent Effects 121 One can observe that the shift difference for H2 is the largest ones, i.e. ∆δ = 0.323 ppm, while that for H8 is ∆δ = 0.287 ppm and that of the methyl group is ∆δ = 0.141 ppm. This result seems most reasonable because H2 is on the one hand spatially close to the N1 site, and on the other hand, it is also part of the aromatic purine system. Therefore the deprotonation of N1H is also more strongly reflected, whereas the methyl group protons are at the aliphatic side chain and thus are less exposed to the acid-base reaction at the purine ring. Finally, the pKa/D2O,av values determined in D2O were also converted to aqueous solution by applying equation (3) giving the result: pK9-MeAH = 3.73 ± 0.07 This result is in agreement with expectations, according to which a lower pKa value is expected with acetone added. 2.8.1.2 80% Acetone / 20% D2O The acidity constant for the deprotonation of the N1H position of the 9- methyladenine in a solution of a mixture of acetone and D2O in a ratio 80:20 was also determined via pD dependent 1H NMR measurements in D2O. The resonances of H8 as well as of H2 and of the aliphatic protons of the methyl group at N9 were used for the determination (Figure 93). The individual pKa values calculated from the different protons are given in Table 19. The final result was then obtained by calculating the mean of the pKa values, giving pKa/D2O,av = 3.11 ± 0.08. As expected, and in agreement with the pKa value, which was obtained below, the pKa value in this case is lower. 2.8. Solvent Effects 122 Table 19: Negative logarithmus of the acidity constant (pKa) of 9-MeA in a solution 20% D2O and 80% Acetone. pKa/D2O determined from the shift of pKa/D2O,av pKa/H2O,av CH3 H2 H8 N1H+ 3.61 ± 0.08 3.61 ± 0.08 3.61 ± 0.08 3.61 ± 0.08 3.11 ± 0.08 0 2 4 6 8 10 12 14 16 3,8 3,9 4,0 8,1 8,2 8,3 8,4 8,5 8,6 H2 H8 CH 3 (δ) / pp m pD Figure 93: pH dependence of the 1H chemical shifts of 9-MeA in the pD range from 2 to 11. For the exact chemical shifts of the species see Tables of the pD dependences. 2.8.2 Mixture of Methanol and D2O Next, methanol was used as a cosolvent with D2O. Methanol is a polar solvent, more polar than acetone, with a dielectric constant of 33.7.[151] 9- methyladenine was dissolved in a mixture of 80% of methanol and 20% D2O. The determination of the pKa value corresponding to deprotonation of N1 position of the 9-MeA was followed by 1H NMR spectroscopy. Table 20 contains the various pKa values obtained for the different protons. 2.8. Solvent Effects 123 Table 20: Negative logarithmus of the acidity constant (pKa) of 9-MeA in a solution 20% D2O and 80% Methanol. pKa/D2O determined from the shift of pKa/D2O,av pKa/H2O,av CH3 H2 H8 N1H+ 3.68 ± 0.02 3.70 ± 0.01 3.69 ± 0.02 3.69 ± 0.02 3.19 ± 0.02 Figure 94 shows the different curves corresponding to the different protons. The final result, pKa/D2O,av = 3.19 ± 0.02, is slightly higher than the pKa/D2O,av = 3.11 ± 0.08 for the mixture 80% acetone and 20% D2O. This finding is in agreement with qualitative conclusions, according to which a lowering of the dielectric constant of the solvent lowers the pKa value. 0 2 4 6 8 10 12 14 3,8 3,9 8,1 8,2 8,3 8,4 H2 H8 CH 3 (δ) / pp m pD Figure 94: pD dependence of 9-MeA in a mixture of methanol/D2O (80%/20%). In Table 21, the acidity constants pKa/D2O,av and its corresponding one in water of the different experiments are summerized. It is evident that these values agree well within expectations pointed out at the beginning of this chapter. By comparing the pKa/H2O,av, one can conclude that the lower the acidity 2.9. Acidity of Aqua Ligands in [(NH3)2Pt(nucleobase)(H2O)]n+ 124 constant is (εD2O > εMethanol > εAcetone), the more acidic the cationic specie is (see A in Figure 91). Therefore, there is a decrease in pKa value. Table 21: Summary of the pKa value of 9-MeA in different solutions. Solution pKa/D2O,av pKa/H2O,av D2O 4.81[149] or 5.02[150] 4.30[149] or 4.50[150] D2O / Aceton (80/20) 4.24 3.73 ± 0.07 D2O / Methanol (20/80) 3.69 3.19 ± 0.02 D2O / Aceton (20/80) 3.61 3.11 ± 0.08 2.9 Acidity of Aqua Ligands in [(NH3)2Pt(nucleobase)(H2O)] n+ The simplest reaction of an aqua-cation in solution is the loss of a proton to give a hydroxo-aqua-species, according to: [M(OH2)x] n+ [M(OH2)x-1(OH)] (n-1)+ + H+aq This behaviour leads to the complicated field of oligomerization. Most of the hydroxo-aqua cations display pronounced tendencies to form hydroxo-, and eventually even oxo- bridged species, for example: M OH OH2 M O O M H H 2 4+2+ -2 H2O +2 H2O At the simplest level, the coordination of a metal ion to a water molecule will, through electrostatics, make proton loss easier (Figure 95).[152] The greater the positive charge on the ion, the easier it should be for the proton to dissociate from an attached water molecule. 2.9. Acidity of Aqua Ligands in [(NH3)2Pt(nucleobase)(H2O)]n+ 125 Mn+ O H H -H+ Mn+ O H Figure 95: Acidity of a hydrated cation. In this chapter, the acidity of a water molecule coordinated to a platinum ion will be discussed. A possible effect of intramolecular H bonding between the OH– group and the exocyclic amino group of a nucleobase on the acidity and reactivity of a metal-bound hydroxide will also be discussed. 2.9.1 Acidity Constants of trans-[Pt(NH3)2 (H2O)2] 2+ pKa values of the aqua ligands in trans-[Pt(NH3)2 (H2O)2]2+ were reported by Appleton et al[153] applying 195Pt as well as 15N NMR spectroscopy (of 15N labelled NH3 ligands). The NMR spectroscopic changes were smaller in magnitude than the corresponding changes when water coordinated trans to ammine in cis-[Pt(H2O)2(15NH3)2]2+ is deprotonated.[154-158] The acid dissociation constants Ka1 and Ka2 correspond to: Pt NH3H2O H3N OH2 2+ Pt NH3HO H3N OH2 + Pt NH3HO H3N OH -H+ +H+ K1 -H++H+ K2 pKa values were found to be pKa1 = 4.35 and pKa2 = 7.40 for the two steps (no standard deviations given). 2.9. Acidity of Aqua Ligands in [(NH3)2Pt(nucleobase)(H2O)]n+ 126 2.9.2 Comparison with its cis-Isomer In a similar way, pKa values were determined for the cis isomer. With the use of an iterative program,[159] the parameters Ka1 and Ka2 were varied to give the best least-squares fit to the experimental data over the pH range 4.3-9.1. The values obtained were pKa1 = 5.93 ± 0.10 and pKa2 = 7.87 ± 0.10. Pt OH2H3N H3N OH2 2+ Pt OHH3N H3N OH2 + Pt OHH2O H3N OH -H+ +H+ K1 -H++H+ K2 The two acid dissociation constants are more difficult to determine, because of the formation of hydroxo-bridged oligomers like [Pt(NH3)2(µ-OH)]nn+. For trans complexes, formation of μ-OH species is much less pronounced. Comparison of the results of the two isomers reveal that the trans isomer is more acidic, most likely a consequence of differences in trans influence, with that of H2O in the trans-isomer being larger than that of NH3. If chloro species of the two isomers are included in the comparison, viz. cis- and trans-[PtCl(NH3)2(H2O)]+, the following picture emerges (Figure 96): 2.9. Acidity of Aqua Ligands in [(NH3)2Pt(nucleobase)(H2O)]n+ 127 Pt OH2H3N H3N OH2 pKa2~7.87 2+ Pt OH2H3N H2O NH3 pKa1~4.35 pKa2~7.40 2+ Pt OH2H3N H3N Cl pKa~6.85 + Pt OH2H3N Cl NH3 pKa~5.63 + pKa1~5.93 cis - trans- Figure 96: Comparison of the acidity constants of cis- and trans- [Pt(H2O)2(NH3)2]2+with cis- and trans-[PtCl(H2O)(NH3)2]+. In the next chapter, the acidity of a water molecule coordinated to a platinum ion, carrying a nucleobase, will be studied. 2.9.3 Effect of Intramolecular H Bonding on the Basicity of a Metal-Bound Hydroxide In order to study the hydrolysis of biologically important phosphate diesters with poor leaving groups (e.g., DNA), the reactivity of three Cu(II) complexes for hydrolizing 2´,3´-cAMP was studied by Chin et al.[160] It was found by these authors, that one amino group in close proximity to the aqua ligand lowers the pKa of the metal-bound water molecule. It appears that the amino group is acting as a hydrogen bond donor to the metal-bound hydroxide, thereby stabilizing it and lowering the pKa of the H2O ligand. If the amine nitrogen was acting as a hydrogen bond acceptor, the pKa of the coordinated water molecule should have increased. ∆0.92 ∆2.5 ∆1.28 2.9. Acidity of Aqua Ligands in [(NH3)2Pt(nucleobase)(H2O)]n+ 128 It was proposed that the mechanism for promoted hydrolysis of 2´,3´- cAMP involves intramolecular nucleophilic attack by the metal-hydroxide on the coordinated phosphate diester.[161] Hydrogen bonds between the amino group and the OH group of the copper complex, should acidify the OH group, making it easier for this group to deprotonate and thus facilitate the expulsion of the leaving-group (Figure 97). Figure 97: Intramolecular nucleophilic attack by the metal-hydroxide on the coordinated phosphate diester. Hydrogen bonds between the amino group and the OH group are also observed. 2.9.3.1 cis- and trans-[(NH3)2Pt(1-MeC-N3)(H2O)] 2+ In order to study the possibility of hydrogen bonds between the exocyclic amino group of the cytosine and the OH group, selected nucleobase compounds were studied by 1H NMR spectroscopy. Measurement of the pD dependence was carried out using identical samples, in which the pD of the solutions was changed in small increments by addition of small amounts of DNO3 and NaOD, respectively. 1H NMR spectra were recorded immediately after adjusting the pD. pD-dependent 1H NMR spectra (2 < pD < 14) of cis-[(NH3)2Pt(1-MeC- N3)(H2O)]2+ reveal two deprotonation steps. The first one occurs with pKa1 = 5.78 ± 0.03 (calculated for H2O). The second one takes place at pD > 13. The first one is assigned to the deprotonation of the water molecule. In this case, a 2.9. Acidity of Aqua Ligands in [(NH3)2Pt(nucleobase)(H2O)]n+ 129 hydrogen bond between the NH2 of the cytosine and OH– could be possible, in principle (Figure 98). Under strongly basic conditions cis-[(NH3)2Pt(1-MeC- N3)(OH)]+ becomes deprotonated also at the exocyclic amino group of the cytosine. The graphical representation of the pD dependence is depicted in Figure 99 (on the right side). A significant upfield shift of the CH3-signals at basic pH values can be observed, due to the deprotonation of the cation. In order to study the deprotonation of the water molecule, the pKa1 value was compared to its trans-isomer, trans-[(NH3)2Pt(1-MeC-N3)(H2O)]2+. In this case, deprotonation of H2O occurs earlier: pKa1 = 5.17 ± 0.1 (Figure 99, on the left side). As there is no intramolecular stabilization possible of the hydroxo ligand in the trans isomer, yet trans-[(NH3)2Pt(1-MeC-N3)(H2O)]2+ is more acidic, one has to conclude that differences in trans-influence rather than a differential stabilization of the hydroxo species is dominating. It may be added that the crystal structure analysis of cis-[(NH3)2Pt(1-MeC-N3)(OH)]+ provides no evidence for the kind of H bond between the OH– ligand and the exocyclic amino group of cytosine discussed above.[162] N N NH2 OH3C Pt OH2 NH3 NH3 N N NH2 OH3C Pt O NH3 NH3 H N N NH2 OH3C Pt NH3 OH2 NH3 N N N OH3C Pt NH3 O NH3 H H H 2+ 2+ 2+ 2+ -H+ +H+ -H+ +H+ cis-[(NH3)2Pt(1-MeC-N3)(H2O)] 2+ trans-[(NH3)2Pt(1-MeC-N3)(H2O)] 2+ Figure 98: Possibility of intramolecular hydrogen bond between the NH2 of the cytosine and the OH– in cis-[(NH3)2Pt(1-MeC-N3)(H2O)]2+. 2.9. Acidity of Aqua Ligands in [(NH3)2Pt(nucleobase)(H2O)]n+ 130 2 4 6 8 10 12 14 3,38 3,39 3,40 3,41 3,42 3,43 (δ) /p pm pD 2 4 6 8 10 12 14 3,37 3,38 3,39 3,40 3,41 3,42 3,43 3,44 (δ) /p pm pD Figure 99: pD dependence (δ, ppm) of the CH3 resonance in D2O of the cation cis-[(NH3)2Pt(1-MeC-N3)(H2O)]2+ (right) and for trans-[(NH3)2Pt(1-MeC- N3)(H2O)]2+ (left). 2.9.3.2 cis- and trans-[L2Pt(9-MeGH-N7)(H2O)] 2+ In order to get some more insight into the question of a stabilizing hydrogen bond between the exocyclic amino group of a nucleobase and the hydroxo group, a similar experiment was carried out with guanine. In this case, 9-methylguanine was used as nucleobase, because an intramolecular hydrogen bond of the kind discussed is not possible (Figure 100). Pt NH3 H2O NH3 -H+ +H+ H N N N N O H2N H3C Pt NH3 O NH3 H N N N N O H2N H3C H Figure 100: Acid-base equilibrium of cis-[(NH3)2Pt(9-MeGH-N7)(H2O)]2+. 2.9. Acidity of Aqua Ligands in [(NH3)2Pt(nucleobase)(H2O)]n+ 131 The acidity of the water molecule in trans-[(NH3)2Pt(9-MeGH-N7)(H2O)]2+ was determined by 1H NMR spectroscopy (pD dependence of CH3 and H8 of guanine) and found to be 5.27 ± 0.05 (calculated for H2O) (Figure 101). This value is practically identical with the one obtained for trans-[(NH3)2Pt(1-MeC- N3)(H2O)]2+ (pKa1 = 5.17 ± 0.1). Cis-[(NH3)2Pt(9-MeGH-N7)(H2O)]2+ was not studied because of its high reactivity, leading to multiple products.[163] Instead, the pKa1 value in the complex cis-[{NH(CH3)2}2Pt(9-MeGH-N7)(H2O)]2+ was determined (5.81 ± 0.03). Again, this value is almost identical with the one obtained for cis-[(NH3)2Pt(1-MeC-N3)(H2O)]2+ (pKa1 = 5.78 ± 0.03), despite having different am(m)ine ligands. To conclude, the pKa values corresponding to the deprotonation of the water molecule in trans complexes are lower than in cis complexes, due to the trans influence. A definitive effect of a neighbour group effect on H2O ligand acidity via H bond formation between the OH─ ligand and a nucleobase was not observed. Table 22 summerizes the finding results. 0 2 4 6 8 10 12 14 3,66 3,67 3,68 3,69 3,70 3,71 3,72 3,73 3,74 (δ) / pp m pD Figure 101: pD dependence (δ, ppm) of the CH3 resonance of 9-MeGH in trans- [(NH3)2Pt(9-MeGH-N7)(D2O)]2+. The first step corresponds to the deprotonation of the water molecule and the second one to the deprotonation of the N1H position of the 9-methylguanine. 2.10. Pyrazole System 132 Table 22: Negative logarithmus of the acidity constant (pKa1) of a water molecule in different complexes, involving Pt atom and a nucleobase. X corresponds to a nucleobase and L to an am(m)ine ligand. cis-[L2PtX(H2O)]2+ trans-[L2PtX(H2O)]2+ X = 1-MeC L = NH3 X = 9-MeGH L = NH(CH3)2 X = 1-MeC L = NH3 X = 9-MeGH L = NH3 pKa1H2O 5.78 ± 0.03 5.81 ± 0.00 5.17 ± 0.1 5.27 ± 0.05 2.10 Pyrazole System The heterocycle pyrazole (pzH) and the pyrazolate anion (pz) are particularly suitable for bringing together metal centers at close distance, due to their good ligating properties and their geometry. The synthesis of polynuclear transition metal complexes, in which the metals are brought to a fixed distance to each other, is an important area of research for the catalysis in biological and industrial reactions.[164,165] In recent years, several elegant examples of polynuclear systems of bonded pyrazolate anions were described,[166] including that of dinuclear complexe with the structure [{Ru(µ-pz)(CNBut)2}2]. Moreover, these complexes were described as “Supernucleophiles”, when used for the activation of X-CH (with X being a halogen).[167] Apart from the bridging species, pyrazole can still coordinate in chelating or monofunctional fashion. The latter is important for the design of polynuclear compounds. Selected metal binding patterns are shown in the following table (Table 23). The idea to involve pyrazole ligands in a study concerned primarily with nucleobase acidification as a consequence of metal coordination and of neighbour group effects was very simple: The pyrazole should act as a “helper” ligand to bring two different nucleobases into close proximity (Figure 102), thereby facilitating H bonding interactions between functional groups in different protonation states (see, in particular 2.10.1.6). 2.10. Pyrazole System 133 NN L1 L2 ? 12 Figure 102: Pyrazolate as “helper” ligand. L1 and L2 = different nucleobases. Table 23: Different ways of coordination of a metal to the pyrazole ligand. Position of coordination Complexes Lit. N NH M N N M (DPPF)Pt(pz)2 cis-Pt(pzH)2Cl2 [Pt(pz)2(Hpz)2]2 [168] [169] [170] N N M M [(DPPF)Pt(µ-pz)2CdI2] [Pt2Cl2(µ-pz)2(PEt3)2] [Pt(pz)2]3 [168] [171] [172] N N M [(C5H5)2Zr(thf)(η2-pz)]+ [173] Dissociation equilibria of pyrazole are shown in Figure 103. Pyrazole can be protonated at the N1 position in acidic solution (pKa1 = 2.56).[174] Under strongly basic conditions, for example in the reactions with alkali metals,[175,176] the second endocyclic nitrogen N2 is deprotonated (pKa2 = 14.21)[177] When a metal becomes coordinated to the N1 position, the electronic density of the ring system decreases and N2H becomes more acidic, so that the second coordination position can also be metalled under relatively mild conditions.[178] 2.10. Pyrazole System 134 NHHN NHN 12 + -H+ pKa1 -H+ pKa2 +H+ +H + NN Figure 103: Acid-base equilibria of pyrazole. Because of the fast tautomeric equilibrium, the H3 and H5 protons of the free pyrazole are equivalent and therefore appear as only one doublet in the 1H NMR spectrum. When coordinated at the N1 position, the protons are no longer chemically equivalent and therefore give rise to two doublets in the spectrum. The electron withdrawing effect of the metal causes a downfield shift (e.g., in trans-[AaPt(pzH)2]2+; a = ammine, A = n-hexylamine). Further complexation under simultaneous deprotonation of the N2 position causes, however, a weak high field shift of the signals. The following table (Table 24) gives an overview of the observed chemical shifts of the discussed complexes in the case of platinum coordination.[179] Table 24: Chemical shifts in ppm of the resonances in 1H NMR of the free and platinum coordinated pyrazole in MeOD.[180] Complex H4 (t) H3 (d) H5 (d) pzH 6.42 7.62 see H3 trans-[AaPt(pzH)2]2+ 6.67 7.98 8.11 trans-[PtAa(pz-N1,N2)2{PtAaCl}2]2+ 6.52 7.70 see H3 2.10. Pyrazole System 135 2.10.1 trans-[(NH3)2Pt(pzH)Cl](NO3) (28) as Starting Compound Addition of one equivalent of pyrazole ligand (pzH) to transplatin leads to the formation of the 1:1 complex (28) and the 2:1 complex trans- [(NH3)2Pt(pzH)2]2+ in high yield. In order to obtain compound (28), variations of the reaction were carried out in order to improve the yield. The highest content of desired 1:1 product, which makes use of an intermediate PtII-DMF species, which then reacts with pyrazole.[181] The improved yield of (28) in relation to similar conditions reaction in water can be attributed on the one hand to the good solubility of trans-DDP in DMF and on the other hand to the fact that further solvolysis is reduced by the coordinated DMF. Recrystallization of the raw product in water leads to an increase of the concentration of the bis(pyrazole)complex. The same is to be observed upon dissolving the raw product and following complete removal of the water in a rotation evaporator. This behaviour was already described for other 1:1 complexes of transplatinum.[182] Here for aqueous solvents the following equilibrium is accepted: 2 trans-[(NH3)2Pt(pzH)Cl] + trans-[(NH3)2Pt(pzH)2] 2+ + trans-[(NH3)2PtCl2] Both complexes (1:1 and 2:1) were separated following recrystallization from a mixture of isopropanol/water (2:1). Crystals of trans- [(NH3)2Pt(pzH)Cl](NO3) (28) were collected and proved useful for X-ray crystallography. The complex crystallizes in the triclinic P-1 space group. Details concerning the crystal, X-ray measurement, and the refinement of data are listed in Tables A-9 (see Appendix). Compound (28) contains two crystallographically independent cations Pt1 and Pt2. View of both cations are given in Figure 104. In both cases the pyrazole ring and the chloride ligand are trans to each other and are bonded to 2.10. Pyrazole System 136 Pt. The platinum is bonded via N1 to the pyrazole. The geometry about Pt is square-planar. Deviations from the ideal case are very small, as evidenced by values of the N-Pt-N and N-Pt-Cl angles: the angles between Pt and the two trans positioned nitrogen atoms and the chloro ligand range from 176.8(3)° to 179.4(4)°, and between Pt and the cis positioned nitrogen atoms and chloride from 89.1(3)° to 90.9(2)°. The platinum coordination sphere is coplanar with the four donors deviating by ± 0.033 Å from the mean plane. Pt-N distances range from 2.003(11) to 2.067(12) Å. In the unit cell, there are two different molecules of (28), which are differently oriented. The distances and angles in the pyrazole of (28) are not unusual (Table 25). A differentiation between N2 and the C5 of the pyrazole ligands was made by interchanging these atoms and by determining the R values for the two situations. The solution leading to the lower R value was eventually chosen. The two external C-N-Pt1 and C-N-C angles are slightly different. The angle on the side of the carbon atom (Pt1-N1B-C5B, 129.9(9)°) is larger than the one on the side of the nytrogen atom (Pt1-N1B-N2B), 124.4(8)°). The atoms in the pyrazole ring are almost coplanar within ± 0.0076 Å. The metal coordination plane and the pyrazole (B) ring form an dihedral angle of 52.0(4)°. In the case of the other molecule, the two external C-N-Pt2 and C-N-C angles are likewise different. The angle on the side of the carbon atom (Pt2-N1A-C5A, 130.4(9)°) is larger than the one on the side of the nytrogen atom (Pt2-N1A-N2A), 121.4(8)°). The atoms in the pyrazole ring are almost coplanar within ± 0.0137 Å. The metal coordination plane and the pyrazole (A) ring form an dihedral angle of 87.7(4)°. Thus, the two crystallographically independent cations differ primarily in the dihedral angles between Pt and pz planes. Table 25: Selected distances (Å) and angles (º) for non-hydrogen atoms in 28. Pt1-N1B 2.003(11) N1B-Pt1-N12 88.8(5) Pt1-N12 2.041(11) N1B-Pt1-N11 91.3(49 Pt1-N11 2.067(12) N12-Pt1-N11 179.4(4) 2.10. Pyrazole System 137 Pt1-Cl1 2.294(3) N1B-Pt1-Cl1 176.8(3) N1B-N2B 1.340(15) N12-Pt1-Cl1 90.9(3) N1B-C5B 1.347(15) N11-Pt1-Cl1 89.1(3) C5B-C3B 1.365(19) N2B-N1B-C5B 105.6(11) N2B-C4B 1.372(17) N2B-N1B-Pt1 124.4(8) C3B-C4B 1.315(18) C5B-N1B-Pt1 129.9(9) N1B-C5B-C3B 109.7(12) C4B-C3B-C5B 109.5(11) C3B-C4B-N2B 107.4(12) Pt2-N1A 2.044(10) N1A-Pt2-N22 90.0(4) Pt2-N21 2.063(11) N21-Pt1-N22 177.0(4) Pt2-N22 2.031(12) N1A-Pt1-N21 89.8(4) Pt2-Cl2 2.283(3) N22-Pt2-Cl2 89.4(3) N1A-N2A 1.320(13) N1A-Pt2-Cl2 178.1(3) N1A-C5A 1.291(16) N21-Pt2-Cl2 90.9(3) C5A-C3A 1.38(2) N2A-N1A-C5A 108.1(10) N2A-C4A 1.308(17) N2A-N1A-Pt2 121.4(8) C3A-C4A 1.33(2) C5A-N1A-Pt2 130.4(9) N1A-C5A-C3A 108.1(13) C4A-C3A-C5A 106.0(13) C3A-C4A-N2A 108.0(13) 2.10. Pyrazole System 138 Pt2 Pt1 N1B N1A Cl1 Cl2 N1NN2N N12 N22 N21 N11 C5B C3BC4B N2B N2A C4A C3A C5A Figure 104: View of two independent cations trans-[(NH3)2Pt(pzH)Cl](NO3) (28) with NO3─ anions in the unit cell. Cations of (28) are arranged in layers (Figure 105). Pt1 Pt2 Figure 105: Detail of packing of trans-[(NH3)2Pt(pzH)Cl](NO3) (28) with nitrates omitted. 2.10. Pyrazole System 139 There are no intramolecular hydrogen bond interactions possible within the cations of (28). However, there are intermolecular H bonding interactions between ligands of neighbouring cations and also with nitrate anions. Distances between Cl and the ammine ligands of the platinum atom are: Cl-N (1-x, 1-y, 1- z), 3.28(1) Ǻ. This situation is shown in Figure 106. Intermolecular Pt···Cl distances are 3.79 Ǻ, which is considered too long for any bonding interaction. Pt1 Figure 106: Weak interaction between Cl and the NH3 ligands of cations (28). The crystal packing of (28) is based on interactions between the trans- [(NH3)2Pt(pzH)Cl]+ cation and the nitrate counter anions in the unit cell. As shown in Figure 107, the oxygen atoms of the nitrate form hydrogen bonds with the protons of the ammine ligand of the platinum and when the other endocyclic nitrogen atom of the pyrazole ligand: O1N···N12, 2.96(1) Ǻ; O2N···N12, 3.30(2) Ǻ; O7N···N2B, 3.05(2) Ǻ; O7N···N2A, 3.32(2) Ǻ; O5N···N2B, 2.97(2) Ǻ; O5N···N11, 2.93(1) Ǻ and O6N···N21, 3.06(1) Ǻ. 2.10. Pyrazole System 140 O5N O6N O7N N11 N2A O2N O1N N12 N2B Pt1 Pt2 Figure 107: Interactions between the cation and the nitrate anions in (28). 2.10.1.1 NMR Studies The complex trans-[(NH3)2Pt(pzH)Cl]+ was also studied by 1H NMR spectroscopy. In comparison to the bis(pyrazole) complex the 7.90/7.81 ppm resonances for H3/H5 and the signal at 6.53 (H4) ppm (pD = 7.4) are slightly highfield shifted (Figure 108). Due to the coordination of the platinum atom to the N1 site of the pyrazole ligand, the 195Pt satellites of 18.1 Hz of the H5 resonance are observed. During the observation of the signals of (28) over the time, the equilibrium mentioned above is confirmed, since after few hours the signals of the 2:1 complex are detectable.[181] In the IR spectrum, the bands corresponding to the 2:1 complex and (28) differ not so much. Only the band ט (Pt-Cl) at 340 cm-1 was found for the complex trans-[(NH3)2Pt(pzH)Cl]+. 2.10. Pyrazole System 141 6.606.807.007.207.407.607.808.00 H3 H4H5 Figure 108: 1H NMR spectrum of the complex trans-[(NH3)2Pt(pzH)Cl]+ in D2O at pD = 7.4. 2.10.1.2 Reaction of trans-[(NH3)2Pt(pzH)2](NO3)2 with trans- [(NH3)2Pt(1-MeC-N3)(H2O)] 2+ Synthesis of trinuclear trans,trans,trans-{(NH3)2Pt(N1-pz-N2)2[(NH3)2Pt(1- MeC-N3)]2}6+ complexes was achieved using the complex trans- [(NH3)2Pt(pzH)2]2+, which is obtained in a high yield in the preparation of the 1:1 complex (28). Two equivalents of trans-[(NH3)2Pt(1-MeC-N3)(H2O)]2+ were added to a solution containing trans-[(NH3)2Pt(pzH)2]2+ and the mixture was brought to pH 8. Under these conditions, the deprotonation of the endocyclic nitrogen of the pyrazole ligand (N2) takes place. The reaction was carried out during three days at 40°C and the sample was left in the refrigerator. After several days, a precipitate formed which was filtered off and disolved in D2O in order to identify the complex. As can be seen in Figure 109, the 1H NMR spectrum of the product (29) is consistent with a complex containing pz and 1- MeC in a 1:1 ratio (integrals of signals) and with pyrazole resonances being different from those of the bis(pyrazole) complex.[181] As expected from the anticipated structure (Figure 110), H3 and H5 are still inequivalent and therefore give rise to two different sets of resonances, centered at 8.00 (H3) and 7.72 ppm (H5). The H4 resonance of pz is at 6.50 ppm. The 1-MeC resonances are observed at 3.51 (s, CH3), 6.09 ppm (d, 3J = 7.5 Hz, H5) and 7.68 ppm (d, H6). 2.10. Pyrazole System 142 CH -C3 H5-C H4-pzH5-pz H6-C H3-pz 8.00 7.50 7.00 6.50 6.00 5.50 5.00 4.50 4.00 3.50 δ / ppm Figure 109: 1H NMR spectrum of trans,trans,trans-{(NH3)2Pt(N1-pz- N2)2[(NH3)2Pt(1-MeC-N3)]2}6+ (29). Unfortunately, attempts to crystallize this complex were not successful. N N N N NH2O H3C Pt Pt N N N N H2N O CH3 Pt 6+ 1 5 4 3 2 Figure 110: trans,trans,trans-{(NH3)2Pt(N1-pz-N2)2[(NH3)2Pt(1-MeC-N3)]2}6+ (29). 2.10. Pyrazole System 143 2.10.1.3 Reaction of trans-[(NH3)2Pt(pzH)Cl] + with AgNO3 Treatment of trans-[(NH3)2Pt(pzH)Cl]+ with one equivalent of AgNO3 in D2O, pD ~ 9, was carried out on an NMR scale. The objective of this experiment was to observe the possibility of obtaining cyclic pyrazole complexes. Once the N2 position of the ligand is deprotonated, another trans-[(NH3)2Pt(pzH)(H2O)]2+ entity could be coordinated. The reaction was followed by 1H NMR spectroscopy (Figure 111). The spectrum on the top was measured immediately after addition of NaOD. The spectrum in the middle was recorded five days later, and new signals of the protons appeared. The spectrum on the bottom was measured one month later, and as can be seen, the signals of the protons are very complicated. Attempts to isolate any of these new species were not successful. What can be concluded is that there is indeed oligomerization with pz-bridging going on, even though it is unclear whether cyclic species and/or open chains are formed. 8.00 7.80 7.60 7.40 7.20 7.00 6.80 6.60 6.40 6.20 δ / ppm 0.5 h 5 d 30 d Figure 111: Lowfield section of 1H NMR of trans-[(NH3)2Pt(pzH)(H2O)]2+ in basic conditions with time. 2.10. Pyrazole System 144 2.10.1.4 Oxidation of Pt(II) to Pt(IV) in trans-[(NH3)2Pt(pzH)Cl] + When a solution containing the complex trans-[(NH3)2Pt(pzH)Cl]+ (28) was heated at 50°C until dryness and posterior disolution in water, yellow crystals appeared. This new complex was characterized by 1H NMR spectroscopy and by X-ray crystallography. The 1H NMR spectrum of this compound displays two doublets of doublets corresponding to H3/H5 protons and a triplet (H4) (Figure 112, spectrum B). Due to the coordination of Pt to the N1 site of the pyrazole, the 195Pt satellites of 11.5 Hz and 11Hz of the H3 and H5 resonances are observed. The chemical shifts of these signals are downfield related to (28) and the 2:1 complex trans-[(NH3)2Pt(pzH)2]2+. The 195Pt NMR spectrum was recorded, which displayed a single signal at -385 ppm, suggesting the existence of a Pt(IV) complex. The crystal proved of poor quality, but a preliminary the structure corresponding to trans-[(NH3)2Pt(pzH)2Cl2](NO3)2·H2O (30) could be established. In order to obtain crystals of Pt(IV) of better quality, these crystals were disolved in water at 40°C and HClO4 was added. After several days in the refrigerator, red and yellow crystals appeared. The red crystals were also studied by X-ray crystallography, but the structure solution proved very difficult. This complex was also characterized by elemental analysis and 1H NMR spectroscopy. The 1H NMR spectrum shows a mixture of two different complexes, containing two platinum species in a 1:1 ratio. As shown in Figure 112, spectrum C represents a superposition of signals corresponding to the Pt(II) complex trans-[(NH3)2Pt(pzH)2]2+ (spectrum A) and signals corresponding to the Pt(IV) complex trans-[(NH3)2Pt(pzH)2Cl2]2+ (spectrum B). According to the elemental analysis, the composition of the red crystals corresponds to trans,trans-[(NH3)2Pt(pzH)2Cl2][(NH3)2Pt(pzH)2](ClO4)4·H2O (31). The structure of (31) is proposed to consist of chains of trans- [(NH3)2Pt(pzH)2Cl2]2+ entities linked to trans-[(NH3)2Pt(pzH)2]2+, by a weak interaction between the Pt(II) atom and the chloro atom of Pt(IV) (Figure 113). The charge of the smallest unit is 4+. This kind of structure has been already observed in other publications.[183-186] 2.10. Pyrazole System 145 A B C 8.40 8.20 8.00 7.80 7.60 7.40 7.20 7.00 6.80 6.60 δ / ppm (pD = 5.52) (pD = 2.85) (pD = 2.85) Figure 112: Lowfield section of 1H NMR of different compounds: A: trans- [(NH3)2Pt(pzH)2](NO3)2; B: trans-[(NH3)2Pt(pzH)2Cl2](NO3)2·H2O (30) and C: trans,trans-[(NH3)2Pt(pzH)2Cl2][(NH3)2Pt(pzH)2](ClO4)4·H2O (31). Pt Hzp a a pzH Pt Hzp a a pzH Cl Cl Pt Hzp a a pzHn 4n+ Figure 113: Structure of (31) forming chains. 2.10.1.4.1 trans,trans,trans-[(NH3)2Pt(pzH)2Cl2](ClO4)2·H2O (32) Yellow crystals obtained after recrystallization of (30) from dilute HClO4 were obtained. Compound (32) crystallizes in a centrosymmetrical space group. After refinement of the measured crystal data, the molecule was found to be in the P2(1)/c space group, having the platinum atom located in the inversion 2.10. Pyrazole System 146 center. All the hydrogen atoms were found in the Fourier difference and refined isotropically. Details concerning the crystal, X-ray measurement, and the refinement of data are listed in Table A-10 (see Appendix). In (32) the platinum atom is coordinated to the N1 site of two pyrazole ligands, to two chloride ion and to two ammine ligand in a non-distorsioned octahedral coordination. The pyrazole ring, and its symmetry related (-x+1, -y+1, -z+1) ring are almost coplanar (Figure 114). N2 and C5 of the pyrazole were differentiated by the R value of the refinement of the data in the crystallography method. Cl N1 N2 C3 C4 C5 N11 Figure 114: View of the cation trans,trans,trans-[(NH3)2Pt(pzH)2Cl2]2+ of (32) with atom numbering scheme. As shown in Table 26, distances and angles in the complex are not unusual. The Pt-N1 distance is 2.013(10) Ǻ and thus similar to the corresponding distances in the N1 coordinated pyrazole ring of (28) (2.003(11) Ǻ), and comparable to other Pt-N1 coordinated pyrazole ligands.[180] Also, the angle between the Pt-N4 coordination plane and the pyrazole plane is similar to the angle between Pt-N3Cl and the N1 coordinated pyrazole ring B in (28), 54.2(3)° vs. 52.0(4)°. The two pyrazole ligands are coplanar to each other. The plane containing Pt-N and the plane containing Pt-Cl are almost perpendicular (89.7(3)°). Distances and angles are compiled in Table 26. 2.10. Pyrazole System 147 Table 26: Selected distances (Å) and angles (º) for non-hydrogen atoms in 32. Pt1-N1 2.013(10) N1-Pt-Cl 88.7(3) Pt1-N11 2.048(9) N11-Pt-Cl 90.3(3) Pt1-Cl1 2.310(3) Cl-Pt1-Cl(i) 180.0(0) N1-N2 1.347(15) Cl-Pt-N1(i) 91.3(3) N1-C5 1.381(13) Cl-Pt-N11(i) 89.7(3) C5-C4 1.322(15) N11-Pt-N1 90.3(4) N2-C3 1.393(19) N1-Pt-N11(i) 89.7(4) C3-C4 1.354(19) Symmetry code: (i) -x+1, -y+1, -z+1. As required by the centrosymmetry of (32), the two trans-positioned pyrazoles are in a head-tail arrangement (Figure 114). With regard to the intramolecular interactions in trans,trans,trans-[(NH3)2Pt(pzH)2Cl2](ClO4)2·H2O (32), the distance between N2 of the pyrazole and the chloro ligand is 3.24(1) Å, which is suitable for a H-bonding. Cl···N11 (3.09(1) Å) and Cl···C5(i) (3.21(1) Å) contacts are relatively short, but angles are unfavourable for H bonding. Intermolecular hydrogen bonds are also possible. The distance between the water molecule and the ammine group is 2.94(1) Å and the closest intermolecular contact (2.84(1) Å) is between one oxygen atom of the perchlorate anion and the water molecule (Figure 115). 2.10. Pyrazole System 148 Figure 115: View of the intermolecular hydrogen bonds in (32). The molecules are arranged in infinite chains along the a axis. There is no possibility for π-stacking interactions between the pyrazole rings (Figure 116). Figure 116: View of the packing of the cations of (32). A view of the crystal packing of (32) with the nitrate anions and the water molcule is given in Figure 117. 2.10. Pyrazole System 149 Figure 117: View of the packing of (20) along the a axis. 2.10.1.5 Reaction of trans-[(NH3)2Pt(pzH)Cl] + with 9-MeGH Trans-[(NH3)2Pt(pzH)(9-MeGH-N7)](NO3)2 (33) was prepared by reaction of trans-[(NH3)2Pt(pzH)(H2O)]2+ and 9-MeGH during two days at 40°C. The 1H NMR spectrum of a solution containing the cationic trans-[(NH3)2Pt(pzH)(9- MeGH-N7)]2+ entity displays a singlet corresponding to the H8 proton of the guanine, two sets of doublets in corresponding to the H3 and H5 protons of the pyrazole ligand and a triplet corresponding to the H4 proton of the pyrazole in the aromatic region and a singlet corresponding to the methyl group of 9-MeGH. When the solution is in pH range from 2 to 7, the signals corresponding to the protons as well as the CH3 singlet of 9-MeGH have chemical shifts of δ = 8.32, 7.98, 7.94, 6.61, and 3.76 ppm, respectively (Figure 118). 2.10. Pyrazole System 150 9.00 8.50 8.00 7.50 7.00 6.50 6.00 5.50 5.00 4.50 4.00 3.50 H8(G) H4(pz) H3/H5(pz) CH (G)3 Figure 118: Section of the 1H NMR spectrum of (33). The simplicity of the 1H NMR spectrum rules out any slow ligand rotation, and hence the existence of individual rotamers in solution (Figure 119). N H N N N OH2N Pt N N H H3C 2+ Figure 119: View of the cation of (33).Possibility of rotation of the pyrazole ligand around Pt-N1 bond or rotation of the guanine nucloebase around Pt-N7. pD dependent 1H NMR measurements have been performed with (33) with the aim of determining the pKa values of the N1H position of the guanine base and the N2H site of the pyrazole. The pD dependence of the H5 proton of the pyrazole is shown in Figure 120. This resonance is practically unaffected in the pD range from 2 to 7; only above pD 7 upfield shifts are observed. 2.10. Pyrazole System 151 The 1H NMR data for each proton were evaluated with equation (1) (see Section 2.1), which takes into account two deprotonation steps. The first one corresponds to the deprotonation of the N1H position of the 9-methylguanine and the second one corresponds to the deprotonation of the N2H of the pyrazole. The individual acidity constants pKa1 (for the deprotonation of N1H of guanine) and pKa2 (for the deprotonation of N2H of pyrazole) are displayed in Table B-25 (see Tables of pD dependences). The acidity constants, valid for the situation in D2O, were obtained by taking the weighted mean of the individual constants pKa1 and pKa2 to give pKa1/D2O = 8.44 ± 0.09 and pKa2/D2O = 9.61 ± 0.06. The values for the situation in water were then converted to the situation in water: pKa1/H2O = 7.87 ± 0.09 pKa2/H2O = 9.03 ± 0.06 Due to the platination of N1 of the pyrazole ligand, the pKa2/H2O value of N2H [180] drops by ∆pKa = 14.21 – 9.03 = 5.18. This increase in acidity does not ensure a second platination at this site, because even at pH of 9 (where about 50% of the pyrazole ligand will be deprotonated at the N2 position) platinum (II) is still present as the hydroxo species in aqua solution. The known inertness of Pt(II)OH species probably is responsible for this lack. 7 8 9 10 11 12 13 14 6,30 6,35 6,40 6,45 6,50 6,55 6,60 (δ) /p pm pD Figure 120: pD dependent chemical shift of H5 of the pyrazole in the complex trans-[(NH3)2Pt(pzH)(9-MeGH-N7)](NO3)2 (33) as determined by pD dependent 1H NMR measurements in D2O. 2.10. Pyrazole System 152 2.10.1.6 trans,trans-[(NH3)2Pt(9-MeGH-N7)(N1-pz-N2)(NH3)2Pt(1- MeC-N3)](NO3)3 (34) Unlike in the case of metalated adenine complexes, where effects of neighbour groups on the acidity of the exocyclic amino group have now been rationalized, variations in pKa values of the N(1)H proton of N7 platinated guanine ligands are practically not understood. We hypothesized, that in analogy to the situation with deprotonated amino groups of adenine, a suitably positioned H bonding donor might lower the pKa also of guanine – N(1)H. For this purpose, and based on model building, the mixed guanine/pyrazolate/cytosine complex (34) (Figure 121) was designed and synthesized. Thus, one equivalent of trans-[(NH3)2Pt(1-MeC-N3)(H2O)]2+ was added to a solution containing trans-[(NH3)2Pt(pzH)(9-MeGH-N7)]2+ and subsequent was added NaOH until pH ~ 7.5 was reached. At this pH, the pyrazole ligand is not completely deprotonated at the N2 position (pKa2/H2O ~ 9.03), but at higher pH´s there is a competition between the platination at N2 and the formation of substitutionally inert hydroxo species. The reaction was followed by 1H NMR spectroscopy. Complex trans,trans-[(NH3)2Pt(9-MeGH-N7)(N1-pz-N2)(NH3)2Pt(1-MeC-N3)](NO3)3 (34) could be identified by 1H spectroscopy in D2O. The resonances show the existence of rotamers, which are either due to the rotation of the cytosine ligand around bond Pt-N2 (pyrazole) or to the rotation of the guanine ligand Pt-N7 (Figure 121). It was not possible to isolate (34) in pure form, but only in a mixture with the starting compound trans-[(NH3)2Pt(pzH)(9-MeGH-N7)]2+ (33). The resonances of (34) are summerized in Table 27. 2.10. Pyrazole System 153 NH N N N O H2N H3C Pt N N N N H2N O CH3 Pt 3+ Figure 121: Possible rotation of guanine or cytosine ligand, which leads to the formation of rotamers. Table 27: Chemical shifts of the resonances corresponding to the complex (34) in D2O, pD ~ 7.8. Signals ppm CH3(C) 3.49 / 3.47 s CH3(G) 3.78 / 3.75 s H5(C) 6.12 / 6.09 6.08 / 6.06 d d H6(C) 7.68 / 7.65 7.67 / 7.63 d d H5(pz) 6.47 m H6(pz) 7.93 m H8(G) 8.20 / 8.42 s The pD dependent 1H NMR spectra of (34) were recorded (in the mixture with (33)). Although not carried out in an extensive way, it was clearly evident, that there was no significant deprotonation of the guanine ligand in (34) below pH 8 (calculated for H2O). This means that it is in the normal range of N7 platinated guanine ligands, and there is no effect of the cytosine ligand at “the other end” of the pyrazolate ligand. Hence, one has to conclude that there is no 2.10. Pyrazole System 154 close distance between N1 of guanine and N(4)H2 of cytosine. The original hope (see Chapter 2.10) of markedly reducing the pKa of guanine – N(1)H thus did not realize. 3.1. Instrumentation and Measurements 155 3 EXPERIMENTAL SECTION 3.1 Instrumentation and Measurements 3.1.1 Determination of pH- and pD-values The pH (uncorrected pH*) of a D2O solution was determined by use of a glass electrode on a Metrohm 6321 pH meter. pD values were obtained after addition of 0.4 units to the value displayed on the pH meter[28]. Measurement of the pD dependences were carried out using identical samples, in which the pD of the solutions was modified in small increments by addition of small amounts of DNO3 and/or NaOD. Due to the limitations of the AgCl glass electrodes, it was not possible to get reliable measurements at pH values close to 14. 3.1.2 NMR Spectroscopy One- and two-dimensional 1H NMR spectra were recorded on a Varian Mercury 200 FT NMR, on a Bruker DRX 300, on a Bruker DRX 400 and on a Varian Inova 600 MHz by or with the collaboration of Prof. Burkhard Costisella and Anette Danzmann. TSP (sodium 3-trimethylsilyl-propanesulfonate) (δ = 0 ppm) and/or TMA (tetramethylammonium bromide) (δ = 3.18 ppm) were used as internal standards. 195Pt NMR spectra were recorded by Dr. Pablo Sanz on a Bruker DRX 300. Na2PtCl6 and/or K2PtCl4 were used as external and/or internal standards, respectively. In the case of internal standards for 195Pt NMR (in D2O), a capillary tube containing a solution of K2PtCl4 (or Na2PtCl6) in H2O was introduced in the sample acting as standard. To fill up a capillary tube of glass (∅ = 0.5mm), a tube closed at the bottom was slightly heated and face down introduced in a solution containing the standard. After filling, the tube had to be centrifuged and closed at the top. The standard Na2PtCl6 displays a signal at 0 ppm. However, 3.1. Instrumentation and Measurements 156 K2PtCl4 displays rapidly two signals due to hydrolysis in water. These are at - 1625 ppm ([PtCl4]2–) and at -1181 ppm ([PtCl3(H2O)]–). The pKa value was obtained after evaluating the chemical shift (δ) data at different pD with a Newton-Gauss non-linear least-squares curve fitting procedure as described in Section 2.1.[30] 3.1.3 IR Spectroscopy IR spectra were taken on a Perkin-Elmer 580 B FT spectrometer. Measurements (KBr) were performed from 4000 to 250 cm-1. The treatment and evaluation of the spectra was done with the help of the program Opus-IR. 3.1.4 Elemental Analysis The contents of carbon, hydrogen and nitrogen were determined on a Carlo-Erba-Strumentazione 1106 Element Analyzer by Markus Hüffner. 3.1.5 X-Ray Crystallography Data collection was performed on an Enraf-Nonius Kappa CCD diffractometer at the University of Dortmund using graphite-monochromated Mo- Kα radiation (λ= 0.71069 Å)[187]. Data reduction and cell refinement were carried out using the programs DENZO and SCALE-PACK[188]. Intensities of reflections were collected at room temperature, 293(2)º. All the structures were solved by standard Patterson methods[189] and refined by full-matrix least-squares methods based on F2 using the SHELXTL-PLUS[190], SHELXL-97[191] and WinGX[192] programs. In the refinement process of the X-ray data, if not 3.1. Instrumentation and Measurements 157 specified, all non-hydrogen atoms of the crystal were refined anisotropically, and all the hydrogen atoms except those of the water molecules were included in geometrically calculated positions and refined with isotropic displacement parameters according to the riding model. The production of the diagrams was achieved with the programs "Ortep-3 for Windows"[193], and "Persistence of Vision Raytracer (POV-Ray)"[194]. 3.1.6 DFT Calculations The DFT calculations for the gas-phase optimized structures were performed by Patrick Lax using the computer program Gaussian 98.[57] The optimization was followed by a frequency calculation in order to confirm every structure to be a minimum structure. The built in combination of Becke's three- parameter hybrid function [195] with Lee-Yang-Parr's exchange functional[196] was applied. Pt atom was described with the LANL2DZ basis set. For the lighter atoms H, C, N and O a 6-31G* basis set was used. 3.2 Synthesis of Complexes 3.2.1 Materials The model nucleobases 9-methylguanine and 9-ethylguanine were purchased from Fa. Chemogen, Konstanz (Germany). 9-Ethyladenine,[197] 1- methylcytosine,[198] 6,9-Dimethyladenine[134] and 9-methyladenine[46] were synthesized as described. All the other chemicals used (either puriss or pro analysi) were from Merck GmbH, Darmstadt (Germany), Fluka AG, Buchs (Switzerland) or Aldrich-Chemical Co. Ltd., Gillingham-Dorset (UK). K2PtCl4, from which trans-(NH3)2PtCl2,[199] cis-(NH3)2PtCl2,[200] and [dienPtI]I[201] were prepared according to the literature, was from Heraeus 3.2. Synthesis of Complexes 158 GmbH, Hanau (Germany). [dienPdI]I was synthesized according to ref. 201, but using K2PdCl4. The following Pt2+ complexes were needed for further syntheses (see Section 3.2.2): trans-[(NH3)2Pt(1-MeC-N3)Cl]NO3 was obtained in aqueous solution following the procedure described in ref 202. The complex [Pt(1-MeC- N3)3Cl](NO3) was synthesized according to a prescription in ref. 94. The compounds [(dien)Pt(9-MeA-N1)](NO3)2 (1), trans-[(NH3)2Pt(9-EtA-N1)(9-MeGH- N7)](NO3)2 and trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N1,N7)](ClO4)4 (7) were kindly provided by Prof. Dr. Bernhard Lippert. The synthesis of trans- [(NH3)2Pt(9-MeA-N7)2](ClO4)2·2H2O (2) is described in ref. 203. Cis-[(NH3)2Pt(9- MeA-N7)2](NO3)2·2H2O (4) was obtained according to the method given in ref. 204, whereas trans-[(NH3)2Pt(9-MeGH-N7)Cl]Cl was prepared analogously to ref. 205. The compounds (1,9-DimeAH)(ClO4) and 6,9-DimeA were kindly provided by Dr. Michael Roitzsch. Trans-[(NH3)2Pt(pzH)2](NO3)2 and trans- [(NH3)2Pt(pzH)Cl](NO3) (28) were synthesized according to ref. 181. The deuterated solvents DMSO-d6, DMF-d7 and D2O were obtained from Deutero GmbH, Kastellaun (Germany), Cambridge Isotope Laboratories (CIL) and from Andoner (USA), respectively. 3.2.2 Preparation of Compounds 3.2.2.1 cis-[(NH3)2Pt(1-MeC-N3)(N7-9-MeA-N1)(dienPt)](ClO4)4 (5) This compound was prepared on an NMR spectroscopy scale in D2O solution from cis-[(NH3)2Pt(9-MeA-N7)(1-MeC-N3)](ClO4)2·H2O (2) by treating it with [(dien)Pt(D2O)]2+ (1:1, 3 days, 40°C). At this stage formation of a single new species was evident, clearly separted from signals of the starting compound, the resonances of which had dissapeared. 1H NMR: 9-MeA: (D2O, pD = 4.8, δ/ppm): 8.76,brs, H2; 8.70, s, H8; 3.89, s, CH3 3.2. Synthesis of Complexes 159 1-MeC: (D2O, pD = 4.8, δ/ppm): 7.56, d (3J = 7.2 Hz), H6; 6.22, d (3J = 7.2 Hz), H5, 3.36, s, CH3 dienPt: (D2O, pD = 4.8, δ/ppm): 3.35, 3.29, 3.12, 3.09, 2.93, 2.90, m, CH2 The assigment of H8 of 9-MeA was confirmed by a 1D NOE experiment; the relative intensities of all resonances are as expected for the composition. 3.2.2.2 trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 (8) [(dien)Pt(9-MeA-N1)](NO3)2 (1) (760.57 mg, 1.33 mmol) and trans-(NH3)2PtCl2 (798.7 mg, 3.66 mmol) were dissolved in 50 ml of water at pH 4.13. The solution was stirred at 45°C for one day. Then the solution was cooled to 4°C and the precipitated excess of trans-(NH3)2PtCl2 was removed. After addition of 2 eq. of NaClO4·H2O, the solution was concentrated to 20 ml by rotaevaporation. The residue remaining after evaporation was recrystallized from water to give 204 mg (0.21mmol) of (8) in 15.8 % yield. Elemental analysis: C10H26N10Pt2Cl4O12, M = 1010.35 g·mol-1 Calculated: C 11.9 %, H 2.6 %, N 13.9 % Obtained: C 11.8 %, H 2.6 %, N 14.3 % 1H NMR: (D2O, pD = 4.9, δ/ppm): 8.86, s, H8; 8.8, brs, H2; 3.95, s, CH3 3.2.2.3 trans-[(NH3)2Pt(N7-9-MeA)(N7-9-MeA-N1)(dienPt)] 4+ (9) This compound was prepared on an NMR spectroscopy scale in D2O solution from trans-[(NH3)2Pt(9-MeA-N7)2](ClO4)2·2H2O (2) by treating it with [(dien)Pt(D2O)]2+ (1:1). After stirring the solution one day at 40°C, a mixture of three compounds was obtained: (2), (10) and (9). This assignment of the three compounds was done according to the pD dependence of the different 3.2. Synthesis of Complexes 160 compounds. The differentation of species was easy if resonances of the methyl group protons were evaluated, yet difficult for the heteroaromatic protons. 1H NMR: Pt(N7-9-MeA-N1): (D2O, pD = 10.3, δ/ppm): 3.97, s, CH3 Pt(N7-9-MeA): (D2O, pD = 10.3, δ/ppm): 3.88, s, CH3 3.2.2.4 trans-[(NH3)2Pt{(N7-9-MeA-N1)(dienPt)}2] 6+ (10) This compound was prepared on an NMR spectroscopy scale in D2O solution from trans-[(NH3)2Pt(9-MeA-N7)2](ClO4)2·2H2O (2) by treating it with [(dien)Pt(D2O)]2+ (1:2). After stirring the solution three days at 40°C, compound (10) had formed. The relative intensities of all resonances are as expected for the composition. The yield was 85%. 1H NMR: (9-MeA): (D2O, pD = 8.30, δ/ppm): 8.86, s, H8; 8.80, brs; 4.00 s, CH3 3.2.2.5 cis-[(NH3)2Pt{(N1-9-MeA-N7)Pt(NH3)3}2](NO3)6·2H2O (11) [(NH3)3Pt(9-MeA-N7)]Cl(NO3)·H2O[206] (0.62 mmol) was initially converted into the NO3- salt by treatment with AgNO3 (0.62 mmol) in water. After stirring for one day at room temperature, the precipitated AgCl was removed. The solution was combined with cis-[(NH3)2Pt(H2O)2](NO3)2, which was prepared from cis- (NH3)2PtCl2 (0.31 mmol) and AgNO3 (0.62 mmol) in water. In order to isolate this compound, the solution was allowed to evaporate at RT. Thus, colourless crystals were obtained. This compound has previously been characterized by 1H NMR, Raman spectroscopy and X-ray crystallography.[53] 3.2. Synthesis of Complexes 161 1H NMR: 9-MeA: (D2O, pD = 5.97, δ/ppm): 9.10, s, H2; 8.78, s, H8 and 9.00, s, H2 (rotamers); 3.88, s, CH3 3.2.2.6 cis-[(NH3)2Pt{(N7-9-MeA-N1)(dienPt)}2](NO3)6 (12) 4,2 mg of cis-[(NH3)2Pt(9-MeA-N7)2](NO3)2·2H2O (0.0061 mmol), 0.066 g of [(dien)PtI]I (0.12 mmol), and 0.041 g of AgNO3 (0.24 mmol) were stirred in 50 ml of water at 40°C for three days. The AgI formed was eliminated by centrifugation. The resulting solution was brought to dryness by rotary evaporation. The compound was pure according to 1H NMR (relative intensities). The yield was 15.3% (0.0302 g). 1H NMR: 9-MeA: (D2O, pD = 5.6, δ/ppm): 8.80, brs, H2; 8.72, s, H8; 3.84, s, CH3 dienPt: (D2O, pD = 5.6, δ/ppm): 3.34, 3.29, 3.12, 3.09, 2.93, 2.90, m, CH2 The assigment of H8 of 9-MeA was again established by an NOE experiment; at pD > 8.2, the H2 resonance disappears because of isotopic exchange. 3.2.2.7 trans,trans,trans-[(NH3)2Pt(N7-9-MeA-N1)(dienPt)(N7-9-EtA- N1){(NH3)2Pt(9-MeGH-N7)}] 6+ (13) Complex (8) (136.13 mg, 0.13 mmol), trans-[(NH3)2Pt(9-EtA-N1)(9-MeGH- N7)](NO3)2 (91.77 mg, 1 eq) and AgNO3 (22.88 mg, 1 eq) were stirred for five days at 40°C in 80 ml of water. After removal of AgCl, the solution was slowly concentrated at room temperature. A few small colourless needles of (13) were obtained, which were suitable for X-ray analysis. The solution contained also a white precipitate, which according to 1H NMR was also (13). 1H NMR: 9-MeA: (D2O, pD = 8.0, δ/ppm): 4.04, s, CH3 3.2. Synthesis of Complexes 162 9-EtA: (D2O, pD = 8.0, δ/ppm): 4.49, q, CH2; 1.61, t, CH2 9-MeG: (D2O, pD = 8.0, δ/ppm): 3.80, s, CH3 No attempt to assign the heteroaromatic protons was made. 3.2.2.8 trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N7,N6)](ClO4)3·3.5H2O (15) trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N1,N7)](ClO4)4 (7) (50 mg, mmol) was dissolved in water (2 ml, brief heating) and the pH value was raised from 4.4 to 11 by adding 1M NaOD. The sample was lyophilized and subsequently dissolved in D2O (1ml), and then the solution was kept in a closed vial until crystals of (15) appeared after several days. The complex was characterized by X-ray crystallography. 3.2.2.9 [(9-MeA-N7)Pt(NH3)3]Cl2·2H2O (16) 9-MeA (4 mmol) was reacted with K2PtCl4 (4 mmol) in 250 ml of water at 55°C for 20 min.[206] After that, the mixture was stirred for 6 hours at room temperature. The yellow precipitate was removed by filtration and washed with 5 ml of cold water. The yellow compound was [PtCl3(9-MeAH-N7)]. This compound was dissolved in water and 80 ml of NH3 (25%) were added. After stirring for one day, the precipitate was removed and the volume was reduced to 30 ml. The pH of the solution was adjusted to pH 4.0 (HNO3, 0.1N). The solution was allowed to evaporate at 4°C. Colourless crystals of (16) were isolated from it and characterized by X-ray crystallography. The yield was 82%. Elemental analysis: C6N8H20PtCl2O2, M = 502.26 g·mol-1 Calculated: C 14.3 %, H 4.0 %, N 22.3 % Obtained: C 14.3 %, H 4.0 %, N 22.5 % 3.2. Synthesis of Complexes 163 3.2.2.10 cis-[(NH3)2Pt(N1-9-MeA-N7)(N6-9-MeA-N7){Pt(NH3)3}2] 5+ (17) To a solution of cis-[(NH3)2Pt{(N1-9-MeA-N7)Pt(NH3)3}2](NO3)6·2H2O (11) in 1ml of D2O was added 1M NaOH. When the pH was ca. 9, the solution was kept at room temperature. After one month, a 1H NMR spectrum was recorded and the signals observed were assigned to compound (17), due to the two different signals of the methyl groups and their relative intensities. (17) has not been isolated. 3.2.2.11 cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2](NO3)4·6H2O (18) cis-[(NH3)2Pt{(N1-9-MeA-N7)Pt(NH3)3}2](NO3)6·2H2O (11) (60mg) was dissolved in 1.2 ml D2O. The pH value was raised from 5.4 to 10 by adding 1M NaOD. The sample was lyophilized and subsequently dissolved in D2O (1ml), and then the solution was kept in a closed vial until crystals of (18) appeared after several weeks. The composition of (18) was confirmed by X-ray crystallography. 3.2.2.12 [{(dien)Pd}3(9-MeA-N1,N7,N6)]Cl3.5(PF6)1.5·3H2O (19) [dienPdI]I (85.8 mg, 0.18 mmol) and AgPF6 (89 mg, 0.36 mmol) were suspended in 10 ml of water and stirred in the dark for 24 hours at 40°C. After removal of AgI,1/2 eq of 9-MeA (13.8 mg, 0.09 mmol) in 5 ml water was added. The pH of the solution was adjusted to 11 with a solution of NaOH/NaCl. After stirring for one hour at 40°C, another equivalent of [dienPd(H2O)]2+ was added. The mixture was stirred for two more hours at 40°C. The solution was subsequently allowed to crystallize (4°C). Formation of small yellow crystals was observed. After isolation, the crystals were characterized by X-ray crystallography. 3.2.2.13 [Pt(1-MeC-N3)3(OH)](ClO4)0.5(OH)0.5·7H2O (21) [Pt(1-MeC-N3)3Cl](NO3) (160 mg, 0.23 mmol) and 9-MeA (34.3 mg, 0.23 mmol) were combined in 30 ml of water and AgNO3 (39.07 mg, 0.23 mmol) was added. 3.2. Synthesis of Complexes 164 Then 1N HNO3 was added dropwise to reach pH 1-2. The mixture was stirred at 40°C for 7 days in the dark. Then AgCl was filtered off and the resulting solution was concentrated to 10 ml volume by rotary evaporation. Then the pH was adjusted to 10 (1M NaOH) and the solution was kept in a closed vial in the refrigerator. After several days, a small amount of unreacted [Pt(1-MeC- N3)3Cl](NO3) was filtered off. Further evaporation led to colourless needles of (21). 1H NMR 1-MeC (trans to OH group): (D2O, pD = 7.1, δ/ppm): 7.43, d, (3J = 7.4 Hz) H6; 5.89, d, H5; 3.35, s, CH3; 1-MeC (cis to OH group): 7.54, d, (3J = 7.4 Hz) H6; 5.96, d, H5; 3.41, s, CH3 3.2.2.14 trans-[(NH3)2Pt(1,9-DimeAH-N7)(9-MeGH-N7)](NO3)2 (23) trans-[(NH3)2Pt(9-MeGH-N7)Cl]Cl (3.5 mg, 7.5 mmol) was stirred in 60 µl of D2O at 40°C in the dark overnight. After removal of AgCl, (1,9-DimeAH)(ClO4) (1.99 mg, 1 eq) was added, the pH of the solution adjusted to 1.3 (NaOD) and stirred at 40°C for three days. The signals of the resonances in the 1H NMR spectrum were assigned to (23), according to the relative intensities. 1H NMR: 1,9-DimeAH: (D2O, pD = 8.7, δ/ppm): 8.61, s, H8; 8.34, s, H2; , s, 3.89, s, N9- CH3; 3.77, s, N1-CH3 9-MeGH: (D2O, pD = 8.7, δ/ppm): 8.22, s, H8; 3.62, s, CH3 3.2.2.15 trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)](NO3)2 (24) trans-[(NH3)2Pt(1-MeC-N3)Cl](NO3) (145.94 mg, 0.32 mmol), 6,9-DimeA (52.72 mg, 1 eq) and AgNO3 (54.88 mg, 1 eq) were stirred in 50 ml water at 40°C for 3 days in the dark. After filtration of AgCl, the filtrate was evaporated slowly by 3.2. Synthesis of Complexes 165 rotary evaporation to 10 ml volume. The solution was then allowed to evaporate at 4°C. Eventually, colourless crystals were obtained. The yield was 65%. Elemental analysis: C12N12H24PtO9, M = 675.48 g·mol-1 Calculated: C 21.3 %, H 3.6 %, N 24.9 % Obtained: C 21.3 %, H 3.8 %, N 24.8 % 1H NMR: 6,9-DimeA: (D2O, pD = 6.3, δ/ppm): 8.56, s, H8; 8.44, s, H2; , s, 3.94, s, N9-CH3; 3.53, s, N6-CH3 1-MeC: (D2O, pD = 6.3, δ/ppm): 7.69, d, H6; 6.09, d, H5; 3.35, s, CH3 3.2.2.16 trans-[(NH3)2Pt(6,9-DimeA-N7)(9-MeGH-N7)](NO3)2·5H2O (25) trans-[(NH3)2Pt(9-MeGH-N7)Cl]Cl (159.816 mg, 0.34 mmol) was stirred together with 6,9-DimeA (56.06 mg, 0.34 mmol) and AgCl (116.72 mg, 0.68 mmol) in 50 ml of water at 40°C in the dark for three days. The initial pH was 5.8. AgCl was then removed by filtration and the resulting solution was concentrated to 10 ml volume by rotary evaporation. After cooling the solution to 4°C for one week, a white precipitate was filtered off and recrystallized from water. Crystals suitable for X-ray crystallography were obtained. The yield was 32%. 1H NMR: 6,9-DimeA: (D2O, pD = 6.4, δ/ppm): 8.43, s, H8; 8.67, s, H2; , s, 3.94, s, N9-CH3; 3.78, s, N6-CH3 9-MeGH: (D2O, pD = 6.4, δ/ppm): 8.33, s, H8; 3.32, s, CH3 3.2. Synthesis of Complexes 166 3.2.2.17 trans,trans,trans-{(NH3)2Pt(N1-pz-N2)2[(NH3)2Pt(1-MeC- N3)]2} 6+ (29) A solution containing trans-[(NH3)2Pt(pzH)2](NO3)2 (52 mg, 0.11 mmol), trans- [(NH3)2Pt(1-MeC-N3)Cl]NO3 (96 mg, 0.22 mmol) and AgNO3 (36 mg, 0.11 mmol) were brought to pH 8.3 with 1M NaOH. The mixture was then stirred at 40°C in the dark for 7 days in 50 ml of H2O. After removal of AgCl, the solution was concentrated to 10 ml volume and cooled to 4°C. Excess of trans-[(NH3)2Pt(1- MeC-N3)Cl]NO3 was filtered off, and the solution was concentrated by slow evaporation. A precipitate was obtained, which was assigned to complex (29), based on the relative intensities of the 1H NMR resonances. 1H NMR: 1-MeC: (D2O, pD = 5.6, δ/ppm): 7.68, d, (3J = 7.5 Hz) H6; 6.09, d, H5; 3.51, s, CH3 pzH: (D2O, pD = 5.6, δ/ppm): 7.96, d, H3; 7.72, d, H5; 6.50, t, H4 3.2.2.18 trans-[(NH3)2Pt{(pzH)Cl}2](NO3)2·H2O (30) A solution containing trans-[(NH3)2Pt(pzH)Cl](NO3) (28) was heated at 50°C to dryness and subsequently dissolved in water. Yellow crystals were collected by filtration and characterized by X-ray crystallography. The yield was 64%. Elemental analysis: PtCl2C6H16N8O9, M = 596.24 g·mol-1 Calculated: C 12.1 %, H 3.0 %, N 18.8 % Obtained: C 12.0 %, H 3.0 %, N 19.1 % 195Pt NMR: -385 ppm 3.2. Synthesis of Complexes 167 3.2.2.19 trans,trans-[(NH3)2Pt(pzH)2Cl2][(NH3)2Pt(pzH)2](ClO4)4·H2O (31) and trans-[(NH3)2Pt{(pzH)Cl}2](ClO4)2·H2O (32) Compound (30) (20 mg, 0.03 mmol) was dissolved in 30 ml of water at 40°C. After addition of 1M HClO4, the solution was allowed to evaporate at 4°C. Red crystals and subsequently yellow crystals appeared, which were separated mechanically under a microscope. The red ones correspond to compound (31) and the yellow ones to (32). The yield was 71%. Elemental analysis: (31) PtC6H14N6Cl3O9, M = 615.65 g·mol-1 Calculated: C 11.7 %, H 2.3 %, N 13.6 % Obtained: C 11.8 %, H 2.3 %, N 13.7 % Elemental analysis: (32) PtCl4C6H16N6O9, M = 653.12 g·mol-1 Calculated: C 11.0 %, H 2.5 %, N 12.9 % Obtained: C 11.1 %, H 2.7%, N 13.2% 3.2.2.20 trans-[(NH3)2Pt(pzH)(9-MeGH-N7)](NO3)2 (33) trans-[(NH3)2Pt(pzH)Cl](NO3) (28) (142 mg, 0.36 mmol), AgNO3 (61 mg, 0.36 mmol) and 9-MeGH (59.4 mg, 0.36 mmol) were dissolved in 200 ml of water. The pH was brought from 3.5 to 4.5 with 1 NaOH. The mixture was stirred at 40°C in the dark for two days. AgCl was filtered off and the solution was evaporated to near dryness. The yield was about 85%. The white solid was characterized by 1H NMR and elemental analysis. Elemental analysis: C9H17N11O7Pt, M = 586.38 g·mol-1 Calculated: C 18.4 %, H 2.9 %, N 26.3 % Obtained: C 18.2 %, H 3.0 %, N 26.2 % 3.2. Synthesis of Complexes 168 1H NMR: 9-MeGH: (D2O, pD = 4.3, δ/ppm): 8.32, s, H8; 3.76, s, CH3 pzH: (D2O, pD = 4.3, δ/ppm): 7.98, d, (3J = 8.8 Hz) H3; 7.94, d, H5; 6.61, t, H4 3.2.2.21 trans,trans-[(NH3)2Pt(9-MeGH-N7)(N1-pz-N2)(NH3)2Pt(1- MeC-N3)](NO3)3 (34) This compound was prepared on an NMR scale in D2O solution from trans- [(NH3)2Pt(pzH)(9-MeGH-N7)](NO3)2 (33) by treating it with trans-[(NH3)Pt(1- MeC-N3)(D2O)]2+ (1:1). The pH was adjusted to 7.5. After stirring the solution three days at 40°C, signals corresponding to (34) appeared, which relative intensities were as expected. 4. Appendix; X-Ray Tables 169 4 Appendix; X-Ray Tables Table A-1: Crystallographic data for compound 8. Compound trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 Formula C10 H27 Cl4 N10 O12 Pt2 Formula weight (g mol-1) 1011.4 Crystal color and habit yellow blocks Space system triclinic Space group P-1 a (Å) 8.8200(18) b (Å) 10.749(2) c (Å) 28.322(6) α (º) 90.01(3) β (º) 93.32(3) γ (º) 92.02(3) Z 4 V (Å3) 2678.9(9) ρcalc (g cm-1) 2.508 μ (Mo Kα) (mm-1) 10.904 F(000) 1908 θ range (º) 3 - 28 No. reflections collected 11428 No. reflections observed 7412 I>2σ(I) No. parameters refined 685 R1 (obs. data) 0.054 wR2 (obs. data) 0.1079 Goodness-of-fit, S 1.06 Residual ρmax, ρmin (e Å -3) 1.562, -1.281 4. Appendix; X-Ray Tables 170 Table A-2: Crystallographic data for compound 15. Compound trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N7,N6)](ClO4)3·3.5H2O Formula C16 H39 Cl3 N15 O17.5 Pt2 Formula weight (g mol-1) 1211.04 Crystal color and habit colourless blocks Space system monoclinic Space group P21/c a (Å) 11.845(2) b (Å) 15.317(3) c (Å) 21.292(4) α (º) 90.00 β (º) 94.43(3) γ (º) 90.00 Z 2 V (Å3) 3851.5(12) ρcalc (g cm-1) 2.101 μ (Mo Kα) (mm-1) 7.551 F(000) 2348 θ range (º) 3 - 27 No. reflections collected 9662 No. reflections observed 6064 I>2σ(I) No. parameters refined 512 R1 (obs. data) 0.0476 wR2 (obs. data) 0.1097 Goodness-of-fit, S 0.967 Residual ρmax, ρmin (e Å -3) 2.486, -2.141 Table A-3: Crystallographic data for compound 16. Compound [(9-MeA-N7)Pt(NH3)3]Cl2·2H2O Formula C6 H20 Cl2 N8 O2 Pt Formula weight (g mol-1) 502.29 Crystal color and habit colourless blocks Space system monoclinic Space group C2/c a (Å) 28.133(6) b (Å) 7.1110(14) c (Å) 17.814(4) α (º) 90.00 β (º) 118.27(3) γ (º) 90.00 Z 8 V (Å3) 3138.6(11) ρcalc (g cm-1) 2.126 μ (Mo Kα) (mm-1) 9.293 F(000) 1920 θ range (º) 3.08 – 27.48 No. reflections collected 3591 No. reflections observed 2764 I>2σ(I) No. parameters refined 253 R1 (obs. data) 0.0283 wR2 (obs. data) 0.0856 Goodness-of-fit, S 1.105 Residual ρmax, ρmin (e Å -3) 1.560, -2.904 4. Appendix; X-Ray Tables 171 Table A-4: Crystallographic data for compound 18. Compound cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2](NO3)4·6H2O Formula C12 H36 N22 O17 Pt3 Formula weight (g mol-1) 1345.9 Crystal color and habit colourless blocks Space system monoclinic Space group C2/c a (Å) 24.753(5) b (Å) 10.812(2) c (Å) 14.514(3) α (º) 90 β (º) 95.80(3) γ (º) 90 Z 4 V (Å3) 3864.6(14) ρcalc (g cm-1) 2.313 μ (Mo Kα) (mm-1) 10.924 F(000) 2528 θ range (º) 3.12 – 27.5 No. reflections collected 4435 No. reflections observed 3624 I>2σ(I) No. parameters refined 244 R1 (obs. data) 0.0273 wR2 (obs. data) 0.0632 Goodness-of-fit, S 1.057 Residual ρmax, ρmin (e Å -3) 1.112, -0.958 Table A-5: Crystallographic data for compound 19. Compound {[(dien)Pd]3(9-MeA-N1,N7,N6)}Cl3.5(PF6)1.5·3H2O Formula C18 H45 Cl3.5 F9 N14 O3 P1.5 Pd3 Formula weight (g mol-1) 1166.41 Crystal color and habit yellow blocks Space system monoclinic Space group P21/c a (Å) 12.659(3) b (Å) 16.193(3) c (Å) 20.720(4) α (º) 90 β (º) 105.52(3) γ (º) 90 Z 4 V (Å3) 4092.5(14) ρcalc (g cm-1) 1.893 μ (Mo Kα) (mm-1) 1.675 F(000) 2304 θ range (º) 2.09 – 27.08 No. reflections collected 8970 No. reflections observed 4845 I>2σ(I) No. parameters refined 472 R1 (obs. data) 0.035 wR2 (obs. data) 0.0613 Goodness-of-fit, S 0.776 Residual ρmax, ρmin (e Å -3) 1.056, -0.592 4. Appendix; X-Ray Tables 172 Table A-6: Crystallographic data for compound 21. Compound [Pt(1-MeC-N3)3(OH)](ClO4)0.5(OH)0.5·7H2O Formula C15 H37.5 Cl0.5 N9 O14 Pt Formula weight (g mol-1) 780.85 Crystal color and habit colourless prisms Space system monoclinic Space group C2/c a (Å) 30.454(6) b (Å) 14.558(3) c (Å) 13.471(3) α (º) 90 β (º) 99.28(3) γ (º) 90 Z 8 V (Å3) 5894(2) ρcalc (g cm-1) 1.76 μ (Mo Kα) (mm-1) 4.882 F(000) 3112 θ range (º) 2.47 – 27.5 No. reflections collected 6150 No. reflections observed 3319 I>2σ(I) No. parameters refined 356 R1 (obs. data) 0.043 wR2 (obs. data) 0.1026 Goodness-of-fit, S 0.897 Residual ρmax, ρmin (e Å -3) 1.492, -0.861 Table A-7: Crystallographic data for compound 24. Compound trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)](NO3)2·H2O Formula C12 H24 N12 O8 Pt Formula weight (g mol-1) 659.52 Crystal color and habit colourless blocks Space system monoclinic Space group P2/n a (Å) 13.374(3) b (Å) 13.183(3) c (Å) 26.970(5) α (º) 90 β (º) 103.34(3) γ (º) 90 Z 8 V (Å3) 4626.8(16) ρcalc (g cm-1) 1.894 μ (Mo Kα) (mm-1) 6.129 F(000) 2576 θ range (º) 3.09 – 27.59 No. reflections collected 10624 No. reflections observed 4595 I>2σ(I) No. parameters refined 540 R1 (obs. data) 0.082 wR2 (obs. data) 0.2227 Goodness-of-fit, S 0.998 Residual ρmax, ρmin (e Å -3) 2.286, -0.714 4. Appendix; X-Ray Tables 173 Table A-8: Crystallographic data for compound 25. Compound trans-[(NH3)2Pt(6,9-DimeA-N7)(9-MeGH-N7)](NO3)2·5H2O Formula C13 H32 N14 O12 Pt Formula weight (g mol-1) 771.53 Crystal color and habit colourless blocks Space system monoclinic Space group P21/c a (Å) 9.6160(19) b (Å) 13.392(3) c (Å) 20.301(4) α (º) 90 β (º) 86.64(3) γ (º) 90 Z 4 V (Å3) 2609.8(9) ρcalc (g cm-1) 1.938 μ (Mo Kα) (mm-1) 5.461 F(000) 1488 θ range (º) 1.82 – 22.94 No. reflections collected 3545 No. reflections observed 1713 I>2σ(I) No. parameters refined 363 R1 (obs. data) 0.0467 wR2 (obs. data) 0.1556 Goodness-of-fit, S 0.855 Residual ρmax, ρmin (e Å -3) 1.869, -1.004 Table A-9: Crystallographic data for compound 28. Compound trans-[(NH3)2Pt(pzH)Cl](NO3) Formula C3 H10 Cl N5 O3 Pt Formula weight (g mol-1) 394.7 Crystal color and habit yellow plates Space system triclinic Space group P-1 a (Å) 7.9570(16) b (Å) 9.789(2) c (Å) 13.267(3) α (º) 77.22(3) β (º) 85.61(3) γ (º) 77.38(3) Z 4 V (Å3) 983.0(3) ρcalc (g cm-1) 2.667 μ (Mo Kα) (mm-1) 14.533 F(000) 728 θ range (º) 2.18 – 26.73 No. reflections collected 4131 No. reflections observed 3011 I>2σ(I) No. parameters refined 237 R1 (obs. data) 0.0613 wR2 (obs. data) 0.153 Goodness-of-fit, S 1.041 Residual ρmax, ρmin (e Å -3) 3.062, -4.075 4. Appendix; X-Ray Tables 174 Table A-10: Crystallographic data for compound 32. Compound trans-[(NH3)2Pt{(pzH)Cl}2](ClO4)2·H2O Formula C6 H14 Cl4 N6 O10 Pt Formula weight (g mol-1) 667.12 Crystal color and habit yellow blocks Space system monoclinic Space group P21/c a (Å) 9.4960(19) b (Å) 10.621(2) c (Å) 10.474(2) α (º) 90 β (º) 115.67(3) γ (º) 90 Z 2 V (Å3) 952.1(3) ρcalc (g cm-1) 2.327 μ (Mo Kα) (mm-1) 7.989 F(000) 636 θ range (º) 3.06 – 27.5 No. reflections collected 2159 No. reflections observed 1502 I>2σ(I) No. parameters refined 124 R1 (obs. data) 0.055 wR2 (obs. data) 0.1565 Goodness-of-fit, S 1.087 Residual ρmax, ρmin (e Å -3) 5.585, -1.247 5. Summary 175 5 Summary Acid-base equilibria involving nucleobases at physiological pH value have recently been recognized as being possible and biorelevant in catalytic reactions, in particular of RNA. Reasons for “shifted” pKa values, hence for pKa values shifted from high or from low values to ~ 7, are beginning to emerge. It is obviously a combination of effects of the medium, of the microenvironment, and of charge distribution in the vicinity of the site involved in acid-base chemistry, which contributes to the shift. The influence of a coordinated metal ion on acid- base equilibria of nucleobases has been studied since quite some time, but it had been the impression that, within narrow limits, the effect of the metal is “constant”, depending mainly on its oxidation state, its vicinity to the acidic/basic group and on coligands in that they determine the overall charge of the complex. It was the observation of M. S. Lüth in our group in 2001, that twofold Pt coordination to 9-methyladenine (9-MeA) not only acidifies the exocyclic N(6)H2 group, but that apparently the microenvironment (intramolecular H bonding leading to a stabilization of the deprotonated form) had an influence on pKa reaching that of the two metal ions. Based on this observation, it was the aim of the present thesis to place these findings into a wider perspective by systematically studying 9- methyladenine complexes of PtII with different coligands capable or incapable of interacting with the deprotonated species and determining relevant pKa values. These studies have been extended to 1,9-dimethyladenine complexes, hence to compounds, in which the adenine base had been chemically modified. Next, the question of NH2 acidification in complexes of 1-methylcytosine was studied, followed by the question of acidification of the aqua ligand in simple nucleobase compounds of composition cis-[(NH3)2Pt(nucleobase)(H2O)]2+. Moreover, by employing 9-methyladenine, the pKa of the protonated species in dependence of the solvent (water and mixtures of acetone/water as well as methanol/water) was investigated. In the final chapter, a preliminary study was 5. Summary 176 undertaken with the aim of extending this work to the deprotonation of N7- platinated guanine. In order to bring the N1 position of the guanine base into reach of the a second nucleobase capable of stabilizing the guanine anion, the bidentate pyrazolate ligand was applied, which was additionally linked via a trans-a2PtII entity to a cytosine nucleobase. Concerning the most important findings of the present thesis, the following points can be listed. (1) A considerable number of complexes of 9-methyladenine with N1 and N7 sites platinated confirms the concept that not only the positive charge of the metals but that in particular favourable hydrogen bonding between the deprotonated exocyclic NH2 group of 9-MeA is extremely important to lower the pKa from 10 – 11 (effect of two metals only) into the pKa range of 7 – 8. This value is close to physiological pH. For example, in trans,trans,trans- [(NH3)2Pt(N7-9-MeA-N1)(dienPt)(N7-9-EtA-N1){(NH3)2Pt(9-MeGH-N7)}]6+ (13), the deprotonation of the NH2 of the adenine nucleobase occurs at pKa 7.14. The pKa shift is due to the combined effects of twofold metal coordination and a favourable intramolecular hydrogen bond between an anionic and a neutral adenine nucleobase. (2) The most dramatic shifts in pKa values of platinated 9-MeA complexes were observed, when there was formation of a “metal-stabilized” rare tautomer of 9-MeA (A*). By this term we mean that in this case a metal is bonded to N6 of the nucleobase and one of the protons that used to be at N(6)H2, has been transferred to N1. 5. Summary 177 N N N N NH2 R 1 HN N N N NH R 1 A A* HN N N N R 1 M-A* N M H Formation of this M-A* species occurs via a metal migration process from N1 to N6, which is greatly facilitated, if there is a second metal coordinated to N7. Thus, starting from trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N1,N7)]4+ (7), the migration product trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N7,N6)]3+ (15) has been isolated and characterized, with a pKa of 5.0 for N(1)H of adenine. Similarly, the double migration product cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2]4+ (18) has been derived from cis-[(NH3)2Pt{(N1-9-MeA-N7)Pt(NH3)3}2]6+ (11). The first pKa value of (18) is 4.9, as compared to 8.7 in (11). Pt2 N6N6a Pt1a Pt1 N7 N7a N11 N13 N12 View of the cation cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2]4+ of (18) (3) By synthesizing mixed nucleobase complexes containing 6,9-DimeA, trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)]2+ (24) and trans-[(NH3)2Pt(6,9- DimeA-N7)(9-MeGH-N7)]2+ (25) and by structurally characterizing these, it was unambiguously demonstrated that the complications seen in spectra of the 5. Summary 178 corresponding 1,9-DimeA complexes trans-[(NH3)2Pt(1,9-DimeAH-N7)(9-MeGH- N7)]3+ (22) and trans-[(NH3)2Pt(1,9-DimeAH-N7)(9-MeGH-N7)]3+ (23) at alkaline pH was due to Dimroth rearrangements. (4) The effect of the solvent (mixture) on the pKa of 9-MeAH+ was relatively minor, even though it displayed the expected direction, viz. when the dielectric constant of the medium decreases, the pKa decreases as well. The fact that the changes were not dramatical may be due to the fact that even in the solvent mixtures applied, there was still a sufficient number of H2O molecules present to properly solvate all the charged species present in equilibrium. (5) The pKa values for the mono(nucleobase) complexes of cis-(NH3)2PtII, cis-[(NH3)2Pt(nucleobase)(H2O)]2+, did not display the expected difference in pKa of the aqua ligand for nucleobase = guanine-N7 and cytosine-N3, even though from model building a stabilizing H bonding effect between the hydroxo ligand and the exocyclic amino group of cytosine could have been anticipated. 6. Zusammenfassung 179 6 Zusammenfassung Säure-Base-Gleichgewichte von Nukleobasen bei physiologischem pH- Wert wurden erst kürzlich als möglich und als biorelevant in katalytischen Reaktionen angesehen, insbesondere solcher der RNA. Ursachen veränderter pKs-Werte, also Verschiebungen von niedrigen oder hohen Werten zu Werten von 7 ± 1 beginnt man gerade erst zu verstehen. Es ist offensichtlich, dass hier eine Reihe verschiedener Effekte eine Rolle spielen, wie z. B. das Medium, die Mikroumgebung oder die Ladungsverteilung. Säure-Base-Gleichgewichte von Nukleobasen unter dem Einfluss koordinierter Metalle werden schon seit einiger Zeit untersucht, wobei man aber bisher meist davon ausgegangen ist, dass der Effekt des Metalls konstant ist und hauptsächlich von der Oxidationsstuffe und der Nähe zur Säure/Base Gruppe abhängt. M. S. Lüth beobachtete 2001, dass die zweifache Pt-Koordination über N1 and N7 an 9-Methyladenin (9-MeA) nicht nur die exozyklische N(6)H2-Gruppe acider macht, sondern dass anscheinend auch die nähere Umgebung (eine intramolekulare Wasserstoffbrücke führt zur Stabilisierung der deprotonierten Form), einen gravierenden Einfluss auf den pKs-Wert hat. Ziel dieser Dissertation war es, aufbauend auf dieser Beobachtung, zu einem tieferen Verständnis der Einflüsse der genannten Faktoren zu gelangen. Hierfür wurden systematisch 9-Methyladenin-Komplexe von PtII studiert, welche unterschiedliche Co-Liganden enthalten, die mit der deprotonierten Adenin-Base entweder interagieren können oder auch nicht. Diese Studie wurde auch auf 1,9-Dimethyladenin-Komplexe ausgeweitet. Weiterhin wurde der Einfluss von Nachbarliganden auf die Acidität der exocyclischen Aminogruppe in N3-platiniertem 1-Methylcytosin sowie auf die Acidität von Aqua-Liganden in Verbindungen der Zusammensetzung cis- [(NH3)2Pt(Nukleobase)(H2O)]2+ untersucht. Schließlich wurde am Beispiel von protoniertem 9-Methyladenin der Einfluss des Lösungsmittels (Wasser sowie Aceton/Wasser- und Methanol/Wasser-Gemische) auf den pKs-Wert studiert. Im 6. Zusammenfassung 180 abschließenden Kapitel wurde eine erste orientierende Untersuchung hinsichtlich der Übertragbarkeit des Konzepts der intramolekularen Stabilisierung anionischer Nukleobasen auf die N1-Position N7 platinierter Guanin-Liganden durchgeführt. Ziel war es hierbei, eine zweite Nukleobase (hier: Cytosin) über einen verbrückenden Pyrazolat-Hilfsliganden so zu positionieren, dass eine Wechselwirkung zwischen N(4)H2 des Cytosins und N1 des Guanins prinzipiell möglich ist. Nachfolgend seien die wichtigsten Ergebnisse dieser Dissertation noch einmal zusammengefasst: (1) Es wurde eine größere Anzahl ein- und mehrkerniger 9-Methyladenin- Komplexe des Pt mit N1 und N7-Koordination hinsichtlich ihrer N(6)H2-Acidität untersucht. Die Ergebnisse bestätigen das Konzept wonach nicht nur die positive Ladung der koordinierten Metalle, sondern in besonderer Weise auch intramolekulare Wasserstoffbrücken äußerst wichtig sind, um den pKs-Wert der NH2-Gruppe auf ca. 7-8 abzusenken. Zum Beispiel findet in trans,trans,trans- [(NH3)2Pt(N7-9-MeA-N1)(dienPt)(N7-9-EtA-N1){(NH3)2Pt(9-MeGH-N7)}]6+ (13) die Deprotonierung der NH2-Gruppe des einen Adenins mit einem pKs-Wert von 7.14 statt, was neben der zweifachen Platinierung vor allem auch auf die intermolekulare Wasserstoffbrücke der NH- -Funktion mit der NH2-Gruppe der zweiten Adeninbase zurück zu führen ist. (2) Die drastischste Veränderung des pKs-Wertes eines platinierten 9- Methyladenin-Liganden tritt bei Ausbildung eines sog. Metall-stabilisierten Tautomers, M-A*, auf. In diesem Fall ist das Metall an der N6-Position der Nukleobase gebunden und eines der beiden Protonen der NH2-Gruppe ist zum N1 gewechselt. 6. Zusammenfassung 181 N N N N NH2 R 1 HN N N N NH R 1 A A* HN N N N R 1 M-A* N M H Die Bildung der M-A* Spezies geht auf eine Metallwanderung von N1 nach N6 zurück, welche durch ein zweites Metall, koordiniert an N7, deutlich erleichtert wird. Ausgehend von trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N1,N7)]4+ (7), wurde z. B. das Umlagerungsprodukt trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA- N7,N6)]3+ (15) isoliert und charakterisiert. Ein pKs-Wert von 5.0 wurde für N(1)H von Adenin bestimmt. Gleichermaßen wurde das Umlagerungsprodukt cis- [(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2]4+ (18) aus cis-[(NH3)2Pt{(N1-9-MeA- N7)Pt(NH3)3}2]6+ (11) hergestellt. Der pKs-Wert verschiebt sich hierbei von 8.7 (11) nach 4.9 (18). Pt2 N6N6a Pt1a Pt1 N7 N7a N11 N13 N12 Ansicht des Kations cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2]4+ von (18) (3) Durch die Synthese und vollständige strukturelle Charakterisierung der gemischten Nukleobase-Komplexe des 6,9-DimeA, trans-[(NH3)2Pt(6,9- 6. Zusammenfassung 182 DimeA-N7)(1-MeC-N3)]2+ (24) und trans-[(NH3)2Pt(6,9-DimeA-N7)(9-MeGH- N7)]2+ (25) konnte der Nachweis erbracht werden, dass die in Spektren der 1,9- DimeA-haltigen Verbindungen trans-[(NH3)2Pt(1,9-DimeAH-N7)(9-MeGH-N7)]3+ (22) und trans-[(NH3)2Pt(1,9-DimeAH-N7)(9-MeGH-N7)]3+ (23) bei alkalischen pH beobachteten Komplikationen auf Dimroth-Umlagerungen zurück zu führen sind. (4) Der Effekt von Lösungsmittelgemischen auf den pKs-Wert von 9- MeAH+ ist verhältnismäßig gering, bestätigt aber den prognostizierten Trend: Wenn die Dielektrizitätskonstante des Lösungsmittels sinkt, verringert sich auch der pKs-Wert. Der relativ kleine Effekt kann damit erklärt werden, dass der Gehalt an H2O in den verwendeten Gemischen noch immer zu hoch ist, um eine effiziente Solvatisierung der ionogenen Spezies zu gewährleisten. (5) Die pKs-Werte von Aqua-Liganden in Verbindungen des Typs cis- [(NH3)2Pt(Nukleobase)(H2O)]2+ zeigten nicht die erwarteten Unterschiede für Guanin-N7 und Cytosin-N3, obwohl man eigentlich von einem stabilisierenden Effekt durch eine Wasserstoffbrücke zwischen dem Hydroxoliganden und der exozyklischen Aminogruppe von Cytosin hätte ausgehen können. 7. Resumen 183 7 Resumen Los equilibrios ácido-base que implican nucleobases en el rango del pH fisiológico se han estudiado recientemente como posibles y biorelevantes en reacciones catalíticas, en particular en el caso del ARN. Las razones de los cambios en los valores de pKa, desde valores ácidos y básicos a valores próximos a 7, están saliendo a la luz. Una combinación de los efectos del medio, del entorno y de la distribución de la carga en la posición a estudiar, contribuyen a estos cambios. En estos equilibrios ácido-base ha sido estudiada desde hace bastante tiempo, la influencia de un ion metálico coordinado a una nucleobase, pero se tenía la impresión de que, dentro de pequeños límites, el efecto del metal era constante, dependiendo principalmente de su estado de oxidación, su cercanía al grupo ácido/básico y de otros ligandos. M. S. Lüth, en nuestro grupo en 2001, observó que una doble coordinación de Pt a 9- metiladenina (9-MeA), acidifica no solamente el grupo exocíclico N(6)H2, sino que también el entorno (puentes de hidrógeno intramoleculares que conducen a una estabilización de la forma desprotonada) tenía al parecer una influencia semejante en el valor de pKa. De acuerdo con esta observación, el objetivo de esta tesis es ampliar estos resultados, estudiando complejos de PtII con 9-metiladenina y con diversos co-ligandos, capaces o incapaces de interaccionar con especies desprotonadas y la posterior determinación de los valores de pKa. Estos estudios han sido extendidos a complejos de 1,9-dimetiladenina, por lo tanto a compuestos, en los cuales la adenina había sido modificada químicamente. Además, ha sido estudiada la acidificación del grupo NH2 en complejos de 1-metilcitosina y también la del ligando H2O en compuestos con nucleobases simples, de composición cis-[(NH3)2Pt(nucleobase)(H2O)]2+. Por otra parte, ha sido investigado el pKa de especies protonadas en dependencia del solvente (agua y mezclas de acetona/agua así como metanol/agua) empleando 9- metiladenina. En el capítulo final, un estudio preliminar fue llevado a cabo con la 7. Resumen 184 idea de extender este trabajo a la desprotonación de la guanina con Pt coordinado en N7. Para acercar la posición N1 de ésta a otra segunda nucleobase, capaz de estabilizar el anión de la guanina, se utilizó el ligando pirazolato, el cual se unió a través de la entidad trans-a2PtII a una citosina. Los resultados más importantes de esta tesis se enumeran a continuacón: (1) un número considerable de complejos de 9-metiladenina con Pt coordinado en las posiciones N1 y N7 confirma el hecho de que no solamente las cargas positivas de los metales, sino también en particular los puentes de hidrógeno que afectan a grupo desprotonado NH2, son muy importantes a la hora de disminuir el pKa desde 10 - 11 (sólo dos metales) hasta 7 - 8, valor cercano del pH fisiológico. Por ejemplo, en trans,trans,trans-[(NH3)2Pt(N7-9- MeA-N1)(dienPt)(N7-9-EtA-N1){(NH3)2Pt(9-MeGH-N7)}]6+(13),la desprotonación del NH2 de la adenina ocurre a pKa 7.14. Este valor bajo de pKa se debe a la combinación de dos efectos: la coordinación de dos átomos de platino y un puente de hidrógeno intramolecular entre la adenina aniónica y la neutral. (2) los cambios más drásticos en los valores del pKa de complejos de 9- MeA con Pt fueron observados en la formación de un tautómero no muy común de 9-MeA (M-A*), que recibe el nombre de “metal-estabilizado”. Este nombre significa, que un metal está enlazado a la posición N6 de la nucleobase y uno de los protones que suelen estar en N(6)H2 se transfiere a N1. N N N N NH2 R 1 HN N N N NH R 1 A A* HN N N N R 1 M-A* N M H 7. Resumen 185 La formación de esta especie M-A* ocurre a través de un proceso de migración del metal de N1 a N6, que está favorecida por la presencia de un segundo metal coordinado a N7. Partiendo del complejo trans-[{(NH3)2Pt(1- MeC-N3)}2(9-MeA-N1,N7)]4+ (7), el producto trans-[{(NH3)2Pt(1-MeC-N3)}2(9- MeA-N7,N6)]3+ (15), ha sido aislado y caracterizado, obteniendo un pKa de 5.0 para el N(1)H de la adenina. De modo parecido, el producto de la doble migración cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2]4+ (18) se obtuvo a partir del compuesto de partida cis-[(NH3)2Pt{(N1-9-MeA-N7)Pt(NH3)3}2]6+ (11). Sin embargo, el valor de pKa de (18) es 4.9 en comparación con el valor de 8.7 del compuesto (11). Pt2 N6N6a Pt1a Pt1 N7 N7a N11 N13 N12 Vista del catión cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2]4+ de (18) (3) En complejos formados por 1,9-DimeA y otra nucleobase: trans- [(NH3)2Pt(1,9-DimeAH-N7)(9-MeGH-N7)]3+ (22) y trans-[(NH3)2Pt(1,9-DimeAH- N7)(9-MeGH-N7)]3+ (23) se observaron complicaciones de las señales en los espectros a pH básico. Se ha demostrado gracias a la síntesis de compuestos como trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)]2+ (24) y trans-[(NH3)2Pt(6,9- DimeA-N7)(9-MeGH-N7)]2+ (25), que estas complicaciones son debidas a un proceso llamado “Dimroth rearrangement”. 7. Resumen 186 (4) el efecto del disolvente (mezcla) en los valores de pKa en 9-MeAH+ es relativamente pequeño. 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List of abbreviations 197 List of abbreviations Ligands and Chemicals 1-MeC 1-methylcytosine 1-MeUH 1-methyluracil in its neutral form 1-MeU– 1-methyluracilate, deprotonated at N3 9-MeA 9-methyladenine 9-MeAH+ 9-methyladeninium, protonated at N1 9-EtA– 9-ethyladenine, deprotonated at N1 9-EtGH 9-ethylguanine in its neutral form 1,9-DimeA 1,9-dimethyladenine (uncharged form; N6 forms an imino group) 1,9-DimeAH+ 1,9-dimethyladeninium (protonated at N6; N6 forms an amino group) A (C, G, T, U) general abbreviation for adenin- (cytosin-, guanin-, thymin-, uracyl-) derivates cis/trans-DDP cis/trans-diamminodichloroplatinum (II) dien diethylenetriamine [(dien)PtII] [(dien)Pt(H2O)]2+ or [(dien)Pt(D2O)]2+ DMSO-d6 deuterated dimethylsulfoxide DNA desoxyribonucleic acid EDTA Ethylendiaminetetraacetic acid – disodium salt pzH pyrazol pyz pyrazine RNA ribonucleic acid TSP sodium 3-trimethylsilyl-propanesulfonate Spectroscopy NMR nuclear magnetic resonance NOE nuclear Overhauser effect List of abbreviations 198 δ NMR chemical shift in ppm ppm parts per million IR infrared Common abbreviations ca. approximately eq. equivalent et al. and co-workers h hour M molar pD negative logarithms of the deuterium ion concentration pH* uncorrected pH meter reading in D2O pKa negative logarithms of the acidity constant viz. in other words vs. versus s singlet brs broad signal d doublet m multiplet List of compounds 199 List of compounds 1 [(dien)Pt(9-MeA-N1)](NO3)2 2 cis-[(NH3)2Pt(1-MeC-N3)(9-MeA-N7)](ClO4)2·H2O 3 trans-[(NH3)2Pt(9-MeA-N7)2](ClO4)2·2H2O 4 cis-[(NH3)2Pt(9-MeA-N7)2](NO3)2·2H2O 5 cis-[(NH3)2Pt(1-MeC-N3)(N7-9-MeA-N1)(dienPt)](ClO4)4 6 cis-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N1,N7)](ClO4)4 7 trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N1,N7)](ClO4)4 8 trans-[(NH3)2ClPt(N7-9-MeA-N1)(dienPt)](ClO4)3 9 trans-[(NH3)2Pt(N7-9-MeA)(N7-9-MeA-N1)(dienPt)]4+ 10 trans-[(NH3)2Pt{(N7-9-MeA-N1)(dienPt)}2]6+ 11 cis-[(NH3)2Pt{(N1-9-MeA-N7)Pt(NH3)3}2](NO3)6·2H2O 12 cis-[(NH3)2Pt{(N7-9-MeA-N1)(dienPt)}2](NO3)6 13 trans,trans,trans-[(NH3)2Pt(N7-9-MeA-N1)(dienPt)(N7-9-EtA- N1){(NH3)2Pt(9-MeGH-N7)}](ClO4)3(NO3)3 14 trans,trans,trans-{(NH3)2Pt(N7-9-MeA-N1)2[(NH3)2Pt(9-EtGH-N7)]2}6+ 15 trans-[{(NH3)2Pt(1-MeC-N3)}2(9-MeA-N7,N6)](ClO4)3·3.5H2O 16 [(9-MeA-N7)Pt(NH3)3]Cl2·2H2O 17 cis-[(NH3)2Pt(N1-9-MeA-N7)(N6-9-MeA-N7){Pt(NH3)3}2]5+ 18 cis-[(NH3)2Pt(N6-9-MeA-N7)2{Pt(NH3)3}2](NO3)4·6H2O 19 [{(dien)Pd}3(9-MeA-N1,N7,N6)]Cl3.5(PF6)1.5·3H2O 20 [Pt(1-MeC-N3)3(9-MeA-N7)](NO3)2 21 [Pt(1-MeC-N3)3(OH)](ClO4)0.5(OH)0.5·7H2O 22 trans-[(NH3)2Pt(1,9-DimeAH-N7)(1-MeC-N3)](NO3)3 23 trans-[(NH3)2Pt(1,9-DimeAH-N7)(9-MeGH-N7)](NO3)3 24 trans-[(NH3)2Pt(6,9-DimeA-N7)(1-MeC-N3)](NO3)2·H2O 25 trans-[(NH3)2Pt(6,9-DimeA-N7)(9-MeGH-N7)](NO3)2·5H2O 26 trans-[(NH3)2Pt(1-MeC-N3)2]2+ 27 trans-[(NH3)2Pt(1-MeC-N3)(9-MeA-N7)](ClO4)2 28 trans-[(NH3)2Pt(pzH)Cl](NO3) 29 trans,trans,trans-{(NH3)2Pt(N1-pz-N2)2[(NH3)2Pt(1-MeC-N3)]2}6+ List of compounds 200 30 trans-[(NH3)2Pt{(pzH)Cl}2](NO3)2·H2O 31 trans,trans-[(NH3)2Pt(pzH)2Cl2][(NH3)2Pt(pzH)2](ClO4)4·H2O 32 trans-[(NH3)2Pt{(pzH)Cl}2](ClO4)2·H2O 33 trans-[(NH3)2Pt(pzH)(9-MeGH-N7)](NO3)2 34 trans,trans-[(NH3)2Pt(9-MeGH-N7)(N1-pz-N2)(NH3)2Pt(1-MeC-N3)](NO3)3 Tables of the pD dependences 201 Tables of the pD dependences 1. pD dependences of the different compounds studied in this thesis Table B-1: Chemical shifts for compound 1 in D2O in dependence of pD. pD CH3 H8 1.04 3.910 8.610 1.42 3.863 8.343 1.55 3.853 8.283 1.88 3.840 8.205 2.09 3.836 8.180 2.33 3.834 8.167 2.92 3.833 8.156 3.55 3.832 8.153 4.35 3.832 8.153 5.05 3.832 8.153 6.45 3.832 8.153 7.4 3.833 8.153 7.84 3.831 8.150 9.35 3.831 8.150 10.66 3.832 8.152 11.47 3.831 8.149 11.96 3.828 8.141 12.37 3.824 8.128 12.50 3.822 8.124 12.79 3.809 8.091 13.21 3.778 8.011 Table B-2: Chemical shifts for compound 2 in D2O in dependence of pD. pD CH3-A CH3-C H6 H5 H2/8 H2/8 7.12 3.868 3.346 7.556 6.007 8.341 8.562 10.32 3.869 3.346 7.554 6.006 8.341 8.566 10.78 3.869 3.346 7.550 6.003 8.342 8.563 10.94 3.869 3.346 7.549 6.002 8.342 8.561 11.10 3.867 3.344 7.545 6.0015 8.337 Tables of the pD dependences 202 11.25 3.868 3.345 7.544 6.0015 8.339 11.56 3.867 3.345 7.534 5.993 8.339 11.7 3.867 3.344 7.526 5.987 8.336 11.94 3.866 3.338 7.503 5.967 8.338 12.15 3.865 3.326 7.464 5.942 8.335 12.22 3.865 3.318 7.432 5.919 8.336 12.47 3.863 3.294 7.351 5.865 8.333 12.52 3.862 3.295 7.356 5.871 8.331 12.7 3.858 3.267 7.263 5.807 8.327 12.78 3.857 3.256 7.228 5.785 8.324 12.92 3.856 3.227 7.125 5.718 8.320 13.02 3.852 3.231 7.062 5.675 8.315 13.11 3.855 3.221 7.098 5.701 8.316 Table B-3: Chemical shifts for compound 3 in D2O in dependence of pD. pD CH3 0.88 4.059 1.17 4.050 1.78 4.014 1.93 3.996 2.27 3.992 2.64 3.985 4.47 3.976 5.84 3.977 6.78 3.977 8.92 3.977 9.33 3.976 10.46 3.976 11.08 3.976 11.42 3.976 12.20 3.974 12.58 3.969 12.75 3.964 12.87 3.956 13.29 3.947 Tables of the pD dependences 203 Table B-4: Chemical shifts for compound 4 in D2O in dependence of pD. pD CH3 0.15 3.909 0.55 3.907 0.66 3.906 0.96 3.900 1.17 3.900 1.66 3.876 2.05 3.852 2.43 3.829 2.81 3.815 3.06 3.814 3.2 3.813 3.4 3.813 4.15 3.812 4.47 3.812 4.75 3.813 6.2 3.813 8 3.812 10.9 3.813 11.54 3.812 11.82 3.812 12.56 3.809 12.7 3.810 13 3.808 Table B-5: Chemical shifts for compound 5 in D2O in dependence of pD. pD CH3-A CH3-C H5 H6 H2/8 5.90 3.892 3.362 6.021 7.558 8.700 6.05 3.891 3.361 6.021 7.558 8.700 6.40 3.891 3.358 6.026 7.559 8.703 7.05 3.890 3.356 6.025 7.560 8.703 7.90 3.892 3.361 6.024 7.560 8.776 8.25 3.890 3.357 6.026 7.560 8.779 9.15 3.891 3.361 6.024 7.559 8.777 9.66 3.890 3.368 6.022 7.561 8.758 9.9 3.885 3.365 6.020 7.559 8.747 Tables of the pD dependences 204 10.05 3.884 3.364 6.019 7.559 8.743 10.15 3.884 3.359 6.024 7.559 8.757 10.45 3.875 3.368 6.015 7.556 8.705 10.50 3.869 3.366 6.015 7.556 8.694 10.63 3.867 3.366 6.014 7.555 8.685 10.78 3.864 3.366 6.013 7.554 8.674 10.79 3.864 3.372 6.012 7.554 8.663 10.85 3.850 3.373 6.007 7.555 8.607 10.90 3.855 3.369 6.01 7.553 8.641 10.95 3.836 3.377 8.547 11.44 3.795 3.385 5.975 7.537 8.384 11.80 3.778 3.388 5.964 7.527 8.315 12.18 3.752 3.383 5.934 7.493 8.220 12.41 3.740 3.372 5.902 7.449 8.181 12.81 3.730 3.346 5.844 7.363 8.156 13.18 3.717 3.290 5.726 7.186 8.131 Table B-6: Chemical shifts for compound 6 in D2O in dependence of pD. pD CH3-A 3.57 3.874 3.73 3.878 4.77 3.875 5.84 3.878 6.60 3.877 7.84 3.879 9.02 3.876 10.09 3.876 10.64 3.859 11.03 3.842 11.28 3.825 11.41 3.816 11.48 3.814 11.69 3.799 11.77 3.793 11.93 3.766 11.95 3.760 12.20 3.754 Tables of the pD dependences 205 12.45 3.740 12.50 3.735 12.66 3.732 12.98 3.714 13.22 3.700 Table B-7: Chemical shifts for compound 7 in D2O in dependence of pD. pD CH3-A 5.8 4.008 7.3 4.008 8.14 4.005 8.6 4.005 9.25 3.992 9.54 3.991 9.63 3.990 9.74 3.984 9.92 3.980 10.23 3.953 10.48 3.934 10.84 3.894 11.14 3.874 11.86 3.839 12.31 3.836 12.41 3.834 Table B-8: Chemical shifts for compound 8 in D2O in dependence of pD. pD CH3 H8 2.05 3.947 8.860 2.52 3.947 8.860 3.99 3.947 8.862 5.08 3.948 8.861 5.48 3.947 8.861 6.28 3.948 8.862 7.90 3.946 8.861 9.18 3.947 8.861 10.08 3.945 8.857 Tables of the pD dependences 206 10.48 3.935 - 11.36 3.884 8.649 11.97 - 8.389 12.10 3.824 8.332 12.58 - - 12.32 3.812 8.285 12.96 3.783 8.179 13.32 3.771 8.154 Table B-9: Chemical shifts for compound 9 in D2O in dependence of pD. pD CH3 CH3 3.01 4.009 4.001 3.4 4.010 4.000 3.57 4.009 4.000 3.86 4.008 4.000 4.88 4.009 3.999 5.18 4.008 3.999 5.2 4.008 3.999 5.5 4.008 3.999 5.73 4.008 3.999 5.85 4.007 3.999 6.99 4.008 3.998 7.85 4.004 3.999 8.8 3.950 3.980 9.84 - 3.973 10.26 3.879 3.967 10.58 3.854 3.961 11.27 3.829 3.955 11.5 3.826 3.954 11.8 3.822 3.953 12.7 3.822 3.952 13.48 3.818 3.949 Tables of the pD dependences 207 Table B-10: Chemical shifts for compound 10 in D2O in dependence of pD. pD CH3 3.32 4.023 3.88 4.023 5.5 4.022 6.49 4.022 7.07 4.024 7.65 4.019 7.96 4.007 8.30 4.001 8.44 3.994 9.17 3.970 9.42 3.951 9.67 3.939 10.13 3.920 10.60 3.908 10.68 3.903 10.80 3.893 11.33 3.878 11.61 3.871 12.32 3.861 Table B-11: Chemical shifts for compound 11 in D2O in dependence of pD. pD H2/H2’ H8 5,97 9,005/9,056 8,784 6,22 9,006/9,051 8,778 6,71 9,008/9,051 8,782 7,09 9,005/9,046 8,785 7,21 9,006/9,047 8,780 7,51 9,011 8,778 7,64 9,010 8,777 7,75 9,009 8,775 7,92 9,006 8,773 8,03 8,994 8,769 8,21 8,977 8,759 8,68 8,936 8,728 8,95 8,894 8,699 Tables of the pD dependences 208 9,4 8,808 8,638 9,69 8,755 8,599 9,84 8,743 10,1 8,674 10,55 8,632 10,65 8,614 11,1 8,498 11,34 8,441 12,14 8,318 13 8,275 Table B-12: Chemical shifts for compound 12 in D2O in dependence of pD. pD H2 H8 5,63 8,722 6,46 8,723 6,98 8,731 8,802 7,3 8,737 8,801 7,85 8,733 8,801 8,16 8,725 8,780 8,34 8,706 8,758 8,53 8,701 8,747 9,02 8,672 9,44 8,644 9,52 8,620 9,56 8,604 9,84 8,572 10,1 8,554 10,43 8,443 10,66 8,316 11,12 8,247 11,47 8,191 12,23 8,142 13,25 8,114 Tables of the pD dependences 209 Table B-13: Chemical shifts for compound 13 in D2O in dependence of pD. pD CH2(Et) CH3-A CH3-G CH3(Et) 2.38 4.504 4.049 3.806 1.618 3.78 4.507 4.050 3.809 1.613 4.26 4.049 3.805 1.620 4.46 4.504 4.049 3.806 1.619 4.80 4.050 3.806 1.620 5.07 4.049 3.806 1.620 5.64 4.505 4.048 3.807 1.612 6.25 4.048 3.806 1.619 6.59 4.505 4.049 3.806 1.618 6.72 4.498 4.046 3.804 1.615 6.80 4.498 4.047 3.805 1.617 7.09 4.503 4.046 3.805 1.612 7.19 4.502 4.046 3.805 1.617 7.27 4.502 4.046 3.804 1.612 7.46 4.500 4.045 3.803 1.612 7.52 4.501 4.045 3.804 1.612 7.71 4.499 4.044 3.802 1.614 7.81 4.497 4.043 3.801 1.613 7.99 4.492 4.040 3.799 1.609 8.13 4.488 4.038 3.798 1.608 8.20 4.489 4.038 3.798 1.609 8.37 4.028 3.793 1.598 8.53 4.465 4.018 3.791 1.595 8.67 4.438 3.999 3.787 1.582 8.75 4.444 4.003 3.787 1.585 8.94 4.428 3.986 3.785 1.575 9.16 3.978 3.785 1.571 9.27 4.402 3.967 3.783 1.563 9.77 4.372 3.940 3.783 1.549 10.55 4.355 3.924 3.781 1.539 10.73 4.353 3.922 3.781 1.539 Tables of the pD dependences 210 Table B-14: Chemical shifts for compound 14 in D2O in dependence of pD. pD CH2(G) CH3(A) CH3(G) 3.01 4.239 4.087 1.502 3.50 4.240 4.087 1.502 4.70 4.240 4.087 1.502 5.05 4.241 4.088 1.504 5.50 4.240 4.088 1.504 6.13 4.238 4.083 1.503 6.64 4.234 4.078 1.502 6.85 4.230 4.068 1.502 7.13 4.215 4.031 1.497 7.52 4.203 4.006 1.493 7.65 4.201 4.001 1.493 7.85 4.194 3.990 1.489 8.02 4.187 3.980 1.486 8.35 4.175 3.969 1.481 8.73 4.169 3.958 1.476 8.94 4.165 3.953 1.473 9.10 4.165 3.949 1.471 9.20 4.158 3.945 1.469 9.47 4.154 3.941 1.467 10.02 4.147 3.933 1.462 10.13 4.147 3.933 1.461 10.80 4.144 3.930 1.460 11.45 4.143 3.929 1.458 11.70 4.142 3.929 1.459 12.18 4.143 3.928 1.458 Table B-15: Chemical shifts for compound 15 in D2O in dependence of pD. pD H2 2.53 8.34 3.0 8.34 4.15 8.34 4.42 8.33 5.02 8.30 5.25 8.28 5.42 8.25 Tables of the pD dependences 211 5.55 8.24 5.74 8.21 6.03 8.18 6.89 8.14 7.48 8.14 7.94 8.14 Table B-16: Chemical shifts for compound 17 in D2O in dependence of pD. pD CH3 CH3 H8(A) H2(A) 1.97 3.902 3.863 9.105 8.333 2.31 3.905 3.866 9.104 8.335 2.82 3.902 3.863 9.105 8.333 4.04 3.903 3.859 9.105 8.332 4.35 3.902 3.862 9.105 8.332 4.44 3.903 3.853 9.102 8.318 4.90 3.905 3.843 9.099 8.303 5.14 3.906 3.841 9.092 8.308 5.24 3.906 3.843 9.094 8.310 5.64 3.909 3.837 9.087 8.267 5.79 3.911 3.802 9.083 8.242 5.91 3.910 3.802 9.084 8.241 6.53 3.912 3.783 9.077 8.218 6.87 3.913 3.783 9.076 8.213 7.61 3.913 3.782 9.074 8.211 8.06 3.919 3.787 9.080 8.219 8.70 3.914 3.780 9.076 8.209 9.22 3.911 3.779 9.070 8.206 9.59 3.913 3.784 9.064 8.211 10.24 3.909 3.780 9.063 8.205 11.07 3.867 3.779 8.908 8.184 11.56 3.826 3.782 8.756 8.170 12.06 3.799 3.777 8.652 8.154 12.13 3.789 3.779 8.617 8.153 12.46 3.782 3.763 8.517 8.144 12.57 3.777 3.764 8.509 8.138 13.14 3.782 3.744 8.436 8.136 13.27 3.781 3.744 8.432 8.135 Tables of the pD dependences 212 Table B-17: Chemical shifts for compound 18 in D2O in dependence of pD. pD H8/2 2.92 8.36 4.00 8.35 5.10 8.32 5.61 8.30 6.12 8.26 6.60 8.22 7.20 8.18 7.80 8.14 8.74 8.12 10.53 8.12 Table B-18: Chemical shifts for compound 20 in D2O in dependence of pD. pD CH3(A) CH3(C1) CH3(C2) H2(A) H6(C2) H6(C1) H5(C2) H5(C1) 3.30 3.850 3.428 3.336 8.272 7.517 7.568 5.930 6.002 5.88 3.844 3.425 3.331 8.260 7.505 7.562 5.921 5.999 6.40 3.847 3.429 3.337 8.262 7.516 7.568 5.930 6.004 7.54 3.846 3.427 3.335 8.261 7.515 7.566 5.928 6.002 7.60 3.846 3.428 3.336 8.262 7.516 7.567 5.929 6.003 8.28 3.846 3.428 3.336 8.262 7.517 7.568 5.929 6.003 8.97 3.846 3.428 3.336 8.261 7.516 7.567 5.929 6.003 9.82 3.847 3.429 3.336 8.263 7.517 7.568 5.930 6.004 10.32 3.847 3.428 3.336 8.264 7.517 7.569 5.930 6.004 10.67 3.851 3.432 3.340 8.266 7.519 7.572 5.932 6.007 10.72 3.850 3.432 3.339 8.266 7.519 7.571 5.932 6.007 10.92 3.850 3.432 3.339 8.266 7.517 7.571 5.931 6.006 11.17 3.842 3.423 3.330 8.257 7.507 7.561 5.922 5.996 11.20 3.850 3.431 3.338 8.266 7.515 7.570 5.929 6.005 11.45 3.846 3.428 3.336 8.263 7.517 7.568 5.929 6.004 11.55 3.842 3.423 3.328 8.257 7.500 7.559 5.917 5.995 12.30 3.843 3.416 3.315 8.259 7.455 7.546 5.885 5.98 12.75 3.837 3.400 3.287 8.253 7.379 7.515 5.830 5.957 12.86 3.836 3.397 3.280 8.250 7.360 7.507 5.816 5.951 13.08 3.833 3.386 3.263 8.248 7.314 7.488 5.782 5.934 13.38 3.823 3.366 3.229 8.237 7.226 7.447 5.717 5.899 13.52 3.821 3.359 3.217 8.235 7.193 7.433 5.693 5.887 Tables of the pD dependences 213 13.61 3.819 3.354 3.207 8.234 7.164 7.420 5.672 5.876 13.72 3.815 3.340 3.186 8.231 7.103 7.385 5.627 5.850 Table B-19: Chemical shifts for compound 21 in D2O in dependence of pD. pD CH3(1) 2.79 3.423 5.14 3.422 5.48 3.420 5.96 3.418 6.24 3.415 7.0 3.411 7.9 3.409 9.95 3.409 10.37 3.409 12.1 3.408 Table B-20: Chemical shifts for compound 22 in D2O in dependence of pD. pD H2 H8 H6 H5 CH3-N9 CH3-N1 CH3-C 1.5 8.754 8.969 7.683 6.099 4.073 4.047 3.505 2.02 8.753 8.967 7.682 6.097 4.070 4.044 3.503 4.68 8.751 8.982 7.683 6.099 4.069 4.045 3.504 5.92 8.699 8.915 7.681 6.096 4.024 3.501 6.14 8.674 8.897 7.680 6.095 4.018 4.008 3.501 6.24 8.663 8.891 7.679 6.094 4.015 3.999 3.501 7.0 8.461 8.697 7.671 6.086 3.950 3.838 3.494 7.13 8.378 8.627 7.667 6.084 3.925 3.776 3.491 8.22 8.238 8.486 7.663 6.077 3.883 3.488 8.45 8.209 8.478 7.666 6.078 3.875 3.636 3.487 8.76 8.206 8.486 7.665 6.079 3.876 3.635 3.486 9.04 8.202 8.479 7.666 6.080 3.874 3.632 3.487 9.18 8.217 8.466 7.668 6.081 3.875 3.639 3.486 9.20 8.204 8.480 7.666 6.080 3.874 3.632 3.488 9.37 8.202 8.478 7.666 6.079 3.874 3.631 3.488 9.76 8.200 8.471 7.666 6.078 3.873 3.629 3.488 9.86 8.200 8.480 7.668 6.082 3.875 3.629 3.488 10.28 8.200 8.475 7.667 6.080 3.874 3.629 3.488 Tables of the pD dependences 214 11.91 8.201 8.470 7.657 6.072 3.873 3.630 3.486 12.15 8.197 8.430 7.606 6.041 Table B-21: Chemical shifts for compound 23 in D2O in dependence of pD. pD CH3-N9 CH3-N1 CH3-(G) H2(A) H8(A) H8(G) 1.81 4.050 4.017 3.788 8.741 9.089 8.357 2.87 4.055 3.794 8.741 9.120 8.400 3.34 4.051 3.957 3.786 8.739 8.448 5.13 4.048 3.790 6.07 4.046 3.790 8.727 8.410 6.14 4.020 3.980 3.785 8.653 9.001 8.356 7.28 3.980 3.943 3.786 7.62 3.941 3.914 3.779 7.85 3.913 3.899 8.75 3.888 3.771 3.619 8.336 8.611 8.220 9.48 3.878 3.753 3.602 8.235 8.410 8.187 11.53 3.879 3.754 3.598 8.243 8.593 8.188 12.09 3.877 3.749 3.600 12.83 3.878 3.749 3.598 Table B-22: Chemical shifts for compound 24 in D2O in dependence of pD. pD CH3-C CH3-N9 CH3-N6 H8 H2 H6 H5 0.61 3.524 4.043 3.551 8.916 8.640 7.681 6.095 0.76 3.524 4.042 3.548 8.901 8.638 7.681 6.092 0.92 3.525 4.042 3.547 8.911 8.638 7.683 6.095 1.12 3.525 4.039 3.541 8.896 8.634 7.685 6.095 1.28 3.526 4.036 3.534 8.891 8.628 7.687 6.097 1.46 3.521 4.029 3.525 8.867 8.614 7.688 6.096 1.55 3.510 4.024 3.526 8.829 8.591 7.688 6.097 1.68 3.496 4.018 3.526 8.829 8.591 7.689 6.097 1.86 3.472 4.005 3.526 8.790 8.567 7.688 6.097 2.14 3.435 3.986 3.525 8.726 8.530 7.689 6.097 2.43 3.436 3.984 3.521 8.740 8.524 7.677 6.091 2.50 3.433 3.983 3.521 8.726 8.521 7.677 6.092 2.56 3.426 3.980 3.521 8.722 8.514 7.678 6.093 2.60 3.383 3.960 3.525 8.634 8.479 7.688 6.096 Tables of the pD dependences 215 3.03 3.378 3.955 3.520 8.643 8.463 7.675 6.092 3.20 3.373 3.953 3.520 8.637 8.457 7.675 6.093 3.28 3.367 3.949 3.520 8.626 8.451 7.674 6.092 3.40 3.352 3.945 3.524 8.599 8.445 7.686 6.097 3.73 3.350 3.941 3.520 8.602 8.433 7.674 6.093 6.47 3.339 3.935 3.519 8.574 8.423 7.674 6.091 Table B-23: Chemical shifts for compound 26 in D2O in dependence of pD. pD CH3 H5 H6 6.94 3.454 6.073 7.654 10.28 3.453 6.072 7.653 10.56 3.454 6.072 7.653 11.29 3.452 6.069 7.647 11.94 3.439 6.042 7.605 12.24 3.439 6.043 7.606 12.40 3.418 6.0015 7.538 12.62 3.405 5.973 7.494 12.66 3.406 5.976 7.498 12.69 3.420 6.004 7.543 13.05 3.361 5.885 7.355 13.07 3.372 7.459 13.1 3.362 5.887 7.358 Table B-24: Chemical shifts for compound 27 in D2O in dependence of pD. pD CH3-A CH3-C H6 H5 H2/8 3.18 3.951 3.489 7.6805 6.0945 8.419 3.75 3.947 3.489 7.679 6.0945 8.408 6.90 3.946 3.489 7.680 6.095 8.403 8.04 3.946 3.489 7.679 6.095 8.402 10.91 3.945 3.487 7.675 6.090 8.403 11.0 3.945 3.487 7.675 8.404 11.34 3.945 3.482 7.656 6.082 8.402 11.54 3.945 3.479 7.651 6.078 8.402 12.1 3.941 3.441 7.531 6.0005 8.397 12.2 3.941 3.435 7.508 5.985 8.395 12.36 3.939 3.416 7.448 5.949 8.393 12.77 3.935 3.369 7.297 5.852 8.386 Tables of the pD dependences 216 Table B-25: Chemical shifts for compound 33 in D2O in dependence of pD. pD CH3-G H8 H6 H5 7.40 3.757 8.315 7.967 6.592 8.00 3.753 8.297 7.294 6.559 8.21 3.751 8.286 7.903 6.540 8.58 3.744 8.250 7.840 6.491 8.65 3.744 8.256 7.844 6.494 8.80 3.744 8.253 7.839 6.495 8.97 3.738 8.229 7.798 6.458 9.09 3.736 8.217 7.781 6.444 9.29 3.737 8.225 7.795 6.446 9.33 3.732 8.195 7.751 6.425 9.57 3.726 8.169 7.709 6.399 10.26 3.718 8.129 7.669 6.366 10.72 3.714 8.112 7.656 6.353 11.08 3.714 8.111 7.649 6.348 11.57 3.713 8.105 7.649 6.347 12.16 3.713 8.103 7.654 6.345 12.30 3.714 8.109 7.648 6.345 13.63 3.712 8.101 7.645 6.344 Table B-26: Chemical shifts for compound 34 in D2O in dependence of pD. pD CH3 2.23 3.783 5.97 3.783 6.35 3.783 6.84 3.783 7.14 3.783 7.60 3.781 7.90 3.782 8.45 3.779 8.85 3.772 9.05 3.771 Tables of the pD dependences 217 2. pD dependences of 9-MeA in different solvents Mixture of 20% Acetone / 80% D2O pD H2 H8 CH3 2.02 8.489 8.345 3.931 2.5 8.476 8.332 3.925 3.72 8.401 8.269 3.895 4.07 8.353 8.226 3.875 5.12 8.233 8.118 3.824 5.9 8.188 8.081 3.804 6.34 8.183 8.073 3.800 7.8 8.173 8.063 3.793 11.22 8.166 8.058 3.790 Mixture of 80% Acetone / 20% D2O pD H2 H8 CH3 1.63 8.564 8.445 3.978 2.21 8.549 8.433 3.973 2.65 8.538 8.421 3.969 3.13 8.502 8.388 3.954 3.76 8.383 8.278 3.910 4.58 8.262 8.160 3.863 5.09 8.254 8.156 3.861 7.34 8.245 8.147 3.859 8.81 8.244 8.145 3.857 13.31 8.252 8.158 3.861 14.45 8.247 8.152 3.859 Mixture of 80% Methanol / 20% D2O pD H2 H8 CH3 1.39 8.423 8.318 3.920 1.95 8.420 8.316 3.919 2.3 8.414 8.311 3.917 2.66 8.403 8.300 3.913 3.56 8.338 8.226 3.882 4.27 8.261 8.138 3.850 Tables of the pD dependences 218 4.31 8.258 8.136 3.850 4.46 8.250 8.127 3.845 5.23 8.221 8.105 3.832 6.06 8.221 8.101 3.834 6.3 8.222 8.100 3.834 6.85 8.219 8.091 3.834 7.7 8.217 8.091 3.832 8.78 8.218 8.091 3.831 11.32 8.217 8.089 3.832 12.75 8.217 8.090 3.831 12.9 8.217 8.091 3.831 3. pD dpendences of compounds cis-[L2PtX(H2O)]2+ and trans- [L2PtX(H2O)]2+ Compound cis-[(NH3)2Pt(1-MeC-N3)(H2O)]2+ pD CH3 H5 H6 2.19 3.432 6.029 7.622 3.63 3.432 6.026 7.620 4.44 3.432 6.027 7.621 5.24 3.432 6.027 7.619 5.67 3.431 6.024 7.617 6.00 3.430 6.023 7.610 6.22 3.429 6.020 7.605 7.02 3.426 6.016 7.591 8.4 3.425 6.015 7.586 10.29 3.425 6.014 7.585 10.69 3.425 6.015 7.586 11.36 3.425 6.015 7.586 11.54 3.425 6.015 7.586 12.08 3.424 6.015 7.585 12.68 3.420 6.010 7.578 13.03 3.419 6.005 7.570 13.27 3.414 5.995 7.553 13.43 3.407 5.982 7.533 13.64 3.391 5.952 7.485 13.75 3.373 5.915 7.427 Tables of the pD dependences 219 Compound trans-[(NH3)2Pt(1-MeC-N3)(H2O)]2+ pD CH3 H5 H6 2.10 3.427 5.989 7.587 2.83 3.426 5.987 7.584 3.92 3.428 5.990 7.588 5.16 3.425 5.992 7.582 5.22 3.424 5.991 7.582 5.60 3.422 5.992 7.576 5.78 3.416 5.993 7.559 6.32 3.421 5.993 7.572 6.69 3.414 5.993 7.557 6.85 3.415 5.994 7.557 7.02 3.413 5.993 7.554 7.83 3.413 5.994 7.553 9.68 3.413 5.995 7.554 9.92 3.413 5.995 7.554 10.19 3.413 5.994 7.552 10.48 3.413 5.995 7.554 10.84 3.413 5.993 7.552 12.34 3.411 5.991 7.548 12.78 3.405 5.985 7.539 13.21 3.390 5.963 7.507 13.48 3.383 5.944 7.478 Compound trans-[(NH3)2Pt(9-MeGH-N7)(H2O)]2+ pD CH3 H8 1.70 3.731 8.234 2.64 3.732 8.235 3.25 3.731 8.234 3.88 3.728 8.231 4.07 3.730 8.231 4.99 3.724 8.216 5.28 3.726 8.216 5.44 3.721 8.209 5.71 3.715 8.194 6.48 3.702 8.168 8.29 3.693 8.135 8.6 3.663 7.965 Tables of the pD dependences 220 9.75 3.675 8.031 9.77 3.674 8.018 10.02 3.670 8.005 10.37 3.667 7.986 10.86 3.664 7.971 11.53 3.664 7.966 11.78 3.691 8.120 12.55 3.663 7.963 12.88 3.663 7.965 Compound cis-[{NH(CH3)2}2Pt(9-MeGH-N7)(H2O)]2+ pD CH3-G H8 3.26 3.803 8.415 3.28 3.764 8.266 4.16 3.796 8.407 5.10 3.798 8.405 5.40 3.804 8.405 5.60 3.791 8.384 5.87 3.799 8.378 6.14 3.791 8.359 6.18 3.762 8.264 6.23 3.786 8.341 6.68 3.792 8.333 6.88 3.778 8.295 7.37 3.777 8.295 7.74 3.775 8.286 8.05 3.774 8.283 8.77 3.771 8.242 9.46 3.762 8.190 9.47 3.752 8.174 10.68 3.746 8.106