Tris(pyrazolyl)borate-containing Complexes of Ruthenium and Tungsten as Covalent Labels for Biomolecules
Dissertation
zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Chemie und Biochemie der Ruhr-Universität Bochum
vorgelegt von
Master of Science (M. Sc.)
Johannes Zagermann
Bochum, Januar 2011
Diese Arbeit wurde von November 2007 bis Januar 2011 am Lehrstuhl für Anorganische Chemie I der Ruhr-Universität Bochum angefertigt.
Referent: Prof. Dr. Nils Metzler-Nolte Koreferent: Prof. Dr. William S. Sheldrick
Meiner Familie und Janine
You live and learn. At any rate, you live. Douglas Adams
ACKNOWLEDGEMENTS
Ich bedanke mich herzlichst bei:
Nils Metzler-Nolte für die Vergabe eines vielseitigen und interessanten Themas, den Freiraum und die Unterstützung bei der Erstellung dieser Arbeit und die Erfindung von Nils@Nine, Matt Kuchta for introducing me to the world of scorpionates and the CalTex life-style, Klaus Merz und Mariusz Molon für die Lösung und Deutung der Kristallstrukturen, Johanna Niesel für späte CV- und ESI-Messungen und die Kultur in Büro und Stadion, Andrea Ewald für ESI-Messungen und Fußballfachgespräche, Bauke Albada für Hilfe bei der HPLC und „zijn vriendschap“, Christian Gemel für aufschlussreiche Diskussionen und Anregungen zur [TpRu]-Chemie, Kathrin Klein für ihre Mitarbeit und die Ergebnisse ihrer Bachelor-Arbeit, Max Lieb und Malay Patra für ihre Zeit nicht nur am Montagmorgen, Nina, Jessica, Maya, Andrea G. und Jan für die entspannte Atmosphäre im „Kopflabor rechts“ und das Ertragen meiner Musik, Gregor Barchan, Martin Gartmann und Hans-Jochen Hauswald für Hilfe und Antworten rund ums NMR, Jochen Lügger und Gerd Bollmann für „gelebten Ruhrpott“ und Beistand in technischen Fragen, Kerstin Brauner und Veronika Hiltenkamp für Elementaranalysen, allen Mitarbeitern der AC I für die gute Zusammenarbeit und unterhaltsame außeruniversitäre Aktivitäten, meinem Freundeskreis für ein Leben außerhalb der Chemie, sowie den Jungs von milhaven für unvergessliche Momente und Melodien.
Besonderer Dank gilt meiner Familie, die mein Leben und Studium bedingungslos unterstützt und durch ihr reges Interesse bereichert hat, sowie meiner liebsten Janine für alles, was wir in den letzten Jahren zusammen erlebt und erreicht haben.
Ohne euch wäre ich nicht so weit gekommen.
Table of contents
TABLE OF CONTENTS
1. INTRODUCTION 1 1.1. Scorpionate ligands 1 1.2. Tp and Tpm ligand syntheses 4 1.3. Bioorganometallic and bioinorganic chemistry 6 1.3.1. Bioorganometallic chemistry of group 8 metallocenes 6 1.3.2. Bioorganometallic chemistry of carbonyl complexes 8 2. TASK FORMULATION 10 3. RESULTS AND DISCUSSION 11
3.1. Group 1, magnesium and thallium p-BrC6H4Tp transfer agents 11 3.1.1. Syntheses and characterization 11 3.1.2. Solid-state structure of compounds 4, 6, and 10 14 3.2. Syntheses and characterization of ruthenocene analogues 19 3.2.1. Mixed ligand CpR/Tp’ ruthenocene analogues 19 3.2.2. Mixed ligand Tp/Tp‘ ruthenocene analogues 22 3.2.3. Mixed ligand Tpm/Tp’ ruthenocene analogues 25 3.3. Mixed sandwich Cp/Tp‘,Tp/Tp‘ and Tpm/Tp’ bioconjugates 31 3.3.1. Application of acid-functionalized 16 and 20 in SPPS 31 3.3.2. Application of mixed sandwich Tp'/Tpm azide 25 in CuAAC 34 3.4. Seven-coordinate Tp*WI(CO)(η2-alkyne) complexes 36 3.4.1. Syntheses and characterization of tungsten “building blocks” 36 3.4.2. Application of tungsten “building blocks” 29 and 30 in SPPS 39 4. SUMMARY AND CONCLUSION 43 5. EXPERIMENTAL PART 48 5.1. Technical equipment 48 5.2. Methods and materials 49 5.3. Chemicals and solvents 49 5.4. Solid-Phase Supported Peptide Synthesis (SPPS) 49 5.5. Syntheses and characterization 50
5.5.1. Alternative preparation of p-BrC6H4TpNa 1 50
5.5.2. p-BrC6H4TpK 2 51
5.5.3. p-BrC6H4TpRb 3 52
5.5.4. p-BrC6H4TpCs 4 53
5.5.5. p-BrC6H4TpTl 5 54
5.5.6. (p-BrC6H4Tp)2Mg 6 55
Table of contents
Me 5.5.7. p-BrC6H4Tp K 7 56
Me 5.5.8. p-BrC6H4Tp Tl 8 57
5.5.9. p-BrC6H4Tp*K 9 58
5.5.10. p-BrC6H4Tp*Tl 10 59
5.5.11. CpRu(p-BrC6H4Tp) 11 60
Me 5.5.12. CpRu(p-BrC6H4Tp ) 12 61
5.5.13. Cp*Ru(p-BrC6H4Tp) 13 62 5.5.14. CpiPrRuCl(COD) 14 63
iPr 5.5.15. Cp Ru(p-BrC6H4Tp) 15 64
iPr 5.5.16. Cp Ru(p-(CO2H)-C6H4Tp) 16 65
iPr 5.5.17. Cp Ru(p-(CO-Phe-OMe)C6H4Tp) 17 66
iPr 5.5.18. Cp Ru(p-(CO-Tyr-Gly-Gly-Phe-Leu-OH) C6H4Tp) 18 67
5.5.19. TpRu(p-BrC6H4Tp) 19 68
5.5.20. TpRu(p-(CO2H)-C6H4Tp) 20 69
5.5.21. TpRu(p-(CO-Val-OtBu)C6H4Tp) 21 70
5.5.22. TpRu(p-(CO-Tyr-Gly-Gly-Phe-Leu-OH)-C6H4Tp) 22 71
5.5.23. (p-BrC6H4Tp)RuCl(COD) 23 72
5.5.24. [(p-BrC6H4Tp)RuTpm]Cl 24 73
2 5.5.24.1. [(p-BrC6H4Tp)Ru(κ -N,N-Tpm]Cl 24a 74
5.5.25. [(p-N3C6H4Tp)RuTpm]Cl 25 75
5.5.26. HCC(CH2)2CO-Val-OtBu 26 76
5.5.27. [TpmRu((p-C2N3H-(CH2)2-CO-Val-OtBu)-C6H4Tp)]Cl 27 77
5.5.28. [TpmRu((p-C2N3H-(CH2)2-CO-Tyr-Gly-Gly-Phe-Leu-OH)-C6H4Tp)]Cl 28 78
2 5.5.29. [Tp*WI(CO)(η -HCC(CH2)2CO2H)] 29 79 5.5.30. [Tp*W(I)(CO)(η2-Fmoc-Pgl)] 30 80
2 5.5.31. [Tp*WI(CO)(η -HCC(CH2)2CO-NH-Lys-Lys-Pro-Tyr-Ile-Leu-OH)] 31 81
2 5.5.32. [Tp*WI(CO)(η -HCC(CH2)2CO-NH-Tyr-Gly-Gly-Phe-Leu-OH)] 32 82
2 5.5.33. H2N-Tyr-Gly-[Tp*W(I)(CO)(η -Pgl)]-Phe-Leu-OH 33 83 6. REFERENCES 84 7. LIST OF PUBLICATIONS 92 8. SUPPORTING INFORMATION 94 8.1. Crystallographic data 94
Abbreviations
ABBREVIATIONS AND SYMBOLS
2-Cl-Trt 2-chloro-trityl Å Ångström ACN acetonitrile APT attached proton test asym asymmetric ca circa CCDC Cambridge crystallographic data centre CMIA carbonyl metallo immuno assay COD 1,5-cyclooctadiene CO-RM carbon monoxide releasing molecule Cp cyclopentadienyl Cp* pentamethylcyclopentadienyl CpCO2H cyclopentadienyl carboxylic acid CpiPr iso-propylcyclopentadienyl CuAAC Copper Catalyzed Azide-Alkyne Cycloaddition dba dibenzylidenacetone DIPEA N,N-diisopropylethylamine DMF N,N-dimethylformamide dmtc N,N-dimethylthiocarbamate d distance, doublet DMSO dimethylsulfoxide e.g. exempli gratia, for example Enk enkephalin eq equivalent ESI electro spray ionisation Et ethyl et al. et alii
Et2O diethylether fac facial Fc ferrocene Fig figure Fmoc (9H-fluoren-9-yl-methoxy)carbonyl Gly glycine HMBA 4-hydroxymethylbenzoic acid HOBt 1-hydroxy-1H-benzotriazole HPLC high pressure/performance liquid chromatography HSQC heteronuclear single quantum coherence Hz Hertz Ile isoleucine IR infrared iPr iso-propyl J coupling constant KOtBu potassium tert-butoxide L ligand, liter Leu leucine Lys lysine m mass, multiplet M molar mass, metal
Abbreviations
Me methyl mer meridional m/z mass over charge n-Bu Butyl NCPh benzonitrile neg negative
NEt3 triethylamine NMR nuclear magnetic resonance ORTEP Oak Ridge Thermal Ellipsoid Plot p.a. pro analisi p-BrC6H4 para-bromophenyl Pgl propargylglycine Ph phenyl Phe phenylalanine p-IC6H4 para-iodophenyl pNT pseudo-neurotensin pos positive ppm parts per million Pro proline pz pyrazolyl pz- pyrazolide pzH pyrazole q quartet RES resin RP reverse phase RT room temperature Rc ruthenocene s singlet sept septett SPPS solid phase peptide synthesis sym symmetric t triplet TBTU benzotriazol-1-yl-N-tetramethyl-uronium tetrafluoroborate t-Bu tert-butyl t-BuOH tert-butanol tert tertiary TFA trifluoroacetic acid THF tetrahydrofuran TIS triisopropylsilane TlOEt thallium ethoxide TMS tetramethylsilane Tyr tyrosine V volume v/v volume to volume Val valine δ chemical shift Δ temperature increase ρ density ν frequency
Abbreviations
ABBREVIATIONS FOR SCORPIONATE LIGANDS
[1,2] Following the system proposed by Curtis et al., tris(pyrazolyl)borate ligand HB(pz)3 is abbreviated as Tp, tris(pyrazolyl)methane ligand HC(pz)3 as Tpm, and heteroscorpionate ligands
R2B(pz)2 as Bp. The positions of the pyrazole rings are numbered, following IUPAC rules for heterocycles, starting with the nitrogen bound to boron. In general, any non-hydrogen substituent in the pyrazole 3- position, the one closest to the coordinated metal, is denoted as a superscript, e.g. tris(3-methyl-
Me pyrazolyl)borate HB(3-Mepz)3 is abbreviated as Tp . Non-hydrogen 5-substituents follow the 3- substituent as a superscript, separated by a comma. When the 3- and 5-substituents are identical, the superscript R-substituent is followed by 2. In the case of tris(3,5-dimethyl-pyrazolyl)borate
Me2 (HB(3,5-Me2pz)3), the common abbreviation Tp* will be used instead of Tp . Substituents replacing hydrogen on the central boron atom are written preceding “Tp”: for instance, pyrazol-tris(pyrazolyl)borate or tetrakis(pyrazolyl)borate is abbreviated as pzTp. The p-BrC6H4-substituted ligands p-BrC6H4B(pz)3, p-BrC6H4B(3-Mepz)3, and p-BrC6H4B(3,5-Me2pz)3 are abbreviated as Tp’, Tp’Me and Tp’*, respectively.
Introduction
1. INTRODUCTION
1.1. Scorpionate ligands
In the late 1960‘s, S. Trofimenko reported a class of molecules,[3-9] that would soon become one of the most studied ligand systems in coordination chemistry: Poly(pyrazolyl)borate anions.[10] Reminiscent of a scorpions pincers, these tripodal ligands bind a metal with the nitrogen heteroatoms of two pyrazole rings attached to a central boron atom. As outlined in figure 1, a third pyrazole ring (or the R group), also attached to the boron-atom, ”stings” the metal like a scorpions tail, hence the commonly used name ”scorpionates”.[11,12]
Figure 1 The scorpionate ligand system
The most commonly used members of this ligand class are the tris(pyrazolyl)borate ligands
[2] (HB(pz)3 or Tp), which are formally analogous to the cyclopentadienyl (Cp) ligand in that both
[13] [14] are six-electron anions or L2X ligands. While this comparison was found useful in describing Tp in terms of other ligands, Trofimenko rated it ”not to [be] helpful at all in underscoring the unusual and specific features of this ligand class.”[15] Obviously, both ligands differ remarkably in size and in complexation geometry and symmetry, respectively. While Cp, as other typical π-donor ligands, mostly occupies one coordination site in tetrahedral complexes, the weak field hard σ-N donor Tp tends to form of facial (fac) complexes with local C3v-symmetry. Moreover, the steric bulk of Tp usually seems to disfavor higher coordination numbers than six,[16] thus forcing the [TpM] fragment to coordinate three additional ligands, resulting in a six-coordinate, octahedral structure. In contrast, Cp containing complexes are capable of forming seven-coordinated species.[17] However, various Tp compounds have been reported to differ from the usually found κ3-N’,N’’,N’’’-coordination by exhibiting modes from rarely seen “κ0” (e.g. as a uncoordinated counterion)[18] to “κ5” in case the pyrazole rings provide additional donor atoms as outlined in figure 2.[19,20]
1 Introduction
Figure 2 Examples of coordination modes found in Tp complexes
Another important difference between Cp and Tp ligands is found in the number of possible derivatives. Whereas the five postions of the Cp ring allow for a maximum of five substituents, the three pyrazole rings in Tp provide nine possible substitution sites. Consequently, a vast number of so-called "second-generation" Tp ligands (Fig. 3 center) has been reported, in which different steric and electronic effects are attained by varying the nature, number, and position of the substituents on the pyrazole rings.
Figure 3 First (left), second (center), and third (right) generation Tp ligands. "Mutations" of the ligand from generation to generation are highlighted
For example, substituents on the pyrazole 3-position were found to have high impact on the cone angle (ϑ)[21] and the resulting steric and electronic effects exerted onto the Tp-coordinated metal
[22-25] center. Thus, bulky substituents have been shown to favor the formation of fac-TpMLn over
Tp2M complexes. In addition, the reactivity of the metal moiety can be manipulated by altering the hydrophobic pocket around the metal ion.[26] An additional option for substitution is based on replacement of the boron-bound hydride by an alkyl or aryl group. The resulting "third-generation" RTp derivatives (Fig. 3 right) represent the scarcest group of Tp ligands.[27] Presence of an R group is known to extensively effect properties such as solubility, spin-state or solid-state structure of the resulting RTpM complexes.[28-30] Furthermore, suitably functionalized substituents provide a means to either covalently attach other groups and whole molecules,[29] or to access oligotopic scorpionate ligands as scaffolds for the syntheses of oligonuclear metal complexes and metallopolymers, respectively.[31-34]
2 Introduction
Following the wide-spread use of the “classical” boron/pyrazole-based ligands, a variety of derivatives, has been investigated, thus extending the definition and application of scorpionate ligands. As exemplified in figure 4, central atoms other than boron, such as carbon,[35,36] silicon,[37,38] phosphorus,[39-41] and nitrogen[42,43] have been employed.
Figure 4 Non-classical scorpionate ligands obtained by variation of apical atom and/or donating groups
Furthermore, the use of various donor groups, including nitrogen heterocycles other than pyrazole,[42-46] acyclic donors,[47-49] phosphorous,[50] and sulfur[51-53] and their influence on the coordination behavior have been investigated. Most recently, Wagner et al. reported on the synthesis of “fourth-generation” scorpionates,[54] which are able to adopt both fac- and mer- coordination modes. Accordingly, an immense variety of scorpionate-containing complexes have been reported for virtually every metal in the periodic table,[11,12] and such complexes have been employed in vastly different areas of chemistry, in particular enzyme modelling, [21,55-60] catalysis, [61-65] and materials science.[66-69] Additionally, scorpionate-containing complexes of 99mTc and Re have been proposed as radiopharmaceuticals or models thereof.[70,71] Such widespread use of the scorpionate ligand family, and Tp ligands in particular, is derived from two major features. First comes the reliability of Tp as a “spectator” ligand.[72] As such, Tp is found to block certain coordination sites and to modulate the steric and electronic properties of the metal ion, thus determining substrate or co-ligand binding without prohibiting chemistry to occur at the metal centre. A second reason for the popularity of Tp ligands is found in the ease with which those ligands may be synthesized. In the following, the syntheses of the two most popular classes of Tp ligands, tris(pyrazolyl)borate and carbon-based tris(pyrazolyl)methane (Tpm), will be described.
3 Introduction
1.2. Tp and Tpm ligand syntheses
Boron-based first- and second-generation Tp ligands can be prepared in a facile manner by heating a solvent-free mixture of excess pyrazole and a borohydride salt MBH4 (M= alkali metal) as originally reported by Trofimenko et al..[3] As depicted in scheme 1, the grade of pyrazole substitution on the cental boron is controlled by temperature adjustment, thus successively giving access to bis(pyrazolyl)borate (BpR’RM), tris(pyrazolyl)borate (TpR’RM), and tetrakis(pyrazolyl)borate (pzTpR’RM) transfer agents.
Scheme 1 Solvent-free synthesis of first and second-generation Bp, Tp and pzTp ligands
Contrastingly, the synthesis of “third-generation” Tp ligands is highly dependent on the availability of suitable, functionalized boron starting materials. As such, Trofimenko initially reported on the syntheses of the alkali metal (M) derivatives nBuTpM and PhTpM by heating a solvent-free mixture of RBH3M (R= nBu, Ph, M= alkali metal) and pyrazole as depicted in equation 1 of scheme 2.[3]
Scheme 2 Syntheses of "third-generation" Tp ligands
In 1967, Trofimenko reported an alternative synthesis of RTp (R= nBu, Ph) derivatives by the reaction of either an aryl boron dihalide, PhBCl2, with an excess amount of pyrazole (equation 2), or the reaction of the boronic acid derivative n-BuB(OH)2 with sodium pyrazolide and pyrazole in solution (equation 3).[7] The latter method was also applied by White et al., who synthesized
[30] p-BrC6H4TpNa starting from p-BrC6H4B(OH)2. More recently, Reger et al. reported low
Me temperature syntheses of p-IC6H4TpNa and p-IC6H4Tp Na by the reaction of p-IC6H4BBr2 with pyrazole and triethylamine, followed by treatment with sodium tert-butoxide.[28] Significantly,
Reger et al. noted that this is the preferred preparative method for p-IC6H4TpNa rather than the methods reported for p-BrC6H4TpNa (vide supra) and the “standard method” of heating functionalized lithium borohydride, that is p-BrC6H4BH3Li, in the presence of an excess amount of
4 Introduction pyrazole. Reger’s preparative method is reminiscent of that previously reported by Wagner et al. to
[73] synthesize the ferrocene-substituted thallium complex FcTpTl (Fc= C5H5FeC5H5). Both syntheses generate a Brønsted acidic RTpH intermediate, which is subsequently deprotonated by a metal-containing Brønsted base that determines the specific identity of the resultant TpM species. As such, these syntheses demonstrate the potential of a general method for making RTpM complexes by employing the appropriate Brønsted bases.
The first synthesis of tris(pyrazolyl)methane was already reported in 1937 by Hückel and Bretschneider, who reacted potassium pyrazolide with chloroform.[74] However, the isolated yield was low, presumably due to carbene associated side reactions,[75] inducing pyrazole ring expansion.[76] In 1984, an improved multi-gram scale preparation was proposed by Elguero and co-workers, who reacted the appropriate pyrazole with chloroform and K2CO3, employing liquid- liquid phase transfer conditions.[77] As depicted in scheme 3, a further improvement was found in replacing K2CO3 by an excess of Na2CO3, thus granting access to a variety of first- as well as second- generation Tpm ligands.[78] Due to the high reactivity of the apical CH group, various “third-generation” RTpm ligands are easily accessible by deprotonation and subsequent addition of a suitable electrophile.[79]
Scheme 3 Synthesis of first and second generation Tpm ligands (left) and their subsequent functionalization to third generation ligands (right)
5 Introduction
1.3. Bioorganometallic and bioinorganic chemistry
One topic of increasing interest in the field of both organometallic and inorganic coordination chemistry is the labelling of biologically active molecules (e.g. DNA/RNA, peptides, drugs) with metal containing compounds, hence decisively altering or preferably enhancing the properties of the resulting bioconjugates.[80-86] While the labeling with classic coordination compounds has been employed in a manifold of applications ranging from drugs and radiochemical imaging agents to heavy atom probes for X-ray spectroscopy, the use of organometallic compounds for similar purposes is limited due to their lower stability under physiological conditions (aqueous media, oxygen atmosphere). Nevertheless, the field of bioorganometallic chemistry has emerged as an important research topic in recent years offering new opportunities in medicinal and biochemical applications, thus generating a steady quest for metal-containing compounds that are air- and waterstable and allow for the facile attachment of biomolecules.
1.3.1. Bioorganometallic chemistry of group 8 metallocenes
A class of compounds that has been intensively investigated and employed in this field are metallocenes, in particular the original ferrocene (Cp2Fe or Fc). Such great interest is derived from the ease of synthesis of functionalized ferrocene derivatives as well as its favourable electronic properties. Accordingly, ferrocene bioconjugates have found applications ranging from biosensors to novel drugs such as ferroquine or ferrocifene (Fig. 5).[87-99]
Figure 5 Top: Antimalarial drug chloroquine (left) and metallocene derivatives (right). Bottom: Estrogen Receptor Modulator tamoxifen (left) and metallocene derivatives (right)
Given the abundance of ferrocene-based bioorganometallics, surprisingly little work is found on comparable applications of its next heavier congener ruthenocene (Cp2Ru or Rc). Apart from some comments in the context of possible radiolabelling with 103Ru,[100,101] and 97Ru,[102] ruthenocene has
6 Introduction been solely applied in the syntheses of the potential antimalarial drug ruthenoquinone,[89,103] the estradiol analogue ruthenocifen,[97,104,105] and for the covalent labeling of the peptide octreotate in our group.[106] Such rare use is mostly derived from the fact that ruthenocene and ferrocene, albeit that they exhibit similar chemistry and biological activity, differ substantially in their reactivity regarding substitutions. As a case in point, ferrocene is more susceptible to electrophilic addition than ruthenocene,[107] and the ferrocenyl Cp protons are less acidic than the corresponding ruthenocenyl protons,[107,108] hence allowing for more selective mono-functionalization by lithiation or Friedel-Crafts reactions.[109] Similarly, applications of metallocene analogues, whether homoleptic or heteroleptic, have not been studied to any serious extent. Thus, given the analogy of Cp and Tp coordination chemistry, and the successful application of functionalized Tp’ transition metal complexes in the syntheses of peptide bioconjugates in our group (Fig. 6),[110,111] we were interested to employ Tp complexes as analogues of group 8 metallocenes and to exploit their potential applications within bioorganometallic chemistry.
Figure 6 Tp‘-based bioconjugates prepared by Kuchta et al.
Following a literature research on possible target molecules, ruthenium was considered over iron for the ease of synthesis and characterization. For example, the synthesis of mixed ligand ferrocene analogues like Cp*FeTp (Cp*= C5(CH3)5) has been reported to be rather complicated due to a high tendency towards ligand redistribution, resulting in the formation of homoleptic FeL2 (L= Cp*, Tp).[112] In contrast, a rich chemistry is found for analogue ruthenium compounds and half-
[113-115] sandwich starting materials. Furthermore, it has to be noticed that the readily available FeTp2 is known for temperature dependent spin-crossover behaviour,[116] thus aggravating proper characterization, e.g. by NMR spectroscopy, and that this behaviour can be triggered by small
[28] changes to the substituent on the boron-atom, as reported exemplarily for Fe(p-IC6H4Tp)2.
7 Introduction
1.3.2. Bioorganometallic chemistry of carbonyl complexes
Another group of organometallic compounds frequently used in the modification of biomolecules is those of transition metal complexes containing carbonyl ligands. This popularity is mainly caused by the sharp IR-spectroscopic window of their metal-coordinated CO ligands in the 1800–2200 cm−1 region, which is not occupied by other carbonyl groups. For example, acid functionalized
CO2H [117] [118] cymantrene (Cp )Mn(CO)3, and osmium carbonyl clusters, covalently bound to biomolecules, have been shown to make bioconjugates traceable by IR spectroscopy. Likewise, Leong and co-workers have demonstrated intracellular IR imaging using metal-carbonyl vibrations of an osmium carbonyl cluster. More recently, scorpionate manganese compound
[119] [TpmMn(CO)3]Cl has been employed in Raman microspectroscopy of living cells. Jaouen et al. established Carbonyl Metallo Immuno Assays (CMIA),[86,120,121] a method to label and detect commonly applied drugs by coordinative attachment of metal carbonyl fragments such as alkyne-
Co2(CO)6 and cymantrenyl as outlined in figure 7.
Figure 7 Drug-derivatives used for CMIA (Bold lines indicate the original drug structure)
Further, introduction of carbonyl moieties, such as Co2(CO)6, into biomolecules can also render them cytotoxic,[122,123] a feature also found for modified peptides, making them potential anti- proliferative drugs comparable to cis-platin.[124,125] More recent investigations focus on the targeted release of CO, an endogenous signalling molecule,[126,127] by metalcarbonyl fragments to provide
[128,129] new medical applications. For example, complexes bearing [M(CO)3] (M= Fe, Ru, Re,
[130-133] [134] Mn) or [M(CO)5] (M= Cr, W, Mn) moieties (Fig. 8) have been identified as possible CO releasing molecules (CO-RMs) with CO liberation rates tuneable by choice of the accompanying ligands.[135]
Figure 8 Organometallic CO-releasing molecules
8 Introduction
Considering the rich chemistry of their carbonyl complexes in general, little work is found concerning bioorganometallic compounds of the group six metals chromium, molybdenum, and
[136] tungsten. Whereas carbonyl containing fragments of Mo and Cr, in particular fac-M(CO)3, have been thoroughly investigated as metal containing markers for biomolecules,[137,138] similar use of
[139] tungsten organometallic compounds is limited to the CpW(CO)3 entity. Besides its carbonyl ligands as an useful IR tracer, tungsten could serve as a high electron density tool in X-ray crystallography of biomolecules. As such, Curran et al. recently proposed tungsten based metallacyclicpeptides, obtained by double alkyne-coordination to [W(dmtc)2] (dmtc= N,N- dimethylthiocarbamate) fragments as shown in scheme 4,[140,141] as useful covalent restraints for peptide conformation.[142]
Scheme 4 Tungsten-based structural restraints for peptides proposed by Curran et al.
9 Task formulation
2. TASK FORMULATION
As emphasized in the introduction, the aim of this work is to develop synthetic routes towards functionalized Tp-containing complexes that allow the covalent attachment to biomolecules, hence providing conjugates with novel biological applications.
RR’ A promising precursor for such purposes has been found in “third-generation” Tp’ (p-BrC6H4Tp ) ligands. To enhance the use of this ligand-system in the synthesis of transition metal complexes, an array of different Tp’ transfer agents should be synthesized and characterized in the first part. In the second part, the application of Tp’ in the syntheses of mixed ligand ruthenocene analogues, surrogating either one or both Cp ligands with a Tp ligand as outlined in scheme 5, should be investigated.
Scheme 5 Ruthenocene-analogue target structures envisioned by replacement of Cp with Tp ligands
The mixed ligand ruthenocene analogues should be characterized, suitably functionalized, and evaluated regarding their stability and applicability in the labeling of peptides. In the third part, the aim is to investigate Tp* (tris(3,5-dimethylpyrazolyl)borate) tungsten complexes containing carbonyl ligands, that could be applied as labels for IR spectroscopy or X-ray crystallography, respectively. As depicted in scheme 6, suitably “synthetic handles” in form of functionalized alkynyl ligands should be introduced. The functionalized tungsten complexes obtained thereby should be characterized and their applicability in the synthesis of bioconjugates assessed.
Scheme 6 Intended synthetic approach towards tungsten-carbonyl bioconjugates
10 Results and discussion
3. RESULTS AND DISCUSSION
3.1. Group 1, magnesium and thallium p-BrC6H4Tp transfer agents
3.1.1. Syntheses and characterization
Derivatized Tp complexes, suitable for subsequent attachment of biomolecules, may be envisioned retro-synthetically from the corresponding p-BrC6H4Tp species by either a “carboxylation” as
[30] precedented by White et al. for the benzoic acid-Tp sandwich complex (p-(CO2H)C6H4Tp)2Co, or
[143] by a copper catalyzed azidonation of the p-BrC6H4 moiety, as outlined in scheme 7.
Scheme 7 Possible derivatization of the Tp‘ ligand by introduction of acid (left) or azide (right) functions
Since the ability to prepare and purify individual Tp-metal complexes can depend upon the metal of the Tp transfer agent used, it was judged desirable to have an array of different p-BrC6H4Tp compounds and, accordingly, a general method for the preparation of potential p-BrC6H4Tp transfer agents was sought.
[144] Utilizing a slightly modified version of Whites preparation starting from p-BrC6H4BBr2, alkali metal and thallium complexes Tp’M (M= Na (1), K (2), Rb (3), Cs (4), Tl (5)), Tp’MeM (M= K (7), Tl (8)), and Tp’*M (M= K (9), Tl (10)) were synthesized as shown in scheme 8. The magnesium Tp sandwich complex Tp’2Mg 6, originally obtained by drying a Tp’K solution over anhydrous magnesium sulfate, was synthesized in reproducible preparations by ligand exchange between Tp‘Rb and magnesium chloride. One attractive feature of this synthetic method for alkali metal Tp’ compounds is the use of aqueous solutions of alkali metal bases, in particular their carbonates. This not only allows a simple synthesis of the entire series, but could also avoid the use of pyrophoric solutions of metal alkyls
[110] such as n-butyllithium, which was previously employed in the synthesis of p-BrC6H4TpLi. In the work of Wagner and Reger, the Tp intermediates are the “proton-salt” complexes p-IC6H4TpH and FcTpH, by virtue of the stoichiometry. As reported previously for the Tp‘Li salt,[110] we observed in our preparations that the presence of a third equivalent of triethylamine has different effects upon the resultant Tp‘ intermediate depending upon whether the pyrazole is substituted or not. Aliquots taken from the reaction mixture after separation from the triethylammonium bromide byproduct were dried under vacuum and analyzed by 1H NMR spectroscopy. The spectra of the crude products indicate the presence of [p-BrC6H4-Tp]Et3NH,
Me p-BrC6H4Tp H, and p-BrC6H4Tp*H as the intermediates in each respective preparation.
11 Results and discussion
These intermediates were found to be slightly air and moisture sensitive, which is further supported by the generally lower yields obtained in the preparations involving aqueous solutions of
M2CO3 (M= Na, K, Rb, Cs) compared to the preparations using potassium tert-butoxide (KOtBu) or thallium ethoxide (TlOEt).
Scheme 8 Syntheses of p-BrC6H4TpM compounds 1-10
In contrast to the intermediates, all solid alkali metal Tp‘ complexes 1–4, 6, 7, and 9 were found to be stable for at least one month, whereas thallium derivatives 5, 8, and 10 were found to be mildly air sensitive and are best stored under an inert atmosphere. 1H NMR spectroscopic data of 1-10 are in accord with the data found for parent "first-generation" Tp ligands. As expected, C3-symmetrical, tripodal ligation of the metal ion is indicated by the appearance of only one set of signals for the three pyrazole moieties. An additional signal, a typical pair of doublets (J= 8.3 Hz, AA'BB' spin
1 system), is observed for the p-BrC6H4 group. As exemplified by the H NMR spectrum of 2 shown in figure 9, the spectra of compounds 1-6 comprise two doublets of doublets (J= 1.8, 0.5 Hz, a+c) assigned to the hydrogens at 3- and 5-position of the pyrazoles, one triplet (J= 1.8 Hz, b) for the hydrogen at 4-position, and two doublets (d+e) for the AA'BB' system.
12 Results and discussion
1 Figure 9 H NMR spectrum of KTp‘ 2 (250 MHz, DMSO-d6, detailed view 5.8-7.5 ppm)
For the 3-methyl substituted compounds 7 and 8, 1H NMR spectra show two doublets (J= 1.8 Hz, c+d) for the hydrogens at 4- and 5-position, one singlet for the methyl groups at 3-position (a), and two doublets for the AA'BB' system (d+e) as displayed for compound 7 in figure 10.
1 Me Figure 10 H NMR spectrum of KTp’ 7 (250 MHz, DMSO-d6, signal at 3.33 ppm is residual water)
13 Results and discussion
As exemplified for compound 9 in figure 11, the 1H NMR spectra of doubly methyl substituted 9 and 10 comprise a set of three singlets for the hydrogens at 4-position and the two methyl groups at 3- and 5-position, respectively.[145] Additionally, the previously seen set of doublets for the AA'BB' system is found.
1 Figure 11 H NMR spectrum of KTp‘* 9 (250 MHz, DMSO-d6, signal at 3.32 ppm is residual water)
3.1.2. Solid-state structure of compounds 4, 6, and 10
In the course of our investigations, different Tp'M compounds were observed to differ remarkably in their crystallization behaviour. While 10 and Tp’ sandwich compound 6 readily gave stable crystals upon solvent evaporation, crystals of alkali metal ligand salts showed instant disintegration upon warming to room temperature or in the presence of air. Therefore, we were surprised to obtain crystals of 4 by slow evaporation of its acetone solution at -70 °C that showed no decomposition at room temperature. The solid-state structure of Tp'Cs 4, to the best of our knowledge the 3rd ever of a TpCs complex,[146,147] shows several unprecedented features, which may have contributed to the stability of the crystalline solid. Most notably, the central Cs+ ion is coordinated unsymmetrically by three individual Tp' ligands, which occupy two unusual chelating fashions as depicted in the ORTEP plot in figure 12 (In the following detailed description of this structure, we will abbreviate the Tp' ligand incorporating B1, B1A, and B2 as Tp'1, Tp'1A, and Tp'2, respectively.)
14 Results and discussion
Figure 12 ORTEP plot of the solid state structure of Tp’Cs 4 (hydrogens omitted, ellipsoids at 30% probability).
Selected bondlengths [Å]: Cs1–N10 3.131(5), Cs1–N4 3.185(5), Cs1–N5 3.109(4) Cs1–pzcentroid 3.392(5) and 3.400(5), Cs1–C6H4-Brcentroid 3.577, Cs2-N12 3.127
1 Two of the ligands, Tp'1 and Tp'1A, are engaged in κ -coordination through one pyrazolyl ring (Cs1–N 3.109-3.185 Å)[148] as well as η5-interaction with the π-system of a second pyrazolyl ring with average Cs1–C and Cs1–N distances of 3.575 Å and 3.590 Å, respectively. Such an association between alkali metal centers and pyrazolyl π-systems is not uncommon and has been reported for
[149] [150] potassium in polymeric [pzTp]M(H2O) (M= Na, K) and PhTpK, as well as for a polynuclear
CF3,CH3 CF3,CH3 [151] Tp compound [Tp CuK(CO3)KTp ]2. Most probably, it is a consequence of the existence of significant induced dipole interactions between alkali metal cations and the polarized π-electron density.[152] More frequently, this type of interaction is observed in pyrazolide (pz-) complexes,[153] such as the kinetically stabilized ruthenocene analogue Cp*Ru(pz*),[154] or
[155] 5 tetranuclear [Tl4(Ph2pz)4]. In the latter, the η -coordination of pyrazole can also be viewed as a sum of η2-(N2) and η3-(C3) coordination due to the difference between the Tl–N (av. 2.87 Å) and Tl–C (av. 3.46 Å) distances and the resulting inclination of the pyrazole ring. However, the η5-interactions of caesium found in 4 compare best to the Cs-η5-cyclopentadienyl coordination
[156] found in [1,1‘-Fc(BMe2pz)2]Cs (av. 3.508 Å). Noteworthy, in both ligands Tp'1 and Tp'1A the third pyrazolyl moiety is void of any interactions. Thus, what makes 4 structurally unique is, besides the
1 5 previously described κ ,η -chelation, the coordination mode found for the third Tp‘ ligand, Tp'2, which is connected to Cs1 through κ1-coordination (Cs1–N4= 3.130 Å) of one pyrazolyl and
15 Results and discussion
6 η -coordination through the π-face of its p-BrC6H4 moiety, resulting in a chelation-mode best
1 6 described as κ ,η . The distance of Cs1 and the centroid of the p-BrC6H4 ring amounts to 3.577 Å.
6 An equivalent η -coordination is found for the interaction of the Tp'1 and Tp'1A ligands with the neighbouring caesium ions Cs2 and Cs1A, respectively (Cs–η6 centroid av. 3.628 Å). In all three cases, the influence of the electron-withdrawing bromine substituent leads to a tilted orientation of the phenyl ring (Cs–(p-BrC6H4)centroid–C angles 82.09-99.53°), with the shortest Cs–C distance found for the ipso carbon atom (Cs–C= 3.664-3.666 Å; sum of caesium ionic radius and carbon van der Waals radius is 3.70 Å).[157,158] Consequently, the longest Cs–C distance is the one to the carbon atom in the para position (4.040-4.226 Å). Additionally, two other intermolecular connections are found. The first one is conducted by the third pyrazolyl ring of Tp'2, which coordinates to a neighbouring caesium ion in a κ1-fashion (Cs2–N12 3.127 Å). The second interaction bridges two caesium ions involving two pyrazolyl rings, pz[N4#] and pz[N4A#], in a μ2-(σ,π)-fashion reminiscent of that reported for PhTpK.[150] As outlined schematically in figure 13, each respective pyrazole ring is connected to one caesium through its nitrogen lone pair, whereas a second caesium ion interacts with the pyrazolyl π-electron cloud, resulting in the formation of two symmetrical bridges with a Cs···Cs distance of 5.307 Å.
Figure 13 Coordination (top) and bridging modes (bottom) found in the solid state structure of CsTp‘ 4
In contrast to the unusual coordination patterns found in CsTp’ 4, the coordination geometry at magnesium in (Tp’)2Mg 7 is a slightly distorted octahedron (Fig. 14), as expected for a Tp2M compound. Moreover, the magnesium-centred metrical parameters of 7 are similar to those
[159] observed in parent Tp2Mg complexes. For example, the range of Mg–N distances, 2.118-2.200
R,R Å, compares well with those of (Tp )2Mg complexes found in the CCD database (2.143-2.201 Å).[160] The same is true for the N–Mg–N angles ranging from 83.61-85.13° compared to 85.61-
[159] 87.01° for parent Tp2Mg.
16 Results and discussion
Figure 14 ORTEP plot of 7 (hydrogens omitted, ellipsoids at 30% probability). Selected bond lengths [Å]: Mg1–N15A 2.132(5), Mg1–N15 2.132(5), Mg1–N16 2.169(5), Mg1–N16A 2.169(5), Mg1–N14 2.187(5), Mg1–N14A, 2.187(5). N–Mg–N angles are 83.61–85.13°
The solid-state structure of Tp’*Tl 10, like that of 4, is remarkable in several ways. Most notably, a single Tp‘* ligand binds to thallium in a κ2-fashion through only two of its three dimethylpyrazole rings. Whereas such coordination is unprecedented for parent (i.e. TpR,R’Tl) complexes, all of which
3 2 have κ -coordinated, C3-symmetric ligands, κ -type structures are known for “boron-substituted” RTpR,R‘Tl complexes such as MeTp*Tl.[161] In fact, 5 of the 10 RTpTl structures found in the Cambridge crystal database (CCD) exhibit κ2-N,N‘ coordination as observed for 10 (Fig. 15).
Figure 15 ORTEP plot of the dimer of 10 showing intermolecular interactions (hydrogens omitted, ellipsoids at 30% probability). Selected bond lengths [Å]: Tl–BrA 3.8150(6), Tl–N1 2.607(4), Tl–N3 2.559(4), TlA–N5B 3.135(4), N5–TlC 3.135(4). Selected angles [°]: N3–Tl–BrA 62.54(9), N3–Tl–N1 69.63(12), N1–Tl–BrA 102.27(9)
17 Results and discussion
The origin of such non-C3-symmetric ligation is, most probably, a consequence of steric interactions between the boron-bound R group and substituents at the pyrazole 5-position. Notably, in all these structures the third pyrazole is engaged in “secondary” bonding to thallium in which the Tl–N distances are significantly longer than the two “regular” Tl–N bonds. Moreover, with exception of monomeric PhTptBuTl,[162] the third Tl–N interaction is intermolecular, producing aggregated solid-state structures. Thus, 10 is structurally unique in possessing one pyrazole ring that not only shows interaction with the thallium atom of a neighbouring molecule, but additionally shows a van der Waals-type interaction between thallium and the bromine atom of a neighbouring molecule. The Tl–Br distance of 3.8150(6) Å is essentially identical to the sum of the Tl and Br van der Waals radii, 3.81 Å.[157] The Tl–N bond lengths for 10, 2.559(4) and 2.607(4) Å, are within the range found for “normal” Tl–N bonds in RTpR,R‘Tl complexes (2.528–2.780 Å) as well as for two-coordinate BpTl (ca. 2.57–2.80 Å) and parent TpTl (ca. 2.50–2.79 Å) complexes.[163] The same is found for the N–Tl–N angle of 69.63° that matches those found for parent TpTl compounds (ca. 69.0–73.4°).[163] The distance of the thallium atom to the neighbouring pyrazole ring (3.135 Å) is found to be longer than for comparable MeTp*Tl (2.876 Å), probably due to the second interaction of thallium to bromine.[161]
18 Results and discussion
3.2. Syntheses and characterization of ruthenocene analogues
3.2.1. Mixed ligand CpR/Tp’ ruthenocene analogues
Of the two basic options for the assembly of mixed Cp/Tp’ ruthenocene analogues starting from ‘‘half-sandwich” complexes, introduction of the Cp ligand prior to the Tp ligand was found to be favourable. While CpRuCl(COD) is kinetically labile and known to undergo reaction with Tp transfer agents giving CpRuTp species,[164,165] its Tp analogue TpRuCl(COD) is known to be quite inert to ligand substitution.[166,167] As summarized in scheme 9, CpRuTp‘ 11, CpRuTp’Me 12 and CpiPrRuTp’ 15 were obtained by modifying the parent syntheses of CpRuTp from species containing
[114] [168] a [RuCpR] synthon, whereas Cp*RuTp‘ 13 was synthesized starting from {Cp*RuCl4}.
Scheme 9 Syntheses of mixed CpR/Tp’ ruthenium compounds
Quite notably, compounds 11-13, and 15 were found to differ in terms of stability and solubility. For example, CpRuTp' 11 was found to be air stable, but, unlike parent CpRuTp, is poorly soluble in most organic solvents. As a result, 11 did not react with n-BuLi neither in diethylether (Et2O) nor in THF at -78 °C, and thus was rated not suitable for acid-functionalization. Further, the low solubility prevented characterization by 13C NMR within a reasonable time-period and only a 1H NMR spectrum was obtained.
To increase the solubility of the complex, a methyl substituted derivative of p-BrC6H4Tp was considered. Hence, reaction of Tp'MeTl 8 with CpRuCl(COD) at room temperature gave CpRuTp'Me 12. Characterization by 1H NMR showed rapid decomposition of samples prepared in air, indicated by colour change from yellow to green, and shift of the signals of both ligands. Subsequent samples prepared under argon atmosphere showed similar decomposition to unidentified products after a couple of hours which made recording of a 13C NMR spectrum impossible. Due to this instability, no further investigations were made on this compound. Similar observations were made with samples of Cp*RuTp' 13. In this case, decomposition proceeded rapidly even in samples prepared under an argon atmosphere. Monitoring the 1H spectra proved complete decomposition within 1 h. Low stability in solution has been reported for other mixed ligand Cp*MTp (M= Ni, V, Fe) complexes as well,[112] whereas related homoleptic metallocenes are known to be air-stable.[169,170] Regarding the observations that an increasing
19 Results and discussion number of methyl groups raises the solubility, but seems to have a negative effect on the stability of the compounds, less substituted Cp ligands were considered as another option to proceed in terms of a stable compound. Modifying the literature preparation of CpRuCl(COD)[171] by substitution of TlCp with NaCpiPr, CpiPrRuCl(COD) 14 was synthesized in comparable yields. Subsequent reaction of 14 and Tp'Li
iPr 1 gave Cp Ru(p-BrC6H4Tp) 15. Characterization by H NMR showed remarkable changes for the CpiPr ligand compared to the starting material. The formerly singlet signal of the four aromatic protons appears as a pair of pseudo-triplets as outlined in figure 16. This kind of splitting is known
R [172] and has been noted recently for related Cp Ru(L2X) complexes, and is most probably a consequence of virtual couplings in the AA’BB’ spin-system of the mono-substituted Cp ligand.[173]
1 iPr Figure 16 H NMR of Cp Ru(p-BrC6H4Tp) 14 (250 MHz, CDCl3, signal at 1.53 ppm is water). The magnification in the region 3.90-4.40 ppm showing the pseudo-triplets for the CpiPr ligand
In contrast to previous related compounds, 15 was found to be air-stable both in solution and as a solid. Reaction with n-BuLi in THF at -78 °C, followed by addition of solid CO2 and workup with hydrochloric acid,[30] as depicted in scheme 10, gave the benzoic acid-functionalized compound
iPr Cp Ru(p-(CO2H)C6H4Tp) 16. Success of the reaction was proven by appearance of one stretch for
-1 [174] the CO2H group at 1685 cm in IR samples, and further supported by a significant shift of the 1H NMR signals for the AA'BB' system of the Tp’ ligand.
20 Results and discussion
Scheme 10 Introduction of the acid functionality to the mixed ligand sandwich complex 15
The solid-state structure of 16 was determined by X-ray diffraction. As depicted in the ORTEP plot in figure 17, the structure is consistent with the ‘‘piano-stool” like appearance of unsubstituted
[165] CpRuTp. The Tp’ ligand shows local C3v-symmetry with an average Ru–N distance of 2.113(4) Å, which is slightly shorter than that in CpRuTp (2.126 Å). The average N–Ru–N angle of 82.9(4)° is in good accordance to that found in other [TpRu] compounds.[175,176] The Ru–C bonds have an average length of 2.149(5) Å, resulting in a Ru–Cp centroid distance of 1.780(1) Å that is only slightly longer than in the unsubstituted compound CpRuTp (1.777 Å), but still in the range found for other cyclopentadienyl compounds of ruthenium.
Figure 17 ORTEP plot of 16 (Hydrogens omitted, ellipsoids at 30% probability). Selected bond lengths (Å): Ru1–N2 2.123(8), Ru1–N4 2.124(11), Ru1–N6, 2.112(9), Ru1–C58 2.158(11), Ru1–C63 2.155(12), Ru1–C52 2.201(11), Ru1–C20 2.132(12), Ru1–C43 2.165(11), Ru1–CpiPr centroid 1.790. N–Ru–N angles are 81.9(3)-82.5(3)°
21 Results and discussion
3.2.2. Mixed ligand Tp/Tp‘ ruthenocene analogues
Motivated by the synthesis of mixed sandwich Cp/Tp’ ruthenium compounds, an analogous route towards mixed Tp/Tp’ sandwiches starting from suitable [TpRu] half-sandwich compounds was investigated. First attempts were inspired by the work of Onishi et al., who reported the syntheses
R of various mixed ligand sandwich Tp ruthenium complexes starting from Tp RuCl(NCPh)2 (TpR= pzTp, Tp) compounds as outlined in scheme 11.[170,177-179]
Scheme 11 Synthesis of mixed Tp/TpR sandwich ruthenium complexes reported by Onishi et al.
However, in our hands the reported synthesis of TpRuCl(NCPh)2, consisting of the reaction of trans-RuCl2(NCPh)4 with TpM (M= K, Tl), resulted repeatedly in the formation of homoleptic
Tp2Ru species. Thus, we sought a synthetic route starting from a different [TpRu] half-sandwich and considered TpRuCl(COD) as a promising candidate since it is readily available in reasonable
[166,180] yields starting either from [RuH(NH2N(CH3)2)3(COD)][X] (X=PF6, BPh4), or from polymeric
[167] [RuCl2(COD)]x . Although the COD ligand is considered substitutionally inert in this complex,[166,180] which is in contrast to the analogous cyclopentadienyl complexes CpRuCl(COD) and Cp*RuCl(COD),[114,164] it has been shown that RuTpCl(COD) is a valuable precursor in the
+ syntheses of TpRu(L2)X, TpRu(L)2X and [TpRu(L)3] complexes with mono- and bidentate N- and P-donor ligands,[41] usually requiring prolonged heating at T>155 °C (DMF reflux).[166] Interestingly, no procedure in which both the COD and chloride ligand are substituted in a single reaction to give complexes of the general formula TpRu(L2X) (L2X= Cp, Tp) has been reported. Monitoring the reaction of TpRuCl(COD) and Tp’K 2 by thin layer chromatography indicated complete consumption of TpRuCl(COD) after 12 hours of DMF reflux. Removal of the volatiles and column-chromatographic separation of the orange product-mixture afforded TpRu(p-BrC6H4Tp) 19 as a light yellow solid in 19% yield. A deep red solid isolated as the major fraction was identified as a mixture of compounds containing Tp and pyrazole species, possibly formed by thermal decomposition of 19 or the starting materials. To avoid these undesired side-reactions, we considered use of microwave-assisted techniques for further syntheses, since a combination of direct and rapid heating by microwave irradiation, in combination with sealed vessel processing, may reduce the reaction time required compared to conventional conditions.[181] Reaction of TpRuCl(COD) and 2 in THF at 155 °C (p~7-8 bar) for one hour in a microwave reactor afforded 19 in 39% yield after purification by column chromatography. Characterization of compound 19 by 1H
22 Results and discussion
NMR (Fig. 18) revealed two sets of signals for the hydrogens at the pyrazole 3- and 4-position of each respective Tp ligand (d, c, e, b) plus an additional broad singlet assigned to the hydrogens at
[177] pyrazole 5-position (a+f), with chemical shifts comparable to parent unsubstituted Tp2Ru.
1 Figure 18 H NMR of 19 (cutout from 4.00-8.50 ppm, 250 MHz, CDCl3, signal at 5.30 ppm is CH2Cl2)
[178,182] As depicted in figure 19, the solid-state structure of 19 is reminiscent of that of parent RuTp2,
in particular showing a nearly octahedral environment with local C3v-symmetry at ruthenium, an average Ru–N bondlength of 2.056 Å (2.061 Å for Tp2Ru) and an average N–Ru–N angle of 87.00°.
Figure 19 ORTEP plot of 19 (Hydrogens omitted, thermal ellipsoids at 30% probability). Selected bondlengths (Å): Ru(1)–N(9) 2.0430(18); Ru(1)–N(7) 2.0510(18); Ru(1)–N(6) 2.0569(18); Ru(1)–N(11) 2.0611(18); Ru(1)–N(2) 2.0658(19); Ru(1)–N(4) 2.0684(19). N–Ru1–N angles (av.): 87.00°
23 Results and discussion
The likeness of 19 to parent Tp sandwich compounds was further supported by the cyclic voltammogramm (CV) of 19 in dichloromethane, which shows a reversible oxidation for
[182] Ru(II)/Ru(III) at -205 mV (-202 mV for Tp2Ru).
Subsequently, aryl-bromide 19 was converted to benzoic acid functionalized TpRu(p-(CO2H)-
C6H4Tp) 20 by the sequence of lithiation, addition of solid CO2, and workup with hydrochloric acid previously applied in the synthesis of compound 16. Reaction success was indicated by appearance
-1 [174] of one stretch for the CO2H group at 1685 cm in IR samples, and additionally supported by a signal at 168.5 ppm in the 13C NMR and shift of the two doublets of the AA’BB’ system in 1H NMR spectra. Although 19 and 20 were found to be stable to air and water, exposure of their chloroform or dichloromethane solutions to sunlight for several days produced a deep red precipitate, presumably containing ruthenium(III) species with chloride counter anions as reported for other
[178] (RTp)2Ru derivatives.
24 Results and discussion
3.2.3. Mixed ligand Tpm/Tp’ ruthenocene analogues
Inspired by recent developments in the functionalization of biomolecules, in particular peptides, by Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC),[183,184] we were eager to investigate an azide functionalization of mixed sandwich Tp ruthenium complexes. To provide higher solubility of the complexes in the polar solvents usually used in azidonation and CuAAC reactions, we thougth of employing non-charged Tpm as a spectator ligand to obtain a positively charged mixed Tp’/Tpm ruthenocene analogue. Of the two possible options for the assembly of a complex suitable for our purposes, the introduction of the Tp’ ligand prior to the Tpm ligand was found to be the favourable method. While half-sandwich [TpmRuCl(COD)]Cl can be obtained by refluxing a mixture of Tpm and
[185] [RuCl2(COD)]x in ethanol for 7 hours, usually containing ethanol which is hardly removable,
TpRuCl(COD) is accessible in 2 hours by reaction of [RuH(NH2N(CH3)2)3(COD)][X] (X= PF6,
[180] [113,166] BPh4) with a TpM transfer agent and subsequent addition of tetrachloromethane.
Consequently, functionalized p-BrC6H4TpRuCl(COD) 23 was synthesized by modification of a literature preparation of TpRuCl(COD) reported by Kirchner et al..[166] Characterization of 23 by 1H NMR, as outlined in figure 20, shows signals comparable to parent TpRu(COD)Cl, with the signals of the Tp ligand split into two sets (2:1 ratio, c, d, e) due to the unsymmetric coordination environment of the central ruthenium atom.[166]
1 Figure 20 H NMR of 23 (250 MHz, CDCl3, signal at 5.30 ppm is CH2Cl2, 1.52 ppm is water)
25 Results and discussion
This structural resemblence of 23 to parent TpRuCl(COD) is proven by the solid-state structure of 23. As shown in the ORTEP plot in figure 22, the Tp’ ligands occupies three coordination sites at ruthenium, resulting in a local C3v-symmetry with Ru–N bondlengths of 2.120-2.131 Å (2.110-2.164 Å for TpRuCl(COD)), and an average N–Ru–N angle of 84.58° (85.00° for parent compound).[186] The longest Ru–N distance is the one trans to both the COD and the adjacent chlorido ligand. The
COD moiety adopts a typical twisted boat conformation of C2-symmetry, causing the Ru–C bond lengths to be all in a narrow range of 2.226-2.233 Å (2.213-2.229 Å for parent compound). Considering the two π-bound CH=CH of COD as single ligands, the geometry at ruthenium is approximately octahedral with Ru–Cl being the longest bond (2.441 Å).
Figure 21 ORTEP plot of 23*CH2Cl2 (ellipsoids at 30% probability, hydrogens and CH2Cl2 omitted). Selected bond lengths (Å): Ru1–C1 2.232(8), Ru1–C2 2.226(9), Ru1–C5 2.233(7), Ru1–C6 2.239(3), Ru1–N2 2.131(1), Ru1–N4 2.125(1), Ru1–N6 2.120(6), Ru1–Cl1 2.441(3). Selected angles (°): N4–Ru1– C6 79.91, N4–Ru1–C1 82.81, N6–Ru1–C2 93.54, N6–Ru1–N2 87.13, N2–Ru1–C5 93.18, C2–Ru–C5 79.35, C1–Ru1–C6 78.10
In close analogy to the synthesis of Tp'RuTp 19, the Tp half-sandwich compound 23 was reacted with Tpm in THF at 155 °C for one hour, utilizing a microwave reactor. The bright yellow complex salt [(p-BrC6H4Tp)RuTpm]Cl 24 precipitated readily upon cooling the reaction solution to room temperature, and was isolated by filtration in 54% yield. Characterization of 24 by 1H NMR, as depicted in figure 22, shows two sets of signals for the pyrazole groups comparable to
C3v-symmetric compound 19. Additional signals are found for the AA'BB' system and the apical CH moiety of the Tpm ligand, the latter being strongly shifted downfield due to the deshielding effects by the pyrazolyl groups.
26 Results and discussion
1 Figure 22 H NMR of 24 (250 MHz, DMSO-d6, cutout from 6.00-11.00 ppm)
The solid state structure of 24, as outlined in the ORTEP plot given in figure 23, was found to be
[178,182] consistent with that of unsubstituted Tp2Ru as well as that of compound 19, showing local
3 C3v-symmetric κ -N,N’,N’’-coordination of both ligands with an average Ru–N distance of 2.059 Å
(2.061 Å for Tp2Ru, 2.057 for 19) and an average N–Ru–N angle of 86.20° (87.00° for TpRuTp’).
Figure 23 Structural view of 24 (ellipsoids at 30% probability, hydrogens and chloride omitted). Selected bond lengths (Å): Ru1–N1 2.073(2), Ru1–N3 2.042(2), Ru1–N5 2.046(2), Ru1–N7 2.062(2), Ru1–N9 2.074(2), Ru1–N11 2.060(2). Selected angles (°): N–Ru1–N 84.0-88.0
27 Results and discussion
However, in several 1H NMR spectra of crude 24, unidentifiable signals were observed that vanished upon repeated washing with THF. Removal of THF from the combined washing solutions gave a microcrystalline, yellow solid 24a. Since characterization by 1H NMR spectroscopy showed a 2:1 splitting of the pyrazole signals (Fig. 24), being reminiscent of that observed in 23 and other
[166,187] unsymmetric TpRuL2X complexes, one can assume that 24a must contain a similar coordination motive.
1 Figure 24 H NMR of 24a (250 MHz, CDCl3, cutout from 5.50-11.00 ppm)
This assumption was confirmed by X-ray diffraction experiments, which revealed [(p-
2 BrC6H4Tp)RuCl(κ -N,N‘-Tpm]Cl 24a to be a structural, or, more precisely, coordination isomer of 24, presumably representing a stable intermediate of the ligand exchange reaction. As shown in the ORTEP plot in figure 25, the Tpm ligand in the neutral complex 24a is coordinated to ruthenium only in a κ2-fashion with Ru–N bond lengths of 2.060 Å and 2.073 Å, which, however, commensurate with those found for [κ3-N,N’,N’’-TpmRu] compounds containing additional N-donor-ligands (Ru–N 2.063-2.084Å).[188] The third pyrazole ring, pointing away from Ru, is devoid of any interaction. The Tp’ ligand shows the expected C3v-symmetric ligation with Ru–N bond lengths, 2.028-2.062 Å, slightly shorter than those found in 19 (2.043-2.086 Å). The roughly octahedral coordination geometry at ruthenium (N–Ru–N 85.5-87.3°) is completed by a chlorido ligand, bound to ruthenium with almost the the same metrical parameters (Ru–Cl 2.443 Å) as found in 23 (Ru–Cl 2.441 Å) and TpRuCl(COD), respectively.[186]
28 Results and discussion
Figure 25 Structural view of 24a*acetone (hydrogens and acetone omitted, ellipsoids at 30% probability). Selected bond lengths (Å): Ru1–Cl1 2.443(8); Ru1–N7 2.028(2); Ru1–N9 2.042(2); Ru1–N11 2.062(2); Ru1–N2 2.060(2); Ru1–N6 2.073(2); N1–C1 1.466(4); N3–C1 1.425(4); N5–C1 1.463(4). Selected angles (°): N7–Ru1–N9 86.83(9); N7–Ru1–N11 87.30(9); N9–Ru1–N11 85.50(9); N2–Ru1–N6 86.20(9)
Next, 24 was functionalized by a copper iodide catalyzed azidonation as outlined in scheme 12. Reaction progress was monitored by shift of the AA'BB' signals in 1H NMR, and the appearance of
-1 -1 [189] two signals at 2085 cm (νN≡N asym.) and 1258 cm (νN≡N sym.) in IR spectra.
Scheme 12 Synthesis of compound 25 by copper catalyzed azidonation of 24
Just as reported for the syntheses of other phenyl-azides from phenyl-bromides,[143] complete conversion of 24 was only observed upon addition of N,N’-dimethylethane-1,2-diamine (0.3 eq.) and sodium ascorbate (0.03 eq), respectively. While N,N’-dimethylethane-1,2-diamine, like other N-donor ligands,[143, 190] efficiently accelerates the reaction, sodium ascorbate positively effects the stability of the catalytic Cu(I) species.[191]
Azide functionalized [(p-N3C6H4Tp)RuTpm]Cl 25 precipitated from the reaction mixture upon addition of water, was separated by filtration, and obtained as a light yellow solid in 72% yield after drying. Although elemental analysis of 3 indicated the presence of a pure compound with a
29 Results and discussion chloride counterion, the solid state structure obtained by X-ray diffraction showed the existence of both a chloride and a bromide counterion in a 0.4:0.6 ratio. Apart from this partial replacement, presumably occuring during salt metathesis with NaN3, the solid state structure of 25, as depicted in the ORTEP plot in figure 26, indicates little change of the Tp coordination in comparison to the starting material 24. Thus, the Ru–N distances (2.042-2.068 Å) as well as the N–Ru–N angles (85.4-86.7°) remain unaffected of the azidonation. The metrical parameters found for the p-
N3C6H4 group, in particular the C23–N14–N15 (116.7°) and N15–N14–N15 (171.7°) angles, compare well with those of representative phenyl azides (C–N–N 115.2-118.1°; N–N–N 171.3-173.0°).[192-194]
Figure 26 Structural view of 25 (ellipsoids at 30% probability, hydrogens and counterions omitted). Selected bond lengths (Å): Ru1–N1 2.055(2), Ru1–N3 2.068(2), Ru1–N5 2.059(2), Ru1–N7; Ru1–N9 2.042(2), Ru1–N11 2.053(2).
Selected angles (°): NTp–Ru1–NTpm 92.8-94.5, NTp–Ru1–NTp 85.4-86.1, NTpm–Ru1–NTpm 86.5-87.0, C23–N13–N14 116.7(2), N13–N14–N15 171.7(3)
30 Results and discussion
3.3. Mixed sandwich Cp/Tp‘,Tp/Tp‘ and Tpm/Tp’ bioconjugates
3.3.1. Application of acid-functionalized 16 and 20 in SPPS
iPr In order to test the suitability of Cp Ru(p-(CO2H)C6H4Tp) 16 and TpRu(p-(CO2H)-C6H4Tp) 20 in their coupling to peptides, they were attached to carboxy-protected amino acids under standard
iPr coupling conditions to give amide compounds Cp Ru(p-(CO-Phe-OMe)C6H4Tp) 17 and
TpRu(p-(CO-Val-OtBu)C6H4Tp) 21 (Scheme 13). Formation of the amide moiety (NHCO) is, in both compounds, unambigously supported by 1H NMR (d, ca. 6.64 ppm; J= 7.5 Hz), 13C NMR (ca. 167.0 ppm), and IR (ca. 1650 cm-1) spectroscopies, respectively.[174]
Scheme 13 Syntheses of the amide compounds 17 and 21 in solution
After the successful test couplings, compounds 16 and 20 were evaluated regarding their suitability in SPPS methodology by coupling to the pentapeptide enkephalin (Enk= Tyr-Gly-Gly- Phe-Leu)[136] as depicted in scheme 14. Specific conditions had to be chosen concerning the low stability of both 16 and 20 against TFA,[195] a commonly used cleavage reagent. Thus, the base labile HMBA (4-hydroxymethylbenzoic acid) linker was used to avoid the need for acid treatment altogether. Resin-bound enkephalin was synthesized manually according to standard Fmoc-SPPS methods.[196] After removal of the 2-chloro-trityl (2-Cl-Trt) protecting group from tyrosine,
iPr Cp Ru(p-(CO2H)C6H4Tp) 16, or TpRu(p-(CO2H)C6H4Tp) 20, were coupled. Although the presence of a phenolic hydroxy group might interfere with the coupling of the acid functionalized metal complex, it has been shown before that possible by-products, such as phenolic esters, decompose during cleavage, thus having little influence on the yield of the bioconjugate.[137,197]
31 Results and discussion
Scheme 14 Syntheses of mixed sandwich ruthenium bioconjugates (p-(CO-Enk-OH)-C6H4Tp) 18 and TpRu(p-(CO-Enk-OH)-C6H4Tp) 22 by SPPS
iPr The bioconjugates Cp Ru(p-(CO-Enk-OH)-C6H4Tp) 18 and TpRu(p-(CO-Enk-OH)-C6H4Tp) 22 were subsequently cleaved from the resin by treatment with 1M NaOH in 1,4-dioxane, purified by reverse phase high pressure liquid chromatography (RP-HPLC) (18, 62% yield) or flash chromatography (22, 57% yield), and characterized by means of mass and 1H NMR spectroscopies.
Figure 27 ESI-MS (neg. mode) spectrum of 18 showing Ru isotope pattern
32 Results and discussion
The ESI-MS spectra (neg. mode) of 18 (Fig. 27) and 22 (Fig. 28) each revealed one major signal corresponding to the carboxylate form of the respective bioconjugate. The observed isotope patterns clearly indicated the presence of ruthenium in 18 and 22.
Figure 28 ESI-MS (neg. mode) spectrum of 22 showing Ru isotope pattern
Additionally, all 1H NMR signals for both 18 and 20 could be assigned with the help of literature data for parent enkephalin.[198] Integration of the 1H NMR signals matched with the proposed composition, e.g. same intensities were observed for the isopropyl groups on the CpiPr ring and the leucine amino acid in compound 18.
33 Results and discussion
3.3.2. Application of mixed sandwich Tp'/Tpm azide 25 in CuAAC
Suitability of the azide functionalized complex 25 in CuAAC was tested in coupling to pentyne- functionalized HCC(CH2)2-CO-Val-OtBu 26. For reasons of solubility of 26, and ease of synthesis, the use of CuI in acetonitrile/dichloromethane (2:1) over the widely employed CuSO4 in H2O/t- BuOH (2:1) was considered.[183] While the latter system requires a reducing agent, copper(I) salts have been reported to give the desired triazole product in comparable purity upon addition of a nitrogen base,[184] preferrably 2,6-lutidine,[199] and exclusion of oxygen. Triazole conjugate
[TpmRu(p-C2N3H-(CH2)2-CO-Val-OtBu)Tp]Cl 27 was obtained in 58% yield (scheme 15).
Scheme 15 Synthesis of 27 by CuAAC in solution
Formation of 27 was unambigiously supported by 1H NMR spectroscopy, in particular by a singlet for the triazole proton at 8.01 ppm (s, 1H, j) and downfield shift of the AA'BB' doublets (h+i) as shown in figure 29.
1 Figure 29 H NMR of 27 (250 MHz, CDCl3, signals at 5.30 and 1.57 are CH2Cl2 and water, respectively)
34 Results and discussion
Additionally, 25 was coupled to pentyne-functionalized peptide enkephaline (penty-Enk-OH) in solution. Since preliminary experiments showed CuI in acetonitrile/dichloromethane not to catalyze the reaction, presumably due to interaction of Cu(I) with the free carboxy group of
[199] enkephalin, CuAAC was performed in H2O/t-BuOH, utilizing CuSO4 and sodium ascorbate. The resulting conjugate [TpmRu((p-C2N3H-(CH2)2-CO-Enk-OH)-C6H4Tp)]Cl 28 was purified by RP- HPLC (21% yield) and characterized by means of mass and 1H NMR (Fig. 30) spectroscopies.
1 Figure 30 H NMR of 28 (400 MHz, CD3OD)
ESI-MS analysis of 28 (pos. mode) showed two major signals, corresponding to the molecular ion (m/z= 1281.03) and the dication [M+H]2+ (m/z= 641.23). Additional signals with ruthenium isotope patterns result from a b-type fragmentation of the enkephalin moiety (Fig. 31).[200]
Figure 31 ESI-MS (pos. mode) and b-type fragmentation pattern observed for 28
35 Results and discussion
3.4. Seven-coordinate Tp*WI(CO)(η2-alkyne) complexes
3.4.1. Syntheses and characterization of tungsten “building blocks”
A promising entry into studies of tungsten bioorganometallic compounds was found in the rich chemistry of tungsten carbonyl complexes containing Tp* ligands and their ability to form
2 2 η -alkyne complexes. Stability of complexes of the general formula Tp*W(CO)2(η -alkyne) was found to be highly dependent on the nature of the alkyne ligand. For example,
2 [201,202] Seidel et al. reported Tp*W(CO)2(η -bisbenzylthioacetylene) to be air-stable whereas
2 [203] Tp*W(CO)2(η -diphenylethyne) is found to be highly air-sensitive. Exchanging one carbonyl ligand for a halide gives chiral complexes of the type [Tp*W(CO)X(η2-alkyne)] (X= Cl, Br, I), whose stability increases in the order Cl 2 2- [Tp*WI(CO)(η -HCC(CH2)2CO2H)] 29 and [Tp*WI(CO)(η Fmoc-Pgl)] 30 (Pgl= propargylglycine) were prepared by alkyne substitution of two carbonyls in Tp*WI(CO)3 following literature procedures as outlined in scheme 16.[203] Scheme 16 Syntheses of η2-alkyne tungsten complexes 29 and 30 Reaction progress is easily monitored by change of colour from red to dark green and downfield shift of the signal for the terminal alkyne in 1H NMR (singlet, 1 H, ca. 12.20 ppm). Moreover, reaction success is indicated by the disappearance of the IR signal of one CO stretch, representing two symmetrically equal CO ligands, as outlined for 29 in figure 32. 36 Results and discussion Figure 32 IR spectra of Tp*WI(CO)3 (grey) and 29 (black) showing CO loss during reaction Heating a mixture of Tp*WI(CO)3 with a slight excess of 4-pentynoic acid in THF gave 29 as green crystals in 53% yield. Whereas 1H NMR spectra of 29 (Fig. 33) indicated the existence of two enantiomers in 92:8 ratio (12.23, 13.13 ppm, singlet, 1 H each), solid-state structure investigation showed only one isomer with the alkyne proton pointing away from the CO moiety like depicted in the ORTEP plot in figure 34. 1 Figure 33 H NMR of 29 (250 MHz, CDCl3, peak at 5.30 ppm is residual CH2Cl2) 37 Results and discussion Figure 34 ORTEP plot of 29 (thermal ellipsoids at 30%, hydrogens omitted). Only one molecule in the unit cell (Z= 2) is shown, the other being the enantiomer. Selected bond lengths (A°): W1–C16 1.954(9); W1–C17 2.020(9); W1–C18 2.065(8); W1–N6 2.152(7); W1–N4 2.241(7); W1–N1 2.265(7); W1–I1 2.7917(12) Assuming the alkyne moiety occupying one coordination site, the coordination sphere of tungsten in 29 could be described as roughly octahedral. The Tp* ligand, which occupies the three coordination sites trans to the accompanying ligands, shows the expected local C3v-symmetry. Short W–Calkyne distances (2.020 and 2.065 Å) found in 29 are consistent with structural features found in other complexes of tungsten with tightly bound d4-alkynes.[203,205,206] Further analogy to these complexes is found in the parallel orientation of the tungsten-bound alkyne to the W–CO axis, which optimizes both the π-acid and π-donor interactions of the alkyne with dπ orbitals on tungsten, while the CO ligand can interact with both filled dπ orbitals. As outlined previously in sche 16, tungsten η2-Fmoc-propargylglycine (Fmoc-Pgl) complex 30 was synthesized in an analogous fashion by refluxing a mixture of Tp*WI(CO)3 with a slight excess of Fmoc-protected propargylglycine in THF. Similar to 29, two isomers were detected in 90:10 ratio by 1H NMR (12.35, 13.10 ppm, singlets, 1H each). Both compounds 29 and 30 were found to be air stable even after prolonged storage in solution or as solids. Furthermore, stability tests with TFA revealed both compounds to be stable towards TFA concentrations up to 40% in DMF solution.[195] 38 Results and discussion 3.4.2. Application of tungsten “building blocks” 29 and 30 in SPPS Pentynoic acid functionalized building-block 29 was tested in SPPS methodology by coupling to enkephalin and pseudo-neurotensin (pNT= Lys-Lys-Pro-Ile-Leu) in manual SPPS experiments.[196] The functionalized bionconjugates [Tp*WI(CO)(HCC(CH2)2CO-pNT-OH)] 31 (Fig. 35) and [Tp*WI(CO)(HCC(CH2)2CO-Enk-OH)] 32 were obtained by coupling 29 in the last step of Fmoc-SPPS as outlined exemplarily for 32 in scheme 17. Figure 35 N-terminally labelled Tp*WI(CO)-alkyne pseudo-neurotensin derivative 31 2 Side-chain functionalized enkephalin derivative H2N-Tyr-Gly-[Tp*WI(CO)(η -Pgl)]-Phe-Leu-OH 33 was obtained by replacement of one glycine by the tungsten functionalized amino acid 30 and continuation of the sequence as depicted in scheme 17. Scheme 17 Synthesis of the tungsten functionalized peptides 32 and 33 by SPPS 39 Results and discussion All functionalized peptides 31-33 were cleaved from the resin by treatment with TFA (20% v/v in DMF) and purified by RP-HPLC (Fig. 36). The pale blue solids, isolated in 24-59% yield, were unambiguously characterized by mass, 1H NMR, and IR spectroscopies. Figure 36 Analytical HPLC traces of 31 (left), 32 (center), and 33 (right) As outlined in figure 37, ESI-MS (pos. mode) of 31 shows two major signals, that correspond to the molecular ion (m/z= 1477.30), and a doubly charged ion due to loss of the iodine ligand (m/z= 675.33). A third signal, centered around m/z= 739.25, corresponds to the dicationic species [M+2H]2+. All 1H NMR signals for 31 could be assigned by comparison to parent neurotensin,[207] and indicate, in accordance with the isomeric distribution of the starting material 29, two isomers in a 92:8 ratio. Figure 37 ESI-MS (pos. mode) and isotope pattern of 31 40 Results and discussion A likewise isomeric distribution was found in the characterization of 32 by 1H NMR. Comparable to the ruthenium-based enkephalin bioconjugates 18 and 22, ESI-MS (neg. mode, Fig. 38) of 32 displayed two major signals, one for its carboxylate form around m/z= 1270.27 and an additional signal for the double charged molecule ion around m/z=634.37, both matching the calculated isotope pattern. Figure 38 ESI-MS (neg. mode) and isotope pattern of 32 Assignment of 1H NMR signals for compound 33 indicated two isomers in a 90:10 ratio. ESI-MS (pos. mode, Fig. 39) analysis of 33 showed one signal for the molecule ion around m/z= 1230.26. Interestingly, a second signal, centered around m/z= 594.41, missing the expected tungsten isotope pattern, is generated by loss of the [Tp*WI(CO)] moiety. Figure 39 ESI-MS (pos. mode) and isotope pattern of 33 41 Results and discussion Characterization of 31-33 by IR spectroscopy indicated little influence of the peptide coupling to the [Tp*WI(CO)] moiety. As exemplified for compound 31 in figure 40, the strong stretch of the tungsten-coordinated CO ligand at 1904 cm-1 is clearly distinguishable from the signals corresponding to the CO vibrations of the amide group and the carboxylic acid at 1644 cm-1 and 1543 cm-1, respectively. However, preliminary experiments showed the tungsten bioconjugates 31- 33 to be unsuitable for CO-based spectroscopy in living cells, since the single carbonyl ligand provided insufficient Raman intensity upon dilution.[208] Figure 40 IR spectrum (solid) of 31 42 Summary and conclusion 4. SUMMARY AND CONCLUSION Aim of this work was the syntheses of tris(pyrazolyl)borate-containing transition metal complexes, their subsequent functionalization, and evaluation of their suitability as labels for biological molecules, in particular peptides. Promising target structures for this purpose were found in metallocene analogue mixed-sandwich ruthenium as well as in seven-coordinate Tp*WI(CO)(η2- alkyne) compounds. While the latter allowed for the introduction of a synthetic handle, e.g. carboxylic acid, by choice of the coordinated alkyne, the intended ruthenium compounds required the incorporation of a convertible functional group on the Tp ligand. Consequently, the first part of this work was the synthesis of a group of different, p-BrC6H4 functionalized “third-generation” Tp’ ligands.[209] The synthetical approach of reacting substituted boron dihalides RBX2 (X= Cl, Br) with pyrazole and triethylamine, previously applied in the [28] [73] [110] syntheses of functionalized ligands such as p-IC6H4Tp, FcTp, and Tp’Li, was successfully adopted for the syntheses of the alkali metal and thallium p-BrC6H4TpM ligand salts 1-5 and 7-10 depicted in figure 41. Figure 41 p-BrC6H4Tp ligand salts prepared in this work Additionally, the functionalized tris(pyrazolyl)borate sandwich Tp’2Mg 6, was synthesized in reproducible preparations by ligand exchange between Tp’Rb 3 and magnesium chloride. Compounds 1-10 were unambiguously characterized by means of 1H/13C NMR spectroscopy and elemental analysis. Whereas the NMR spectra indicated, comparably to unfunctionalized Tp ligands, the expected local C3v-symmetry at the metal ion for all compounds, the solid-state structures revealed fundamental differences. As such, only the magnesium compound Tp’2Mg 6 displayed an expectable structure with both Tp’ ligands coordinated in a κ3-N,N’,N’’-fashion, [159,160] reminiscent of the one found in parent Tp2Mg. In contrast, Tp’*Tl 10 was found to form coordination polymers in which κ2-N,N’ coordinated thallium shows a van der Waals-type interaction to the bromine of a neighbouring Tp’* ligand. Moreover, the third pyrazole ring is involved in “secondary” coordination of a neighbouring thallium ion.[209] An even more spectacular coordination pattern was found in the solid state structure of Tp’Cs 4,[210] where the central caesium ion is coordinated by three individual Tp’ 43 Summary and conclusion ligands in two chelating fashions involving both κ1-N-pyrazolyl and π-coordination through either a pyrazolyl or the p-BrC6H4 ring, respectively. Additionally, neighbouring caesium ions are connected in two bridging modes, involving κ1-N- pyrazolyl and π-coordination as well, resulting in a polymeric structure. Subsumingly, the different metal-ligand interactions found in 4 and 10 expand the spectrum of known coordination modes for RTpM compounds, hence showing that, as previously reported for other “third-generation” RTp ligands and compounds, in scorpionate chemistry even “innocent” spectator groups can contribute essentially to metal coordination.[150] In the second part of this work, mixed ligand ruthenocene analogues, summarized in figure 42, were synthesized and characterized. Figure 42 Mixed sandwich Cp/Tp‘ (left), Tp/Tp‘ (center) and Tpm/Tp‘ ruthenium compounds Although Cp/Tp’ derivatives with different substitution patterns on both Cp and Tp part were found to be readily accessible by slight modification of literature procedures, only iPr Cp Ru(p-BrC6H4Tp) 15 met with the prerequisites of stability and solubility for further [211] iPr functionalization. Accordingly, an acid-functionalized derivative Cp Ru(p-(CO2H)C6H4Tp 16 of the parent compound 15 was prepared in reasonable yield, and could be characterized in the solid state, thereby showing close structural resemblance to parent, unfunctionalized CpRuTp. The preparative approach of reacting suitable (L2X)RuLn (L2X= Cp, Tp) half-sandwich compounds with Tp’ ligands was further applied in the microwave assisted syntheses of the mixed Tp ligand [212] compounds TpRu(p-BrC6H4Tp) 19 from TpRu(COD)Cl, and [(p-BrC6H4Tp)RuTpm]Cl 24 from [213] (p-BrC6H4Tp)Ru(COD)Cl 23, respectively. Structural and functional characterization of 19 by NMR spectroscopy, cyclovoltammetry, and X- ray diffraction, respectively, showed little influence of the functionalization on the structure and chemistry of 19 in comparison to its parent analogue Tp2Ru. In close analogy to the synthesis of 16, acid-functionalized derivative TpRu(p-(CO2H)C6H4Tp) 20 was readily prepared and characterized. Positively charged ruthenocene analogue 24 was subsequently converted to the azido-functionalized derivative [(p-N3C6H4Tp)RuTpm]Cl 25 by a copper-catalyzed azidonation in t-BuOH/H2O, thus proving additional synthetic scope for p-BrC6H4 functionalized Tp ligands. Functionalized mixed ligand ruthenocene analogues 16, 20 and 25 (Fig. 43) were finally tested on their suitability in the labeling of peptides by coupling to protected amino acids in solution. 44 Summary and conclusion Figure 43 Functionalized mixed sandwich ruthenium complexes Thus, acid-functionalized 16 and 20 were coupled to Phe-OMe and Val-OtBu, respectively, following a standard activation in solution. Azido-functionalized 25 was coupled to alkyne- functionalized HCC(CH2)2CO-Val-OtBu 26 by standard CuAAC methods. Resulting amides iPr Cp Ru(p-(CO-Phe-OMe)C6H4Tp) 17 and TpRu(p-(CO-Val-OtBu)C6H4Tp) 21 as well as triazole conjugate [TpmRu(p-C2N3H-(CH2)2-CO-Val-OtBu)Tp]Cl 27 were characterized, thus showing no influence of the attached amino acids on the structure of the ruthenium complexes. The relative low stability of 16 and 20 to TFA required a peptide synthesis scheme which avoided commonly used acidic cleavage/deprotection methods. Hence, a HMBA resin was used and acid functionalized 16 and 20 were coupled after Tyr deprotection to obtain the amide compounds iPr Cp Ru(p-(CO-Enk-OH)-C6H4Tp) 18 and TpRu(p-(CO-Enk-OH)-C6H4Tp) 22. Charged triazole bioconjugate [TpmRu((p-C2N3H-(CH2)2-CO-Enk-OH)-C6H4Tp)]Cl 28 was synthesized in solution according to standard CuAAC methods. The bioconjugates 18, 22, and 28 were purified and unambiguously characterized by means of ESI-MS and 1H NMR spectroscopies. Figure 44 Bioconjugates containing ruthenocene analogue mixed ligand sandwich complexes In summary, mixed ligand ruthenium sandwich compounds shown in figure 44 not only represent an extension of ruthenium compounds applicable in bioconjugate chemistry, but also give an insight to the versatile functionalization and applicability of Tp’ ligands as functionalized Cp surrogates in biological applications. 45 Summary and conclusion A different role of Tp ligands was investigated in the synthesis of tungsten compounds in the third part of this work.[214] While the Tp’ ligand served as a synthetic precursor in ruthenium compounds, the Tp* ligand in tungsten compounds is best described as a spectator ligand, nevertheless contributing decisively to the stability of the complexes. As a consequence, functional groups had to be attached by coordination of suitable alkynes, thus introducing η2-alkyne tungsten complexes 2 2- [Tp*WI(CO)(η -HCC(CH2)2CO2H)] 29 and [Tp*WI(CO)(η Fmoc-Pgl)] 30 as new compounds for the labeling of biomolecules. Figure 45 Tp*WI(CO)-η2-coordinated acid (left) and Fmoc-Pgl (right) Owing to the excellent stability of the Tp*WI(CO)-alkyne fragment, the use of either alkyne functionalized acids or amino acids permits introduction of this metal fragment at any stage during 2 SPPS. As such, the N-terminally labeled peptides [Tp*WI(CO)(η -HCC(CH2)2CO-pNT-OH)] 31 and 2 [Tp*WI(CO)(η -HCC(CH2)2CO-Enk-OH)] 32 were obtained by coupling 29 in the last step of standard SPPS experiments. Figure 46 N-terminally Tp*WI(CO)-labelled peptides Although N-terminal labeling has been established as one of the most commonly used method for metal carboxylates,[110,111,117,196,197,215,216] it sometimes still poses a problem when functionalized 46 Summary and conclusion amino acids require special and/or rather harsh conditions for subsequent side-chain deprotection, like shown in the syntheses of the ruthenium bioconjugates 18 and 22. In comparison, internal peptide modification with organometallics is rather limited.[124,125,217] As exemplified by the 2 synthesis of H2N-Tyr-Gly-[Tp*W(I)(CO)(η -Pgl)]-Phe-Leu-OH 33 (Fig. 47), internal peptide modification in this work has been made possible via a tungsten η2-propargylglycine complex 30, which withstands the conditions of all subsequent steps in SPPS. Figure 47 Sidechain-labelled Enk-derivative 33 Such metal modification at internal positions is particularly important if a free N-terminal amino group is required for receptor binding, which is indeed the case for enkephalin. Moreover, previous structure-activity studies on this peptide have shown that there should be sufficient space to accommodate a bulky substituent on the third amino acid (Gly3).[218,219] The syntheses and application of complexes such as 29 and 30 mark the road ahead for the use of heavy atom peptide derivatives with additional possibilities for IR detection in biochemical and receptor binding studies. Notably, propargyl-glycine has been identified in naturally occurring proteins of some bacteria.[220,221] It may thus be possible to design a specific label for this unusual amino acid by employing metal fragments such as the one previously described. Conclusively, this work has shown a successful approach on the application of Tp-containing complexes in the field of bioorganometallic chemistry, thus introducing mixed ligand ruthenocene analogues and Tp*WI(CO)(η2-alkyne) complexes as novel labels for biomolecules. Both compound classes have been shown to be readily accessible by modification of known synthetic methods, including the introduction of p-BrC6H4-functionalized Tp’ ligands as versatile synthetic precursors. Thus, given the number of known Tp-containing compounds, it is considered likely that similar approaches will contribute decisively to the progress of bioconjugate chemistry. 47 Experimental part 5. EXPERIMENTAL PART 5.1. Technical equipment NMR spectra were recorded at ambient temperature on Bruker DPX-250 and DRX-400 spectrometers. Chemical shifts (δ) are reported in parts-per-million (ppm) relative to the residual proton chemical shifts of the solvents set relative to external tetramethylsilane (TMS). Absolute values of the coupling constants (J) are given in Hertz (Hz). 13C{1H} assignments were obtained from standard Attached Proton Test (APT) and Heteronuclear Single Quantum Coherence (HSQC) experiments. Abbreviations: singlet (s), doublet (d), triplet (t), quartet (q), septet (sept), multiplet (m). Electrospray ionisation mass spectra were recorded on a Bruker Esquire 6000 spectrometer (4 kV, nebulizer pressure 10-20 psi, capillary temperature 300 °C, flow rate 240 µL/min). Infrared spectra of solid samples were recorded on a Bruker Tensor 27 spectrometer equipped with a Pike MIRacle Micro ATR accessory. Microwave supported syntheses were performed on a CEM Discover LabMate Synthesizer. Cyclic voltammetry was performed using a Princeton Applied Research Potentiostat in + dichloromethane solutions of 0.1 M [Bu4N][PF6]. Fc/Fc (0.001 M) was used as an external reference. The measuring cell consisted of a 1.6 mm platinum wire working electrode, a silver wire reference electrode (0.01 M AgNO3 + 0.1 M [Bu4N][PF6] and a glassy carbon auxiliary electrode. All samples were prepared under nitrogen/argon atmosphere in 0.001 M concentrations and purged with argon before measurement. All scan rates were 50 mV/s. Analytical and preparative HPLC were carried out on either a Knaur or a Varian Dynamax Prostar instrument, both using a RP Varian Dynamax column (C18 microsorb 60 Å, diameter 10 mm, length 250 mm). Eluents were water/acetonitrile (95 %/5 %, buffer A) and acetonitrile/water (95%/5%, buffer B), both containing 0.1% v/v TFA, using a linear gradient of 0%-100% buffer B for 25 min at a flow rate of 1.0 (analytical), or 7.5 (preparative) ml/min. X-ray diffraction measurements were performed on Bruker AXS and Oxford Excalibur II diffractometers (MoKα radiation 0.71073 Å, ω scan, T<-60°C), respectively. All structures were solved with direct methods (SHELXS97)[222] and refined against F2 with all measured reflections (SHELXL97 and Platon/Squeeze).[222,223] Elemental analysis of ruthenium-containing compounds were carried out at the laboratory for microanalytics and thermal analysis, University of Essen (Inorganic Chemistry Department). All other elemental analysis were carried out at the RUBiospek Biospectroscopy Department, Ruhr- University Bochum. 48 Experimental part 5.2. Methods and materials Unless noted otherwise, all preparations were carried out under an inert atmosphere of argon or nitrogen utilizing standard Schlenck techniques and a MBraun glove box. 5.3. Chemicals and solvents All chemicals and anhydrous solvents used in the described preparations were purchased from commercial sources in p.a. quality and used without further purification. Air and moisture sensitive compounds were stored under an atmosphere of nitrogen/argon or in the glove-box. Anhydrous solvents were stored over molecular sieve under an inert atmosphere of argon. [110] [110] [180] p-BrC6H4BBr2, p-BrC6H4TpLi, [Ru(H)(COD)(NH2NMe2)3][X] (X= PF6, BPh4), [171] [168] [224] [203] [166,167] CpRuCl(COD), {Cp*RuCl}4, [Tp*W(CO)3][NEt4], Tp*WI(CO)3, TpRuCl(COD) , TpK,[6] Tpm,[78,130] and pentynoic acid functionalized enkephalin (penty-Enk-OH)[124,140,141] were prepared and purified according to published procedures. 5.4. Solid Phase Supported Peptide Synthesis (SPPS) All peptides/bioconjugates were synthesized manually in filter-syringe reactors open to air following literature procedures.[196] Details on the coupling of the metal compounds and cleavage of the bioconjugates are given in each respective preparation. Tentagel-S resin containing a HMBA linker and Tentagel-S resin containing a 2-Cl-Trt linker were obtained from NovaBiochem (Germany). Fmoc-propargylglycine, HOBt, TBTU, and DIPEA were purchased from Iris Biotech (Germany). Enantiomerically pure Fmoc-protected L-amino acids were purchased from NovaBiochem (Germany) and Iris Biotech (Germany). 49 Experimental part 5.5. Syntheses and characterization [30] 5.5.1. Alternative preparation of p-BrC6H4TpNa 1 A solution of p-BrC6H4BBr2 (0.85 g, 2.61 mmol) in dichloromethane (5 mL) was added dropwise over ca. 90 minutes to a cooled (0 °C) and rapidly stirred solution of pyrazole (0.55 g, 8.09 mmol) in dichloromethane (10 mL). At the end of the addition, the mixture was stirred for 10 more minutes on the ice bath, triethylamine (0.82 g, 8.09 mmol) was added and the mixture was stirred overnight at room temperature. The volatiles were removed from the mixture under reduced pressure leaving a white solid which was extracted with THF (20 mL), filtered and cooled on an ice bath. An aqueous solution (5 mL) of sodium carbonate (0.15 g, 1.44 mmol) was added to the extract and the mixture was vigorously stirred at room temperature for 2 hours after which the solvents were removed under reduced pressure. The residue was washed with Et2O (3×5 mL) and hexane (1×5 mL), then dried under reduced pressure at 110 °C leaving 1 as a white solid (0.26 g, 0.68 mmol, 24%). Anal. calc. for C15H13BBrN6Na (M= 391.01): C, 46.08; H, 3.35; N, 21.49. Found C, 45.57; H, 3.82; N, 1 21.28%. H NMR (DMSO-d6, 250 MHz) δ 7.41 (dd, J= 0.5, 1.8, 3H, pz-H5), 7.34 (d, J= 8.3, 2H, part of C6H4), 7.22 (d, J= 8.3, 2H, part of C6H4), 6.80 (dd, J= 0.5, 1.8, 3H, pz-H3), 6.01 (t, J= 1.8 Hz, 13 3H, pz-H4); C NMR (DMSO-d6, 62.5 MHz) δ 138.8 (pz-C3), 136.3 (pz-C5), 133.5 (CH of C6H4), 128.4 (CH of C6H4), 102.6 (pz-C4) ppm. 50 Experimental part 5.5.2. p-BrC6H4TpK 2 A solution of p-BrC6H4BBr2 (1.06 g, 3.25 mmol) in dichloromethane (5 mL) was added dropwise over ca. 90 minutes to a cooled (0 °C) and rapidly stirred solution of pyrazole (0.69 g, 10.10 mmol) in dichloromethane (10 mL). At the end of the addition, the mixture was stirred for 10 more minutes on the ice bath, triethylamine (1.02 g, 10.10 mmol) was added and the mixture was stirred overnight at room temperature. The volatiles were removed from the mixture under reduced pressure leaving a white solid which was extracted with THF (20 mL), filtered and cooled on an ice bath. An aqueous solution (5 mL) of potassium carbonate (0.25 g, 1.79 mmol) was added to the extract and the resulting mixture was vigorously stirred at room temperature for 30 minutes after which the solvents were removed under reduced pressure. The residue was dissolved in THF (20 mL), stirred with potassium hydroxide pellets, filtered and the solvent was removed from the filtrate under reduced pressure. The residual solid was washed with Et2O (3×5 mL) and hexane (5 mL), then dried under reduced pressure at 110 °C leaving 2 as a white solid (0.27 g, 0.66 mmol, 20%). Anal. calc. for C15H13BBrKN6 (M= 407.12): C, 44.25; H, 3.22; N, 20.64. Found C 43.37, H 3.17, N 1 19.46%. H NMR (DMSO-d6, 250 MHz) δ 7.41 (dd, J= 2.2, 0.5, 3H, pz-H5), 7.35 (d, J= 8.3, 2H, part of C6H4), 7.22 (d, J= 8.3 Hz, 2H, part of C6H4), 6.81 (dd, J= 0.5, 1.8, 3H, pz-H3), 6.01 (t, J= 1.8 Hz, 13 3H, pz-H4); C NMR (DMSO-d6, 62.5 MHz) δ 138.6 (pz-C3), 136.4 (pz-C5), 133.3 (CH of C6H4), 128.3 (CH of C6H4), 102.4 (pz-C4) ppm. 51 Experimental part 5.5.3. p-BrC6H4TpRb 3 A solution of p-BrC6H4BBr2 (3.38 g, 10.35 mmol) in dichloromethane (10 mL) was added dropwise over ca. 90 minutes to a cooled (0 °C) and rapidly stirred solution of pyrazole (2.11 g, 31.06 mmol) in dichloromethane (10 mL). At the end of the addition, the mixture was stirred for 10 more minutes on the ice bath then triethylamine (3.14 g, 31.06 mmol) was added and the mixture was stirred overnight at room temperature. The volatiles were removed from the mixture under reduced pressure leaving a white solid which was extracted with THF (30 mL), filtered and cooled on an ice bath. An aqueous solution (2 mL) of rubidium carbonate (0.75 g, 5.15 mmol) was added to the extract. The resulting mixture was vigorously stirred at room temperature for 3 hours after which the solvents were removed under reduced pressure. The residue was washed with Et2O (3×10 mL) and dried under reduced pressure. The residue was extracted with warm acetone (100 mL, ca. 50 °C), filtered and the solvent removed under reduced pressure leaving 3 as a white solid. The temperature was raised to 110 °C and the 3 was dried overnight (1.22 g, 2.69 mmol, 26%). Anal. calc. for C15H13BBrN6Rb (M= 453.49): C, 39.73; H, 2.89; N, 18.53. Found C, 39.74; H, 2.43; 1 N, 18.09%. H NMR (DMSO-d6, 250 MHz) δ 7.41 (dd, J= 0.5, 1.8, 3H, pz-H5), 7.37 (d, J= 8.3, 2H, part of C6H4), 7.24 (d, J= 8.3, 2H, part of C6H4), 6.80 (dd, J= 0.5, 1.8, 3H, pz-H3), 6.01 (t, J= 1.8 13 Hz, 3H, pz-H4). C NMR (DMSO-d6, 62.5 MHz) δ 138.6 (pz-C5), 136.5 (pz-C3), 133.3 (CH of C6H4), 128.2 (CH of C6H4), 102.5 (pz-C4) ppm. 52 Experimental part 5.5.4. p-BrC6H4TpCs 4 A solution of p-BrC6H4BBr2 (0.50 g, 1.53 mmol) in dichloromethane (5 mL) was added dropwise over ca. 90 minutes to a cooled (0 °C) and rapidly stirred solution of pyrazole (0.35 g, 4.60 mmol) in dichloromethane (ca. 10 mL). At the end of the addition, the mixture was stirred for 10 more minutes on the ice bath then triethylamine (0.47 g, 4.60 mmol) was added and the mixture was stirred overnight at room temperature. The volatiles were removed from the mixture under reduced pressure leaving a white solid which was extracted with THF (10 mL), filtered and cooled on an ice bath. An aqueous solution (2 mL) of cesium carbonate (0.15 g, 0.77 mmol) was added to the extract, which produced a mild effervescence. The resulting mixture was vigorously stirred at room temperature for 1 hour after which the solvent was removed under reduced pressure. The residue was washed with Et2O (2×10 mL) and hexane (5 mL), then dried under reduced pressure at 110 °C leaving 4 as a white solid (0.20 g, 0.40 mmol, 26%). Anal. calc. for C15H13BBrCsN6 (M= 500.92): C, 35.97; H, 2.62; N, 16.78. Found C, 35.54, H, 2.86, N, 1 15.81%. H NMR (DMSO-d6, 250 MHz) δ 7.41 (dd, J= 0.5, 1.8, 3H, pz-H5), 7.34 (d, J= 8.3, 2H, part of C6H4), 7.22 (d, J= 8.3, 2H, part of C6H4), 6.81 (dd, J= 0.5, 1.8 Hz, 3H, pz-H3), 6.01 (t, J= 1.8 Hz, 13 3H, pz-H4); C NMR (DMSO-d6, 62.5 MHz) δ 138.5 (pz-C5), 136.5 (pz-C3), 133.3 (CH of C6H4), 128.2 (CH of C6H4), 102.6 (pz-C4) ppm. Solid state structure determination: A crystal of 4 (colourless needle), obtained by slow evaporation of an acetone solution, was placed on a glass capillary in perfluorinated oil and measured in a cold gas flow. C15CsH13BBrN6, M= 500.92, triclinic, a= 8.540(4) Å, b= 15.045(6) Å, c= 15.879(7) Å, α= 65.853(8)°, β= 88.457(8)°, γ= 75.056(8)°, V= 1791.4(13) Å3, space group P , 2 Z= 4, 6215 reflections collected, 5461 unique (Rint= 0.0555), wR(F )= 0.1369 (all data). CCDC-797015 contains the supplementary crystallographic data for 4. 53 Experimental part 5.5.5. p-BrC6H4TpTl 5 A solution of p-BrC6H4BBr2 (0.56 g, 1.72 mmol) in dichloromethane (5 mL) was added dropwise over ca. 90 minutes to a cooled (0 °C) and rapidly stirred solution of pyrazole (0.35 g, 5.15 mmol) in dichloromethane (10 mL). At the end of the addition, the mixture was stirred for 10 more minutes on the ice bath then triethylamine (0.52 g, 5.15 mmol) was added and the mixture was stirred overnight at room temperature. The volatiles were removed from the mixture under reduced pressure leaving a white solid which was extracted with THF (15 mL) and filtered into a flask containing a solution of thallium ethoxide (0.43 g, 1.72 mmol) in THF (2 mL). The mixture was stirred overnight to produce a white precipitate which was isolated by filtration, washed with methanol (3×10 mL), and dried giving 5 as a white solid (0.18 g, 0.31 mmol, 18 %). THF was removed from the filtrate, the residue washed, then dried under reduced pressure giving another 0.18 g (0.31 mmol, 18 %) of 5. Anal. calc. for C15H13BBrN6Tl (M= 572.40): C, 31.47; H, 2.29; N, 14.68. Found: C, 31.18; H, 2.76; N, 1 14.41%. H NMR (DMSO-d6, 250 MHz) δ 7.64 (dd, J= 2.3, 0.4, 3H, pz-H5), 7.49 (d, J= 8.2, 2H, part of C6H4), 7.44 (dd, J= 2.3, 0.4, 3H, pz-H4), 7.05 (d, J= 8.2, 2H, part of C6H4), 6.28 (t, J= 2.3, 13 3H, pz-H4); C NMR (DMSO-d6, 62.5 MHz) δ 139.3 (pz-C3), 135.9 (pz-C5), 134.1 (CH of C6H4), 129.2 (CH of C6H4), 103.1 (pz-C4) ppm. 54 Experimental part 5.5.6. (p-BrC6H4Tp)2Mg 6 An aqueous solution (2 mL) of magnesium chloride (20 mg, 0.21 mmol) in water was added slowly to a solution of p-BrC6H4TpRb (200 mg, 0.44 mmol) in THF (4 mL) and the mixture was stirred for 1 hour. After removal of the volatiles, the colourless residue was extracted with dichloromethane (2×4 mL) and the extract filtered over a silica plug. Removal of the solvent yielded a colourless residue that was washed with water (2×2 mL) and Et2O (2×2 mL), then dried under lowered pressure at 110 °C over night to give 6 as a colourless solid (112 mg, 0.15 mmol, 71%). Anal. calc. for C30H26B2Br2MgN12 (M= 760.38): C, 47.39; H, 3.45; N, 22.11. Found C, 47.34; H, 3.66; 1 N, 22.12. H NMR (DMSO-d6, 250 MHz) δ 7.58 (dd, J= 1.8, 0.5, 6H, pz-H5), 7.54 (d, J= 8.3, 4H, part of C6H4), 7.41 (d, J= 8.3, 4H, part of C6H4), 7.18 (dd, J= 1.8, 0.5, 6H, pz-H3), 6,19 (t, J= 1.8, 13 6H, pz-H4); C NMR (DMSO-d6, 62.6 MHz) δ 140.9 (pz-C3), 135.1 (pz-C5), 133.1 (CH of C6H4), 128.3 (CH of C6H4), 103.9 (pz-C4) ppm. Solid state structure determination: A crystal of 6 (colourless needle), obtained by slow evaporation of a chloroform solution, was placed on a glass capillary in perfluorinated oil and measured in a cold gas flow. C30H26B2Br2MgN12, M= 760.38, triclinic, a= 10.957(3) Å, b= 12.144(4) Å, c= 12.517(4) Å, α= 97.219(7)°, β= 99.152(7)°, γ= 91.674(6)°, V= 1629.3(8) Å3, space group P , 2 Z= 2, 9090 reflections collected, 5706 unique (Rint= 0.0388), wR(F )= 0.1905 (all data). CCDC-741193 contains the supplementary crystallographic data for compound 6. 55 Experimental part Me 5.5.7. p-BrC6H4Tp K 7 A solution of p-BrC6H4BBr2 (3.47 g, 10.6 mmol) in dichloromethane (15 mL) was added dropwise to a cooled (0 °C) and well stirred solution of 3-methylpyrazole (2.71 g, 33.0 mmol) in dichloromethane (8 mL) over 60 minutes. After complete addition, triethylamine (4.57 mL, 33.0 mmol) was added and the solution was stirred at ambient temperature for 12 hours. The volatiles were removed under lowered pressure and the remaining colourless solid was extracted with THF (2x20 mL). Solid KOtBu (1.20 g, 10.6 mmol) was added portionwise to the extract and the resulting solution was stirred for 30 minutes. Removal of the volatile components gave a pale yellow solid, which was washed with cold Et2O (-20 °C, 2×35 mL). Drying under lowered pressure gave 7 as a colourless solid (1.95 g, 4.34 mmol, 41%). Anal. calc. for C18H19BBrKN6 (M= 449.20): C, 48.13; H, 4.26; N, 18.71. Found: C, 47.33; H, 4.31; N, 1 18.10. H NMR (DMSO-d6, 250 MHz) δ 7.37 (d, J= 8.2, 2H, part of C6H4), 7.22 (d, J= 8.2, 2H, part 13 of C6H4), 6.70 (d, J= 1.8, 3H, pz-H5), 5.75 (d, J= 1.8, 3H, pz-H4), 2.12 (s, 9H, pz-CH3); C NMR (DMSO-d6, 62.5) δ 146.2 (pz-C5), 136.6 (CH of C6H4), 134.3 (CH of C6H4), 102.0 (pz-C4), 13.9 (pz- CH3) ppm. 56 Experimental part Me 5.5.8. p-BrC6H4Tp Tl 8 A solution of p-BrC6H4BBr2 (2.00 g, 6.12 mmol) in dichloromethane (10 mL) was added over 60 minutes to a cooled (0 °C) and rapidly stirred solution of 3-methylpyrazole (1.56 g, 18.97 mmol) and triethylamine (2.66 mL, 21.64 mmol) in dichloromethane (4 mL). After stirring for 12 hours at room temperature, the solvent was removed and the remaining white solid was extracted with THF (2x15 mL) and thalliumethoxide (1.85 g, 6.98 mmol) was added to the extract. After stirring for 12 hours, the solvent was removed to give a colourless solid, which was washed with Et2O (2×10 mL). Drying under lowered pressure yielded 8 as a colourless solid (2.05 g, 3.34 mmol, 55%). Anal. calc. for C18H19BBrN6Tl (M= 614.48): C, 35.18; H, 3.12; N, 13.68. Found C, 35.81; H, 3.57; N, 1 13.03%. H NMR (CDCl3, 250 MHz) δ 7.51 (d, J= 8.4, 2H, part of C6H4), 7.31 (d, J= 8.4, 2H, part of 13 C6H4), 7.25 (d, J= 2.0, 3H, pz-H5), 5.99 (d, J= 2.0, 3H, pz-H4), 2.36 (s, 9H, pz-CH3); C NMR (CDCl3, 62.5 MHz) δ 149.6 (pz-C5), 137.1 (CH of C6H4), 136.5 (CH of C6H4 ), 130.9 (C-B), 122.3 (C- Br), 104.9 (pz-C4), 13.7 (pz-CH3) ppm. 57 Experimental part 5.5.9. p-BrC6H4Tp*K 9 A solution of p-BrC6H4BBr2 (3.50 g, 10.7 mmol) in dichloromethane (15 mL) was added dropwise to a cooled (0 °C) and well stirred solution of 3-methylpyrazole (3.19 g, 33.2 mmol) in dichloromethane (10 mL) over 60 minutes. After complete addition, triethylamine (4.54 mL, 33.2 mmol) was added and the solution was stirred at ambient temperature for 12 hours. The volatiles were removed under lowered pressure and the remaining colourless solid was extracted with THF (2×20 mL). Solid KOtBu (1.33 g, 11.8 mmol) was added portionwise to the extract and the resulting solution was stirred for 30 minutes. Removal of the volatile components gave a pale yellow solid, which was purified by washing with cold Et2O (-20 °C, 2×35 mL). Drying under lowered pressure gave 9 as a colourless solid (2.95 g, 6.0 mmol, 56%). Anal. calc. for C21H25BBrKN6 (M= 491.28): C, 51.34; H, 5.13; N, 17.11. Found: C, 50.83; H, 4.97; N, 1 17.53. H NMR (DMSO-d6, 250 MHz) δ 7.15 (d, J= 8.2, 2H, part of C6H4), 6.89 (d, J= 8.2, 2H, part 13 of C6H4), 5.55 (s, 3H, pz-4H), 1.98 (s, 9H, pz-CH3), 1.41 (s, 9H, pz-CH3); C NMR (DMSO-d6, 62.5 MHz) δ 136.9 (CH of C6H4), 128.0 (CH of C6H4), 104.8 (pz-C4), 14.0 (pz-CH3), 12.7 (pz-CH3) ppm. 58 Experimental part 5.5.10. p-BrC6H4Tp*Tl 10 A solution of p-BrC6H4BBr2 (0.50 g, 1.53 mmol) in dichloromethane (5 mL) was added dropwise over ca. 90 minutes to a cooled (0 °C) and rapidly stirred solution of 3,5-dimethylpyrazole (0.50 g, 5.20 mmol) in dichloromethane (10 mL). At the end of dropwise addition, the mixture was stirred for 10 more minutes on the ice bath then triethylamine (0.51 g, 5.04 mmol) was added and the mixture was stirred for 4 hours at room temperature. The volatiles were removed from the mixture under reduced pressure leaving a white solid which was extracted with hexane (20 mL), then THF (10 mL) and filtered. TlOEt (0.80g, 3.21 mmol) was added to the extract and the mixture was stirred overnight to produce a white precipitate which was isolated by filtration, washed with hexane (5×10 mL) and dried under reduced pressure giving 10 as a white solid (0.25 g, 25%). Anal. calc. for C21H25BBrN6Tl (M= 656.56): C, 38.42; H, 3.84; N, 12.80. Found: C, 39.14; H, 4.18; 1 N, 13.29%. H NMR (CDCl3, 250 MHz) δ 7.35 (d, J= 8.2, 2H, part of C6H4), 7.18 (d, J= 8.2, 3H, part 13 of C6H4), 5.84 (s, 3H, pz-H4), 2.19 (s, 9H, pz-CH3), 1.67 (s, 9H, pz-CH3); C NMR (CDCl3, 62.5 MHz) δ 136.8 (CH of C6H4), 130.3 (CH of C6H4), 108.25 (pz-C4), 14.1 (pz-CH3), 13.6 (pz-CH3) ppm. Solid state structure determination: A crystal of 10 (colourless needle), obtained by slow evaporation of a dichloromethane solution, was placed on a glass capillary in perfluorinated oil and measured in a cold gas flow. C21H25BBrN6Tl, M= 656.56, triclinic, a= 8.0989(6) Å, b= 8.3437(6) Å, c= 17.0443(12) Å, α= 85.7240(10)°, β= 78.4870(10)°, γ= 76.5310(10)°, V= 1097.07(14) Å3, space 2 group P , Z= 2, 7632 reflections collected, 4917 unique (Rint= 0.0157), wR(F )= 0.0983 (all data). CCDC-741194 contains the supplementary crystallographic data for compound 10. 59 Experimental part 5.5.11. CpRu(p-BrC6H4Tp) 11 CpRuCl(COD) (0.11 g, 0.36 mmol) and p-BrC6H4TpLi (0.15 g, 0.39 mmol) were mixed and dissolved in THF (10 mL) and the solution was stirred for 2 hours. The solvent was removed under lowered pressure and the residue was extracted with boiling chloroform (50 mL). Filtration of the light yellow extract followed by removal of the solvent yielded 11 as a pale yellow solid (85.5 mg, 0.16 mmol, 45 %). 1 H NMR (CDCl3) δ 8.11 (dd, J= 2.2; 0.5, 3H, pz-H5), 7.75 (d, J= 8.4, 2H, part of C6H4), 7.63 (d, J= 8.4, 2H, part of C6H4), 7.40 (d, J= 2.2; 0.5, 3H, pz-H3), 6.13 (t, J= 2.2, 3H, pz-H4), 4.30 (s, 5H, C5H5) ppm. 60 Experimental part Me 5.5.12. CpRu(p-BrC6H4Tp ) 12 Me CpRuCl(COD) (0.17 g, 0.55 mmol) and p-BrC6H4Tp Tl (0.29 g, 0.46 mmol) were mixed and THF (10 mL) was added. Stirring for 1 hour gave a pale green solution, that was filtered. The remaining insolubles were extracted with THF (3x5 mL). Removal of the solvent from the combined extracts and filtrate gave 12 as a yellow solid (0.12 g, 0,21 mmol, 45%). 1 H NMR (CDCl3, 250 MHz) δ 7.69 (d, J= 8.4, 2H, part of C6H4), 7.56 (d, J= 8.4, 2H, part of C6H4), 7.28 (d, J= 2.2, 3H, pz-H5), 5.93 (d, J= 2.2, 3H, pz-H4), 4.71 (s, 5H, C5H5), 2.50 (s, 9H, pz-CH3) ppm. 61 Experimental part 5.5.13. Cp*Ru(p-BrC6H4Tp) 13 THF (4 mL) was slowly added to a mixture of {Cp*RuCl4} (0.31 g, 1.15 mmol) and p-BrC6H4TpLi (0.43 g, 1.15 mmol). The resulting orange solution was stirred at room temperature for 2 hours. Upon removal of the solvent, a yellow solid precipitated from the solution. It was isolated by filtration, washed with Et2O (2x15 mL) and dried under lowered pressure to give 13 as a yellow solid (0.21 g, 0.35 mmol, 30%). 1 H NMR (C6D6, 250 MHz) δ 8.07 (dd, J= 2.2; 0.5, 3H, pz-H5), 7.52 (d, J= 8.4, 2H, part of C6H4), 7.32 (d, J= 8.4, 2H, part of C6H4), 7.27 (dd, J= 2.2; 0.5, 3H, pz-H3), 5.97 (t, J= 2.2, 3H, pz-H4), 1.59 (s, 15H, C5(CH3)5) ppm. 62 Experimental part 5.5.14. CpiPrRuCl(COD) 14 iPr A solution of [Ru(H)(COD)(NH2NMe2)3][PF6] (2.14 g, 4.0 mmol) and NaCp (0.57 g, 4.4 mmol) in THF (30 mL) was heated to reflux for 45 minutes. Removal of the volatiles gave a brown solid which was extracted with hexane (2x35 mL). Dropwise addition of tetrachloromethane (0.5 mL, 4.0 mmol) to the extract produced an orange precipitate, which was separated by filtration at 0 °C and dried under lowered pressure to yield 14 as a golden orange solid (1.20 g, 3.41 mmol, 85 %). 1 Anal. calc. for C16H23ClRu (M= 366.91): C, 54.61; H, 6.59. Found C, 54.76; H, 6.63%. H NMR iPr (CDCl3, 250 MHz) δ 5.14 (m, 2H, olefinic H of COD), 4.78 (s, 4H, Cp ), 4.26 (m, 2H, olefinic H of COD), 2.63-2.54 (m, 3H, aliphatic H of COD, CH(CH3)2), 2.07 (m, 4H, aliphatic H of COD), 1.97 13 (m, 2H, aliphatic H of COD), 1.08 (d, J= 6.9, 6H, CH(CH3)2); C NMR (CDCl3, 62.5 MHz) δ 88.3 iPr iPr (olefinic C of COD), 86.2 (olefinic C of COD), 80.7 (Cq of Cp ), 78.7 (CH of Cp ), 32.4 (aliphatic C of COD), 28.1 (aliphatic C of COD), 25.8 (CH(CH3)2), 22.7 (CH3) ppm. 63 Experimental part iPr 5.5.15. Cp Ru(p-BrC6H4Tp) 15 p-BrC6H4TpLi (0.23 g, 0.60 mmol) and 14 (0.19 g, 0.53 mmol) were mixed and dissolved in THF (15 mL). The solution was stirred for 45 minutes at room temperature and filtered to give a clear light green solution which was reduced to ca. 5 mL under lowered pressure. Cooling the solution to -20 °C over night yielded a yellow precipitate that was isolated by filtration and dried under lowered pressure to give 15 as a yellow microcrystalline solid (0.23 g, 0.40 mmol, 76%). Anal. calc. for C23H24BBrN6Ru (M= 576.26): C, 47.94; H, 4.20; N, 14.58. Found: C, 47.95; H, 4.74; 1 N, 14.82%. H NMR (CDCl3, 250 MHz) δ 8.09 (dd, 3H, J= 2.2; 0.5, pz-H5), 7.77 (d, J= 8.4, 2H, part of C6H4), 7.61 (d, J= 8.4, 2H, part of C6H4), 7.39 (dd, J= 2.2; 0.5, 3H, pz-H3), 6.12 (t, J= 2.2, 3H, pz-H4), 4.30 (pseudo t, J= 1.6, 2H, part of CpiPr), 4,06 (pseudo t, J= 1.6, 2H, part of CpiPr), 2.60 13 (sept, 1H, J= 6.8, CH(CH3)2), 1.18 (d, 6H, J= 6.8, CH(CH3)2); C NMR (CDCl3, 62.5 MHz) δ 145.0 (pz-C3), 136.5 (pz-C5), 134.9 (C-B), 136.5 (CH of C6H4), 134.0 (CH of C6H4), 127.9 (C-Br), 105.1 (pz- iPr iPr iPr C4), 85.5 (Cq of Cp ), 73.1 (CH of Cp ), 64.8 (CH of Cp ), 26.7 (CH(CH3)2), 24.1 (CH3) ppm. ESI- + iPr + + MS (pos.) m/z 432.12 ([M-C6H4Br+H] ), 498.46 ([M-Cp +Na] ), 577.32 ([M+H] ), exact mass for C23H24BBrN6Ru= 576.04. 64 Experimental part iPr 5.5.16. Cp Ru(p-(CO2H)-C6H4Tp) 16 A solution of 15 (0.10 g, 0.17 mmol) in THF (25 mL) was cooled to -70 °C. n-BuLi (0.12 mL, 1.6 M in hexane, 0.19 mmol) was added dropwise and the resulting mixture was stirred for one hour. After addition of excess solid CO2 (~5g), the solution was allowed to warm to room temperature. The volatile components were removed under lowered pressure to obtain a pale yellow solid. On the benchtop open to air, it was slurried in water (5 mL) and treated with HCl (2 mL, 2 N) to give a yellow precipitate that was washed with water (2x10 ml) and dried under lowered pressure to give 16 as a bright yellow solid (38 mg, 0.07 mmol, 43%). Anal. calc. for C24H25BN6O2Ru (M= 542.12): C, 53.25; H, 4.65; N, 15.52. Found: C, 53.23; H: 4.80; 1 N, 15.09%. H NMR (DMSO-d6, 250 MHz) δ 12.9 (s, 1H), 8.26 (d, J= 2.2, 3H, pz-H5), 8.07 (d, J= 8.4, 2H, part of C6H4), 7.95 (d, J= 8.4, 2H, part of C6H4), 7.37 (d, J= 2.2, 3H, pz-H3), 6.19 (t, J= 2.2, 3H, pz-H4), 4.54 (pseudo t, 2H, part of CpiPr), 4.13 (pseudo t, 2H, part of CpiPr), 2.57 (sept, J= 13 6.8, 1H, CH(CH3)2), 1.11 (d, J= 6.8, 6H, CH(CH3)2); C NMR (DMSO-d6, 62.5 MHz) δ 167.4 (CO2H), 145.2 (pz-C3), 136.8 (C-B), 134.6 (pz-C3), 133.7 (CH of C6H4), 128.6 (CH of C6H4), 128.0 iPr iPr iPr (C-CO2H), 105.4 (pz-C4), 84.3 (Cq of Cp ), 73.6 (CH of Cp ), 64.7 (CH of Cp ), 25.9 (CH(CH3)2), -1 23.9 (CH3) ppm. IR (solid) 1685 cm (CO2H) Solid state structure determination of 16: A crystal of 16 (yellow needle), obtained by slow evaporation of a dichloromethane solution, was placed on a glass capillary in perfluorinated oil and measured in a cold gas flow. C24H25BN6O2Ru M= 542.12, triclinic, a= 9.38(3) Å, b= 13.62(4) Å, c= 20.44(7) Å, α= 88.10(8)°, β= 78.75(7)°, γ= 79.44(13)°, V= 2517(13) Å3, space group P , Z= 4, 11644 2 reflections collected, 8442 unique (Rint= 0.0739), wR(F )= 0.2462 (all data). CCDC-699278 contains the supplementary crystallographic data for compound 16. 65 Experimental part iPr 5.5.17. Cp Ru(p-(CO-Phe-OMe)C6H4Tp) 17 TBTU (112 mg, 0.35 mmol) and 16 (190 mg, 0.35 mmol) were mixed in dichloromethane (12 mL) and DIPEA (0.41 mL, 2.45 mmol) was added to the slurry. After 10 minutes, phenylalanine- methylester hydrochloride (76 mg, 0.35 mmol) was added and the resulting solution was stirred over night. Removal of the solvent gave a brown residue, that was extracted with a minimum amount of dichloromethane. The solution was eluted from a silica column (60 Å, dichloromethane) to give 17 as a yellow solid (130 mg 0.19 mmol, 53%) after removal of the solvent. Anal. calc. for C34H36BN7O3Ru (M= 702.58): C, 58.12; H, 5.16; N, 13.96. Found: C, 58.11; H, 5.34; 1 N, 13.87 %. H NMR (CDCl3, 250 MHz) δ 8.10 (d, J= 2.2, 3H, pz-H5), 7.98 (d, J= 8.4, 2H, part of C6H4), 7.83 (d, J= 8.4, 2H, part of C6H4), 7.39 (d, J= 2.2, 3H, pz-H3), 7.33-7.16 (m, 5H, C6H5 of Phe), 6.64 (d, J= 7.5, 1H, NH), 6.12 (t, J= 2.2, 3H, pz-H4), 5.16 (m, 1H, α-CH), 4.31 (pseudo t, iPr iPr J=1.6, 2H, part of Cp ), 4.06 (pseudo t, J=1.6, 2H, part of Cp ), 3.30 (m, 2H, β-CH2), 2.61 (sept, 13 J= 6.8, 1H, CH(CH3)2), 1.18 (d, J= 6.8, 6H, CH(CH3)2); C NMR (CDCl3, 62.5 MHz) δ 171.5 (CO2Me), 167.0 (CONH), 147.7 (pz-C3), 140.0 (Cq of C6H4), 135.2 (C-B), 134.2 (CH of C6H4), 129.5- iPr iPr iPr 126.4 (CH of C6H4 and Phe), 105.3 (pz-C4), 85.4 (Cq of Cp ), 73.1 (CH of Cp ), 64.8 (CH of Cp ), 53.7 (α -CH), 52.6 (OCH3), 38.1 (β-CH2), 26.6 (CH(CH3)2), 24.3 (CH3) ppm. ESI-MS (pos.) m/z: + iPr + 704.09 ([M+H] ), 443.19 ([Cp Ru(pz)3+Na] ), exact mass for C34H36BN7O3Ru= 703.20. IR (solid): -1 1740 (CO2Me), 1653 (CONH) cm . 66 Experimental part iPr 5.5.18. Cp Ru(p-(CO-Tyr-Gly-Gly-Phe-Leu-OH)C6H4Tp) 18 Resin-bound enkephalin Tyr(2-Cl-Trt)-Gly-Gly-Phe-Leu-(HMBA-RES) was obtained by standard Fmoc-SPPS starting from Fmoc-Leu loaded Tentagel S resin with a HMBA linker (500 mg, loading 0.24 mmol/g). After 2-Cl-Trt deprotection of the tyrosine phenolic hydroxy group with 5% TFA v/v / 5% TIS v/v in dichloromethane the metal complex 16 was coupled to the peptide as described here: 16 (140 mg, 0.23 mmol), TBTU (150 mg, 0.47 mmol), HOBt (65 mg, 0.48 mmol) and DIPEA (121 μL, 0.72 mmol) were mixed in DMF (3 mL) and stirred for 5 minutes. The homogenous solution was then added to the resin-bound peptide and the mixture was shaken for ca. 20 hours. After filtering the reaction mixture, the resin-bound product was washed with DMF (5x2 mL) and dichloromethane (5x2 mL) and dried under reduced pressure for one hour. The bioconjugate was cleaved from the resin by shaking it with a cooled (0 °C) mixture of aqueous NaOH solution (2.5 mL, 1M) and 1,4-dioxane (7.5 mL) for 10 minutes. The resulting yellow solution was filtered and adjusted to pH= 7 by addition of dilute hydrochloric acid. Removal of the solvents under lowered pressure gave a mixture of colourless NaCl and a dark yellow solid. It was taken into a minimum volume of methanol, filtered from NaCl and purified by RP-HPLC to give 18 as a yellow solid (80 mg, 0.07 mmol, 62% based on resin load). 1 H NMR (CD3OD, 400 MHz) Note: The resonances from one α-CH and two β-CH2 groups were obscured by the solvent peaks. Assignments are based, in part, upon comparison to literature [198] data. δ 8.04 (d, J= 8.0, 2H, part of C6H4), 7.96 (d, J= 8.0, 2H, part of C6H4), 7.84 (d, J= 2.2, 3H, pz-H5), 7.63 (d, J= 2.2, 3H, pz-H3), 7.26-7.14 (m, 5H, C6H5 of Phe), 7.15 (d, J= 8.0, 2H, part of Tyr p-phenol), 6.75 (d, J= 8.0, 2H, part of Tyr p-phenol), 6.32 (d, J= 2.2, 3H, pz-H4), 4.66 (dd, J= 9.0, 5.5, 1H, α-CH), 4.62 (s, 2H, part of CpiPr), 4.47 (s, 2H, part of CpiPr), 4.40 (dd, J= 9.0, 5.5, 1H, α- CH), 3.86 (m, 4H, α-CH2 of Gly), 3.24 (dd, J= 13.8, 9.0, 2H, β-CH2), 2.97 (dd, J= 13.8, 9.0, 2H, β- iPr CH2), 1.70-1.58 (m, 4H, α-CH, β-CH2, CH(CH3)2 of Cp ), 1.19 (d, J= 6.3 Hz, 6H, CH(CH3)2), 0.88 (d, J= 6.3, 3H, Leu-CH3), 0.83 (d, J= 6.3, 3H, Leu-CH3) ppm. ESI-MS (neg. mode) 1078.29 [M- + - H ] , exact mass for C52H60BN11O8Ru= 1079.38. 67 Experimental part 5.5.19. TpRu(p-BrC6H4Tp) 19 Conventional Synthesis TpRuCl(COD) (680mg, 1.49 mmol) and 2 (667 mg, 1.63 mmol) were mixed and DMF (8 mL) was added. The resulting yellow solution was heated to 155 °C for 12 hours. Removal of the volatiles under lowered pressure gave a dark orange solid. Open to air, it was taken into a minimal amount of dichloromethane, filtered from insoluble KCl and loaded onto a silica column. Elution with hexane/dichloromethane (10:1) spread a yellow band that was washed down with hexane/dichloromethane (5:1). Removal of the solvents and drying under lowered pressure yielded 19 as a light yellow solid (191 mg, 0.28 mmol, 19%). Microwave-assisted synthesis TpRuCl(COD) (492mg, 1.08 mmol) and 2 (481 mg, 1.18 mmol) were mixed and THF (5 mL) was added. The resulting mixture was heated to 155 °C for 1 hour in a microwave reactor. The resulting red solution was filtered, open to air, over a celite plug and the volatiles were removed to obtain a deep red solid that was taken into a minimal amount of dichloromethane and loaded onto a silica column. Elution with hexane/dichloromethane (10:1) spread a yellow band that was washed down with hexane/ dichloromethane (5:1). Removal of the solvents and drying under reduced pressure yielded 19 as a light yellow solid (290 mg, 0.42 mmol, 39%). An analytically pure sample of 19·hexane was obtained by recrystallization from dichloromethane/hexane. Anal. calc. for C30H37B2BrN12Ru (M= 682.12): C, 46.90; H, 4.85; N, 21.88. Found: C 46.60; H, 4.38; 1 N, 22.11 %. H NMR (CDCl3, 250 MHz,): δ 8.11 (d, J= 8.4, 2H, part of C6H4), 7.85 (d, J= 1.9, 3H, pz- H5), 7.74 (d, J= 8.4, 2H, part of C6H4), 7.68 (d, J= 1.9, 3H, pz-H5), 6.89 (s, 6H, pz-H3), 6.16 (t, J= 13 1.9, 3H, pz-H4), 6.12 (t, J= 1.9, 3H, pz-H4); C NMR (CDCl3, 62.5 MHz,): δ 144.1 (pz-C3), 143.3 (pz-C3), 137.1 (CH of C6H4), 135.4 (pz-C5), 135.1 (pz-C5), 131.32 (CH of C6H4), 128.9 (C-Br), 105.6 (pz-C4), 105.5 (pz-C4) ppm. Solid state structure determination of 19: A crystal of 19 (yellow cube), obtained by slow evaporation of a hexane solution, was placed on a glass capillary in perfluorinated oil and measured in a cold gas flow. C24H23B2BrN12Ru, M= 682.12, monoclinic, a= 9.1476(2) Å, b= 3 17.3437(3) Å, c= 17.1771(3) Å, α= γ= 90°, β= 99.939(2)°, V= 2684.30(9) Å , space group P21/c, Z= 2 4, 9542 refelctions collected, 4723 unique (Rint= 0.0228), wR(F )= 0.0565 (all data). CCDC-781625 contains supplementary crystallographic data for compound 19. 68 Experimental part 5.5.20. TpRu(p-(CO2H)-C6H4Tp) 20 A solution of 19 (840 mg, 1.22 mmol) in THF (10 mL) was cooled to -70 °C. n-BuLi (0.84 mL, 1.6 M in hexane, 1.34 mmol) was added dropwise and the reaction mixture was stirred for 1 hour. Excess solid CO2 (~5 g) was added and the mixture was allowed to warm to room temperature. After removal of the volatiles under reduced pressure, the remaining bright yellow solid was slurried in water (15 mL) open to air. Dropwise addition of 1 M HCl (~3 mL) produced a dark yellow precipitate, that was separated by filtration, washed with water (2×10 mL) and pentane (2×15 mL) and dried under reduced pressure to give 20 as a pale yellow solid (580 mg, 0.89 mmol, 72%). Recrystallization from Et2O gave an analytically pure sample of 20·Et2O. Anal. calc. for C29H34B2N12O3Ru (M= 647.23): C, 48.29; H, 4.75; N, 23.30. Found: C, 48.00; H, 1 4.25; N, 23.93 %. H NMR (DMSO-d6, 250 MHz): δ 13.2 (broad s, 1H, CO2H), 8.22 (d, J= 8.4, 2H, part of C6H4), 8.17 (d, J= 8.4, 2H, part of C6H4), 8.02 (d, J= 1.9, 3H, pz-H5), 7.72 (d, J= 1.9, 3H, pz- 13 H5), 6.72 (broad s, 6H, pz-H3), 6.25 (broad s, 6H, pz-H4); C NMR (DMSO-d6, 62.5 MHz): δ 168.5 (CO2H), 143.3 (pz-C3), 142.6 (pz-C3), 135.7 (pz-C5), 135.1 (pz-C5), 134.8 (CH of C6H4), 133.5 (CH of -1 C6H4), 106.0 (pz-C3), 105.8 (pz-C3) ppm. IR (solid) 1685 (CO2H) cm . 69 Experimental part 5.5.21. TpRu(p-(CO-Val-OtBu)C6H4Tp) 21 To a solution of 20 (301 mg, 0.47 mmol) in dichloromethane (10 mL), TBTU (151 mg, 0.47 mmol) was added. After stirring the mixture for 10 minutes, DIPEA (0.56 mL, 3.29 mmol) was added. After 10 minutes, Val-OtBu·HCl (97 mg, 0.47 mmol) was added and the resulting solution was stirred over night. Removal of the volatiles gave a brown oil, which was dissolved in a minimal amount of dichloromethane, loaded onto a small silica column and eluted with dichloromethane to obtain a yellow solution. Removal of the solvent under lowered pressure gave 21 as a bright yellow solid (321 mg, 0.40 mmol, 85%). An analytically pure sample of 21 was obtained by recrystallization from Et2O/hexane. Anal. calc. for C34H41B2N13O3Ru (M= 802.47): C, 50.89; H, 5.15; N, 22.69. Found: C, 50.71; H, 5.10; 1 N, 23.13 %. H NMR (CDCl3, 250 MHz): δ 8.49 (d, J= 8.4, 2H, part of C6H4), 8.12 (d, J= 8.4, 2H, part of C6H4), 7.57 (broad s, 3H, pz-H5), 7.39 (broad s, 3H, pz-H5), 6.90 (d, J= 6.3, NH), 6.00 (broad s, 6H, pz-H3), 5.90 (broad s, 6H, pz-H4), 4.81 (m, 1H, α-CH), 2.38 (m, 2H, OCH(CH3)3 and 13 CH(CH3)2), 1.55 (s, 12H, OCH(CH3)3), 1.09 (d, J= 6.9, CH(CH3)2), 1.07 (d, J= 6.9, CH(CH3)2); C NMR (CDCl3, 62.5 MHz): δ 171.7 (CO2tBu), 167.6 (CONH), 143.2 (pz-C3), 143.1 (B-C), 142.24 (pz- C3), 136.4 (CH of C6H4), 136.0 (pz-C5), 135.9 (C-CONH), 135.8 (pz-C5), 126.7 (CH of C6H4), 104.8 (pz-C4), 104.6 (pz-C4), 82.5 (OC(CH3)3), 57.9 (α-CH), 32.3 (CH(CH3)2), 28.3 (CH(CH3)3), 19.3 -1 (CH(CH3)2), 18.1 (CH(CH3)2) ppm. IR (solid): 1654 (NHCO), 1728 (CO2tBu) cm . 70 Experimental part 5.5.22. TpRu(p-(CO-Tyr-Gly-Gly-Phe-Leu-OH)-C6H4Tp) 22 Resin-bound enkephalin Tyr(2-Cl-Trt)-Gly-Gly-Phe-Leu-(HMBA-Resin) was obtained by Fmoc- SPPS starting from Fmoc-Leu loaded Tentagel-S resin with a HMBA linker (773 mg, loading 0.24 mmol/g). After 2-Cl-Trt deprotection of the tyrosine hydroxy group with TFA 5% v/v / TIS 5 % v/v in dichloromethane, ruthenium compound 20 was coupled to the peptide as described here: 20 (240 mg, 0.37 mmol), TBTU (116 mg, 0.37 mmol), HOBt (57 mg, 0.42 mmol) and DIPEA (93 µL, 0.55 mmol) were mixed in DMF (3 mL) and stirred for 5 min. The homogenous solution was then added to the resin-bound peptide and the mixture was shaken for ca. 12 hours. After filtration, resin-bound product was washed with DMF (5×2 mL) and dichloromethane (5×2 mL) and dried under reduced pressure for one hour. The product was cleaved from the resin by shaking it with a cooled (0 °C) mixture of aqueous NaOH (2.5 mL, 1M) and 1,4-dioxane (7.5 mL) for 15 min. The resulting yellow solution was filtered and adjusted to pH 7 by addition of dilute hydrochloric acid. Removal of the volatiles gave a mixture of colourless NaCl and a yellow solid. It was taken into a minimal volume of methanol, filtered from insolubles and purified by flash-chromatography to give 22 as a yellow solid (125 mg, 0.11 mmol, 57% based on resin load) upon removal of the solvent. 1 H NMR (CD3OD, 400 MHz) Note: The resonances from one α-CH and two β-CH2 groups were obscured by the solvent peaks. Assignments are based, in part, upon comparison to literature [198] data. δ 8.34 (d, J= 8.4, 2H, part of AA'BB'), 8.07 (d, J= 8.4, 2H, part of C6H4), 7.72 (broad s, 3H, pz-H5), 7.49 (broad s, 3H, pz-H5), 7.29-7.19 (m, 5H, C6H5 of Phe), 7.15 (d, 2H, CH of Tyr p-phenol), 6.71 (d, 2H, CH of Tyr p-phenol), 6.21 (broad s, 6H, pz-H3), 6.04 (s, 3H, pz-H4), 6.01 (s, 3H, pz-H4), 4.66 (m, H, α-CH of Phe), 4.41 (m, 1H, α-CH of Leu) 3.84 (m, 4H, α-CH2 of Gly), 3.23- 3.19 (m, 2H, β-CH2 of Tyr, Phe), 2.99 (m, 2H, β-CH2 of Tyr, Phe), 1.65 (m, 2H, β-CH2 of Leu), 1.48 (m, 1H, γ-CH of Leu), 0.91 (d, J= 6.3, 3H, Leu-CH3), 0.86 (d, J= 6.3, 3H, Leu-CH3) ppm. ESI-MS - (neg. mode) m/z 1184.17 ([M-H] ), exact mass for C53H59B2N17O8Ru= 1185.84. 71 Experimental part 5.5.23. (p-BrC6H4Tp)Ru(COD)Cl 23 A mixture of 2 (3.44 g, 8.45 mmol) and [RuH(NH2NMe2)3(COD)][BPh4] (6.00 g, 8.45 mmol) in acetone (70 mL) was heated to reflux for two hours. CCl4 (15 mL) was added and the mixture refluxed for additional 45 minutes. Cooling to -20 °C resulted in the formation of a yellow precipitate of 23 and KCl, which was isolated by filtration and extracted with dichloromethane (2×30 mL). Removal of the solvent and drying under reduced pressure gave analytically pure 23 as orange microcrystals (3.23 g, 5.27 mmol, 62%). Anal. calc. for C23H25BBrClN6Ru (M= 612.72): C, 45.09; H, 4.11; N, 13.72. Found: C, 45.05 ; H, 4.19; 1 N, 13.70 %. H NMR (CDCl3, 250 MHz): δ 8.27 (d, J= 2.2 Hz, 1H, pz-H3), 7.88 (d, 2H, J= 8.4 Hz, part of C6H4), 7.72-7.68 (m, 6H, pz-H3, pz-H5 and part of C6H4), 7.40 (d, J= 2.2 Hz, 1H, pz-H5), 6.27 (t, J= 2.2 Hz, 1H, pz-H4), 6.22 (t, J= 2.2 Hz, 2H, pz-H4), 4.94 (m, 2H, olefinic H of COD), 4.14 (m, 2H, olefinic H of COD), 2.97 (m, 2H, COD), 2.63 (m, 2H, COD), 2.45 (m, 2H, COD), 2.28 (m, 13 2H, COD); C NMR (CDCl3, 62.5 MHz): δ 146.1 (pz-C5), 143.5 (pz-C5), 138.6 (C-B), 137.0 (pz-C3), 135.5 (pz-C3), 131.6 (part of AA'BB'), 123.4 (C-Br), 106.2 (pz-C4), 106.0 (pz-C4), 95.48 (olefinic C of COD), 87.6 (olefinic C of COD), 30.7 (aliphatic C of COD), 29.9 (aliphatic C of COD) ppm. Solid state structure of 23·CH2Cl2: A crystal of 23·CH2Cl2 (light orange needle), obtained by slow evaporation of a dichloromethane solution, was placed on a glass capillary in perfluorinated oil and measured in a cold gas flow. C24H27BBrCl3N6Ru (M= 697.66), triclinic, a= 10.226(5) Å, b= 10.664(5) Å, c= 12.763(6) Å, α= 72.565(9)°, β= 87.041(8)°, γ= 83.228(8)°, V= 1318.4(10) Å3, space 2 group P , Z= 2, 10468 reflections collected, 4595 unique (Rint= 0.0437), wR(F )= 0.1350 (all data). CCDC-797019 contains the supplementary crystallographic data for compound 23. 72 Experimental part 5.5.24. [(p-BrC6H4Tp)RuTpm]Cl 24 Tpm (214 mg, 1 mmol), 23 (613 mg, 1 mmol) and THF (5 mL) were mixed in a microwave vial. The mixture was heated to 155 °C by microwave irradiation for 1 hour. Cooling to room temperature resulted in the formation of a bright yellow solid, that was isolated by filtration open to air, washed with pentane (2×5 mL) and dried under reduced pressure to give analytically pure 24 (385 mg, 0.54 mmol, 54%). Anal. calc. for C25H23BBrClN12Ru (M= 718.77): C, 41.78; H, 3.23; N, 23.38. Found: C, 41.85; H, 1 3.25; N, 23.30 %. H NMR (250 MHz, DMSO-d6): δ 10.51 (s, 1H, (pz)3CH), 8.71 (d, J= 2.2, 3H, pz- H3), 8.01 (d, J= 8.4, 2H, part of AA'BB'), 7.82 (d, J= 8.4, 2H, part of AA'BB'), 7.77 (d, J= 2.2, 3H, pz-H3), 7.26 (broad s, 3H, pz-H5), 6.91 (broad s, 3H, pz-H5), 6.63 (t, J= 2.2, 3H, pz-H4), 6.34 (t, 13 J= 2.2, 3H, pz-H4); C NMR (DMSO-d6, 62.5 MHz): δ 146.0 (CH, pz-C5), 143.6 (CH, pz-C5), 136.9 (CH, pz-C3), 136.2 (CH, pz-C3), 134.9 (CH of C6H4), 131.2 (CH of C6H4), 122.4 (C-Br), 108.9 (CH, pz-C4), 106.8 (CH, pz-C4), 75.7 (CH of Tpm) ppm. Solid state structure of 24·THF: A crystal of 24·THF (yellow needle), obtained by slow diffusion of THF into an acetone solution of 24, was placed on a glass capillary in perfluorinated oil and measured in a cold gas flow. Note: The solvent molecule could not be modeled within the structure solution due to partially high disorders. Thus, it was not taken into account for the structure refinement. C25H23BBrClN12Ru (M= 718.79), triclinic, a= 7.7245(2) Å, b= 14.4789(3) Å, c= 17.0407(4)Å, α= 91.823(2)°, β= 101.008(2)°, γ= 103.716(2)°, V= 1811.51(7)Å3, space group P , Z= 2 2, 5394 reflections collected, 4991 unique (Rint= 0.0262), wR(F )= 0.0586 (all data). CCDC-804163 contains the supplementary crystallographic data for compound 24. 73 Experimental part 2 5.5.24.1. [(p-BrC6H4Tp)Ru(κ -N,N-Tpm]Cl 24a 2 One of the major byproducts in the synthesis of 24 was identified as [(p-BrC6H4Tp)Ru(κ -N,N- Tpm]Cl 24a, probably formed as an intermediate of the reaction. Hence, it was obtained in variable yields (5-15%) from the THF filtrate. 1 H NMR (CDCl3, 250MHz) δ 10.75 (s, 1H, (pz)3CH), 8.12 (d, J =2.2, 1H, pz-H3), 8.10 (s, 1H, pz-H3), 8.00 (d, J= 8.4, 2H, part of C6H4), 7.90 (broad s, 2H, pz-H3), 7.73-7.68 (m, 5H, pz-H3, pz-H5, part of C6H4), 7.59 (d, J= 2.2, 1H, pz-H5), 7.51 (broad s, 1H, pz-H5), 7.06 (broad s, 2H, pz-H5), 6.64 (t, 13 J= 2.2, 1H, pz-H4), 6.29 (t, J= 2.2, 3H, pz-H4), 5.92 (broad s, 2H, pz-H4); C NMR (CDCl3, 62.5 MHz) δ 146.3 (CH, pz-C5), 145.2 (CH, pz-C5), 144.7 (CH, pz-C5), 143.8 (CH, pz-C5), 136.9 (CH, pz- C3), 135.9 (CH, pz-C3), 134.8 (CH of C6H4), 132.1 (CH, pz-C3), 131.4 (CH of C6H4), 123.1 (C-Br), 108.3 (CH, pz-C4), 107.57 (CH, pz-C4), 106.21 (CH, pz-C4), 105.8 (CH, pz-C4), 80.6 (CH of Tpm) ppm. Solid state structure of 24a: A crystal of 24a·acetone (yellow needle), obtained by slow evaporation of an acetone solution, was placed on a glass capillary in perfluorinated oil and measured in a cold gas flow. C28H29BBrClN12ORu (M= 776.85), triclinic, a= 10.7878(5) Å, b= 11.8810(7) Å, c= 12.4405(7) Å, α= 75.555(5)°, β= 82.523(4)°, γ= 89.662(4)°, V= 1530.38(14) Å3, space group P , Z= 2 2, 5372 reflections collected, 4218 unique (Rint= 0.0382), wR(F )= 0.0639 (all data). CCDC-797020 contains the supplementary crystallographic data for compound 24a. 74 Experimental part 5.5.25. [(p-N3C6H4Tp)RuTpm]Cl 25 Sodium azide (112 mg, 1.72 mmol), 24 (385mg, 0.54 mmol) and copper(I) iodide (10 mg, 0.05 mmol) were mixed and ethanol/water (7:3, 10 mL) was added. The solution was degassed and backfilled with argon. After addition of N,N’-dimethylethane-1,2-diamine (17 µL, 0.30 mmol) and sodiumascorbate (3 mg, 0.01 mmol), the flask was covered with aluminium foil and the mixture was heated to 90 °C over night. Addition of water (20 mL) produced a yellow precipitate that was isolated by filtration and washed with water (2×5 mL) and hexane (2×5 mL). Drying under reduced pressure gave 25 as a light yellow solid (265 mg, 0.39 mmol, 72%). An analytically pure sample of 25·CH2Cl2 was obtained by recrystallisation from dichloromethane. Anal. calc. for C26H25BCl3N15Ru (M= 680.89): C, 40.78; H, 3.29; N, 27.43. Found: C, 40.53 ; H, 1 3.26; N, 27.35%. H NMR (DMSO-d6, 250 MHz): δ 10.08 (s, 1H, (pz)3CH), 8.66 (d, J= 2.2, 3H, pz- H3), 8.10 (d, J= 8.4, 2H, part of C6H4), 7.77 (d, J= 2.2, 3H, pz-H3), 7.37 (d, J= 8.4, 2H, part of C6H4), 7.27 (broad s, 3H, pz-H5), 6.92 (broad s, 3H, pz-H5), 6.64 (t, J= 2.2, 3H, pz-H4), 6.34 13 (J=2.2, 3H, pz-H4); C NMR (DMSO-d6, 62.5 MHz): δ 145.9 (pz-C5), 143.5 (pz-C5), 139.5 (C-N3), 136.4 (pz-C3), 136.2 (pz-C3), 134.8 (CH of C6H4), 119.0 (CH of C6H4), 108.8 (pz-C4), 106.7 (pz-C4), 75.7 (CH of Tpm) ppm. IR (solid): 2085 (azide N=N asym.), 1258 (azide N=N sym.). ESI-MS (pos. + + mode): 645.95 ([M-Cl] ), 620.02 ([M-Cl-N2+2H] ), exact mass for C25H23BN15Ru= 646.14. Solid state structure of 25: A crystal of 25 (yellow needle), obtained by slow evaporation of an acetone solution of 25, was placed on a glass capillary in perfluorinated oil and measured in a cold gas flow. C25H23BBr0.6Cl0.4N15Ru (M=707.59), monoclinic, a= 15.0766(2) Å, b= 13.5560(2) Å, c= 3 16.1782(2) Å, α= γ= 90°, β= 102.555(1)°, V= 3227.41(8) Å , space group P21/c, Z= 4, 5652 2 reflections collected, 5211 unique (Rint= 0.0324), wR(F )= 0.0829 (all data). CCDC-804164 contains the supplementary crystallographic data for compound 25. 75 Experimental part 5.5.26. HCC(CH2)2CO-Val-OtBu 26 4-pentynoic acid (980 mg, 10 mmol) and TBTU (3.21 g, 10 mmol) were stirred in dichloromethane (30 mL) for 15 minutes. Triethylamine (9.7 mL, 70 mmol) was added and the resultant homogenous solution stirred for 10 minutes. H-Val-OtBu·HCl (2.10 g, 10 mmol) was added and the mixture stirred over night at room temperature. After removal of the volatiles, the remaining oil was dissolved in ethyl acetate (40 mL), and the resulting solution was washed with aqeous KHSO4 (1 M, 2×40 mL), NaHSO4 (5%, 2×50 mL) and brine (2×50 mL). The organic layer was dried over MgSO4 and evaporated to dryness to give 26 as an off-white solid (2.03 g, 8 mmol, 80%). Anal. calc. for C14H23NO3 (M= 253.34): C, 66.37; H, 9.15; N, 5.53. Found: C, 66.14; H, 9.06 ; N, 1 5.51%. H NMR (CDCl3, 250 MHz): δ 6.15 (d, J= 6.6, 1H, NHCO), 4.48 (m, 1H, α-CH), 2.58-2.42 (m, 4H, (CH2)2), 2.14 (m, 1H, CH(CH3)2), 2.00 (s, 1H, HCC(CH2)2), 1.46 (s, 9H, OC(CH3)3), 0.93 (d, 13 J=6.8, 3H, CH(CH3)2), 0.90 (d, J= 6.8, 3H, CH(CH3)2); C NMR (CDCl3, 62.5 MHz): δ 171.4 (CONH), 170.9 (CO2tBu), 83.1 (HCCCH2), 82.2 (C(CH3)3), 69.5 (HCCCH2), 57.5 (α-CH), 35.7 (HCCCH2CH2), 31.7 (CH(CH3)2), 28.2 (C(CH3)3), 19.1 (CH(CH3)2), 17.8 (HCCCH2CH2 ) ppm. 76 Experimental part 5.5.27. [TpmRu((p-C2N3H-(CH2)2-CO-Val-OtBu)-C6H4Tp)]Cl 27 To a mixture of 25 (396 mg, 0.58 mmol), 26 (174 mg, 0.69 mmol) and copper(I) iodide (3 mg, 0.03 mmol), acetonitrile/dichloromethane (2:1, 9 mL) was added. After addition of 2,6-lutidine (20 µL, 0.30 mmol), the solution was degassed and backfilled with argon and the reaction mixture stirred for 12 hours in the dark. Removal of the volatiles gave a light green solid, that was dissolved in methanol (3 mL). Addition of water (~20 mL) produced a yellow precipitate, that was isolated by filtration, washed with water (2×5 mL) and hexane (2×5 mL) and dried under lowered pressure to yield 27 as a yellow powder (314 mg, 0.34 mmol, 58%). An analytically pure sample of 27·CH2Cl2 was obtained by recrystallisation from dichloromethane. Anal. calc. for C40H48BCl3N16O3Ru (M= 934.22): C, 47.14; H, 4.75; N, 21.99. Found: C, 47.92; H, 1 5.11; N, 21.52%. H NMR (CDCl3, 250 MHz): δ 12.09 (s, 1H, HC(pz)3), 9.06 (broad s, 3H, pz-H3), 8.29 (d, J=8.4, 2H, part of C6H4), 8.01 (s, 1H, triazole), 7.98 (d, J= 8.4, 2H, part of C6H4), 7.77 (d, J= 2.2, 3H, pz-H3), 7.20 (d, J= 2.2, 3H, pz-H5), 6.92 (d, J= 2.2, 3H, pz-H5), 6.46 (broad s, 3H, pz- H4), 6.24 (t, J= 2.2, 3H, pz-H4), 6.08 (d, J=6.6, 1H, NH), 4.46 (m, 1H, α-CH), 2.83-2.44 (m, 4H, (CH2)2), 2.14 (m, 1H, CH(CH3)2), 1.47 (s, 9H, OC(CH3)3), 0.92 (d, J= 6.8, 3H, CH(CH3)2), 0.88 (d, 13 J= 6.8, 3H, CH(CH3)2); C NMR (CDCl3, 62.5 MHz): note: signals for Tpm-CH and Phenyl-C- Ntriazole were not observed. δ 171.8 (CONH), 171.3 (CO2tBu), 145.8 (pz-C5), 143.9 (pz-C5), 137.5 (C- B), 136.5 (pz-C3 and CH of AA'BB'), 134.8 (pz-C3) 134.4 (CH of C6H4), 120.2 (triazole-CH), 120.1 (CH of C6H4), 108.8 (pz-C4), 106.6 (pz-C4), 82.2 (C(CH3)3), 57.6 (α-CH), 35.9 (HCCCH2CH2), 31.5 (CH(CH3)2), 28.2 (C(CH3)3), 21.4 (HCCCH2CH2), 19.1 (CH(CH3)2), 17.8 (C(CH3)3) ppm. ESI-MS + (pos. mode): 899.13 ([M-Cl] ), exact mass for complex cation C39H46BN16O3Ru= 899.31. 77 Experimental part 5.5.28. [TpmRu((p-C2N3H-(CH2)2-CO-Tyr-Gly-Gly-Phe-Leu-OH)-C6H4Tp)]Cl 28 To a mixture of penty-Enk-OH (85 mg, 0.14 mmol), 25 (93 mg, 0.14 mmol), and CuSO4×5 H2O (4 mg, 0.014 mmol) in t-BuOH (1 mL), a freshly prepared solution of sodium ascorbate (14 mg, 0.07 mmol) in water (2 mL) was added. The resulting solution was degassed, backfilled with argon, and stirred in the dark for 12 hours. Subsequent removal of the solvents yielded a greenish solid, that was washed with water (2×4 mL), and dried under reduced pressure. Purification by RP- HPLC yielded 28 as a light yellow solid (35 mg, 0.03 mmol, 21%). 1 H NMR (400 MHz, CD3OD): note: The resonances from one α-CH and two β-CH2 groups were obscured by the solvent peaks. Assignments are based, in part, upon comparison to literature [198] data. δ 9.73 (s, 1H, HC(pz)3), 8.56 (d, J= 2.2, 3H, pz-H3), 8.38 (s, 1H, triazole-H), 8.30 (d, J=8.4, 2H, part of AA'BB'), 8.10 (d, J= 8.4, 2H, part of AA'BB'), 7.87 (d, J= 2.2, 3H, pz-H3), 7.30 (d, J=2.2, 3H, pz-H5), 7.26-7.13 (m, 5H, Phe), 7.05 (d, J=8.4, 2H, AA'BB' of Tyr), 6.97 (d, J= 2.2, 3H, pz-H5), 6.70 (d, J= 8.4, 2H, AA'BB' of Tyr), 6.63 (t, J= 2.2, 3H, pz-H4), 6.31 (t, J= 2.2, 3H, pz- H4), 4.66 (dd, J= 9.0; 5.5, 1H, α-CH), 4.52 (m, 1H, α-CH), 4.41 (m, 1H, α-CH of Leu), 3.90 (m, 2H, α-CH2 of Gly), 3.90 (m, 2H, α-CH2 of Gly), 3.75 (m, 2H, α-CH2 of Gly), 3.21-3.04 (m, 4H, β-CH2 and CH2CH2-triazole), 2.99-2.86 (m, 2H, CH2CH2-triazole), 2.70 (m, 2H, β-CH2), 1.63 (m, 3H, β- CH2 and γ-CH of Leu), 0.91 (d, J=6.3, CH3 of Leu), 0.87 (d, J=6.3, CH3 of Leu) ppm. ESI-MS (pos. mode): 1281.03 ([M]+), 1151.06 ([M-Leu]+), 1004.06 ([M-(Phe-Leu)]+), 947.07 ([M-(Gly-Phe- Leu)]+), 890.09 ([M-(Gly-Gly-Phe-Leu)]+), 641.23 ([M+H]2+), exact mass for C58H64BN20O8Ru= 1281.44. 78 Experimental part 2 5.5.29. [Tp*WI(CO)(η -HCC(CH2)2CO2H)] 29 4-pentynoic acid (88 mg, 0.89 mmol) and [Tp*WI(CO)3] (560 mg, 0.81 mmol) were dissolved in THF (15 mL) and the dark red solution was heated to reflux for 12 hours open to a bubbler. Removal of the solvent yielded a green solid, which was dissolved in Et2O (10 mL). Addition of cold pentane (0 °C, 10 mL) gave a pale green solid, which was isolated by filtration, washed with pentane (2×5 mL), and dried under lowered pressure (315 mg, 0.43 mmol, 53%). An analytically pure sample was obtained by slow evaporation of a dichloromethane solution as the adduct 29·CH2Cl2. Anal. calc. for C22H30BCl2IN6O3W (M= 819.98): C, 32.22; H, 3.81; N, 10.25. Found C, 32.22; H, 1 2.93; N, 10.24%; H NMR (CDCl3, 250 MHz): major isomer (92%): δ 12.23 (s, 1H, HCC(CH2)2), 6.10, 5.81, 5.67 (s, 1:1:1, pz-H4), 4.12 (m, 1H, part of CH2CH2), 3.85 (m, 1H, part of CH2CH2), 3.12 (m, 1H, part of CH2CH2), 2.98 (m, 1H, part of CH2CH2), 2.80, 2.62, 2.53, 2.40, 2.32, 1.59 (s, 3:3:3:3:3:3, CH3 of Tp*); minor isomer (8%) δ 13.13 (s, 1H, HCC(CH2)2), 6.11, 5.78, 5.66 (s, 1:1:1, pz-H4), 4.16 (m, 1H, part of CH2CH2), 3.80 (m, 1H, part of CH2CH2), 3.17 (m, 1H, part of CH2CH2), 13 2.93 (m, 1H, part of CH2CH2), 2.83, 2.62, 2.55, 2.41, 2.29, 1.66 (s, 3:3:3:3:3:3, CH3 of Tp*); C NMR (CDCl3): major isomer δ 233.9 (CO), 206.89 (HCCR), 204.39 (HCCR), 177.38 (CO2H), 155.17- 143.38 (pz-C3 and pz-C5), 108.46-107.24 (pz-C4), 34.43 (CH2CO2H), 32.87 (CH2CH2CO2H), 18.43- -1 12.56 (Tp*CCH3) ppm; IR (solid): 1907 (CO), 1714 (CO2H) cm . Solid state structure determination of 29: A crystal of 29 (green needle), obtained by slow evaporation of the pentane washing solution, was placed on a glass capillary in perfluorinated oil and measured in a cold gas flow. C21H28BIN6O3W (M= 734,04), triclinic, a= 8.236(4) Å, b= 10.387(5) Å, c= 17.389(7) Å, α= 82.035(9)°, β= 84.714(10)°, γ= 78.758(9)°, V= 1441.7(11) Å3, space 2 group P , Z= 2, 7975 reflections collected, 4952 unique (Rint= 0.0459), wR(F )= 0.1495 (all data). CCDC-722677 contains the supplementary crystallographic data for compound 29. 79 Experimental part 5.5.30. [Tp*W(I)(CO)(η2-Fmoc-Pgl)] 30 Tp*WI(CO)3 (340 mg, 0.49 mmol)) and Fmoc-Pgl (180 mg, 0.54 mmol) were dissolved in THF (15 mL) and the red solution was heated to reflux for 12 hours open to a bubbler. After cooling to room temperature, the green solution was filtered and the solvent removed under reduced pressure. The resulting green solid was washed with pentane (2×5 mL) and recrystallized from Et2O/hexane to give dark green crystals of 30 (321 mg, 0.33 mmol, 67%). Analytical samples were found to consist of 30·Et2O even after prolonged drying. Anal. calc. for C40H49BIN7O6W (M= 972.30): C, 45.96; H, 4.72; N, 9.38. Found: C, 46.15; H, 4.27; 1 N, 9.61%. H NMR (CDCl3, 250 MHz): major isomer (90%) δ 12.34 (s, 1H, HCC(CH2)2), 7.74-7-31 (m, 8H, C6H4 of Fmoc), 6.08, 5.81, 5.66 (s, 1:1:1, pz-H4), 5.14 (m, 1H, α-CH), 4.55-4.18 (m, 5H, 13 Fmoc CH and CH2, β-CH2), 2.80, 2.61, 2.53, 2.41, 2.32, 1.55 (s, 3:3:3:3:3:3, CH3 of Tp*); C NMR (CDCl3, 62.5 MHz): δ 232.8 (CO), 208.1 (HCCR), 203.8 (HCCR) 175.7 (CO2H), 155.2 (NHCO2), 155.0-141.4 (pz-C3, pz-C5, Cq of Fmoc), 127.8-125.4 (CH), 108.8-107.6 (pz-C4), 67.7 (Fmoc-CH2), 47.3 (Fmoc-CH), 38.8 (HCCCH2), 18.7-12.8 (CH3 of Tp*) ppm. IR (solid): 1902 (CO), 1716 (CO2H) cm-1. 80 Experimental part 2 5.5.31. [Tp*WI(CO)(η -HCC(CH2)2CO-NH-Lys-Lys-Pro-Tyr-Ile-Leu-OH)] 31 Resin bound pseudo-neurotensin Lys(IvDde)-Lys(IvDde)-Pro-Tyr(2Cl-Trt)-Ile-Leu-(2Cl-Trt-RES) was obtained by standard SPPS starting from Fmoc-Leu loaded 2-Cl-Trt resin (116 mg, loading 0.86 mmol/g). After Fmoc deprotection of the second lysine, the tungsten complex 29 was coupled to the peptide as detailed here: 29 (173 mg, 0.21 mmol), HOBt (54 mg, 0.40 mmol), TBTU (125 mg, 0.39 mmol) and DIPEA (101 μL, 0.60 mmol) were mixed in DMF (3 ml) and the solution was stirred for 5 minutes, then added to the resin-bound peptide and the mixture was shaken for ca. 20 hours. Resin-bound product was washed with DMF (5×2 mL) and dichloromethane (5×2 mL) and dried under reduced pressure. IvDdE protecting group of lysine was removed by shaking the resin with 3% v/v hydrazine in DMF (4 mL) for 15 minutes followed by washing with DMF (5×2 mL). Subsequently, 31 was cleaved from the resin by treatment with 20% v/v TFA in DMF (4 mL) for two hours. The resulting green solution was filtered and the resin washed with methanol (2×2 mL). The solutions were combined and TFA and methanol were removed under lowered pressure. Bioconjugate 31 precipitated as a pale green-blue solid upon addition of cold Et2O, was isolated by filtration, dried, and purified by RP-HPLC (57 mg, 0.04 mmol, 56% based on resin load). 1 H NMR (CD3OD, 400 MHz) Note: Assignments are based, in part, on comparison with literature [207] data for neurotensin. major isomer (92%): δ 12.36 (s, 1H, HCC(CH2)2), 6.98 (d, J= 8.0, 2H, Tyr), 6.62 (d, J= 8.0, 2H, Tyr), 6.08, 5.82, 5.69 (s, 1:1:1, pz-H4), 4.44-4.01 (m, 8H, α-CH and CH2), 3.72 (m, 2H, CH2), 3.24 (m, 2H, β-CH2), 3.04 (m, 4H, Lys ε-CH2), 2.88, 2.85, 2.68, 2.52, 2.50 (s, 3:3:3:3:3, CH3 of Tp*), 1.90 (m, 4H, Lys β-CH2), 1.79-1.73 (m, 5H, Lys γ-CH2 and Ile β-CH), 1.68 (m, 4H, Lys NH2), 1.60 (s, 1H), 1.62-1.59 (m, 6H, Leu β-CH2, γ-CH and CH3 of Tp*), 1.16 (m, 2H, + Ile γ-CH2), 0.92-0.78 (m, 12H, CH3 of Ile and Leu) ppm. ESI-MS (pos.): 1477.30 ([M+H] ), 739.25 2+ 2+ ([M+2H] ), 675.33 ([M-I+H] ), exact mass for C59H90BIN14O10W= 1476.56. IR (solid): 1904 (CO), 1643 (CONH) cm-1. 81 Experimental part 2 5.5.32. [Tp*WI(CO)(η -HCC(CH2)2CO-NH-Tyr-Gly-Gly-Phe-Leu-OH)] 32 Resin-bound enkephalin Tyr(2-Cl-Trt)-Gly-Gly-Phe-Leu(2Cl-Trt-resin) was obtained by standard SPPS starting from 2-Cl-Trt resin (82 mg, loading 0.86 mmol/g) preloaded with leucine. After Fmoc deprotection of tyrosine, metal complex 29 was coupled to the peptide as detailed here: 29 (78 mg, 0.11 mmol), HOBt (44 mg, 0.28 mmol), TBTU (89 mg, 0.28 mmol) and DIPEA (72 μL, 0.43 mmol) were mixed in DMF (3 mL) and stirred for 5 minutes. The homogenous green solution was then added to the resin-bound peptide and the mixture was shaken for ca. 20 hours. After filtering the reaction mixture, resin-bound product was washed with DMF (5×2 mL) and dichloromethane (5×2 mL) and dried under reduced pressure over night. The product was cleaved from the resin by treatment with 20% v/v TFA in DMF (4 mL) for two hours. The resulting green solution was filtered and the resin was washed with methanol (2×2 mL). The solutions were combined and TFA and methanol were removed under lowered pressure. Product 32 precipitated as a pale green-blue solid upon addition of cold Et2O, was isolated by filtration, dried under lowered pressure, and purified by RP-HPLC (44 mg, 0.034 mmol, 29% based on resin load). 1 H NMR (CD3OD, 400 MHz) Note: Assignments are based, in part, on comparison with literature [198] data. major isomer (92%) δ 12.37 (s, 1H, HCC(CH2)2), 7.27-7.19 (m, 5H, C6H5 of Phe), 6.63 (d, J= 8.0, 2H, Tyr), 6.01 (d, J= 8.0, 2H, Tyr), 6.14, 5.87, 5.73 (s, 1:1:1, pz-H4), 4.67 (dd, J= 9.0, 5.5, 1H, α-CH), 4.50-4.31 (m, 3H, α-CH and CH2), 3.86-3.62 (m, 6H, CH2 and Gly α-CH2), 3.22 (m, 2H, β- CH2), 2.98 (m, 3H, β-CH2 and CH3 of Tp*), 2.85, 2.75, 2.69, 2.57 (s, 3:3:3:3, CH3 of Tp*), 1.67- 1.59 (m, 3H, α-CH, β-CH2), 1.56 (s, 1H, CH3 of Tp*), 0.88 (d, J= 6.3, 3H, CH3 of Leu), 0.84 (d, J= - 2- 6.3, 3H, CH3 of Leu) ppm. ESI-MS (neg.): 1270.27 ([M-H] ), 634.37 ([M-2H] ), exact mass for -1 C49H64BIN11O9W= 1271.35; IR (solid): 1904 (CO), 1634 (CONH) cm . 82 Experimental part 2 5.5.33. H2N-Tyr-Gly-[Tp*W(I)(CO)(η -Pgl)]-Phe-Leu-OH 33 Resin-bound enkephalin functionalized with a tungsten-propargylglycine moiety was obtained by standard SPPS starting from Fmoc-Leu loaded 2-ClTrt resin (123 mg, load 0.86 mmol/g). For the second coupling, 30 (154 mg, 0.16 mmol), HOBt (65 mg, 0.42 mmol) TBTU (133 mg, 0.41 mmol) and DIPEA (107 μL, 0.64 mmol) were dissolved in DMF (3 mL). After stirring the solution for 5 minutes, it was added to the resin and the mixture was shaken for 20 hours. After filtration, washing with DMF (5×2 mL), and Fmoc-cleavage, the peptide sequence was completed by coupling of glycine and tyrosine. Crude 33 was cleaved from the resin by shaking it with 20% v/v TFA in DMF (4 mL) for two hours. The resulting green solution was filtered and the resin was washed with methanol (2×2 mL). The solutions were combined and TFA and methanol were removed under lowered pressure. Side-chain functionalized 33 precipitated from the solution as a pale turquoise solid upon addition of cold Et2O and was isolated by filtration, dried under lowered pressure and purified by RP-HPLC (25 mg, 0.02 mmol, 24% based on resin load). 1 H NMR (CD3OD, 400 MHz): note: Assignments are based, in part, on comparison with literature [198] data. major isomer (90%) δ 12.35 (s, 1H, HCC(CH2)2), 7.29-7.16 (m, 5H, C6H5 of Phe), 7.13 (d, J= 8.0, 2H, Tyr), 6.78 (d, J=8.0, 2H, Tyr), 6.14, 5.88, 5.74 (s, 1:1:1, pz-H4), 4.72 (dd, J= 9.0, 5.5, 1H, α-CH), 4.41 (dd, J= 9.0, 5.5, 1H, α-CH), 4.31 (dd, J= 9.0, 5.5, 1H, α-CH), 3.89 (m, 2H, α-CH2 of Gly), 3.21 (m, 3H, β-CH2), 2.97 (m, 2H, β-CH2), 2.74, 2.58, 2.55, 2.41, 2.36 (s, 3:3:3:3:3 CH3 of Tp*), 1.61-1.55 (m, 6H, β-CH2 γ-CH of Leu, CH3 of Tp*), 0.88 (d, J= 6.3, 3H, δ-CH3 of Leu), 0.83 + (d, J= 6.3, 3H, δ-CH3 of Leu) ppm. ESI-MS (pos.) m/z: 1230.26 ([M+H] ), 594.41 ([M- + (Tp*WICO)+H] ), exact mass for C47H61BIN11O8W= 1229.34. 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C.; Merz, K.; Metzler-Nolte, N.: para-Bromophenyl- [tris(pyrazolyl)]borate Complexes of Group 1 Metals, Thallium and Magnesium: Synthesis and Characterization of Transfer Agents for “Third-Generation” Tp Ligands, Eur. J. Inorg. Chem. 2009, 5407-5412. Zagermann, J.; Kuchta, M. C.; Merz, K.; Metzler-Nolte, N.: Ruthenium-based bioconjugates: iPr Synthesis and X-ray structure of the mixed ligand sandwich compound RuCp (p-(CO2H)C6H4Tp) and labelling of amino acids and the neuropeptide enkephalin, J. Organomet. Chem. 2009, 694, 862-867. Zagermann, J.; Merz, K.; Metzler-Nolte, N.: Labeling of peptides with halocarbonyltungsten complexes containing functional η2-alkynyl ligands, Organometallics 2009, 28, 5090-5095 Zagermann, J.; Molon, M.; Metzler-Nolte, N.: Microwave-assisted synthesis of the Tp sandwich compound TpRu(p-Br-C6H4Tp) and application of its benzoic acid derivative TpRu(p-(CO2H)-C6H4Tp) in the covalent labelling of biomolecules, Dalton Trans. 2010, DOI: 10.1039/c0dt01121e. Zagermann, J.; Merz, K.; Metzler-Nolte, N.: Unusual coordination modes in "third-generation" tris(pyrazolyl)borate p-BrC6H4TpCs, manuscript submitted to Inorg. Chem., December 2010. Zagermann, J.; Klein, K.; Molon, M.; Merz, K.; Metzler-Nolte, N.: Synthesis and characterization of the azido-functionalized ruthenocene analogue [TpmRu(p-N3C6H4)Tp]Cl and its attachment to biomolecules by copper catalyzed azide-alkyne cycloaddition, manuscript submitted to Eur. J. Inorg. Chem., January 2010. TALKS Zagermann, J.: Functionalization of biomolecules with transition metal complexes, Section Day “Natural Sciences & Engineering” RUB Research School, Bochum, 2009 POSTER PRESENTATIONS Zagermann, J.; Kuchta, M. C.; Merz, K.; Metzler-Nolte, N.: Synthesis and characterization of ruthenium(II) mixed sandwich complexes with functionalized tris(pyrazolyl)borate ligands, Koordinationschemietreffen, Gießen, 2008. 92 List of publications Zagermann, J.; Kuchta, M. C.; Merz, K.; Metzler-Nolte, N.: Tris(pyrazolyl)borates as versatile ligands in the synthesis of bioorganometallic compounds, International Conference on Bioinorganic Chemistry (ICBIC 14), Nagoya, 2009. Zagermann, J.; Kuchta, M. C.; Merz, K.; Metzler-Nolte, N.: Tris(pyrazolyl)borates as versatile ligands in the synthesis of bioorganometallic compounds, International Symposium on Bioorganometallic Chemistry (ISBOMC ‘10), Bochum, 2010. 93 Supporting information 8. SUPPORTING INFORMATION 8.1. Crystallographic data CsTp’ Mg(Tp’)2 TlTp’* 4 6 10 CCDC number 797015 741193 741194 Formula C15CsH13BBrN6 C30H26B2Br2MgN12 C21H25BBrN6Tl Formula weight [g/mol] 500.92 760.38 656.56 Crystal size [mm] 0.2×0.1×0.1 0.2×0.2×0.1 0.12×0.06×0.06 -3 ρcalc. [g/cm ] 1.859 1.550 1.988 Temperature 223(2) K 213(2) 243(2) space group P P P Z 4 2 2 -1 μ [mm ] (MoKα) 4.306 2.550 9.203 a [Å] 8.540(4) 10.957(3) 8.0989(6) b [Å] 15.045(6) 12.144(4) 8.3437(6) c [Å] 15.879(7) 12.517(4) 17.0443(12) α [°] 65.853(8) 97.219(7) 85.7240(10) β [°] 88.457(8) 99.152(7) 78.4870(10) γ [°] 75.056(8) 91.674(6) 76.5310(10) V [Å3] 1791.4(13) 1629.3(8) 1097.07(14) F(000) 962 764 628 θmax [°] 25.00 25.24 28.23 No of reflections (total) 6215 9090 7632 No of reflections (unique) 5461 5706 4917 No of parameters 433 427 272 R1(F) all data 0.0555 0.0918 0.0414 2 wR2(F ) all data 0.1369 0.1905 0.0983 Goodness of fit on F2 1.074 1.055 1.090 Diff. Four. Peaks 1.423/-2.053 1.006/-0.615 2.263/-1.105 max/min, [eÅ-3] 94 Supporting information iPr Cp Ru(p-CO2H)-C6H4Tp) TpRuTp’ Tp’Ru(COD)Cl 16 19 23·CH2Cl2 CCDC number 699278 781625 797019 Formula C24H25BN6O2Ru C24H23B2BrN12Ru C24H27BBrCl3N6Ru Formula weight [g/mol] 542.12 682.12 697.66 Crystal size [mm] - 0.2×0.2×0.1 0.1×0.1×0.1 -3 ρcalc. [g/cm ] 1.424 1.688 1.757 Temperature 213(2) 113(2) 223(2) space group P P21/c P Z 4 4 2 -1 μ [mm ] (MoKα) 0.654 2.550 2.441 a [Å] 9.38(3) 9.1476(2) 10.226(5) b [Å] 13.62(4) 17.3437(3) 10.664(5) c [Å] 20.44(7) 17.1771(3) 12.763(6) α [°] 88.10(8) 90 72.565(9) β [°] 78.75(7) 99.939(2) 87.041(8) γ [°] 79.44(13) 90 83.228(8) V [Å3] 2517.0(13) 2684.30(9) 1318.4(10) F(000) 1098 1360 696 θmax [°] 25.00 25.00 25.00 No of reflections (total) 11644 9542 4595 No of reflections (unique) 8442 4723 4199 No of parameters 614 427 325 R1(F) all data 0.1587 0.0292 0.0533 2 wR2(F ) all data 0.2462 0.0565 0.1350 Goodness of fit on F2 1.054 0.979 1.117 Diff. Four. Peaks 1.867/-1.785 0.462/-0.346 0.926/-1.893 max/min, [eÅ-3] 95 Supporting information [Tp’RuTpm]Cl [Tp’RuClTpm] 24·THF 24a·acetone CCDC number 804163 797020 Formula C25H23BBrClN12Ru C28H29BBrClN12ORu Formula weight [g/mol] 718.79 776,85 Crystal size [mm] 0.5×0.4×0.2 0.4×0.2×0.1 -3 ρcalc. [g/cm ] 1.318 1.686 Temperature 105(2) 113(2) space group P P Z 2 2 -1 μ [mm ] (MoKα) 1.641 1.951 a [Å] 7.7245(2) 10.7878(5) b [Å] 14.4789(3) 11.8810(7) c [Å] 17.0407(4) 12.4405(7) α [°] 91.823(2) 75.555(5) β [°] 101.008(2) 82.523(4) γ [°] 103.716(2) 89.662(4) V [Å3] 1811.51(7) 1530.38(14) F(000) 716 780 θmax [°] 25.00 25.00 No of reflections (total) 6363 5372 No of reflections (unique) 5821 4218 No of parameters 370 408 R1(F) all data 0.0314 0.0382 2 wR2(F ) all data 0.0825 0.0639 Goodness of fit on F2 1.054 0.933 Diff. Four. Peaks 0.740/-0.605 0.683/-0.809 max/min, [eÅ-3] 96 Supporting information 2 [p-N3C6H4TpRuTpm]Cl [Tp*WI(CO)(η -HCC(CH2)2CO2H)] 25 29 CCDC number 804164 722677 Formula C21H28BIN6O3W C25H23BBr0.6Cl0.4N15Ru Formula weight [g/mol] 734.04 707.59 Crystal size [mm] 0.2×0.2×0.1 0.4×0.3×0.1 -3 ρcalc. [g/cm ] 1.802 1.456 Temperature 213(2) 105(2) space group P P21/c Z 2 4 -1 μ [mm ] (MoKα) 5.117 1.303 a [Å] 8.236(4) 15.0766(2) b [Å] 10.387(5) 13.5560(2) c [Å] 17.389(7) 16.1782(2) α [°] 82.035(9) 102.555(1) β [°] 84.714(10) 90 γ [°] 78.758(9) 90 V [Å3] 1441.7(11) 3227.41(8) F(000) 756 1419 θmax [°] 25.00 25.00 No of reflections (total) 4931 5652 No of reflections (unique) 4531 5211 No of parameters 326 397 R1(F) all data 0.0592 0.0324 2 wR2(F ) all data 0.1482 0.0829 Goodness of fit on F2 1.061 1.137 Diff. Four. Peaks 1.871/-2.722 0.724/-0.372 max/min, [eÅ-3] 97