Tyrosinase Model Systems and Biohybrid Conjugates with Copper Bis(Pyrazolyl)Methane Complexes Selbstständig Und Ohne Unzulässige Fremde Hilfe Erbracht Habe

Home , Methane monooxygenase

Tyrosinase Model Systems

and Biohybrid Conjugates with

Copper Bis(pyrazolyl)methane Complexes

Dissertation

von

Patricia Liebhäuser

aus

Tegernsee

Tyrosinase Model Systems

and Biohybrid Conjugates with

Copper Bis(pyrazolyl)methane Complexes

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Patricia Liebhäuser, M. Sc.

aus Tegernsee

Berichter: Prof. Dr. Sonja Herres-Pawlis

Prof. Dr. Jun Okuda

Tag der mündlichen Prüfung: 21.12.2018

Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek verfügbar.

Eidesstattliche Versicherung

Ich, Patricia Liebhäuser, versichere hiermit an Eides Statt, dass ich die vorliegende Dissertation mit dem Titel:

Tyrosinase Model Systems and Biohybrid Conjugates with Copper Bis(pyrazolyl)methane Complexes selbstständig und ohne unzulässige fremde Hilfe erbracht habe. Ich habe keine anderen als die angegebenen Quellen und Hilfsmittel benutzt. Für den Fall, dass die Arbeit zusätzlich auf einem Datenträger eingereicht wird, erkläre ich, dass die schriftliche und die elektronische Form vollständig übereinstimmen. Die Arbeit hat in gleicher oder ähnlicher Form noch keiner Prüfungsbehörde vorgelegen.

______Ort, Datum Unterschrift

Belehrung

§ 156 StGB: Falsche Versicherung an Eides Statt

Wer vor einer zur Abnahme einer Versicherung an Eides Statt zuständigen Behörde eine solche Versicherung falsch abgibt oder unter Berufung auf eine solche Versicherung falsch aussagt, wird mit Freiheitsstrafe bis zu drei Jahren oder mit Geldstrafe bestraft.

§ 161 StGB: Fahrlässiger Falscheid; fahrlässige falsche Versicherung an Eides Statt

1. Wenn eine der in den §§ 154 bis 156 bezeichneten Handlungen aus Fahrlässigkeit begangen worden ist, so tritt Freiheitsstrafe bis zu einem Jahr oder Geldstrafe ein. 2. Straflosigkeit tritt ein, wenn der Täter die falsche Angabe rechtzeitig berichtigt. Die Vorschriften des § 158 Abs. 2 und 3 gelten entsprechend.

Die vorstehende Belehrung habe ich zur Kenntnis genommen:

______Ort, Datum Unterschrift

List of Publications, Projects, Presentations and Posters

First Author Publications

1. P. Liebhäuser, A. Hoffmann, S. Herres-Pawlis, Tyrosinase Models: Synthesis, Spectroscopy, Theory, and Catalysis. In: Elsevier Reference Module in Chemistry, Molecular Science and Chemical Engineering, Elsevier, Waltham, MA, 2016. 2. P. Liebhäuser, Bioanorganisches Symposium 2016 in Aachen, Nachr. Chem. 2016, 64, 1208. 3. P. Liebhäuser, K. Keisers, A. Hoffmann, T. Schnappinger, I. Sommer, A. Thoma, C. Wilfer, R. Schoch, K. Stührenberg, M. Bauer, M. Dürr, I. Ivanović-Burmazović, S. Herres-Pawlis, Record Broken: A Copper Peroxide Complex with Enhanced Stability and Faster Hydroxylation Catalysis, Chem. Eur. J. 2017, 23, 12171-12183.

Further Publications

1. C. Wilfer, P. Liebhäuser, H. Erdmann, A. Hoffmann, S. Herres-Pawlis, Biomimetic Hydroxylation Catalysis Through Self-Assembly of a Bis(pyrazolyl)methane Copper- Peroxo Complex, Eur. J. Inorg. Chem. 2015, 2015, 494-502. 2. C. Wilfer, P. Liebhäuser, A. Hoffmann, H. Erdmann, O. Grossmann, L. Runtsch, E. Paffenholz, R. Schepper, R. Dick, M. Bauer, M. Dürr, I. Ivanović-Burmazović, S. Herres-Pawlis, Efficient Biomimetic Hydroxylation Catalysis with a Bis(pyrazolyl)imidazolylmethane Copper Peroxide Complex, Chem. Eur. J. 2015, 21, 17639-17649. 3. A. Hoffmann, M. Wern, T. Hoppe, M. Witte, R. Haase, P. Liebhäuser, J. Glatthaar, S. Herres-Pawlis, S. Schindler, Hand in Hand: Experimental and Theoretical Investigations into the Reactions of Copper(I) Mono- and Bis(guanidine) Complexes with Dioxygen, Eur. J. Inorg. Chem. 2016, 2016, 4744-4751. 4. D. Schurr, F. Strassl, P. Liebhäuser, G. Rinke, R. Dittmeyer, S. Herres-Pawlis, Decay kinetics of sensitive bioinorganic species in a SuperFocus mixer at ambient conditions, React. Chem. Eng. 2016, 1, 485-493. 5. S. F. Hannigan, A. I. Arnoff, S. E. Neville, J. S. Lum, J. A. Golen, A. L. Rheingold, N. Orth, I. Ivanović-Burmazović, P. Liebhäuser, T. Rösener, J. Stanek, A. Hoffmann,

S. Herres-Pawlis, L. H. Doerrer, On the Way to a Trisanionic {Cu3O2} Core for Oxidase Catalysis: Evidence of an Asymmetric Trinuclear Precursor Stabilized by Perfluoropinacolate Ligands, Chem. Eur. J. 2017, 23, 8212-8224.

6. J. Moegling, A. Hoffmann, F. Thomas, N. Orth, P. Liebhäuser, U. Herber, R. Rampmaier, J. Stanek, G. Fink, I. Ivanović-Burmazović, S. Herres-Pawlis, Designed to React: Terminal Copper Nitrenes and Their Application in Catalytic C-H Aminations, Angew. Chem. Int. Ed. 2018, 57, 9154-9159; Maßgeschneiderte terminale Kupfernitrene für katalytische C-H-Aminierungen, Angew. Chem. 2018, 130, 9294- 9299. 7. S. E. N. Brazeau, E. E. Norwine, S. F. Hannigan, N. Orth, I. Ivanović-Burmazović, D. Rukser, F. Biebl, B. Grimm-Lebsanft, G. Praedel, M. Teubner, M. Rübhausen, P. Liebhäuser, T. Rösener, J. Stanek, A. Hoffmann, S. Herres-Pawlis, L. H. Doerrer,

Dual Oxidase/Oxygenase Reactivity and Resonance Raman Spectra of {Cu3O2} Moiety with Perfluoro-t-butoxide Ligands, Dalton Trans. 2019, accepted.

DFG Projects

1. SPP1740: Reaktive Blasenströmungen (engl.: Reactive Bubbly Flows), subproject: Steuerung der Bildung und Reaktion von Kupfer-Sauerstoff-Adduktkomplexen in Mehrphasenströmungen 2. FOR1405: Biological and Inorganic Charge Transfer Dynamics (BioCTDyn) 3. IRTG 1628: Selectivity in Chemo- and Biocatalysis (SeleCa), subproject: Organo-/ Biohybrid Catalysis

Presentations

1. SPP1740 Annual Colloquium, Hamburg, 2015: Kinetics of the Activation and Transport of Oxygen with Bis(pyrazolyl)methane Copper Complexes 2. FOR1405 Meeting, Aachen, 2015: Sauerstoffübertragung mit Bis(pyrazolyl)imidazolyl- methankupferkomplexen 3. SPP1740 Joint Colloquium, Aachen, 2015: Kinetics of the Activation and Transport of Oxygen with Bis(pyrazolyl)methane Copper Complexes 4. Bioinorganic Symposium, Aachen, 2016: Kinetics of the Activation and Transport of Oxygen with Bis(pyrazolyl)methane Copper Complexes 5. SeleCa Joint Symposium, Aachen, 2016: Kinetics of the Activation and Transport of Oxygen with Bis(pyrazolyl)methane Copper Complexes 6. Tag der Chemie, Aachen, 2017: Effiziente biomimetische Hydroxylierungskatalyse mit Kupfer-Peroxo-Komplexen

7. Biotechnology and Chemistry for GREEN GROWTH (SeleCa Joint Symposium), Osaka, 2017: Phenol Oxidation with Copper Bis(pyrazolyl)methane Complexes 8. Joint Workshop of CaSuS (Catalysis for Sustainable Synthesis) and SeleCa, Bielefeld, 2017: Hydroxylation Catalysis with a Peroxide Dicopper(II) Complex 9. Biotechnology and Chemistry for GREEN GROWTH (SeleCa Joint Symposium), Osaka, 2018: Maleimide-linked Bis(pyrazolyl)methane Ligands for Oxygen Activating Reactions 10. 255th ACS National Meeting & Exposition, New Orleans, 2018: Tyrosinase Model Systems with Efficient Catalytic Activity 11. SeleCa Final Symposium, Aachen, 2018: Oxygen Activation with Maleimide-linked Bis(pyrazolyl)methane Ligands

Posters

1. 6th EuCheMS Conference on Nitrogen Ligands, Beaune, 2015: Kinetics of the Activation and Transfer of Oxygen with Bis(pyrazolyl)methane Copper Complexes 2. 12. Koordinationschemie-Treffen, Kiel, 2016: Bis(pyrazolyl)methane Copper Complexes and their Application as Tyrosinase Models 3. 6th EuCheMS Chemistry Congress, Sevilla, 2016: Efficient Hydroxylation Catalysis Mimicking Tyrosinase 4. SeleCa Joint Symposium, Aachen, 2017: Bis(pyrazolyl)methane Copper Complexes in Hydroxylation Catalysis 5. SeleCa Final Symposium, Aachen, 2018: Oxygen Activation with Maleimide-linked Bis(pyrazolyl)methane Ligands

Abstract

In the last decades, small molecule transition metal complexes became useful models for analysing enzymatic mechanisms, substrate reactivities and active site structures. This doctoral thesis describes two applications of bis(pyrazolyl)methane ligands in the field of enzyme modelling. Copper complexes of these N-donor ligands are known to serve excellently as structural and functional models for the dinuclear copper enzyme tyrosinase. Furthermore, the use of bis(pyrazolyl)methane copper complexes in the field of biohybrid catalysis opens up the possibility of modelling mononuclear copper enzymes like particulate methane monooxygenase.

The development of a bis(pyrazolyl)methane ligand with a substituted pyridinyl moiety

(HC(3-tBuPz)2(4-CO2MePy)) established a new tyrosinase model system. The reaction with molecular oxygen displayed that the peroxide dicopper(II) complex with this substituted pyridinyl ligand exceeds the stability of the system with the former pyridinyl ligand. Simultaneously, conversion of the phenolic substrate 8-hydroxyquinoline showed an improved catalytic reactivity compared to the reported imidazolyl system with regard to yield and reaction time. The hydroxylation of sodium phenolates resulted in saturation kinetics. Moreover, an electrophilic aromatic substitution mechanism was found, which resembles that of the enzyme tyrosinase. The ligand HC(3-tBuPz)2(4-CO2MePy) was furthermore used for the synthesis and structural characterisation of copper(I) and copper(II) complexes. The stabilisation of a mononuclear superoxide copper(II) species with bis(pyrazolyl)methane ligands is rather difficult due to the mostly transient nature of superoxide complexes, which tend to form dinuclear species. This might be overcome with the help of a biohybrid conjugate. The design of maleimide-bearing ligands led to the establishment of a new application field for bis(pyrazolyl)methanes. The optimised synthesis of two maleimido-bis(pyrazolyl)methane ligands is described. Copper complexes of both ligands were analysed concerning oxygen activation and transfer reactions. This was performed with the phenolic substrates 8-hydroxyquinoline and 4-methoxyphenol. Furthermore, these maleimide ligands were conjugated into variants of the -barrel protein nitrobindin. The formation of copper complexes inside of the protein as well as the incorporation of molecular oxygen were analysed spectroscopically. Reactivity was investigated with oxygenation, oxidation or H-atom transfer reaction substrates, but no activity could be detected yet.

Kurzzusammenfassung

In den letzten Jahrzehnten wurden zunehmend Übergangsmetallkomplexe mit kleinen Ligand- molekülen als Modelle verwendet, um den Reaktionsmechanismus, Substratreaktionen und die Struktur des aktiven Zentrums von Enzymen zu untersuchen. Diese Dissertation beschreibt zwei Anwendungen von Bis(pyrazolyl)methanliganden im Bereich der Modellierung von Enzymen. Kupferkomplexe dieser N-Donorliganden sind bereits als exzellente strukturelle und funktionelle Modelle des zweikernigen Kupferenzyms Tyrosinase bekannt. Weiterhin eröffnet die Verwendung von Bis(pyrazolyl)methankupferkomplexen im Bereich der Biohybridkatalyse die Möglichkeit zur Modellierung von einkernigen Kupferenzymen wie der partikulären Methan- monooxygenase.

Die Entwicklung eines Bis(pyrazolyl)methanliganden mit einem substituierten Pyridinylrest

(HC(3-tBuPz)2(4-CO2MePy)) führte zu einem neuen Tyrosinasemodellsystem. Die Reaktion mit molekularem Sauerstoff resultierte mit diesem substituierten Pyridinylliganden in einem Peroxiddikupfer(II)-Komplex, der die Stabilität von früheren Pyridinylsystemen übertrifft. Gleichzeitig wurde festgestellt, dass die katalytische Umsetzung des phenolischen Substrats 8-Hydroxychinolin hinsichtlich der Ausbeute und Reaktionszeit verglichen mit einem früheren Imidazolylsystem verbessert werden konnte. Die Kinetik der Hydroxylierung von Natrium- phenolaten entspricht einer Sättigungskinetik. Zudem wurde ein elektrophiler aromatischer Substitutionsmechanismus nachgewiesen, der den Mechanismus der Tyrosinase widerspiegelt. Der Ligand HC(3-tBuPz)2(4-CO2MePy) wurde außerdem zur Synthese und strukturellen Charakterisierung von Kupfer(I)- und Kupfer(II)-Komplexen verwendet. Die Stabilisierung einkerniger Superoxidkupfer(II)-Spezies ist mit Bis(pyrazolyl)methanliganden schwierig zu realisieren, da Superoxidkomplexe meist kurzlebig sind und zur Ausbildung zweikerniger Spezies neigen. Ermöglicht werden könnte dies durch den Einsatz von Biohybrid- verbindungen. Mit der Entwicklung von Liganden, die eine Maleimidgruppe aufweisen, wurde ein neuer Anwendungsbereich für Bis(pyrazolyl)methane eröffnet. Die optimierte Synthese zweier Maleimido-Bis(pyrazolyl)methane wird beschrieben. Die entsprechenden Kupfer- komplexe wurden mittels der phenolischen Substrate 8-Hydroxychinolin und 4-Methoxyphenol auf die Aktivierung und Übertragung von Sauerstoff hin untersucht. Des Weiteren wurden die Maleimidliganden in Varianten des Fassproteins Nitrobindin konjugiert. Die Bildung von Kupferkomplexen innerhalb des Proteins und die Aufnahme von Sauerstoff wurden spektroskopisch untersucht. Reaktivitätsstudien wurden mit unterschiedlichen Substraten durchgeführt. Bisher konnte keine Reaktivität im Hinblick auf Oxygenierungs-, Oxidations- oder H-Atom-Transferreaktionen ermittelt werden.

Danksagung

An erster Stelle möchte ich mich bei meiner Doktormutter Prof. Dr. Sonja Herres-Pawlis für das interessante und spannende Dissertationsthema bedanken. Ich danke ihr für die Diskussionen und Denkanstöße, sowie die Freiheiten im praktischen Ausführen dieser Arbeit. Des Weiteren bedanke ich mich dafür, dass ich die Möglichkeit hatte, zahlreiche nationale und internationale Konferenzen zu besuchen, um so meinen wissenschaftlichen Horizont zu erweitern. Weiterhin möchte ich mich für die ermöglichte Mitarbeit in den Projekten SPP1740, FOR1405 und IRTG 1628 (SeleCa) bedanken, wodurch ich neue interdisziplinäre Sichtweisen kennen lernen konnte. Besonders danke ich ihr außerdem für die Ermöglichung des zweimonatigen Aufenthalts in einem Forschungslabor an der Osaka University in Japan im Rahmen des SeleCa-Projekts.

Bei Herrn Prof. Dr. Jun Okuda bedanke ich mich für die Übernahme des Zweitgutachtens dieser Arbeit. Zudem möchte ich ihm für die Möglichkeit zur Teilnahme am internationalen Graduiertenkolleg SeleCa danken.

Herrn Prof. Dr. Takashi Hayashi von der Osaka University danke ich für die Möglichkeit des zweimonatigen Aufenthalts in seiner Arbeitsgruppe, um meine Forschungsarbeiten im Bereich der Biotechnologie auszuüben. Für anregende Diskussionen in diesem Bereich danke ich außerdem Prof. Dr. Akira Onoda von der Osaka University. Für die Möglichkeit zur Weiterführung der biotechnologischen Experimente in seinem Forschungslabor danke ich Prof. Dr. Ulrich Schwaneberg von der RWTH Aachen.

Bei Dr. Alexander Hoffmann möchte ich mich für rege Diskussionen während der praktischen Ausführung dieser Arbeit bedanken. Ferner danke ich ihm für das Finalisieren der Kristall- strukturen und das Korrekturlesen des Experimentalteils dieser Arbeit.

Mein Dank gilt zudem den Mitarbeitern des Arbeitskreises von Sonja Herres-Pawlis, meinen derzeitigen und ehemaligen Kollegen. Für inspirierende Diskussionen und hilfreiche Kritik danke ich im Besonderen Thomas Rösener, Kristina Keisers, Dr. Julian Moegling, Dr. Julia Stanek und Dr. Claudia Wilfer. Katharina Beyer danke ich für die Vorarbeiten in meinem Forschungsthema. Thomas Rösener danke ich für das intensive Korrekturlesen dieser Arbeit.

Des Weiteren bedanke ich mich bei Claudia Nelleßen für die stete Hilfsbereitschaft in organisatorischen Fragen. Mein Dank gilt außerdem Kiyomi Lee von der Osaka University, die mir mit größtem Einsatz jegliche Frage zum Forschungsaufenthalt in Osaka beantwortete.

Für die Zusammenarbeit im Gebiet der Biohybridkatalysatoren möchte ich mich bei Alexander Grimm (Arbeitsgruppe von Prof. Dr. U. Schwaneberg, RWTH Aachen) bedanken. Ebenso gilt mein Dank den Studenten der Arbeitsgruppe von Prof. Dr. T. Hayashi (Osaka University) für jegliche Unterstützung während des Forschungsaufenthalts an der Osaka University.

Weiterhin bedanke ich mich bei Prof. Dr. Michael Rübhausen (CFEL, Universität Hamburg) und seinen Mitarbeitern für die Möglichkeit der Messung von Resonanzramanspektren. Prof. Dr. Ivana Ivanović-Burmazović (FAU Erlangen-Nürnberg) und ihren Mitarbeitern danke ich für die Messung der UHR-CSI-Massenspektren. Ich bedanke mich zudem bei Prof. Dr. Matthias Bauer (Universität Paderborn) und seinen Mitarbeitern für die durchgeführten XAS-Messungen am ESRF in Grenoble.

Ich danke allen Mitarbeitern des Instituts für Anorganische Chemie (RWTH Aachen) und im Besonderen den Analytikabteilungen des IAC und des IOC für die Durchführung der analytischen Messungen. Prof. Dr. Ullrich Englert und seinen Mitarbeitern danke ich außerdem für die röntgenkristallographischen Messungen.

Ich möchte mich bei meinen Praktikanten und Bacheloranden für die stets motivierte Mitarbeit an meinem Forschungsprojekt bedanken. Besonders danke ich Regina Schmidt, die als meine Nachfolgerin zukünftig mein Forschungsthema weiterbearbeiten wird.

Bei meinen Freunden Thomas, Julian, Kristina, Julia, Angi, Claudia und Uli möchte ich mich für die wunderschöne Zeit und die zahlreichen Ausflüge und Unternehmungen neben dem Laboralltag bedanken. Ich danke ihnen dafür, dass sie mir das Leben in Aachen, in weiter Entfernung zur Heimat, erleichterten.

Mit größter Herzlichkeit möchte ich mich bei meiner Familie bedanken, die mich trotz der weiten Entfernung zum bayerischen Oberland und nach Shanghai bestmöglich unterstützte. Mein besonderer Dank gilt meiner Schwester Lisa, die immer genau weiß, wie sie mich motivieren und aufheitern kann.

Meinen größten Dank möchte ich Thomas aussprechen, der mich in allen Lebenslagen bedingungslos unterstützte. Er wusste es, mich auch in schwierigen Zeiten der Promotion immer wieder zu motivieren und für die Arbeit zu begeistern. Ich bedanke mich für die zahlreichen Erlebnisse in und um Aachen. Durch ihn habe ich mich in Aachen zuhause gefühlt.

An essential aspect of creativity is not being afraid to fail.

Edwin Herbert Land US-amerikanischer Physiker und Erfinder

Table of Contents

1. Introduction ...... 1

1.1. Copper Enzymes ...... 1

1.1.1. The Dinuclear Copper Enzyme Tyrosinase ...... 2

1.1.2. Mononuclear Copper Enzymes ...... 5

1.1.3. Enzyme Modelling ...... 6

1.2. Tyrosinase Model Systems ...... 7

1.2.1. N-Donor Ligands ...... 7

1.2.2. Oxygen Activation ...... 8

1.2.3. Hydroxylation Reactions ...... 9

1.3. Particulate Methane Monooxygenase Model Systems ...... 13

1.3.1. Structural Models ...... 14

1.3.2. Substrate Reactivity ...... 15

1.4. Biohybrid Catalysts ...... 15

1.4.1. Protein Scaffolds ...... 16

1.4.2. Anchoring Strategies ...... 18

1.4.3. Copper-Based Systems ...... 19

1.5. Poly(pyrazolyl)methanes...... 19

1.5.1. Bis(pyrazolyl)methane Ligands ...... 20

1.5.2. Bis(pyrazolyl)methane Complexes ...... 21

2. Objective and Outline ...... 23

2.1. Objective ...... 23

2.2. Outline ...... 24

3. Tyrosinase Models ...... 27

3.1. Bis(pyrazolyl)methanes ...... 27

3.1.1. Synthesis of 2-(4-Methoxycarbonylpyridinyl)bis(3-tert-butylpyrazolyl)methane (L1) ...... 30

3.1.2. Characterisation of 2-(4-Methoxycarbonylpyridinyl)bis(3-tert-butylpyrazolyl)- methane (L1) ...... 31

3.2. Copper Bis(pyrazolyl)methane Complexes ...... 32

3.2.1. Synthesis and Characterisation of [Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1) .. 33

3.2.2. Synthesis and Characterisation of [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2) ... 36

3.2.3. Synthesis and Characterisation of [Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3) ...... 38

3.2.4. Synthesis and Characterisation of [Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4) ...... 40

3.3. Oxygen Activation ...... 42

3.3.1. Formation and Stability of the Peroxide Dicopper(II) Species P1, P2 and P3 .. 43

3.3.2. Characterisation of the Peroxide Dicopper(II) Complex P1 ...... 53

3.4. Hydroxylation Reactions ...... 60

3.4.1. Hydroxylation Kinetics...... 61

3.4.2. Catalytic Conversions ...... 69

3.5. Conclusion ...... 75

4. Biohybrid Conjugates ...... 77

4.1. Bis(pyrazolyl)methanes ...... 77

4.1.1. Synthesis of 2-(4-Hydroxymethylenepyridinyl)bis(3-tert-butylpyrazolyl)methane (L4) ...... 78

4.1.2. Synthesis of 2-(4-Maleimidoethylenecarboxymethylenepyridinyl)bis(3-tert-butylpyrazolyl)methane (L5) ...... 79

4.1.3. Synthesis of 2-(4-Maleimidomethylenepyridinyl)bis(3-tert-butylpyrazolyl)- methane (L6) ...... 80

4.1.4. Characterisation of the Bis(pyrazolyl)methanes L4, L5 and L6 ...... 81

4.2. Tyrosinase Reactions ...... 82

4.2.1. Oxygen Activation with L5 and L6 ...... 83

4.2.2. Catalytic Hydroxylation Reactions ...... 85

4.3. Nitrobindin Conjugates ...... 89

4.3.1. Conjugation of L5 and L6 to NB4 and NB4exp ...... 89

4.3.2. Copper Complexation in the Conjugated Protein ...... 93

4.3.3. Reduction of Copper ...... 98

4.3.4. Oxygen Activation ...... 99

4.3.5. Reaction with Hydrogen Peroxide ...... 101

4.3.6. Substrate Oxidation Reactions ...... 101

4.4. Conclusion ...... 106

5. Conclusion and Outlook ...... 109

5.1. Conclusion ...... 109

5.2. Outlook ...... 113

6. Experimental Part ...... 115

6.1. General Procedures ...... 115

6.2. Chemicals ...... 115

6.3. Methods ...... 117

6.4. Ligand Syntheses ...... 121

6.4.1. 2-(4-Methoxycarbonylpyridinyl)bis(3-tert-butylpyrazolyl)methane (L1) ...... 121

6.4.2. 2-(4-Hydroxymethylenepyridinyl)bis(3-tert-butylpyrazolyl)methane (L4) ...... 123

6.4.3. 2-(4-Maleimidoethylenecarboxymethylenepyridinyl)bis(3-tert-butylpyrazolyl)- methane (L5) ...... 124

6.4.4. 2-(4-Maleimidomethylenepyridinyl)bis(3-tert-butylpyrazolyl)methane (L6) ..... 126

6.5. Complex Syntheses ...... 127

6.5.1. [Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1) ...... 127

6.5.2. [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2) ...... 128

6.5.3. [Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3) ...... 129

6.5.4. [Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4) ...... 130

6.6. Formation of Peroxide Dicopper(II) Complexes ...... 131

2 2 6.6.1. [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) ...... 132

2 2 6.6.2. [Cu2{HC(3-tBuPz)2(1-MeIm)}2(- : -O2)](SbF6)2 (P2) ...... 133

2 2 6.6.3. [Cu2{HC(3-tBuPz)2(Py)}2(- : -O2)](SbF6)2 (P3) ...... 133

2 2 6.6.4. [Cu2{HC(3-tBuPz)2(4-Mal1Py)}2(- : -O2)](SbF6)2 (P5) ...... 134

2 2 6.6.5. [Cu2{HC(3-tBuPz)2(4-Mal2Py)}2(- : -O2)](SbF6)2 (P6) ...... 135

6.7. Hydroxylation Reactions with Peroxide Dicopper(II) Species ...... 135

6.7.1. Stoichiometric Hydroxylation of Sodium Phenolates ...... 135

6.7.2. Catalytic Conversions of Phenolic Substrates ...... 137

6.8. Biohybrid Catalysts ...... 138

6.8.1. Protein Conjugation with Bis(pyrazolyl)methane Ligands ...... 138

6.8.2. Incorporation of Copper ...... 139

6.8.3. Reduction of Copper ...... 140

6.8.4. Reaction with Oxygen ...... 141

6.8.5. Reaction with Hydrogen Peroxide ...... 141

6.8.6. Hydroxylation or Oxidation Reactions ...... 142

7. Literature ...... 145

8. Appendix ...... 157

8.1. Crystallographic Data ...... 157

8.2. Extinction Coefficient of P1 ...... 161

8.3. Protein Structure ...... 162

Table of Compounds

All compounds marked with an asterisk (*) were resynthesised.

Ligands

L1 HC(3-tBuPz)2(4-CO2MePy) L4 HC(3-tBuPz)2(4-CH2OHPy)

L2 HC(3-tBuPz)2(1-MeIm)* L5 HC(3-tBuPz)2(4-Mal1Py)

L3 HC(3-tBuPz)2(Py)* L6 HC(3-tBuPz)2(4-Mal2Py)

Complexes

C1 [Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2]

C2 [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br]

C3 [Cu{HC(3-tBuPz)2(1-MeIm)}Cl]

C4 [Cu{HC(3-tBuPz)2(1-MeIm)}I]

Peroxide complexes have the same numbers as the associated ligands.

2 2 P1 [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2

2 2 P2 [Cu2{HC(3-tBuPz)2(1-MeIm)}2(- : -O2)](SbF6)2*

2 2 P3 [Cu2{HC(3-tBuPz)2(Py)}2(- : -O2)](SbF6)2*

2 2 P5 [Cu2{HC(3-tBuPz)2(4-Mal1Py)}2(- : -O2)](SbF6)2

2 2 P6 [Cu2{HC(3-tBuPz)2(4-Mal2Py)}2(- : -O2)](SbF6)2

Figure 1: Overview of the synthesised and characterised bis(pyrazolyl)methane ligands L1-L6. Resynthesised ligands L2 and L3 are marked with an asterisk.

Figure 2: Overview of the synthesised and characterised copper bis(pyrazolyl)methane complexes C1-C4.

Table of Abbreviations

ABTS 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) aliph. aliphatic arom. aromatic calcd. calculated CT charge transfer DCM dichloromethane Cys cysteine ddH2O double-distilled water DEAD diethyl azodicarboxylate

DEAD-H2 diethyl hydrazinedicarboxylate DNA deoxyribonucleic acid DTBP-H 2,4-di-tert-butylphenol DTT dithiothreitol EPR electron paramagnetic resonance eq. equivalents Et ethyl EXAFS extended X-ray absorption fine structure GC gas chromatography His histidine HV high vacuum Im imidazolyl

L-DOPA L-3,4-dihydroxyphenylalanine Mal maleimido Me methyl MeCN acetonitrile MeOH methanol Met methionine MOPS 3-(N-morpholino)propanesulfonic acid MTP microtiter plate

NB4 nitrobindin variant 4 NB4exp nitrobindin variant 4 expanded PDB protein database Py pyridinyl Pz pyrazolyl RNA ribonucleic acid r.t. room temperature tBu tert-butyl TCEP tris(2-carboxyethyl)phosphine TD-DFT time-dependent density functional theory TON turnover number Tyr tyrosine XANES X-ray absorption near edge structure XAS X-ray absorption spectroscopy

Mass Spectrometry

CSI cryospray ionisation EI electron ionisation ESI electrospray ionisation FAB fast atom bombardment HRMS high resolution mass spectrometry ICP-MS inductively coupled plasma mass spectrometry LC liquid chromatography M molecular ion peak MALDI-TOF matrix assisted laser desorption ionisation-time of flight UHR ultrahigh resolution

NMR Spectroscopy

 chemical shift d doublet J coupling constant

m multiplet NMR nuclear magnetic resonance s singlet t triplet

IR Spectroscopy

ATR attenuated total reflection IR infrared m medium ṽ wavenumber  stretching vibration s strong vs very strong vw very weak w weak

Introduction

1. Introduction

1.1. Copper Enzymes

As biological catalysts, enzymes enable the conversion of demanding substrates by decreasing the activation energy. Therefore, most of the enzymes possess a metal ion as cofactor in the catalytic pocket. These enzymes are called metalloenzymes.[1] Metal centres can promote electron transfer reactions or act as substrate binding sites. In most cases, these metal ions are transition metals like copper, cobalt, iron, zinc, nickel, manganese or molybdenum. The metal cation is coordinated by nitrogen, oxygen or sulphur donors of amino acid side chains of the surrounding protein scaffold. Furthermore, water molecules often complete the coordination sphere.

In copper enzymes,[2] the metal ion is found in oxidation states +I, +II or +III in some intermediates. In the field of copper enzymes, different types are classified according to the active site structure, spectroscopic properties and catalytic reactions.[3] Overall, seven classes of copper enzymes are defined.[4] The main classes are named type I, type II and type III copper enzymes (figure 3). Moreover, type IV (or multicopper sites), CuA, CuB and CuZ sites are known.

Figure 3: Catalytic centres of copper enzyme types. Type I: plastocyanin, type II: galactose oxidase, type III: oxy-tyrosinase.[4]

Introduction

Considering spectroscopic features and due to the intense blue colour, type I copper enzymes are known as blue copper proteins. Examples are azurin or plastocyanin, where the active site consists of a mononuclear copper centre.[5] This metal ion is coordinated by S- and N-donor amino acids in a highly distorted geometry. Blue copper proteins are mostly involved in electron transfer reactions. Type II or normal copper enzymes exhibit also a monocopper catalytic centre but do not show intense colour. The metal ion is coordinated by histidine and/or tyrosine residues. Enzymes like galactose oxidase,[6] Cu/Zn superoxide dismutase[7] or particulate methane monooxygenase[8] are involved in alcohol oxidation, superoxide degradation or C-H bond activation reactions. In contrast to type I and II, type III copper enzymes possess a dinuclear copper complex in the catalytic pocket. The copper ions are coordinated by histidine residues. This class consists of the enzymes tyrosinase,[9] hemocyanin[10] and catechol oxidase.[11] They transport molecular oxygen or activate and transfer it to phenolic substrates.

In the following, the type III copper enzyme tyrosinase and type II copper enzymes such as methane monooxygenase or galactose oxidase are presented in detail.

1.1.1. The Dinuclear Copper Enzyme Tyrosinase

The type III copper enzyme tyrosinase is an important enzyme in the biosynthesis of the pigment melanin. It catalyses the hydroxylation of the amino acid L-tyrosine to L-DOPA

(L-3,4-dihydroxyphenylalanine) and the subsequent dehydrogenation to L-dopaquinone (scheme 1).[12] Further reaction steps including polymerisation result in eumelanin.[13]

Scheme 1: Hydroxylation of L-tyrosine to L-DOPA and subsequent dehydrogenation to L-dopaquinone.

Crystal Structure

The molecular structure of tyrosinase was resolved by Matoba et al. in 2006,[9] much later than the structures of the type III copper enzymes hemocyanin[14] and catechol oxidase.[11] The active site of tyrosinase consists of a dinuclear copper centre, where each copper ion is coordinated by three histidine residues (figure 4). The incorporation of molecular dioxygen results in the formation of a bridging side-on -2:2-peroxide dicopper(II) core. Here, the

Introduction copper-copper distance is 3.4 Å, whereas in the oxygen-free active site it is reported to 4.1 Å.[9] Furthermore, in its oxygenated form, tyrosinase shows an absorption at 350 nm, which is characteristic for side-on peroxide dicopper(II) species.[15]

Figure 4: Molecular structure of the active site of oxy-tyrosinase of streptomyces castaneoglobisporus. The crystal structure was determined in the presence of the caddie protein ORF378. Resolution: 1.5 Å. PDB entry: 1WX4.[9,16]

Mechanism of the Catalytic Activity

In contrast to catechol oxidase, tyrosinase exhibits catecholase and phenolase activity.[17] The hydroxylation of phenolic substrates and the oxidation of catecholic substrates is depicted in scheme 2. Starting from the oxygen-free deoxy-tyrosinase, oxygen incorporation leads to the oxy form by forming the characteristic -2:2-peroxide dicopper(II) core. Subsequently, the phenolase pathway describes the coordination of a phenolic substrate to one of the copper centres. After hydroxylating the phenolate to a catecholate, the D-met species with catecholate-bound copper centres is formed. The presence of protons leads to the release of water, the oxidation of catecholate to the quinone product and the re-formation of deoxy- tyrosinase. With a catecholic substrate, tyrosinase shows catecholase activity. First, the catechol coordinates to both copper centres. This D-oxy form of tyrosinase oxidises the catecholate and releases water and the quinone product in the presence of protons. Subsequently, met-tyrosinase incorporates another catecholic substrate to give the D-met form. In the presence of protons, this results again in water, the quinone product and deoxy- tyrosinase.

Introduction

Scheme 2: Phenolase and catecholase activity of tyrosinase. Proposed mechanism of Tuczek et al.[18] This scheme is drawn according to literature.

Controversially discussed is the nature of the catalytically active species in the hydroxylation reaction of tyrosinase. Although the crystal structure displays a -2:2-peroxide dicopper(II) core in the active site,[9] the possibility of an O-O bond cleavage and thus the formation of the isomeric bis(-oxide) dicopper(III) species is given (scheme 3). For a few N-donor ligand tyrosinase model systems, the O-O bond cleavage is reported to be prior to the substrate hydroxylation.[19,20] Thus, this mechanism has to be considered for the enzyme as well.[17]

Scheme 3: Equilibrium of the isomeric -2:2-peroxide dicopper(II) and bis(-oxide) dicopper(III) species.

Introduction

1.1.2. Mononuclear Copper Enzymes

Reactive copper oxygen species are also known for several type II copper enzymes. For example, in galactose oxidase (GO),[21] Cu/Zn superoxide dismutase (SOD)[22] or peptidylglycine -hydroxylating monooxygenase (PHM),[23] a superoxide ligand is found to coordinate the copper centre in the active site. Whereas substrate reactions of GO and SOD are not further discussed in this thesis, the reactivity of PHM and other monooxygenases is of high interest. PHM, lytic polysaccharide monooxygenase (LPMO) and particulate methane monooxygenase (pMMO) activate unreactive C-H bonds and therewith hydroxylate aliphatic substrates. In PHM, the active site consists of a mononuclear copper centre that is coordinated by two histidine residues, one methionine and one water molecule (figure 5). Upon oxygen incorporation, the formation of an end-on bound superoxide species is observed.

Figure 5: Left: molecular structure of the active site of peptidylglycine -hydroxylating monooxygenase (PHM) of rattus norvegicus. The crystal structure was determined with bound peptide and dioxygen. Resolution: 1.85 Å. PDB entry: 1SDW.[23,24] Right: molecular structure of the active site of particulate methane monooxygenase (pMMO) of methylomicrobium alcaliphilum 20Z. Resolution: 2.704 Å. PDB entry: 6CXH.[25,26]

The active sites of LPMO and pMMO are not as well characterised as that of PHM. Whereas numerous oxygen-free structures of LPMO were crystallised,[27] the nature of the active site in pMMO is still controversially discussed. Both a mononuclear and a dinuclear copper centre are reported, however spectroscopic features indicate pMMO to be a type II copper enzyme.[28,29] In 2011, X-ray crystallographic analysis revealed a dinuclear copper centre with a short copper-copper distance.[28] In recent studies, the original X-ray data were re-resolved with quantum crystallographic methods. There, the active site of pMMO was best described as mononuclear copper centre.[30] The latest publication of the crystal structure of pMMO proves the indication of a mononuclear centre (figure 5).[25] The metal ion is coordinated by three histidine residues, one amine function and one water molecule. The amine function is part of one of the histidine residues, which is the N-terminal amino acid of the protein. This

Introduction coordination motif was reported as histidine brace[31] and is also found in LPMO.[32] The structural and spectroscopic similarities between pMMO and LPMO underline the assumption of the mononuclear copper centre in pMMO.

Whereas LPMO promotes the degradation of polysaccharides by cleaving glycosidic bonds oxidatively, pMMO catalyses the oxidation of methane to methanol. For both monooxygenases, the detailed catalytic mechanism and the reactive copper oxygen species are still unknown. The possibility of a superoxide intermediate species in the active sites of LPMO and pMMO is given, as this is observed for above-mentioned type II copper enzymes. Nevertheless, a superoxide could not be determined yet and other copper oxygen species cannot be neglected.[8,29,33]

1.1.3. Enzyme Modelling

With the conversion of sophisticated substrates at ambient temperatures, enzymes are highly efficient biocatalysts. Thus, two major reasons encouraged synthetic (bio)chemists to mimic natural structures and/or substrate reactivities. First, mimicking the structure of the enzyme helps to better understand enzymes, especially the catalytic reaction. Second, modelling of reactivities leads to the opportunity of a broader substrate range with synthetic systems. With the knowledge of mechanistic details, substrate conversions and scope can be improved.

The crystal structure of tyrosinase was resolved in 2006.[9] Until then, model systems were highly necessary to gain insights into structural and spectroscopic properties of the enzyme. With the structural data of the active site at hands, the targeted synthesis of even more efficient catalysts for aromatic C-H bond activation is now possible.

In contrast to tyrosinase, particulate methane monooxygenase allows the activation of more inert C-H bonds. The conversion of methane to methanol is of great industrial and environmental significance. Methanol represents one of the most important bulk chemicals.[34] On the other hand, methane is a very potent greenhouse gas.[35] Thus, the chemical industry seeks for methods to efficiently convert methane into methanol. Mimicking the active site of pMMO might enable the ecologic and economic production of methanol with dioxygen under ambient conditions.

Introduction

1.2. Tyrosinase Model Systems

Before 2006, tyrosinase model systems helped to better understand the structure of the enzyme’s active site. With the synthesis of oxygenated N-donor ligand copper complexes, a comparison of spectroscopic features of the models and the enzyme resulted in the indication of the active site structure. Furthermore, model systems support the detailed analysis of the hydroxylation mechanism. Synthetic models show a slower hydroxylation of phenolic substrates. Moreover, these models are handled in organic solvents, which can be cooled to temperatures down to -145 °C, in certain cases. Both properties lead to the opportunity of characterising and/or stabilising intermediate species within the hydroxylation reaction at low temperatures.

1.2.1. N-Donor Ligands

The active site of tyrosinase displays a dinuclear copper centre, where each copper ion is coordinated by three histidine residues (figure 4).[9] The amino acid histidine consists of an imidazole moiety that is responsible for the copper coordination. Thus, tyrosinase model systems with a huge diversity of N-donor ligands were developed.[15,18,36–39] A selection of these ligands is shown in figure 6.

Figure 6: Selected N-donor ligand systems for tyrosinase model complexes. Mononucleating systems: A) monodentate,[40] B) bidentate,[41] C) tridentate;[42,43] dinucleating system: D) hexadentate.[44]

Besides the histidine-like 5-membered ring donor moieties imidazole, benzimidazole or pyrazole, the 6-membered heterocycle pyridine, simple imine or amine donors are applied. In tyrosinase, the protein scaffold holds the histidine residues in close proximity. In model systems, this is accomplished by polydentate ligands, where two or three N-donor moieties form a single bi- or tridentate ligand (B) or C)). Furthermore, the linkage of two tridentate

Introduction ligands (D)) induces the formation of two copper centres within one complex molecule. Moreover, single monodentate imidazoles (A)) can serve as ligands for tyrosinase models, although they possess no connection between the N-donor moieties.

1.2.2. Oxygen Activation

Copper Oxygen Species

The incorporation of dioxygen into deoxy-tyrosinase results in the formation of a -2:2-peroxide dicopper(II) species in the active site.[9] However, the reaction of copper complexes with dioxygen can lead to different copper oxygen species including mono-, di- or trinuclear complexes (scheme 4).[15]

[15] Scheme 4: Copper oxygen binding motifs in CuO2, Cu2O2 and Cu3O2 species.

Binding of dioxygen to a copper(I) complex results in the first step in a 1:1 Cu:O2 species, where copper is oxidised and molecular oxygen is reduced. The superoxide coordination is either found in an end-on or in a side-on fashion. Furthermore, the isomerisation to a peroxide copper(III) species is conceivable. These CuO2 species are rather short-lived, as they quickly react with a second copper(I) complex to give Cu2O2 species. The peroxide ligand is then bound either in a -1,2- or in a -2:2-way. The latter is also known as side-on peroxide dicopper(II) complex, which can isomerise into the bis(-oxide) dicopper(III) complex by O-O bond cleavage. -1,2-peroxide dicopper(II) species are mostly stabilised with tetradentate ligands, whereas with tridentate or bidentate ligand systems, -2:2-peroxide dicopper(II) or

Introduction bis(-oxide) dicopper(III) species are favoured.[15] The reaction of the peroxide or the oxide complex with another copper(I) complex can result in a Cu3O2 species, which displays one copper(III) and two copper(II) centres.

Structural Tyrosinase Models

The first crystallographically characterised side-on -2:2-peroxide dicopper(II) species was published by Kitajima et al. in 1989.[45] Here, the metal centres are each coordinated by a tridentate tris(pyrazolyl)hydroborate ligand (figure 7). This complex showed high physico- chemical similarities to oxy-hemocyanin and oxy-tyrosinase and thus, the first evidence of a side-on peroxide dicopper(II) species in the active site of these enzymes was found.

Figure 7: Side-on -2:2-peroxide dicopper(II) species. Left: first crystallographically characterised complex.[45] Right: most stable model complex.[46]

From then on, numerous crystal structures of -2:2-peroxide dicopper(II) complexes were published.[15,39,46,47] However, the crystallisation of model systems, which show catalytic reactivity (section 1.2.3.) was not achieved yet. The most stable side-on peroxide dicopper(II) species is to date a system published by Scarborough et al. with the N-donor ligand tBu3tacn (figure 7). The half-life of this peroxide complex is reported to 14 h at r.t. in the solvent

[46] methanol. Furthermore, it is stable for several days in aqueous solution containing Na2HPO4 or NaH2PO4.

1.2.3. Hydroxylation Reactions

Many of the tyrosinase model systems mimic the enzyme not only structurally, but also functionally. The conversion of phenolic substrates to catechols and/or quinones is reported for a variety of substrates.[18,36–39] It is mentioned that although there exists a large number of

Introduction known reactivity models, only selected systems display the ability of hydroxylating substrates catalytically.[41–43,48–57]

The reaction of side-on peroxide dicopper(II) complexes with phenolic substrates can result in different product species (scheme 5). The biomimetic ortho-hydroxylation of a phenol leads to an ortho-catecholic compound (pathway a) and further oxidation results in an ortho-quinone product (pathway b). Furthermore, the reaction of a quinone with a phenol can result in a C-O coupled product (pathway c).[57,58] On the other hand, phenols can be converted into C-C coupled compounds via phenoxyl radicals (radical mechanism, pathway d).[17] This radical pathway is promoted, when using phenols instead of phenolates without addition of an auxiliary base.

Scheme 5: Possible reaction pathways for phenolic substrates. Hydroxylation to catechol (a), further oxidation to quinone (b), oxidative C-O coupling of phenol with quinone (c), C-C coupling through radical mechanism (d).

Stoichiometric Substrate Hydroxylation

The hydroxylation of phenolic substrates can be divided into two different reaction types: the intramolecular hydroxylation of the ligand framework and the intermolecular hydroxylation of exogeneous phenolic substrates. Oxygen transfer to the ligand was observed in several model systems[18,38,39] but will not be further discussed.

The phenolic hydroxylation is characterised by two important parameters (cf. scheme 18 in chapter 3.4.). The equilibrium constant Keq describes the phenolate coordination to the copper centre. The subsequent oxygenation to the catecholate is given by the hydroxylation constant kox. The determination of these reaction constants is performed via stoichiometric reactions of peroxide dicopper(II) complexes with sodium or lithium phenolates. The nature of the hydroxylation is analysed with para-substituted phenolates. A faster reaction with electron-

Introduction richer substrates indicates an electrophilic aromatic substitution mechanism, whereas a nucleophilic hydroxylation is proven when electron-poor substrates are converted faster.

The enzyme tyrosinase is reported to hydroxylate phenolates in an electrophilic aromatic

[59–61] substitution mechanism. Hydroxylation constants kcat for para-substituted phenolates are in the range of 10-1-102 s-1.[61] The electron-poor substrate 4-hydroxybenzaldehyde is

-1 hydroxylated with kcat = 0.19 s , whereas the electron-rich 4-hydroxyanisole displays a kcat of 274.87 s-1. The fastest hydroxylation model with a peroxide dicopper(II) complex is to date a system with the bis(pyrazolyl)methane ligand HC(3-tBuPz)2(1-MeIm) (figure 8). For

-1 [42,43] bis(pyrazolyl)methane systems, kox values between 0.3 and 4.3 s are reported. The bis(pyridinyl)amine system LPy2Bz of Itoh et al. displays comparable velocities (0.08-0.76 s-1), considering that these hydroxylation constants were determined at -94 °C.[62] Other tyrosinase model systems show much slower reactions with phenolates (10-4-10-3 s-1).[41,63] Besides polydentate N-donor ligands, Stack et al. described the hydroxylation of phenolates even with a peroxide dicopper(II) system containing simple imidazole ligands.[40] In this case, the addition of a crown ether was necessary to determine hydroxylation constants of para-substituted 2-tBu-phenolates. A few years later, the same group published reactions at -145 °C and found an intermediate bis(-oxide) species during the conversion of phenolates with the side-on peroxide dicopper(II) complex exhibiting 1-methylimidazole ligands.[64] This represents an evidence of an intermediate bis(-oxide) species as catalytically active species in tyrosinase model systems and thus in the enzyme as well.

Figure 8: Left: side-on peroxide dicopper(II) model complex displaying the fastest phenolate hydroxylation.[43] Right: ligands providing systems with the most efficient catalysis towards phenolic substrates.[50,65]

Catalytic Substrate Conversion

In the field of catalytically active tyrosinase models, the number of potent ligand systems was more than duplicated in the last five years. Most systems were published by Tuczek et al., who

Introduction screened a series of bidentate ligands, which exhibit imine, pyridine, benzimidazole, imidazole, pyrazole or triazole N-donor functions (two examples in figure 8).[41,50–53,55,57] Within tridentate ligand systems it was found that one donor moiety must be different to the other donors. Whereas tris(pyrazolyl)methane ligands do not show tyrosinase-like activity, systems with bis(pyrazolyl)(pyridinyl/imidazolyl)methane ligands hydroxylate substrates catalytically.[42,43] This behaviour was also proven for bidentate ligand systems. In 2010, Tuczek et al. published the first catalytically active mononuclear copper complex.[41] This complex exhibits an imine- pyridine ligand. Furthermore, they synthesised complexes with two imine or two pyridine functions. Neither with the imine nor with the pyridine ligand, tyrosinase activity was achieved. On the other hand, the application of ligands, which consist only of pyrazole or imidazole donor moieties, resulted in the ability to catalytically convert phenolic substrates.[52,55]

As tyrosinase catalyses the hydroxylation of tyrosine, model complexes are also analysed concerning the conversion of phenolic substrates. Such substrates are different para- substituted phenols (p-X = OMe, Me, H, tBu), 3-tert-butylphenol, 2,4-di-tert-butylphenol (DTBP-H), hydroxyquinoline or the more biologically relevant estrone or N-acetyltyrosine ethyl ester (figure 9).

Figure 9: Phenolic substrates for tyrosinase activity analyses of model systems (R = OMe, tBu, Me, H).

The first catalytically active tyrosinase model system was published in 1990.[48] With the dinucleating ligand BiPh(impy)2, the formation of a side-on peroxide dicopper(II) system was reported. The conversion of DTBP-H was achieved with a turnover number (TON) of 16 in 1 h. The catalysis stopped after a reaction time of 1 h according to unknown reasons. The fastest

[43] catalytic reaction is reported with the bis(pyrazolyl)methane ligand HC(3-tBuPz)2(1-MeIm). Here, a TON of 14 was obtained after 20 min when converting the substrate 8-hydroxyquinoline. The highest substrate conversion is reported with the benzimidazole-imine

[50] [65] ligand Lbzm1 (conversion of DTBP-H, TON = 31) and the triazole-pyridine ligand TMP3 (conversion of 4-methoxyphenol, TON = 48).

Besides the expected quinone, the hydroxylation of phenols displays also the formation of C-C or C-O coupled products (cf. scheme 5).[41,50–52,55,57] The quinones with 4-methoxyphenol and 3-tert-butylphenol are reactive species and hence the C-O coupled product is obtained instead

Introduction of the simple quinone.[55,57] With 4-methoxyphenol, the C-O coupled compound 4-methoxy-5-(4-methoxyphenoxy)benzoquinone was reported to exhibit an absorption feature at the same wavelength than the uncoupled quinone.[57] Thus, the differentiation between 4-methoxybenzoquinone and the C-O coupled product via UV/Vis spectroscopy is difficult.

Lumb et al. intensively studied C-C and C-O coupled products making use of a simple di-tert-butylethylenediamine (DBED) ligand system. They analysed hydroxylation and subsequent coupling reactions with ortho-, meta- or para-substituted phenols with a variety of substituents[66,67,68] and furthermore, intermediates like semiquinone radicals.[58] The use of heteroatom-bearing substituents led to the possibility of further cyclisation reactions.[69] Thus, the formation of 5-membered rings resulted in indole systems. Starting from tyrosine esters, the bioinspired synthesis of a variety of substituted indoles is reported by the cyclisation reaction of the amine function of tyrosine.[70] Amine compounds can also condensate with the O-atom of the quinone to give an imine, which subsequently reacts with the second quinone O-atom and results in a benzoxazole product.[71]

The exact mechanism for the termination of the catalytic hydroxylation with tyrosinase model systems is still unclear. One hypothesis is the irreversible formation of unreactive copper species, for example a bis(-hydroxido) dicopper(II) complex. Conceivably, water formed during the catalytic reaction can enable hydrolytic reactions and destroy the catalyst. One possible decomposition product represents a -fluorido dicopper(II) complex with the ligand

[57] - dmPMP. Here, one fluoride anion is abstracted from the counterion PF6 to form the irreversibly inactivated fluoride-bridged complex.

1.3. Particulate Methane Monooxygenase Model Systems

The oxygenation of methane to methanol is one of the most important research topics. Thus, it is of high interest to obtain more structural details about the active site in particulate methane monooxygenase (pMMO). Furthermore, deeper insights into the mechanism of the oxygenation reaction are required. Model systems should therefore mimic the enzymatic active site structurally and should moreover act as reactivity models.

Introduction

1.3.1. Structural Models

The structure of particulate methane monooxygenase was controversially discussed, since both a dinuclear and a mononuclear copper centre might have been possible.[28,29] Latest investigations, however, reveal the evidence of a mononuclear copper centre,[8,30] which is proven by the newest crystal structure.[25] The copper coordination by three histidine residues and one amine function (section 1.1.2. figure 5) led to the development of synthetic models containing several N-donor moieties. A superoxide species is proposed to be the catalytically active oxygenation species, as this was reported for the peptidylglycine -hydroxylating monooxygenase (PHM, section 1.1.2. figure 5). Thus, efforts were made to synthesise superoxide copper model complexes.[15,36,39]

The binding mode of oxygen to the mononuclear copper centre can be in a side-on (2) or an end-on (1) fashion.[15] Whereas synthetic complexes with both coordination motifs are known,[39] end-on superoxide copper(II) species are reported in enzymes like galactose oxidase[21] or PHM.[23]

The first and only crystal structure of a 1-superoxide copper model complex is reported with

[72,73] the ligand tris(tetramethylguanidino)tris(2-aminoethyl)amine (TMG3tren) (figure 10). This very basic and sterically demanding ligand leads to the stabilisation of a stable superoxide copper(II) species. Even the reversible formation of the CuO2 species could be spectroscopically analysed. By warming a cold solution of [Cu(TMG3tren)O2]SbF6 to r.t., molecular oxygen was released from the coordination sphere. Re-cooling to low temperatures resulted again in the formation of the superoxide complex.

Figure 10: First and only crystallised end-on superoxide copper(II) model complex with the ligand [73] tris(tetramethylguanidino)tris(2-aminoethyl)amine (TMG3tren).

Besides the crystallographically characterised model system, several synthetic end-on superoxide copper(II) complexes were characterised according to their spectroscopic features.[39] Besides two tridentate ligand systems containing pyridine and amine/amide N-donor functions,[74,75] systems with tetradentate TMPA (tris(2-pyridinylmethyl)amine)[76–78] or tren (tris(2-aminoethyl)amine)[79,80] derivatives display spectroscopic properties of end-on

Introduction superoxide copper(II) species. Furthermore, one ligand system with three N- and one S-donor[81] and one threefold negative ligand (ONO pincer)[82] are known.

1.3.2. Substrate Reactivity

The first example of a superoxide copper(II) model complex that shows reactivity towards external substrates was reported by Karlin et al. in 2007.[78] The reactive oxygen species is supported by a NMe2-substituted TMPA ligand. The addition of 4-OMe-2,6-DTBP-H at -85 °C resulted in three different products (scheme 6). UV/Vis spectroscopy displays the formation of the 1,4-benzoquinone (405 nm), whereas EPR spectroscopic measurements show the occurrence of a stabilised phenoxyl radical and GC-MS characterises the arylhydroperoxide product. The detection of the phenoxyl radical led the authors to the suggestion that the initial step for this reactivity must be a H-atom abstraction (hydrogen atom transfer, HAT). Later studies proved the HAT and gave more insights into the oxidation mechanism.[76]

Scheme 6: Products of the conversion of 4-OMe-2,6-DTBP-H with the superoxide copper(II) complex NMe2 + [78] [Cu(TMPA )O2] in THF at -85 °C.

Aliphatic hydroxylation activity as in PHM or pMMO was observed with a tridentate pyridine- amine ligand.[75] Here, the decomposition of the end-on superoxide copper(II) species is explained with the simultaneous hydroxylation of a phenethyl moiety of the ligand framework. Another reactivity study shows the first intermolecular C-H bond activation using the substrate 1-benzyl-1,4-dihydronicotinamide (BNAH, analogous to the natural NADH).[77] Further studies were published with the substrates 1,4-cyclohexadiene, triphenylphosphane or thioanisole.[79]

1.4. Biohybrid Catalysts

Biohybrid catalysts combine the advantage of biological protein scaffolds with the catalytic activity of synthetic transition metal complexes.[83–85] By using such a catalyst, substrate reactions can be enhanced to a higher substrate and product (enantio)selectivity. The host

Introduction protein is therefore of high importance by providing a second coordination sphere for the metal centre.[85] Protein scaffolds can improve the reaction environment by changing its polarity and make it for instance more accessible for hydrophobic substrates. Furthermore, highly instable synthetic complexes can be stabilised through amino acids of the second coordination sphere. Biohybrid catalysts perform a variety of substrate reaction types including reductions,[85–87] oxidations,[85–87] C-C bond formations,[85–87] olefin metatheses,[86,88,89] C-H bond activations[88,90] and allylic alkylations and aminations.[85]

Several principles have to be considered when developing a biohybrid catalyst.[85,91] The reactivity of the transition metal complex must be orthogonal to the protein. Thus, the metal centre should not be affected or coordinated by amino acid side chains unless it is desired. On the other hand, the biomolecule should not be affected by the reactivity of the transition metal complex. Another important fact is that enzymatic reactions are performed in aqueous buffer solution and thus, the transition metal complex must tolerate this medium. In some cases, the used complex is rather water soluble and the usage of organic solvent is required. Then, the protein needs to tolerate this solvent, at least in low concentrations. Furthermore, the cavity of the artificial metalloenzyme should have an appropriate size: on one hand, small enough to provide a second coordination sphere for the metal centre and on the other hand, the cavity must be large enough for substrates to enter.

1.4.1. Protein Scaffolds

During the last decade, a large variety of host biomacromolecules was developed.[87] Besides polypeptide (protein) scaffolds, the use of nucleotides (DNA or RNA) as environment for transition metal complexes is also reported.[92] With the choice of the appropriate host scaffold, it must be considered that DNA- or RNA-based host molecules are rather inapplicable for oxidation reactions. The oxidative cleavage of the polynucleotide strand caused by the transition metal can occur.[93] As protein scaffolds examples like myoglobin,[94] streptavidin[95] and nitrobindin[96–98] are reported.

The proteins FhuA and NB4

Two proteins that are frequently used in biohybrid catalysis are the ferric hydroxamate uptake protein component A (FhuA) and the heme protein nitrobindin (NB). Both are -barrel proteins that consist of a hydrophilic exterior and a hydrophobic core.[97,99] This provides the possibility

Introduction of hydrophobic substrate reactions in an aqueous environment. FhuA and NB are mutated with a cysteine residue at a distinct position for anchoring synthetic transition metal complexes into the protein (see section 1.4.2.). FhuA is a transmembrane protein to which a Grubbs-Hoveyda (GH)-type catalyst can be covalently attached. A change in enantioselectivity is reported by using this type of biohybrid catalysts in the ring-opening metathesis polymerisation (ROMP) of a water-soluble oxanorbornene derivative.[100] A related but more enhanced reactivity is displayed with Grubbs-Hoveyda-type catalysts and two variants of the protein nitrobindin (NB4 and NB11).[101] In this study, it was observed that NB4 exhibits a too small cavity for the GH-type complex. Thus, the variant NB11, which has a larger cavity, was developed with the help of theoretical calculations. Nitrobindin finds also application in the polymerisation of phenylacetylene.[96,102] Here, the biohybrid catalyst reversed the stereoselectivity from cis- poly(phenylacetylene) (PPA) without the protein to trans-PPA with the protein.

NB4 and NB4exp

As mentioned above, the cavity size of the nitrobindin variant NB4 is rather small for the incorporation of large synthetic transition metal complexes. NB11 was reported as nitrobindin variant that exhibits more space for a Grubbs-Hoveyda-type catalyst, for instance.[101] An even larger -barrel displays the variant NB4exp (expanded nitrobindin variant 4).

Figure 11: Structures of two variants of the protein nitrobindin. a) Crystal structure of NB4; b) calculated model of NB4exp. Duplicated -strands are coloured in orange. The figure is reprinted from literature[103] with the permission of A. R. Grimm.

Introduction

The -barrel of NB4 consists of 10 -strands and one loop, which closes one site of the barrel.[102] The cavity size is reported to be 855 Å3. Compared to NB4, NB4exp exhibits two additional -strands, since 29 amino acids are duplicated (orange -strands in figure 11 b)).[103] Thus, the cavity is enlarged to 1389 Å3 and applicable for larger transition metal complexes. In figure 11 (right depiction), the larger cavity size of NB4exp is clearly seen.

Both nitrobindin variants (NB4 and NB4exp) exhibit an essential mutation at position 96. The amino acid glutamine is exchanged to cysteine. This is crucial for the application as protein scaffold for the above-mentioned biohybrid catalysts. The transition metal complex exhibits a maleimide moiety, which anchors covalently to Cys96 of the protein.

1.4.2. Anchoring Strategies

The introduction of a transition metal complex into a protein scaffold can proceed via four major methods (figure 12): supramolecular assemblies, dative anchoring, covalent attachment or metal substitution.[84,85,91,104] The anchoring via supramolecular assemblies (a)) is based on strong and highly specific interactions between the protein scaffold and small molecules (e.g. ligands). Thus, the incorporation of the transition metal complex can easily fail if the interaction of the small molecule with the protein scaffold is too weak. An example for supramolecular assemblies is the strong and specific interaction between biotin and streptavidin.[105] Another method is the coordination of the metal centre to functional groups of the biomacromolecule (dative anchoring, b)). The advantage here is the exact location of the catalytic centre in the protein cavity. This is found for instance in Fe/Mn corrole systems.[106] The approach of a covalent attachment (c)) leads to high regioselectivity towards the anchoring position. Mostly a cysteine residue (sometimes also lysine or serine) reacts irreversibly with a functional group of the ligand. An important example is the addition of the cysteine thiol group to a maleimide moiety.[101] The fourth method is based on the substitution of the native metal centre (d)) with an unnatural transition metal.

Figure 12: Anchoring strategies for protein scaffolds with transition metal complexes. a) Supramolecular assemblies, b) dative anchoring, c) covalent attachment, d) metal substitution.[85,87]

Introduction

1.4.3. Copper-Based Systems

Copper biohybrid systems were developed to mimic the active site of type I copper enzymes structurally and functionally. The most biohybrid catalytic systems containing copper as transition metal make use of the biotin-Sav (streptavidin) technology.[107,108] This is based on the strong interaction between the protein streptavidin and the vitamin biotin.[109] Pyridinyl- amine based N-donor ligands that are known from copper oxygen chemistry are modified with a biotin moiety.[107] Thus, the ligand can coordinate at one side a copper ion and at the other side, the biotin moiety interacts strongly with the protein scaffold of streptavidin. The reaction with H2O2 results in a hydroperoxide species that is capable of oxidising 4-chlorobenzylamine.[108]

Another study describes the use of copper biohybrid systems with the peptide hormone bovine pancreatic polypeptide as host scaffold.[110] Here, the introduction of the nonproteinogenic amino acids 3- and 4-pyridylalanine leads to an active site that provides a copper coordination site. These artificial metalloenzymes can perform Diels-Alder or Michael addition reactions.

1.5. Poly(pyrazolyl)methanes

Poly(pyrazolyl) Ligands

With the tris(pyrazolyl)methane (figure 13, left), Hückel and Bretschneider described the first synthesis of a poly(pyrazolyl)methane in 1937.[111] Since their synthetic procedure proceeded only with low yield, these ligands were neglected for the next three decades. In 1966, the development of the isoelectronic hydroborates (figure 13, right) led to the establishment of a new class of N-donor ligands.[112]

Figure 13: Left: tris(pyrazolyl)methane; right: tris(pyrazolyl)hydroborate.

The synthesis was described as the reaction of pyrazole with an alkali metal borohydride. The variation of substituents at the pyrazole moieties and the boron atom led to a plenty of new ligands,[113] which were used for the coordination of transition metals. Furthermore, poly(pyrazolyl)alkanes were synthesised via the conversion of pyrazole with the alkyl

Introduction bromide.[114] The analysis of these ligands regarding coordination ability was of interest because of the structural and electronic similarities to the poly(pyrazolyl)hydroborates. Inspired by the exchange of a boron to a carbon atom, the apical atom was replaced by further elements. The result was the development of bis(pyrazolyl)silanes,[115] amines[116] or transition metalates,[117] as well as tris(pyrazolyl) ligands with aluminium, indium, gallium,[118] silicon,[119] germanium or tin.[120]

Synthesis of Poly(pyrazolyl)methanes

As mentioned above, the first synthesis of a tris(pyrazolyl)methane yielded a rather small amount of desired product (34 %).[111] The chloroform, which was reacted with potassium pyrazole, was consequently changed to diiodomethane to result in bis(pyrazolyl)methane with better yields. The usage of an autoclave with 150 °C reaction temperature further increased the amount of product (45 %). However, the best yield was achieved when synthesising the bis(pyrazolyl)propane ligand (75 %).[114] In further studies, Elguero et al. reported on the application of a phase transfer catalyst, which resulted in the successful synthesis of bis(pyrazolyl)methane ligands with substituted pyrazolyl moieties.[121] The preparation of tris(pyrazolyl)methane failed due to the formation of dichlorocarbene, which reacts with unsubstituted pyrazole.[122] The formation of dichlorocarbene was prevented by replacing KOH

[123] in the phase transfer catalysis with K2CO3.

In 1990, Canty et al. reported on bis(pyrazolyl)methanes, that exhibit other functional groups, such as a pyridinyl moiety.[124] The synthesis of these ligands is divided into two steps. First, the pyrazole reacts with triethylamine and phosgene to result in bis(pyrazolyl)methanone. The second step is based on a Peterson rearrangement,[125] where the ketone is reacted with pyridine-2-aldehyde to give the bis(pyrazolyl)(pyridinyl)methane ligand. By using this synthetic procedure, other bis(pyrazolyl)methanes with different substituents were developed.[126] Further studies on improving this synthesis led to the exchange of phosgene to thionyl chloride.[127] In 2010, a general procedure for the preparation of substituted bis(pyrazolyl)- methanes was published.[128]

1.5.1. Bis(pyrazolyl)methane Ligands

Bis(pyrazolyl)methanes belong to the class of scorpionate ligands.[129] The name of these ligands originates from the tridentate, facial coordination motif towards a metal ion. Two donor

Introduction moieties coordinate the metal centre from both sides, like the pincers of a scorpion. The third donor moiety comes from above and coordinates the metal ion like the scorpion’s sting. The coordination of the metal ion results in a 6-membered chelate ring (M-Npz-Npz-C-Npz-Npz), that displays boat conformation (figure 14).

Figure 14: Coordination of a scorpionate ligand to the metal centre M. X = heteroatom of the third donor moiety.

The scorpionate ligands are divided into two ligand groups. Homoscorpionates are ligands, which display three similar donor moieties. On the other hand, heteroscorpionates possess at least one different donor moiety (figure 15). Another N-atom (pyridinyl, quinolinyl), as well as an O-atom (phenolate, carboxylate) or a S-atom (thiophene) can function as third donor.

Figure 15: Heteroscorpionate bis(pyrazolyl)methane ligands.

Bis(pyrazolyl)methanes were furthermore substituted at the pyrazolyl moieties to change the steric demand of the ligand. Different substituents including methyl, ethyl, iso-propyl, tert-butyl, phenyl or mesityl are reported. Thus, a large variety of bis(pyrazolyl)methane ligands is developed to date.[130–133]

1.5.2. Bis(pyrazolyl)methane Complexes

Bis(pyrazolyl)methane complexes are known with numerous transition metals, like copper, cobalt, iron or zinc. Generally, these complexes are more stable than the isoelectronically related complexes with bis(pyrazolyl)hydroborate ligands. This fact is based on the sensibility of the B-N bond towards hydrolysis.[134]

Bis(pyrazolyl)methanes exhibit three heteroatom donor moieties and are thus tridentate ligands. Depending on the transition metal and the framework of the ligand, two basic coordination motifs are possible (figure 16). With bulky substituents, a facial coordination of the metal ion is preferred. A bisfacial coordination fashion, where two bis(pyrazolyl)methanes

Introduction coordinate the metal centre, is however obtained with ligands exhibiting unsubstituted pyrazolyl moieties or small substituents.

Figure 16: Coordination motifs of copper complexes with bis(pyrazolyl)methane ligands.[43,54,130]

Within one ligand, different donor moieties display different donor strengths. Regarding N-donors of copper complexes, an imidazolyl donor is reported to be stronger than a pyrazolyl donor and a pyridinyl donor is the weakest in this series.[43] This difference in donor strengths and the influence of the counterion can lead to the observation that a tridentate ligand participates the coordination sphere only with two donor moieties (figure 16)[43] or even only with one.[135]

For characterising the geometry of the coordination sphere around the metal centre in tetra- and pentacoordinated complexes, two geometrical factors 4 and 5 are defined (equation 1 and 2). These values were established by Reedijk et al.[136] and reformulated by Houser et al.[137] In both calculations, the two largest bond angles including the central ion,  and , are important. Tetracoordinated complexes with a 4 value of 1 exhibit an ideal tetrahedral coordination geometry, whereas an ideal square plane is characterised by 4 = 0.

Pentacoordinated complexes with 5 = 1 show an ideal trigonal-bipyramidal coordination sphere and ideal square-pyramidal complexes exhibit a 5 value of 0.

360° - ( + ) 4 = (1) 141°

 -  5 = (2) 60°

Objective and Outline

2. Objective and Outline

2.1. Objective

In the field of bioinorganic chemistry, the structure of the active site and the catalytic activity of enzymes are modelled with small molecule transition metal complexes. Whereas, numerous of structural models for the copper enzyme tyrosinase are reported, only few catalytically active systems capable of converting phenolic substrates have been developed so far.[38] Bis(pyrazolyl)methane copper complexes are known to serve as excellent models for tyrosinase.[42,43] In the last years, a stable -2:2-peroxide dicopper(II) species with a bis(pyrazolyl)(pyridinyl)methane ligand was observed. A highly reactive bis(pyrazolyl)- (imidazolyl)methane system showed the fastest phenolate hydroxylation.

One aim of this work was to develop a new tyrosinase model system, which combines the stability of the side-on peroxide dicopper(II) complex with the high activity towards the hydroxylation of phenolic substrates. As the crystallisation of peroxide dicopper(II) complexes with these ligands is rather difficult, bis(pyrazolyl)methane complexes of the newly developed ligand should be crystallised with simple copper salts. These complexes can give an indication about the coordination mode of the ligand in tyrosinase model complexes. The new model system, as well as already published systems should be analysed concerning formation kinetics. Phenolic substrate hydroxylation should be performed in stoichiometric reactions for the determination of binding kinetics. Thus, insights into the hydroxylation mechanism will be obtained. Furthermore, the catalytic conversion of phenolic substrates should prove this system as a functional tyrosinase model.

The stabilisation of superoxide copper(II) species with bis(pyrazolyl)methane ligands is rather difficult and thus, mimicking of mononuclear copper enzymes with this ligand class is impossible to date.

The second objective of this work was the development of maleimide-bearing bis(pyrazolyl)- methane ligands, which can be anchored to a cysteine residue of a protein scaffold. The oxygenation or oxidation ability of copper complexes with maleimido ligands should be analysed by the formation of -2:2-peroxide dicopper(II) species and subsequent catalytic conversions of phenolic substrates. Furthermore, these ligands should be conjugated into

Objective and Outline variants of the protein nitrobindin. Copper incorporation and subsequent reduction should lead to a copper-containing biohybrid catalyst. The addition of molecular oxygen should lead to the stabilisation of a superoxide copper(II) species. The presence of the second coordination sphere of the protein should stabilise this species at least temporarily. Substrate oxygenation, as well as oxidation and H-atom transfer reactions should be performed with the biohybrid catalyst to prove its functionality as an enzyme model.

2.2. Outline

The results of these doctoral studies are divided into two parts.

The first part (chapter 3) describes the development of a new bis(pyrazolyl)methane ligand system. This ligand and an already reported related ligand were used to synthesise and structurally characterise copper(I) and copper(II) complexes. Features of the molecular structures are analysed and discussed. Moreover, the newly synthesised bis(pyrazolyl)methane ligand was applied in oxygen activation reactions. The formation kinetics of the side-on peroxide dicopper(II) species were determined at different temperatures. Kinetics of related ligand systems were also analysed and compared to the new tyrosinase model. For all three systems, the activation enthalpy and entropy were calculated from the kinetic measurements at different temperatures. Furthermore, kinetics of the hydroxylation of phenolic substrates with the new tyrosinase model system were determined. Herefore, substrate binding kinetics were performed with different sodium phenolates. The resulting hydroxylation constants kox were brought in relation to the substitution parameters of the phenolates and the Hammett’s constant was determined. Additionally, the catalytic ability was analysed with the phenolic substrate 8-hydroxyquinoline. Turnover numbers were calculated from UV/Vis spectroscopic measurements. The catalytic reaction was performed at several temperatures and various conditions.

The second part of this thesis (chapter 4) describes the design of two bis(pyrazolyl)methane ligands for the use in biohybrid catalysis. These ligands possess a maleimide moiety, that can be anchored to a protein scaffold. The application in oxygen activation is described with both maleimide-bearing ligands. Using UV/Vis spectroscopy, the catalytic conversion of the phenolic substrates 8-hydroxyquinoline and 4-methoxyphenol was analysed and turnover numbers were calculated. Both bis(pyrazolyl)methane ligands were conjugated to different variants of the protein nitrobindin and the conjugation was analysed via mass spectrometry.

Objective and Outline

Copper ions were incorporated into these biohybrid conjugates and the coordination was analysed via UV/Vis and EPR spectroscopy. Furthermore, oxygenation of the biohybrid conjugates was performed and investigated using stopped-flow UV/Vis spectroscopy. The ability to serve as a biohybrid catalyst was evaluated by the conversion of oxygenation, oxidation or H-atom transfer reaction substrates.

Although chapter 3 and 4 can be read independently, it is recommended to read chapter 3 prior to chapter 4. General aspects of oxygen activation reactions with bis(pyrazolyl)methane ligands and catalytic conversions are described in detail only in chapter 3.

Tyrosinase Models

3. Tyrosinase Models

In literature, tyrosinase model complexes are known for several decades and many attempts were made to design new ligand systems for a higher activity and more stable complexes.[15,18,36–38] Based on the latest publications of Herres-Pawlis et al.,[42,43,54] a new tyrosinase model system was developed. The first part of this chapter explains the synthesis of a new bis(pyrazolyl)methane ligand, followed by the synthesis and characterisation of copper complexes. Here, properties and influences of different N-donor moieties are discussed. The next section deals with the formation, stability and characterisation of peroxide dicopper(II) complexes with the new ligand and with bis(pyrazolyl)methane ligands that are already published. The influence of a modified N-donor moiety on properties of the peroxide complex is evaluated. In the last part of this chapter, reactions of the newly developed tyrosinase model system with phenolic substrates are summarised. Investigations with respect to catalytic abilities and to the kinetics of the hydroxylation were performed.

Parts of this chapter are already published in:

P. Liebhäuser, K. Keisers, A. Hoffmann, T. Schnappinger, I. Sommer, A. Thoma, C. Wilfer, R. Schoch, K. Stührenberg, M. Bauer, M. Dürr, I. Ivanović-Burmazović, S. Herres-Pawlis, Chem. Eur. J. 2017, 23, 12171-12183.

3.1. Bis(pyrazolyl)methanes

The bis(pyrazolyl)methanes used within these doctoral studies are heteroscorpionate ligands with three N-donor moieties. Besides two pyrazolyl units, the third donor is either an imidazolyl unit (L2) or a pyridinyl unit (L3) which can also possess an ester substituent in para-position to the N-donor (L1) (figure 17). A general synthetic pathway to bis(pyrazolyl)methanes was published in 2010 by Herres-Pawlis et al.[128] and can be applied to a variety of donor moieties.[130–133]

Tyrosinase Models

Figure 17: Bis(pyrazolyl)methane ligands with a pyridinyl (L3), a substituted pyridinyl (L1) or an imidazolyl (L2) moiety. N-donor atoms are marked in blue.

In scheme 7, the general synthesis of bis(pyrazolyl)methanes with different third donor moieties (R2) and distinct substituents (R1) at the pyrazolyl unit is shown. R1 can be methyl, iso-propyl, tert-butyl or phenyl, for instance and R2 is a heterocyclic compound such as imidazole, pyridine or quinoline.

Scheme 7: General bis(pyrazolyl)methane synthesis. R1 = alkyl or aryl substituent, R2 = heterocyclic compound.[128]

This one-pot ligand synthesis is divided into two parts. First, pyrazole is deprotonated with sodium hydride and the resulting pyrazolide is reacted with thionyl chloride. A second pyrazolide molecule transforms the intermediate into a bis(pyrazolyl)sulfoxide. This sulfoxide reacts with an aldehyde in a Co(II)-catalysed reaction to form the desired bis(pyrazolyl)- methane. The Co(II) catalysis is a modified Peterson rearrangement,[138] of which the detailed mechanism is given in scheme 8.

With the coordination of Co(II) by the two pyrazolyl N-donors of the bis(pyrazolyl)sulfoxide, electron density is shifted towards the metal ion and the S-N bond is weakened and even broken. Subsequently, the pyrazolyl N-atom attacks the aldehyde carbonyl function. After a rearrangement, SO2 and Co(II) are released and the bis(pyrazolyl)methane is obtained.

Tyrosinase Models

Scheme 8: Modified Peterson rearrangement: Co(II)-catalysed reaction of the bis(pyrazolyl)sulfoxide with an aldehyde.

Within the tyrosinase models published by Herres-Pawlis et al., the peroxide dicopper(II) complex with L2 (HC(3-tBuPz)2(1-MeIm)) (P2) is the most reactive one towards phenolic

[43] substrates, but it shows low stability. In contrast, the complex with L3 (HC(3-tBuPz)2(Py)) (P3) exhibits the highest stability, but substrate reactions are slow and have small turnover numbers (TONs).[42] Thus, it was targeted to assemble these two properties and develop a new tyrosinase model system, which displays high stability in combination with high reactivity.

Based on the bis(pyrazolyl)(pyridinyl)methane system (P3), substitution at the pyridinyl moiety should influence the stability of this system. As imidazolyl is a stronger donor than pyridinyl[43] and the resulting peroxide dicopper(II) complex (P2) is less stable, efforts were made to develop a ligand system with a weaker donor. Substituents in ortho- or para-position to the N-donor atom have the largest effect on the donor ability. As the ortho-position of the pyridinyl moiety in L3 is at one side occupied by the bond to the apical carbon atom and at the other side substitution would affect the metal coordination sterically, the para-position is the position of choice. To weaken the donor strength, electron-withdrawing groups like ester, nitro or cyano substituents must be introduced. Here, an ester substituent is chosen because of the opportunity of performing further reactions at the ester group.

Introducing the ester group at the pyridinyl moiety of the completed bis(pyrazolyl)methane selectively in para-position to the N-donor is rather difficult. Hence, the aldehyde starting material is prepared bearing an ester substituent (scheme 9).[139]

Tyrosinase Models

Scheme 9: Synthesis of methyl 2-formylisonicotinate starting from methyl isonicotinate.

Starting from methyl isonicotinate, hydroxymethyl is formally added at the ortho-position to the nitrogen atom via an electrophilic substitution followed by an oxidation. This alcohol function is then oxidised to the aldehyde via a Dess-Martin oxidation.[140]

3.1.1. Synthesis of 2-(4-Methoxycarbonylpyridinyl)bis(3-tert-butylpyrazolyl)- methane (L1)

The synthesis of the bis(pyrazolyl)methane HC(3-tBuPz)2(4-CO2MePy) (L1), where the pyridinyl moiety has an ester substituent in para-position to the N-donor, follows basically the general protocol[128] and is shown in scheme 10. After deprotonation of 3-tert-butylpyrazole with sodium hydride, thionyl chloride is added to form the sulfoxide intermediate. With the addition of methyl 2-formylisonicotinate and catalytic amounts of cobalt(II) chloride, the reaction results under reflux in the desired bis(pyrazolyl)methane L1 within two days.

Scheme 10: Synthesis of HC(3-tBuPz)2(4-CO2MePy) (L1).

In the general protocol, purification is performed by suspending the bis(pyrazolyl)methane in hexane and after filtration, a solid is obtained.[128] This method worked well in the case of reported syntheses of L2 (HC(3-tBuPz)2(1-MeIm)) or L3 (HC(3-tBuPz)2(Py)), but failed for L1.

Tyrosinase Models

Usually, the general synthesis proceeds with yields of 50-95 % and hence unreacted pyrazole remains in the product. If the amount of pyrazole is too large, the purification method with hexane does not work anymore. An alternative way of purification represents the distillation in high vacuum (HV). Although it is conducted in HV, temperatures of 150-180 °C are needed to receive pure L1.[56] The problem of these high temperatures is that a not negligible amount of L1 is lost by degradation and thus the yield is considerably decreased (59 %). The second disadvantage of distillation is the facilitated isomerisation of L1 with heat. Here,

HC(3-tBuPz)2(4-CO2MePy) isomerises to the thermodynamically favoured HC(3-tBuPz)-

(5-tBuPz)(4-CO2MePy) or even to HC(5-tBuPz)2(4-CO2MePy). These isomerisation reactions occur also during the synthetic procedure and can be reduced to a minimum by changing reaction conditions like temperature, reaction time or amount of catalyst. Considering the dis- advantages of a distillation, the best method to purify L1 is via column chromatography (87 % yield). Here, the starting materials 3-tert-butylpyrazole and methyl 2-formylisonicotinate, as well as parts of the desired L1 are separated from the isomerised bis(pyrazolyl)methane. The subsequent step is an acid-catalysed isomerisation to transform 5-tBuPz substituents back into 3-tBuPz substituents.[141,142] Under the chosen reaction conditions (high temperature), parts of L1 break down and 3-tert-butylpyrazole is regained. Consequently, the re-isomerised product must be also purified via column chromatography. L1 is received as orange, highly viscous oil and at this point it is suspended in hexane as described in the general protocol to obtain a pale yellow solid. The overall yield of the synthesis of L1 is calculated to 52 %.

3.1.2. Characterisation of 2-(4-Methoxycarbonylpyridinyl)bis(3-tert-butylpyrazolyl)methane (L1)

HC(3-tBuPz)2(4-CO2MePy) (L1) is characterised via NMR, IR spectroscopy and mass spectrometry (EI, ESI). NMR signals of L1 are mainly similar to the unsubstituted bis(pyrazolyl)methane L3, apart from the proton signal where the substituent in L1 (4-py(H)) is located and the signals of the ester substituent. Most of the proton and carbon signals are insignificantly shifted. Discrepancies of this are listed in table 1. Considering the 1H-NMR spectrum, the ester substitution at the C4 of the pyridinyl moiety results in an about 0.6 ppm higher chemical shift of both neighbouring protons (3-py(H) and 5-py(H)). This is explained by the electron-withdrawing character of the ester substituent which leads to deshielding of the neighbouring protons. In the 13C-NMR spectrum, the signal of the ester substituent-bearing C4 is hence found at a 1.7 ppm higher chemical shift and the one of C2 is 1.4 ppm shifted.

Tyrosinase Models

1 13 Table 1: Selected H- and C-NMR signals of HC(3-tBuPz)2(4-CO2MePy) (L1) and HC(3-tBuPz)2(Py) (L3).

1H-NMR 13C-NMR 3-py(H) 5-py(H) 2-py(C) 4-py(C) L1 7.45 7.81 157.4 138.6 L3 6.86 7.25 156.0 136.9

The IR spectrum of L1 shows the characteristic pattern of a bis(pyrazolyl)methane spectrum.[128] Except for the intense vibration at 1736 cm-1, it is similar to the IR spectrum of L3. Vibrations around 1700 cm-1 give indication of a carbonyl group of an aldehyde, ketone or ester.[143] Hence, the IR frequency at 1736 cm-1 is ascribed to the C=O stretching vibration of the ester substituent in L1.

In the EI mass spectrum, the mass peak and several degradation products of L1 are found. The most abundant fragment has a m/z value of 272 and refers to the cation

+ [HC(3-tBuPz)(4-CO2MePy)] , where one pyrazolyl moiety is cleaved off. This fragment is characteristic for bis(pyrazolyl)methanes due to their weak C-Npz bond.

3.2. Copper Bis(pyrazolyl)methane Complexes

As tyrosinase model complexes with bis(pyrazolyl)methane ligands were not crystallisable yet, attempts were made to crystallise copper complexes with these ligands without bound oxygen. It is of general interest to study the coordination situation in copper(II) and copper(I) complexes. Copper(II) complexes give hints to understand the metal coordination in peroxide dicopper(II) species, whereas copper(I) complexes reveal information about the coordination sphere in precursor complexes.

The newly developed bis(pyrazolyl)methane ligand HC(3-tBuPz)2(4-CO2MePy) (L1) as well as the resynthesised ligand HC(3-tBuPz)2(1-MeIm) (L2) were reacted with copper(I) and copper(II) salts. One copper(II) and one copper(I) complex with L1 and two copper(I) complexes with L2 were crystallised.

Tyrosinase Models

3.2.1. Synthesis and Characterisation of [Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1)

Synthesis

The reaction of L1 with CuCl2 in THF immediately results in the precipitation of

[Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1) as green solid. After dissolving this solid in MeCN and overlaying the solution with diethyl ether and n-pentane, green crystals are obtained at -25 °C after several weeks with a yield of 62 %. The synthesis of C1 is shown in scheme 11.

Scheme 11: Synthesis of [Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1).

Characterisation

[Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1) is characterised via mass spectrometry, IR spectroscopy and X-ray crystallography. Furthermore, the structural properties are compared to related complexes, where the third donor moiety is either a pyridinyl (CC1)[54] or a methylimidazolyl (CC2)[43] unit.

Figure 18: Molecular structure of the complex [Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1). H-atoms are omitted for clarity.

Tyrosinase Models

The complex C1 crystallises monoclinic in the space group Cc and the unit cell consists of four complex molecules. The molecular structure of C1 is given in figure 18 and selected bond lengths and angles for C1, CC1 and CC2 are depicted in table 2.

In C1, one ester-substituted pyridinyl and two pyrazolyl donor moieties of the ligand L1 as well as two chlorido counterions form the coordination sphere of the metal centre. The central copper ion, which has an oxidation state of +II, is hence pentacoordinated. The complex CC1, where the pyridinyl moiety has no ester substituent, exhibits the same coordination pattern. The geometry of the coordination sphere can be further characterised with the geometrical factor 5, which is calculated to 0.35 for C1. As a 5-value of 0 characterises a square-pyramidal coordination geometry and 5 = 1 is ascribed to a trigonal-bipyramidal coordination motif, complex C1 is a highly distorted square pyramid. This is also expressed in one elongated

Cu-Npz bond (Cu-Npz(4): 2.313(3) Å), whereas the other two Cu-N bonds are similar (Cu-Npz(2):

2.072(3) Å and Cu-Npy: 2.071(4) Å). In CC1, the Cu-Npy bond (2.058(4) Å) does not significantly differ from the Cu-Npy bond in C1 and thus, the ester substitution here has no influence. A greater difference is given in the Cu-Npz bonds, which are equal in CC1 (2.233(3) Å). With

5 = 0.71, the coordination geometry in CC1 is characterised as a highly distorted trigonal bipyramid. This large difference between the coordination geometries in C1 and CC1 is also obvious from the bond angles. Whereas two angles in C1 are larger than 150° and one of them is even larger than 170° (Npy-Cu-Cl(2): 173.6(1) Å, Npz(2)-Cu-Cl(1): 152.8(1) Å), only one angle in

CC1 is nearly 180° (Npy-Cu-Cl(2): 176.0(2) Å). This explains mainly the difference in the trigonal- bipyramidal character of CC1 compared to the square pyramid of C1. In the case of C1, the remaining angles should display values of about 90° for a square-pyramidal structure. The residual angles in CC1 should show 90 or 120° for a trigonal bipyramid. The relatively high deviations from these ideal angles (up to 27.2°) result in such a high distortion in the coordination geometries of both complexes, which is described by the 5-values. It has to be further mentioned that C1 and CC1 did not crystallise in the same space group (Cc vs. Pnma). This leads to the statement that molecular structures which crystallise in different space groups are hardly comparable. One important difference in CC1 is that the centre of the molecule is located on a mirror plane. This results in a high symmetry and hence varies significantly from the structure of C1. Thus, the different space groups exclude the discussion of concrete differences between the complex with an ester-substituted pyridinyl moiety (C1) and the complex with an unsubstituted pyridinyl moiety (CC1).

Tyrosinase Models

Nevertheless, regarding the coordination sphere of the central metal ion, C1 can be compared to the bis(pyrazolyl)(methylimidazolyl)methane copper(II) chloride complex CC2. Here, copper(II) is tetracoordinated by only two N-donor moieties of the ligand and two chlorido counterions. The stronger methylimidazolyl N-donor leads to a significantly shortened Cu-N bond (Cu-Nim: 1.957(3) Å) and thus one of the pyrazolyl moieties does not participate in the coordination sphere. In contrast to that, the weaker donor pyridinyl allows the coordination of all three N-donors of the bis(pyrazolyl)methane ligand. Hence, both pyridinyl-bearing complexes C1 and CC1 exhibit pentacoordinated metal centres.

Table 2: Selected bond lengths, angles and -values of the complex [Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1) and [54] [43] the comparison complexes [Cu{HC(3-tBuPz)2(Py)}Cl2] (CC1) and [Cu{HC(3-tBuPz)2(1-MeIm)}Cl2] (CC2).

C1 CC1 CC2 space group Cc Pnma P1̅ bond lengths [Å]

Cu-Npy/Nim* 2.071(4) 2.058(4) 1.957(3)*

Cu-Npz(2) 2.072(3) 2.233(3) 2.044(3)

Cu-Npz(4) 2.313(3) 2.233(3) -

Cu-Cl(1) 2.250(2) 2.226(2) 2.225(1)

Cu-Cl(2) 2.263(2) 2.267(2) 2.251(1) bond angles [°]

Npy/Nim*-Cu-Npz(2) 81.7(2) 83.8(1) 89.3(2)*

Npy-Cu-Npz(4) 86.3(2) 83.8(1) -

Npz(2)-Cu-Npz(4) 93.1(2) 92.2(2) -

Npy/Nim*-Cu-Cl(1) 91.5(1) 93.2(2) 150.8(1)*

Npy/Nim*-Cu-Cl(2) 173.6(1) 176.0(2) 93.4(1)*

Npz(2)-Cu-Cl(1) 152.8(1) 133.6(1) 98.8(1)

Npz(2)-Cu-Cl(2) 92.2(1) 93.5(1) 141.7(1)

Npz(4)-Cu-Cl(1) 112.9(1) 133.6(1) -

Npz(4)-Cu-Cl(2) 96.3(1) 93.5(1) -

Cl(1)-Cu-Cl(2) 93.0(1) 90.8(1) 97.3(1)

5/4* 0.35 0.71 0.48*

Tyrosinase Models

3.2.2. Synthesis and Characterisation of [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2)

Synthesis

Reacting L1 with CuBr in a warm mixture of acetone and THF leads to a red brown solution, which indicates copper complex formation. At r.t., [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2) precipitates as brown solid. The addition of MeCN redissolves the solid and overlaying with diethyl ether leads to red brown crystals after several weeks with 24 % yield. The synthesis of C2 is illustrated in scheme 12.

Scheme 12: Synthesis of [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2).

Characterisation

Figure 19: Molecular structure of the complex [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2). H-atoms are omitted for clarity.

Tyrosinase Models

The characterisation of [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2) is performed via mass spectrometry, IR spectroscopy and X-ray crystallography.

The complex C2 crystallises monoclinic in the space group P21/c with eight formula units in the unit cell. The asymmetric unit consists of two complex molecules C2a and C2b, which are very similar. The molecular structure of C2a is illustrated in figure 19 and selected bond lengths and angles for C2a and C2b are depicted in table 3.

Table 3: Selected bond lengths, angles and -values of the complex [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2). The asymmetric unit of C2 consists of two different complex molecules C2a and C2b.

C2a C2b

space group P21/c bond lengths [Å]

Cu-Npy 2.153(3) 2.152(3)

Cu-Npz(2) 2.088(3) 2.063(3)

Cu-Npz(4) 2.066(3) 2.090(3) Cu-Br 2.310(1) 2.310(1) bond angles [°]

Npy-Cu-Npz(2) 91.4(1) 90.0(1)

Npy-Cu-Npz(4) 89.0(1) 89.8(1)

Npz(2)-Cu-Npz(4) 88.5(1) 89.3(1)

Npy-Cu-Br 107.2(1) 108.6(1)

Npz(2)-Cu-Br 130.9(1) 137.3(1)

Npz(4)-Cu-Br 135.3(1) 127.5(1)

4 0.67 0.68

As in C1, all three N-donor moieties of the bis(pyrazolyl)methane ligand L1 coordinate the metal centre in C2. Additionally, one bromido counterion coordinates the central ion. Hence, the copper centre, which is in oxidation state +I, is tetracoordinated. This displays the tridentate character of L1 not only in copper(II), but also in copper(I) complexes. The coordination sphere in C2 is characterised with the geometrical factor 4, where a value of 1 reveals an ideal tetrahedron and a value of 0 an ideal square plane. With 4 = 0.67 (C2a) and 0.68 (C2b), both complex molecules exhibit a highly distorted tetrahedral geometry and among themselves they differ only slightly. This distortion is also indicated by considering the bond angles around the copper ion. Compared with the ideal tetrahedral angle of 109.5°, the Npz-Cu-Br angles are larger (up to 27.8°) because of the large bromido ion. In contrast to that, N-Cu-N angles are

Tyrosinase Models

around 20° smaller than 109.5°. In both C2a and C2b, the Cu-Npy bond (Cu-Npy: 2.153(3) /

2.152(3) Å) is longer than the Cu-Npz bonds (Cu-Npz: 2.088(3), 2.066(3) / 2.063(3), 2.090(3) Å), which indicates that the ester-substituted pyridinyl moiety is the weaker donor compared to the pyrazolyl moieties.

3.2.3. Synthesis and Characterisation of [Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3)

Synthesis

By the reaction of L2 with CuCl in a warm mixture of MeCN and MeOH, colourless crystals of

[Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3) result at r.t. within three months. The yield is calculated to 15 %. The synthesis of C3 is illustrated in scheme 13.

Scheme 13: Synthesis of [Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3).

Characterisation

The complex [Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3) is analysed via mass spectrometry, IR spectroscopy and X-ray crystallography. Furthermore, structural properties of C3 are compared to the related CuCl complexes, which possess bis(pyrazolyl)methane ligands with a quinolinyl (CC3)[54] or a pyridinyl (CC4)[54] moiety.

The complex C3 crystallises orthorhombic in the space group Pnma and the unit cell consists of four asymmetric units. The molecular structure of C3 is given in figure 20 and selected bond lengths and angles for C3, CC3 and CC4 are summarised in table 4.

Tyrosinase Models

Figure 20: Molecular structure of the complex [Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3). H-atoms are omitted for clarity.

In C3, the central metal is coordinated by three N-donor units of the bis(pyrazolyl)- (methylimidazolyl)methane ligand L2 and one chlorido counterion. Hence, the copper centre, which is in oxidation state +I, is tetracoordinated. This ligand coordination motif is contrary to the one observed in the comparable CuCl2 complex (CC2). There, only two N-donor moieties of L2 bound the copper ion, since with the strong imidazolyl and the two chloride donors, the coordination sphere is electronically saturated in this case. The coordination geometry is described with the geometrical factor 4, which is calculated to 0.73 and thus, C3 shows a highly distorted tetrahedral structure. This distortion is similar in complexes, where the third donor moiety is quinolinyl (CC3: 0.76) or pyridinyl (CC4a: 0.73, CC4b: 0.71; the asymmetric unit of CC4 consists of two complex molecules). In general, N-Cu-N bond angles are smaller than the ideal tetrahedral angle, whereas N-Cu-Cl bond angles are larger than 109.5°. This is because of the ring strain within the six-membered rings Cap-C/N-N-Cu-N-N of the ligand L2.

A huge difference is found in the Cu-Nim/qu bonds. With the strong imidazolyl donor, the Cu-Nim bond in C3 is 2.078(2) Å, whereas the Cu-Nqu bond is with 2.149(2) Å much longer and thus shows the weakness of the quinolinyl donor in consequence of its large, steric demand. The pyridinyl moiety in CC4 is also a weaker donor than imidazolyl, but this effect is less pronounced in bond lengths, particularly because complexes crystallised in different space groups are hardly comparable (P21/c vs. Pnma). One important difference to CC4 is that C3 and CC3 display a high symmetry due to the location of the molecule centre on a mirror plane.

Tyrosinase Models

Table 4: Selected bond lengths, angles and -values of the complex [Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3) and the [54] [54] comparison complexes [Cu{HC(3-tBuPz)2(Qu)}Cl] (CC3) and [Cu{HC(3-tBuPz)2(Py)}Cl] (CC4). The asymmetric unit of CC4 consists of two different complex molecules CC4a and CC4b.

C3 CC3 CC4a CC4b

space group Pnma Pnma P21/c bond lengths [Å]

Cu-Nim/Nqu*/Npy** 2.078(2) 2.149(2)* 2.120(2)** 2.132(2)**

Cu-Npz(2) 2.220(2) 2.124(2) 2.106(2) 2.082(2)

Cu-Npz(2)’ 2.220(2) 2.124(2) 2.183(2) 2.120(2) Cu-Cl 2.234(1) 2.215(1) 2.199(1) 2.187(1) bond angles [°]

Nim/Nqu*/Npy**-Cu-Npz(2) 88.4(1) 89.1(1)* 88.5(1)** 90.8(1)**

Nim/Nqu*/Npy**-Cu-Npz(2)’ 88.4(1) 89.1(1)* 86.0(1)** 88.7(1)**

Npz(2)-Cu-Npz(2)’ 88.1(1) 89.1(1) 90.5(1) 86.6(1)

Nim/Nqu*/Npy**-Cu-Cl 135.0(1) 129.2(1)* 121.5(1)** 118.1(1)**

Npz(2)-Cu-Cl 121.8(1) 124.2(1) 130.0(1) 130.9(1)

Npz(2)‘-Cu-Cl 121.8(1) 124.2(1) 127.2(1) 129.3(1)

4 0.73 0.76 0.73 0.71

3.2.4. Synthesis and Characterisation of [Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4)

Synthesis

The reaction of L2 and CuI in a warm mixture of THF and acetonitrile results in a red solution, which indicates copper complex formation. After solvent reduction and overlay of the solution with diethyl ether and n-pentane, red brown crystals of [Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4) are obtained within several weeks in 33 % yield. The synthesis of C4 is illustrated in scheme 14.

Scheme 14: Synthesis of [Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4).

Tyrosinase Models

Characterisation

[Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4) is characterised via mass spectrometry, IR spectroscopy and X-ray crystallography. Furthermore, C4 is compared to the above-mentioned complex C3 regarding structural properties.

The complex C4 crystallises orthorhombic in the space group Pnma with four complex molecules in the unit cell. The molecular structure of C4 is illustrated in figure 21 and selected bond lengths and angles for C4 are depicted in table 5.

Figure 21: Molecular structure of the complex [Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4). H-atoms are omitted for clarity.

As in C3, all three N-donor moieties of the bis(pyrazolyl)methane ligand L2 and one iodido counterion coordinate the metal centre. This tetracoordinated copper ion exhibits the oxidation state +I. The geometry of the copper centre is described with 4, which is calculated to 0.74 and hence differs barely from the 4-value of C3. The large discrepancy of an ideal tetrahedron is displayed in the bond angles. N-Cu-I bond angles are much larger (up to 22.6°), whereas N-Cu-N bond angles are smaller (up to 20.8°) than the ideal tetrahedral angle of 109.5°. As in

C3, the Cu-Nim bond (2.076(4) Å) is shorter than Cu-Npz bonds (2.199(3) Å) because of the good donor ability of the imidazolyl moiety. Furthermore, C4 exhibits the high symmetry with the central mirror plane as it is found in C3. Considering all these properties, the complex with an iodido counterion does not show large differences to the CuCl complex. The only difference is the much greater ionic radius of the iodido counterion in comparison with chlorido, which leads to a longer Cu-I bond and wider N-Cu-I angles.

Tyrosinase Models

Table 5: Selected bond lengths, angles and the -value of the complex [Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4).

C4 space group Pnma bond lengths [Å] bond angles [°]

Cu-Nim 2.076(4) Nim-Cu-Npz(2) 88.7(2)

Cu-Npz(2) 2.199(3) Nim-Cu-Npz(2)’ 88.7(2)

Cu-Npz(2)’ 2.199(3) Npz(2)-Cu-Npz(2)’ 89.0(2)

Cu-I 2.521(1) Nim-Cu-I 132.1(2)

Npz(2)-Cu-I 123.0(1)

4 0.74 Npz(2)’-Cu-I 123.0(1)

3.3. Oxygen Activation

The reaction of copper(I) precursor complexes with oxygen results in the activation of molecular dioxygen by the formation of copper oxygen species. Due to the nature of the ligand

(bidentate, tridentate, tetradentate), different CunO2 (n = 1-3) stoichiometries and various oxidation states are possible (see section 1.2.2.).[15] Bis(pyrazolyl)methane ligands stabilise

Cu2O2 species of peroxide dicopper(II) character and thus serve as excellent ligands for tyrosinase model systems.[42,43,54] The tridenticity of bis(pyrazolyl)methanes makes the peroxide dicopper(II) species to be energetically more favoured than the isomeric bis(-oxide) dicopper(III) coordination motif.[15]

2 2 With the newly synthesised ligand HC(3-tBuPz)2(4-CO2MePy) (L1) the - : -peroxide dicopper(II) complex P1 could be stabilised as one of the most stable tyrosinase models with catalytic activity. P1 was characterised via UV/Vis, resonance Raman and X-ray absorption spectroscopy, as well as UHR CSI mass spectrometry. Furthermore, formation kinetics of P1 and the literature-known peroxide complexes P2[43] and P3[42] were determined.

Tyrosinase Models

3.3.1. Formation and Stability of the Peroxide Dicopper(II) Species P1, P2 and P3

Precursor Complex Synthesis

2 2 For the synthesis of [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1), the precursor complex [Cu{HC(3-tBuPz)2(4-CO2MePy)}]SbF6 has to be prepared. It is indispensable that the

- - - copper(I) precursor complex comprises a non-coordinating counterion like SbF6 , PF6 or BF4 to provide a copper coordination site for dioxygen binding. Moreover, simple copper(I) salts with non-coordinating counterions cannot be used as such copper salts are in most cases tetrakis(acetonitrile) salts. The disadvantage of present acetonitrile is the high propensity to coordinate the copper ion. If the coordination site of the metal centre is blocked by acetonitrile ligands, oxygen cannot bind the copper ion.[144] Thus, the precursor complex [CuL1]+ is

- [42] synthesised with SbF6 as counterion through an indirect route (scheme 15).

Scheme 15: Synthesis of [Cu{HC(3-tBuPz)2(4-CO2MePy)}]SbF6.

In the first step, [CuL1]+ is prepared as chlorido complex by the addition of L1 to CuCl in dichloromethane. Copper complex formation is indicated by a colour change from yellow to dark red. Within 1 h, [CuL1Cl] is formed completely as tetracoordinated copper(I) complex, comparable to the complex C3 (section 3.2.3.). Subsequently, AgSbF6 is added as THF- solution to the complex solution to exchange the chlorido ligand with the non-coordinating

- counterion SbF6 . Insoluble AgCl precipitates in the solution and can be filtered off. This red precursor complex solution can be stored in a Schlenk tube at r.t. for two days. Compared to

Tyrosinase Models

[43] precursor complex solutions with the ligands L2 (HC(3-tBuPz)2(1-MeIm)) or L3

[42] (HC(3-tBuPz)2(Py)), [CuL1]SbF6 is more stable. [CuL3]SbF6 decays within one day and the instable complex [CuL2]SbF6 even within few hours.

Peroxide Dicopper(II) Complex Synthesis

The synthesis of P1 was performed according to syntheses of previously reported peroxide dicopper(II) species.[42,43]

The reaction of [CuL1]SbF6 with dioxygen in dichloromethane at -78 °C results in the dinuclear

2 2 peroxide complex [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) (scheme 16). Dark violet colouring of the solution indicates the formation of a side-on peroxide dicopper(II) complex. The formation of P1 is completed within 40 min and thus is slower than the formation of peroxide dicopper(II) species with the ligands L2 (15 min)[131] or L3 (3 min),[42] but much faster than a P species containing bis(pyrazolyl)(quinolinyl)methane ligands (8 h).[54]

When saturating dichloromethane with oxygen and injecting the precursor complex at r.t., the generation of P1 is possible at r.t. as well. Although the formation is limited, P1 is formed in 70 % yield within 3.5 min at r.t. The formation of P2 and P3 can also be performed in tetrahydrofuran, acetone or methanol. This is impossible for P1. P1 can exclusively be generated in the non-coordinating solvent dichloromethane. In this case, the feasibility of tetrahydrofuran, acetone and methanol to coordinate the copper centre is too high.

2 2 Scheme 16: Synthesis of [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1).

It is literature-known that the addition of dioxygen to a copper(I) precursor complex results in the first step in a superoxide intermediate species (scheme 17), where oxygen binds the copper centre either in an end-on or side-on fashion.[15] Immediately, a second molecule of copper(I) precursor complex reacts with the superoxide species so that this intermediate is not

Tyrosinase Models observed. Even with stopped-flow measurements at -80 °C, where the first spectrum is received after 50 ms, no evidence of a superoxide species is found with the ligand L1. Whereas superoxide species with bis(pyrazolyl)methane ligands are instable and short-lived, superoxide complexes that are actually stable enough to crystallise are reported with strong (sometimes negative) N-donor ligands.[73,145]

Scheme 17: Reaction of the copper(I) precursor complex with dioxygen to the side-on peroxide dicopper(II) complex through superoxide intermediates either with side-on- or end-on-bound oxygen.[15]

Formation Kinetics

Reaction rates of the formation of the peroxide dicopper(II) complexes P1

(HC(3-tBuPz)2(4-CO2MePy) ligand), P2 (HC(3-tBuPz)2(1-MeIm) ligand) and P3

(HC(3-tBuPz)2(Py) ligand) are determined by stopped-flow UV/Vis spectroscopic measurements. Using this device, equal volumes of a precursor complex solution with 4 mmol L-1 and oxygen-saturated dichloromethane are mixed in the sample chamber. Thus, the final

-1 concentration of P is 1 mmol L . For the determination of the reaction rates kobs, the increase of the characteristic absorption at ~550 nm was monitored (see section 3.3.2. for characteristic absorptions of peroxide dicopper(II) complexes). In first studies, attempts were made to monitor the more intense absorption at ~350 nm, as the signal-to-noise ratio would be better. However, with the stopped-flow device, the optical path length is 10 mm and thus 10-fold higher than in measurements using the UV/Vis immersion probe (1 mm optical path length). To obtain comparable absorption values, the concentration of P must be 10-fold lower (related to Beer-Lambert law: A =  · c · d). With a concentration of 0.1 mmol L-1, the formation of P is no longer comparable to the formation in a 1 mmol L-1 solution as dilution leads to a significant slowdown of the reaction velocity. Hence, it was decided to monitor the less intense absorption at ~550 nm with the comparable concentration of 1 mmol L-1. In figure 22, an exemplary formation of P1 is shown as increase of the 550 nm absorption with time.

Tyrosinase Models

Figure 22: UV/Vis spectroscopic monitoring of the formation of the peroxide dicopper(II) complex 2 2 -1 [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) in a 1 mmol L solution at -80 °C in dichloromethane. The absorption trace at 550 nm is depicted.

Within the formation of a peroxide dicopper(II) species, the reaction of dioxygen with the copper(I) precursor complex is reported to be the rate-determining step.[146] The subsequent reaction with a second molecule of copper(I) precursor complex is very fast and undetectable. Thus, pseudo-first-order conditions can be applied for the second-order process of peroxide dicopper(II) complex formation by using oxygen in excess. In the oxygenation reaction of

[CuL]SbF6, oxygen is provided by the oxygen-saturated solvent. The solubility of oxygen in dichloromethane is reported to be 5.8 mmol L-1 at 20 °C.[147] As this concentration is at least 3-fold higher than the copper concentration, the kinetics of the reaction can be analysed with a pseudo-first-order rate law. Therefore, the increase of the absorption intensity at 550 nm is fitted with a first-order exponential equation (equation 3). The fit curve is drawn in red in figure 22. This exponential fit provides the observable reaction rate constant kobs, which was determined for several temperatures for P1, P2 and P3 (table 6). The monitored 550 nm absorption band of P1 and P3 is shifted to 534 nm in P2.[43]

k·x y = A · e + y0 (3) with the pre-exponential factor A, the observed reaction rate constant k and the y-intercept y0.

Tyrosinase Models

Table 6: kobs values for the formation of the peroxide dicopper(II) complexes P1, P2 and P3 at different temperatures -1 in 1 mmol L solutions. P1 exhibits the ligand HC(3-tBuPz)2(4-CO2MePy), P2 the ligand HC(3-tBuPz)2(1-MeIm) and P3 the ligand HC(3-tBuPz)2(Py).

-1 T [°C] kobs [s ] P1 P2 P3 -90 - - (0.48 ± 0.14) · 10-2 -80 (0.30 ± 0.16) · 10-2 (0.10 ± 0.02) · 10-2 (0.90 ± 0.15) · 10-2 -70 (0.38 ± 0.01) · 10-2 (0.22 ± 0.05) · 10-2 (1.49 ± 0.13) · 10-2 -60 (0.72 ± 0.15) · 10-2 (0.37 ± 0.11) · 10-2 (2.23 ± 0.36) · 10-2 -50 (0.85 ± 0.10) · 10-2 (0.61 ± 0.10) · 10-2 (3.00 ± 0.40) · 10-2 -40 (1.49 ± 0.38) · 10-2 (1.05 ± 0.41) · 10-2 3.70 · 10-2# -30 1.05 · 10-2# (2.61 ± 0.22) · 10-2 3.35 · 10-2 (-34 °C)# #Only one measurement und thus no standard deviation is calculated.

Considering the different peroxide dicopper(II) systems P1, P2 and P3, it is observed that the reaction of the respective precursor complex [CuL]SbF6 with dioxygen becomes faster with higher temperature. Measurements with temperatures higher than -40 °C, however, are difficult to perform and kobs values are not reliable. Here, P formation is limited and occurs with less than 100 % yield. Moreover, the decay of P is significantly noticeable. A comparison between the three peroxide dicopper(II) systems confirms the faster formation of P3 in contrast to P1. On the other hand, the slow formation of P2 is surprising. P2 is reported to fully generate within 15 min at -78 °C[43] and hence is more than twice as fast as P1 (40 min). The essential difference is however, that the reported procedure uses the precursor complex as THF- solution, whereas in this thesis, the formation of P2 is performed with a DCM-based precursor solution.

As is seen from kobs values, the formation of P complexes is temperature-dependent. By applying the Eyring equation,[148] thermodynamic values such as the enthalpy of activation H≠

≠ and the entropy of activation S can be calculated from the reaction rate constant kobs. The Eyring theory[149] was established by H. Eyring and M. Polanyi in the 1930s and is based on the Arrhenius equation[150] and the transition state theory.[151] In the following, theoretical basics on the Eyring theory are given.[148]

Eyring Theory

The following constants, variables and factors are used in equations 4-16: reaction rate constant k, pre-exponential factor A, activation energy EA, general gas constant R, temperature T, reaction rate r, frequency of vibration v, equilibrium constant K≠,

Tyrosinase Models concentrations of the reactants [A] and [B] as well as the transition state [AB]≠, Boltzmann

≠ ≠ constant kB, Planck constant h, Gibbs free energy G , activation enthalpy H , activation entropy S≠.

The Eyring equation displays the temperature dependence of the reaction rate, as it was established by Arrhenius in 1889 (equation 4).

EA - k = A · e RT (4)

The reaction of A and B results in a transition state AB, which reacts to the product C. The rate of this reaction is defined via two equations (5 and 6):

r = k · [A] · [B] (5)

[AB]≠ r = v · [AB]≠ = v · [A] · [B] · K≠ with K≠ = (6) [A] · [B]

Equalising these two equations gives the reaction rate constant k in dependence of v and K≠:

k = v · K≠ (7)

With the definition of v, equation 7 can be formulated as follows:

k · T k · T k = B · K≠ with v = B (8) h h

To introduce the thermodynamic values H≠ and S≠, the van’t Hoff equation 9 and the Gibbs- Helmholtz equation 10 are formulated as follows:

G≠ = - R · T · ln K≠ (9)

G≠ = H≠ - T · S≠ (10)

- R · T · ln K≠ = H≠ - T · S≠ (11)

H≠ S≠ ln K≠ = - + (12) R T R

After equalising (11) and rearranging, equation 12 is transferred to equation 8. This results in the Eyring equation 13 and its logarithmised form (equation 14).

≠ ≠ H S kB · T - k = · e R T · e R (13) h

Tyrosinase Models

k k H≠ 1 S≠ ln = ln B - · + (14) T h R T R

k 1 From the Eyring equation (14) it becomes apparent that with the plot of ln vs. (Eyring plot), T T the activation enthalpy H≠ and the activation entropy S≠ can be calculated. H≠ results from the slope of the Eyring plot (15), whereas S≠ from the y-intercept (16).

H≠ m = - → H≠ = - m · R (15) R

k S≠ k y = ln B + → S≠ = (y - ln B) · R (16) h R h

Eyring plots for the reaction of precursor complexes [CuL]SbF6 (L = L1, L2, L3) with oxygen in dichloromethane are illustrated in figures 23, 24 and 25.

With the Eyring plots of P1, P2 and P3 and equations 15 and 16, the thermodynamic values H≠ and S≠ are calculated and depicted in table 7.

Figure 23: Eyring plot for the reaction of [Cu{HC(3-tBuPz)2(4-CO2MePy)}]SbF6 with oxygen in dichloromethane. 2 2 Final concentration of the peroxide dicopper(II) complex [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1): 1 mmol L-1. Averaged experimental data points are drawn with error bars. The red data point is excluded for the calculation of the linear fit.

Tyrosinase Models

Figure 24: Eyring plot for the reaction of [Cu{HC(3-tBuPz)2(1-MeIm)}]SbF6 with oxygen in dichloromethane. Final 2 2 concentration of the peroxide dicopper(II) complex [Cu2{HC(3-tBuPz)2(1-MeIm)}2(- : -O2)](SbF6)2 (P2): 1 mmol L-1. Averaged experimental data points are drawn with error bars. The red data point is excluded for the calculation of the linear fit.

Figure 25: Eyring plot for the reaction of [Cu{HC(3-tBuPz)2(Py)}]SbF6 with oxygen in dichloromethane. Final 2 2 -1 concentration of the peroxide dicopper(II) complex [Cu2{HC(3-tBuPz)2(Py)}2(- : -O2)](SbF6)2 (P3): 1 mmol L . Averaged experimental data points are drawn with error bars. The red data points are excluded for the calculation of the linear fit.

Tyrosinase Models

≠ ≠ Table 7: Thermodynamic values H and S for the reaction of [CuL]SbF6 (L = L1, L2, L3) with oxygen in dichloromethane.

P1 P2 P3 P3* H≠ [kJ mol-1] 14.6 ± 1.5 19.9 ± 0.8 13.1 ± 1.0 14.8 S≠ [J mol-1 K-1] -216 ± 7 -195 ± 4 -213 ± 5 -202 *The Eyring plot for P3 was already determined with another stopped-flow device (errors were not reported).[135]

Considering the activation enthalpy H≠, all peroxide dicopper(II) systems display a positive value, which indicates that the transition state is on a higher energy level than the reactants. P2 reveals the highest activation enthalpy and thus at the same temperature, the reaction of

[CuL2]SbF6 with oxygen is slower than the reaction with L1 or L3 (cf. above). By comparing P1 and P3, P1 shows an insignificantly higher H≠ value, as the error of the calculated activation enthalpy is about 1 kJ mol-1. The activation entropy is significantly negative in all three peroxide dicopper(II) formation reactions. This reveals the transition state to be a more ordered system than the reactants, as the entropy is a measure for disorder. Moreover, it indicates that three molecules combine to a single entity.

The Eyring plot of P3 was previously determined with another stopped-flow device (P3*).[135] The activation parameters obtained from the new measurements confirm the former calculated values.

Stability

2 2 The peroxide dicopper(II) complex [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) shows a high stability, as it is stable for at least half a year at -78 °C. At this temperature P3[42] is stable for several weeks, whereas P2[43] decays within six days. This stability is counter- intuitive, as P2 exhibits an imidazolyl moiety which is a stronger donor than the pyridinyl moieties in P1 and P3. One explanation would be that with the better donation of imidazolyl, both peroxide donor atoms are less donating and thus, the peroxide complex is more instable. The stability of P1 is analysed at r.t. as well. In figure 26, the decay of the peroxide dicopper(II) absorption at 350 nm is depicted. The experimental data show a peak in the beginning of the decay, which is ascribed to a baseline shift due to the temperature increase from -78 °C to r.t. The baseline shift is characteristic for this type of UV/Vis spectroscopic setup with an immersion probe. The experimental curve is fitted first-order exponentially (drawn in red in figure 26), where the red-marked data points of the peak are excluded.

Tyrosinase Models

Figure 26: UV/Vis spectroscopic monitoring of the decay of the peroxide dicopper(II) complex 2 2 [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) at r.t. in dichloromethane. The absorption trace at 350 nm is depicted. The red data points (peak) are excluded for the calculation of the exponential fit.

With this exponential fit, the reaction constant k for the decay of P1 is obtained. The half-life

ln 2 [42] t1/2 is defined to be and is calculated to 44 min at r.t. (table 8). Compared to P3, P1 is one k and a half times more stable at r.t., although the difference is only an ester substituent at the pyridinyl moiety. P2 is less stable and the half-life is not reported for r.t., but for 0 °C to be 3.5 min.[43]

2 2 Table 8: Decay constant k for [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) at r.t. and half-life values 2 2 [43] 2 2 t1/2 for P1, P2 ([Cu2{HC(3-tBuPz)2(1-MeIm)}2(- : -O2)](SbF6)2) and P3 ([Cu2{HC(3-tBuPz)2(Py)}2(- : -O2)]- [42] (SbF6)2) at different temperatures.

P1 P2 P3 r.t.: k [s-1] 2.65 · 10-4 - -

r.t.: t1/2 [min] 44 - 30

0 °C: t1/2 [min] - 3.5 -

-78 °C: t1/2 half a year six days several weeks

Both P2 and P3 are to a certain degree resistant to other solvents like tetrahydrofuran, acetone or methanol, even the formation is possible in these solvents. In contrast to that, there is no possibility to perform the formation of P1 in these solvents. P1 exhibits no such stability, as the addition of 1 mL of THF, acetone or methanol leads to the decay of the peroxide dicopper(II) complex. It was found that a 1 mmolar solution of P1 in 10 mL of dichloromethane tolerates amounts of 250 L of methanol. This is important for substrate hydroxylation reactions, which are presented in section 3.4.1.

Tyrosinase Models

3.3.2. Characterisation of the Peroxide Dicopper(II) Complex P1

UV/Vis Spectroscopy

2 2 The formation of P1 ([Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2) is monitored via time-resolved UV/Vis spectroscopic measurements with an immersion probe setup. Although

-1 the precursor complex [CuL1]SbF6 is red in a 30 mmol L solution, UV/Vis absorptions are negligible small as the concentration is diluted to 2 mmol L-1 in the UV/Vis measurement cell. Thus, oxygen-saturated dichloromethane is suitable as baseline and the formation of P1 is observed after injecting [CuL1]SbF6 into the solution. The sequentially recorded UV/Vis spectra with the increasing characteristic absorptions of P1 are depicted in figure 27.

Figure 27: UV/Vis spectra of the reaction of [Cu{HC(3-tBuPz)2(4-CO2MePy)}]SbF6 with oxygen in dichloromethane 2 2 at -78 °C. Final concentration of the peroxide dicopper(II) complex [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)]- -1 (SbF6)2 (P1): 1 mmol L .

The UV/Vis spectrum shows two more intense absorptions (350 and 280 nm), one less intense band (550 nm) and one very weak shoulder (~430 nm). The shoulder at ~430 nm stems from an electron transition within the bis(pyrazolyl)methane copper complex. This is also the case for the more intense absorption at 280 nm. Both absorptions are not found in bis(pyrazolyl)(imidazolyl)methane copper complexes.[43] TD-DFT (time-dependent density

2 2 functional theoretical) calculations on P3 ([Cu2{HC(3-tBuPz)2(Py)}2(- : -O2)](SbF6)2) show that the 412 nm absorption stems from a pyrazole/pyridine * → copper dxy charge transfer transition.[42] The intense absorption at 350 nm and the less intense one at 550 nm have an intensity ratio of 13:1 and are characteristic for -2:2-peroxide dicopper(II) complexes.[15,37] In most of the cases, side-on peroxide dicopper(II) species display an absorption intensity ratio of about 20:1. Typically, these absorption bands are found in the range of 340-380 and

Tyrosinase Models

510-560 nm and feature extinction coefficients of 18000-25000 and 1000 L mol-1 cm-1, respectively. The extinction coefficients of the absorptions in the UV/Vis spectrum of P1 are calculated to 23800 and 1900 L mol-1 cm-1 (figures 91 and 92 in the appendix). Both features are LMCT (ligand-to-metal charge transfer) transitions, where an electron moves from an oxygen-bound orbital to a copper-bound orbital. The high-energy CT at 350 nm originates from an in-plane * → dxy transition, whereas the 550 nm absorption from an out-of-plane v* → dxy transition (figure 28).[152] The latter causes the intensive dark violet colour of P1 in solution.

Figure 28: Molecular orbital diagram of a side-on peroxide dicopper(II) complex. Scheme adapted from Stack et al.[15]

Mass Spectrometry

The solution of P1 is temporarily stable at r.t., nevertheless, electrospray ionisation mass spectrometric (ESI-MS) measurements could not be performed at r.t. Hence, ultrahigh resolution cryospray ionisation mass spectra (UHR CSI-MS) were recorded in cooperation with Prof. Dr. Ivanović-Burmazović at the FAU in Erlangen-Nürnberg.[153] Figure 29 shows the UHR CSI mass spectrum of P1 (spray gas: -80 °C), where calculated peaks are drawn in red. In table 9, found and calculated m/z values for P1 are depicted.

Tyrosinase Models

2 2 Figure 29: UHR CSI mass spectrum of [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) in dichloromethane. Spray gas temperature: -80 °C. Black: experimental spectrum, red: calculated values. This mass spectrum was recorded by the group of Prof. Dr. Ivanović-Burmazović at the FAU in Erlangen-Nürnberg.[153]

Table 9: Found (UHR CSI-MS) and calculated m/z values for the isotopic distribution of the monocationic complex 2 2 + ([Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)]SbF6) .

formula m/z (found) m/z (calculated)

63 121 C44H58 Cu2F6N10O6 Sb 1183.2029 1183.2069 13 63 121 C43 CH58 Cu2F6N10O6 Sb 1184.2066 1184.2106 63 65 121 C44H58 Cu CuF6N10O6 Sb and 63 123 1185.2056 1185.2069 C44H58 Cu2F6N10O6 Sb 13 63 65 121 C43 CH58 Cu CuF6N10O6 Sb and 13 63 123 1186.2049 1186.2106 C43 CH58 Cu2F6N10O6 Sb 65 121 C44H58 Cu2F6N10O6 Sb and 63 65 123 1187.2047 1187.2069 C44H58 Cu CuF6N10O6 Sb 13 65 121 C43 CH58 Cu2F6N10O6 Sb and 13 63 65 123 1188.2049 1188.2069 C43 CH58 Cu CuF6N10O6 Sb 65 123 C44H58 Cu2F6N10O6 Sb 1189.2055 1189.2069 13 65 123 C43 CH58 Cu2F6N10O6 Sb 1190.2066 1190.2069

In the UHR CSI mass spectrum of P1, the dicationic complex [Cu2{HC(3-tBuPz)2-

2 2 2+ (4-CO2MePy)}2(- : -O2)] is found with the highest intensity. Furthermore, the mono-

2 2 + cationic complex ([Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)]SbF6) is detected, which isotopic distribution fits to the calculated values (figure 29 and table 9). The analogous monocationic complex was found for the peroxide dicopper(II) species P2 as well.[43]

Tyrosinase Models

Resonance Raman Spectroscopy

2 2 As for ESI-MS, the characterisation of P1 ([Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)]-

(SbF6)2) via resonance Raman (rR) spectroscopy could not be performed at r.t. Hence, measurements were conducted at low temperature (-80 °C) in cooperation with Prof. Dr. Rübhausen at the CFEL in Hamburg.[154] The major challenge with these measurements is the interfering rR signals of the peroxide dicopper(II) species and the used solvents. O-O stretching vibrations are found in the range of 730-760 cm-1,[15] which is at lower energy than frequencies for typical peroxide ions (~850 cm-1). The reason for this is the back-bonding of electron density from the copper centre into the antibonding * orbital of the peroxide (figure 28), which weakens the O-O bond.[155] Dichloromethane exhibits a strong feature around 720 cm-1 that spans a 50 cm-1 broad region in this area.[156] Thus, it interferes with upcoming O-O vibrations of peroxide dicopper(II) species. To overcome this problem, P3 is reported to be analysed in an acetone solution, where a strong solvent vibration appears at 787 cm-1.[42] The formation of P1 cannot be performed in acetone, because of the instability of P1 towards other solvents than dichloromethane. Regarding the rR spectrum of CD2Cl2 (826 (vw), 716 (w), 677 (vs), 282 (s) cm-1), the important vibration frequencies are shifted to lower energy compared to

-1 [156] CH2Cl2 (893 (vw), 713 (s), 282 (m) cm ). By changing the solvent to deuterated dichloromethane no loss in the formation of P1 was observed and thus rR measurements were performed in CD2Cl2 (figure 30).

The rR spectrum of P1 shows the characteristic vibrational frequencies of a side-on peroxide dicopper(II) complex (figure 30, blue spectrum). The O-O stretching vibration of the peroxide unit appears at 757 cm-1 and is shifted towards lower energy when oxygenation is performed

18 18 18 with O2. In the rR spectrum, this O- O band is not observed (figure 30, red spectrum) because the shift is reported to be ~40 cm-1[15] and thus the peroxide vibration is overlapped by the large solvent band of CD2Cl2. The second characteristic peroxide dicopper(II) feature is

-1 found at ~280 cm , which overlaps with the vibration of CD2Cl2 (visible through the shoulder

18 in the peroxide spectra and the much higher intensity). The oxygenation with O2 results in no frequency shift. The assignment for this feature is discussed controversially, as on one hand a

[157] Cu(II)-N(His)axial stretching mode is reported. On the other hand, it is described as a fundamental symmetric Cu2O2 core vibration. The latter was proven by Solomon et al., who describe this vibrational band composed predominantly of Cu-Cu motion.[158] Furthermore, a

-1 weaker, isotope-insensitive feature at ~330 cm is observed and associated with a Cu-Neq motion.

Tyrosinase Models

2 2 Figure 30: Resonance Raman spectrum of [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) in CD2Cl2 16 at -80 °C. Black: precursor complex [Cu{HC(3-tBuPz)2(4-CO2MePy)}]SbF6, blue: oxygenated complex with O2, 18 red: oxygenated complex with O2. Solvent vibration bands are marked with an asterisk. This rR spectrum was recorded in the group of Prof. Dr. Rübhausen at the CFEL in Hamburg.[154]

-1 -1 18 One additional vibrational band is found at 566 cm , which shifts by about 20 cm with O2. This feature is not characteristic for a -2:2-peroxide dicopper(II) species, but for the isomeric bis(-oxide) dicopper(III) complex. A vibration in the range of 560-590 cm-1, associated to this bis(-oxide) species, is reported in rR spectra of other side-on peroxide dicopper(II) complex solutions.[62,159] Solomon et al. performed measurements at different excitation wavelengths to compare intensity changes of a feature at 577 cm-1 with that of a peroxide dicopper(II) feature at 257 cm-1.[159] While the intensity at 257 cm-1 stays constant, the intensity of the 577 cm-1 feature decreases with lower excitation wavelengths. This indicates the occurrence of two different species in the Cu2O2 sample. By combining this with rR spectra of pure side-on peroxide dicopper(II) and bis(-oxide) dicopper(III) complexes, the amount of oxide species in

[159] the Cu2O2 sample is calculated to 5-20 %.

However, UV/Vis spectroscopic measurements (described above) and XAS analyses (described below) of the P1 solution reveal exclusively formed peroxide dicopper(II) complex. Thus, the origin of the feature at 566 cm-1 has to be further investigated.

Tyrosinase Models

X-ray Absorption Spectroscopy

As peroxide dicopper(II) complexes with bis(pyrazolyl)methane ligands are hardly

2 2 crystallisable, a solution of P1 ([Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2) was additionally analysed via X-ray absorption spectroscopy (XAS). These measurements were performed in cooperation with Prof. Dr. Bauer (University of Paderborn) at the ESRF in Grenoble.[160] To gain more information concerning the oxidation state and the coordination sphere of the copper centre, XANES and EXAFS analyses were conducted. In figure 31, the Cu K-edge X-ray absorption near-edge structure (XANES) spectrum of P1 is depicted. This spectrum shows the K-edge of copper at 8986.5 eV (lit: 8980.5 eV[161]) with a very weak pre- edge signal at 8978 eV. The low energy and intensity exclude copper to be in oxidation state +I. This weak feature is characteristic for a dipole-forbidden 1s → 3d quadrupole transition of Cu(II).[162–164] As a consequence, the characteristic 1s → 4p simultaneous with a LMCT shakedown transition is only found as very weak shoulder at 8987 eV.[165] The XANES spectrum of a Cu(III) complex exhibits these pre-edge features around these energy values as well, but sharper and with higher intensity as there are more empty d-states available.[166] Thus, an oxidation state of +III is also excluded and copper(II) is proven for P1. Furthermore, the white line morphology closely resembles that of literature known -2:2-peroxide dicopper(II) complexes.[167,168]

2 2 Figure 31: Cu K-edge XANES spectrum of [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1). This spectrum was recorded in cooperation with Prof. Dr. Bauer at the ESRF in Grenoble.[160]

Tyrosinase Models

The higher energetic region of the XAS spectrum (figure 31) contains the extended X-ray absorption fine structure (EXAFS). Therewith, the type, number and distance of neighbouring atoms are evaluated. The Fourier transformed EXAFS spectrum of P1 is shown in figure 32 and therewith resulting structural parameters are depicted in table 10. Fitting of the experimental EXAFS functions reveals four distance values between the X-ray absorbing atom and the backscattering atoms. First coordination sphere neighbouring atoms, namely the two oxygen atoms of the peroxide unit and the three nitrogen atoms of the bis(pyrazolyl)methane ligand, are found at an averaged distance of 1.99 Å to the copper centre. The two shells at 2.87 and 3.52 Å belong to combined C/N-backscattering atoms from the ligand. The low backscattering amplitude leads to a larger error in coordination numbers of C- and N-atoms in a distance greater than 3 Å.[169] At these distances, not negligible interference of multiple scattering effects can occur as well. The dimeric structure of this copper oxygen species in solution is proven by the obtained Cu-Cu contribution at 3.57 Å, which reveals one backscattering atom within the error margin. A Cu-Cu distance of ~3.6 Å is reported for tyrosinase model systems[15] and for the enzyme itself.[9] Thus, it is confirmed one more time

2 2 that P1 exhibits a - : -Cu2O2 core, since bis(-oxide) dicopper(III) complexes reveal shorter Cu-Cu distances (~2.8 Å).[15] By Fourier filtering, the multiple scattering shells at higher distances were removed.[162,167] The atom distance values obtained from the EXAFS measurement are in good agreement with distances of the calculated structure of P1.[56]

Figure 32: Fourier backtransformed function of the filtered Cu K-edge EXAFS spectrum of 2 2 [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1). Black solid line: experimental data; grey dashed line: fitted function. This spectrum was recorded in cooperation with Prof. Dr. Bauer at the ESRF in Grenoble.[160]

Tyrosinase Models

2 2 Table 10: Structural parameters for [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) obtained by fitting the experimental EXAFS functions with theoretical models. The fitting was performed in the group of Prof. Dr. Bauer.[160]

Abs-Bs[a] N(Bs)[b] r(Abs-Bs)[c] [Å] [d] [Å] R[e] [%] Cu-N/O 5.0 ± 0.5 1.99 ± 0.02 0.050 ± 0.006 Cu-N 1.6 ± 0.2 2.87 ± 0.03 0.039 ± 0.004 15.54 Cu-C/N 4.7 ± 0.3 3.52 ± 0.03 0.039 ± 0.008 Cu-Cu 0.5 ± 0.4 3.57 ± 0.04 0.063 ± 0.019 [a]Abs = X-ray absorbing atom, Bs = backscattering atom (neighbour). [b]Number of neighbour atoms. [c]Distance between Abs and Bs. [d]Debye-Waller-like factor to account for disorder. [e]Quality of fit.

3.4. Hydroxylation Reactions

2 2 With the peroxide dicopper(II) complexes P2 ([Cu2{HC(3-tBuPz)2(1-MeIm)}2(- : -O2)]-

2 2 (SbF6)2) and P3 ([Cu2{HC(3-tBuPz)2(Py)}2(- : -O2)](SbF6)2), it is shown that bis(pyrazolyl)- methane copper complexes serve excellently as tyrosinase model systems.[42,43] Not only structurally but also functionally, these complexes mimic features and reactivities of the enzyme. Tyrosinase catalyses the hydroxylation of the phenolic substrate L-tyrosine to L-DOPA and the subsequent oxidation to L-dopaquinone (see section 1.1.1.). The peroxide dicopper(II) complexes P2 and P3 hydroxylate phenolic substrates as well (scheme 18).

Scheme 18: Hydroxylation of phenolic substrates with peroxide dicopper(II) complexes.

Generally, the hydroxylation of phenolic substrates can be analysed concerning the kinetics of the hydroxylation step. The first step consists of the phenolate coordination to the copper centre, which is described through the equilibrium constant Keq. Subsequently, the phenolate is hydroxylated to the catechol with the hydroxylation rate constant kox. With the application of sodium phenolates, the reaction stops with the formation of the catecholic intermediate and the further oxidation to the quinone is prevented. Thus, the pure kinetics of the hydroxylation step can be analysed. On the other hand, the use of phenols instead of phenolates leads to the formation of quinone products. With the addition of an auxiliary base, the catalytic cycle is

Tyrosinase Models maintained and the turnover number of a peroxide dicopper(II) species concerning a phenolic substrate can be determined.

2 2 Thus, the newly developed complex [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) is analysed regarding hydroxylation kinetics (with sodium phenolates) and catalytic hydroxylation reactions (with phenolic substrates).

3.4.1. Hydroxylation Kinetics

Stoichiometric Hydroxylation of Sodium Phenolates

To analyse the kinetics of the rate-determining hydroxylation step (kox, scheme 18), the peroxide dicopper(II) complex P1 is reacted with sodium phenolates. The phenolates are substituted in para-position to the hydroxyl group to make use of substrates with different electronic properties. Therefore, electron-donating substituents (OMe, Me) as well as an electron-withdrawing ester moiety (CO2Me) are applied. The substrate binding kinetics are studied by using different amounts of the phenolate (2-30 eq. based on the Cu2O2 species).

P1 is synthesised in an UV/Vis measurement cell at -78 °C as described in section 3.3.1. After complete formation of the peroxide dicopper(II) complex, excess oxygen is removed in order to avoid undesired substrate overoxidation. Subsequently, sodium 4-X-phenolate (X = OMe,

Me, CO2Me) is added as MeOH solution in one portion by use of a Hamilton syringe. For every number of equivalents (2-30, based on the Cu2O2 species), a distinct stock solution is prepared and hence the added volume is equal in every experiment. This is an important aspect when analysing reactions with P1, because of the instability of P1 towards other solvents than dichloromethane (vide supra). In previous studies, no substrate binding kinetics were obtained due to the addition of the phenolate in different amounts of solvent which led to a mixture of substrate binding kinetics and P1 decay kinetics. It was proven that a 1 mmolar solution of P1 in 10 mL of dichloromethane tolerates 250 L of methanol without significant degradation of the complex in the first few minutes after addition. On the other hand, P2 and P3 tolerate the addition of THF or methanol.[42,43] Thus, there is no difficulty concerning the addition of sodium phenolates in different solution volumes.

The hydroxylation is monitored indirectly through the decrease of the characteristic absorption of the peroxide dicopper(II) species at 350 nm. One sample decay curve for the reaction of P1 with 30 eq. of sodium CO2Me-phenolate in 250 L methanol is illustrated in figure 33.

Tyrosinase Models

Figure 33: UV/Vis spectroscopic monitoring of the decay of the characteristic absorption of 2 2 [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) at 350 nm after the addition of 30 eq. sodium 4-CO2Me-phenolate in 250 L MeOH at -78 °C.

By fitting the experimental curve with a first-order exponential decay (red line in figure 33), the observable rate constant kobs is obtained. The reaction of P1 with substrates was performed for sodium OMe-, Me- and CO2Me-phenolate, in stoichiometries of 2-30 eq. phenolate per

Cu2O2 complex. For every reaction, the kobs value was received. The plot of the kobs values against the equivalents of substrate results in saturation curves (figures 34, 35 and 36). This is a Michaelis-Menten-like behaviour of substrate binding, which was established for enzymatic reactions by L. Michaelis and M. L. Menten in 1913.[170] They analysed the dependence of the substrate concentration on the velocity of the enzyme-catalysed reaction.

Michaelis-Menten Theory

Scheme 19: General reaction of enzyme E with substrate S forming in an equilibrium the substrate-bound enzyme complex ES. The conversion into the product P is irreversible and releases free enzyme E.

The reaction of an enzyme E with a substrate S leads to the formation of a substrate-bound enzyme complex ES. This complex reacts to the product P and releases free enzyme E (scheme 19). Based on this general reaction, the Michaelis-Menten equation was formulated as follows:[171,172]

Tyrosinase Models

v [S] v = max (17) KM + [S] with the velocity v, the maximum velocity at maximum substrate concentrations vmax, the substrate concentration

[S] and the Michaelis-Menten constant KM.

1 K 1 1 = M · + (18) v vmax [S] vmax

Furthermore, the Michaelis-Menten equation (17) can be reciprocally transformed to a

[173] 1 Lineweaver-Burk-type description (equation 18). The linear regression of the plot of v vs. 1 1 1 K provides the y-intercept , the x-intercept - and the slope M . [S] vmax KM vmax

The Michaelis-Menten constant KM reveals the substrate concentration at half-maximal reaction velocity (v = ½ vmax). At this substrate concentration, half of the enzyme molecules are occupied by substrate molecules and thus KM is an inverse measure for the enzyme-

[171] substrate affinity. KM is defined as:

k2 + k1' KM = (19) k1 using the reaction rate constants k of the enzyme reaction in scheme 19. In the case of Michaelis-Menten kinetics, the so-called rapid equilibrium approximation defines the catalytic reaction to be the rate-determining step and thus much slower than the equilibrium reaction

(k2 << k1’). Hence, KM is approximately the dissociation constant of the enzyme-substrate equilibrium and described as:[171]

k1' KM = (20) k1

For the equilibrium in scheme 19, the equilibrium constant Keq is defined as:

k1 Keq = (21) k1'

Regarding equations 20 and 21, the following correlation is given for KM and Keq:

1 KM = (22) Keq

Moreover, in the case of hydroxylation reactions, vmax = k2 = kox and v = kobs. The exchange of these characters in the Lineweaver-Burk-type description of the Michaelis-Menten equation (18) leads to the following equation:

Tyrosinase Models

1 1 1 1 = · + (23) kobs kox Keq [S] kox

1 1 With plotting vs. , Itoh et al. were able to calculate the equilibrium constant Kf (= Keq) kobs [S] [62] and the rate constant of the hydroxylation kox.

More exact than the linear fit of a Lineweaver-Burk plot is the direct fit of the substrate binding saturation curve. The saturation curve, obtained from kobs vs. the substrate concentration, was fitted with the backtransformed Michaelis-Menten-like equation 24 (red lines in figures 34, 35 and 36), where kox is the rate-determining intrinsic hydroxylation rate constant and Keq is the equilibrium constant of the pre-equilibrium (cf. scheme 18). Keq and kox values for P1 and for the peroxide dicopper(II) complexes P2[43] and P3[42] are depicted in table 11.

kox Keq [S] kobs = (24) 1 + Keq [S]

2 2 Figure 34: Substrate binding kinetics of the reaction of [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) -1 with sodium 4-CO2Me-phenolate at -78 °C. Initial concentration of P1: 1 mmol L . Averaged experimental data points are drawn with error bars.

Tyrosinase Models

2 2 Figure 35: Substrate binding kinetics of the reaction of [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) with sodium 4-Me-phenolate at -78 °C. Initial concentration of P1: 1 mmol L-1. Averaged experimental data points are drawn with error bars.

2 2 Figure 36: Substrate binding kinetics of the reaction of [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) with sodium 4-OMe-phenolate at -78 °C. Initial concentration of P1: 1 mmol L-1. Averaged experimental data points are drawn with error bars.

Tyrosinase Models

Table 11: Hydroxylation constants kox and equilibrium constants Keq for the reaction of the peroxide dicopper(II) 2 2 complexes P1 ([Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2), P2 ([Cu2{HC(3-tBuPz)2(1-MeIm)}2- 2 2 [43] 2 2 [42] (- : -O2)](SbF6)2) and P3 ([Cu2{HC(3-tBuPz)2(Py)}2(- : -O2)](SbF6)2) with sodium 4-X-phenolates.

X = P1 P2 P3

-1 kox [s ] OMe 2.87 4.27 1.33 Me 1.61 3.36 0.87

CO2Me 0.68 2.28 0.36 -1 Keq [L mol ] OMe 0.011 0.062 0.04 Me 0.045 0.096 0.14

CO2Me 0.034 0.066 1.00

The reaction rate constants kox for the hydroxylation of sodium 4-X-phenolates (X = OMe, Me,

2 2 CO2Me) with P1 ([Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2), P2

2 2 [43] ([Cu2{HC(3-tBuPz)2(1-MeIm)}2(- : -O2)](SbF6)2) and P3 ([Cu2{HC(3-tBuPz)2(Py)}2-

2 2 [42] (- : -O2)](SbF6)2) display that electron-rich substrates (methoxy- or methylphenolate) are hydroxylated faster than electron-deficient phenolates (CO2Me) (table 11). The more electron density is shifted from the substituent in para-position to the phenolic oxygen atom into the aromatic system, the faster the hydroxylation is. This confirms an electrophilic aromatic substitution mechanism, which is reported for hydroxylation reactions with peroxide dicopper(II) species[20,40,62,174,175] and even with the enzyme tyrosinase itself.[60,61] The aromatic ring system is hydroxylated selectively in ortho-position to the phenolic oxygen atom. This is explained by the overlap of the peroxide * orbital with the  orbital of the aromatic ring in ortho-position (figure 37).[18]

Figure 37: Overlap of the peroxide * orbital of a peroxide dicopper(II) species with the  orbital of the aromatic ring of a phenolic substrate in ortho-position. Figure adapted from Tuczek et al.[18]

Tyrosinase Models

Regarding kox values of phenolate hydroxylations with the peroxide dicopper(II) complexes P1, P2 and P3, it is observed that P2 (2.28-4.27 s-1) is the fastest hydroxylating system. With the introduced ester substituent at the pyridinyl moiety in P1 in para-position to the N-donor, higher

-1 -1 kox values (0.68-2.87 s ) are achieved compared to reactions with P3 (0.36-1.33 s ), where the pyridinyl moiety is unsubstituted. Theoretical studies on donor abilities displayed the ester- substituted pyridinyl to be a weaker donor than the unsubstituted pyridinyl moiety, which correlates to the faster phenolate hydroxylation with P1.[56] On the other hand, the imidazolyl moiety in P2 is demonstrated as a stronger N-donor than pyridinyl or pyrazolyl and nevertheless, phenolic hydroxylation is the fastest with P2.[43] These observations are contrary and not yet fully understood.

-1 Keq values are <1 for reactions with P1 (0.011-0.045 L mol ) and in most of the cases also for P2 (0.062-0.096 L mol-1) and P3 (0.04-1.00 L mol-1). This means that the coordination equilibrium of the phenolic substrate to the copper centre is on the phenolate-unbound side. In the case of methoxyphenolate, the substrate binding kinetics with P1 show an almost linear

-1 behaviour. Here, Keq exhibits the small value of 0.011 L mol , which reveals that the phenolate binding is very weak. Thus, the influence of the substrate concentration is remarkably high and even with high substrate concentrations, almost no saturation in the substrate binding kinetics is achieved. Regarding equation 24, the small Keq value leads to a linear correlation between kobs and kox.

Concerning other peroxide dicopper(II) species with pyridinyl or benzimidazolyl and benzylamine or imine donor moieties, comparable or smaller hydroxylation rate constants for the conversion of para-substituted phenolates are reported. Whereas rate constants of the

BzIm2Bz [63] hydroxylation of CO2Me-phenolate with the ligand L (-85 °C) and DTBP with the ligand LImPy (r.t.)[41] are in the range of 10-4-10-3 s-1, the ligand system LPy2Bz, published by Itoh et al.,[62]

-1 displays hydroxylation rate constants comparable to P1 (4-X-phenolates: X = Cl: kox = 0.76 s ,

-1 X = CO2Me: kox = 0.083 s (-94 °C)). Although the direct comparison of the kox values with

-1 Py2Bz -1 CO2Me-phenolate (P1: 0.68 s , L : 0.083 s ) shows an eight-fold faster reaction with P1, it must be taken into account that the reaction with LPy2Bz is reported at -94 °C, whereas P1 hydroxylates the phenolate at -78 °C. Thus, both hydroxylation reactions are in the same velocity range. The hydroxylation of para-X-phenolates with the natural enzyme tyrosinase results in 10-100-fold faster reactions (X = Me: 14.76 s-1, X = OMe: 274.87 s-1 (r.t.)).[59,61] Here, it has to be considered that the reaction proceeds at ambient temperature. Hence, the low- temperature model complexes might approach the velocity of natural tyrosinase, if kinetics could be measured at r.t.

Tyrosinase Models

The natural logarithm of the calculated kox values for the hydroxylation of sodium

+[176] para-X-phenolates with P1 are plotted against the substituent constants p of the phenolates (figure 38). This so-called Hammett plot displays the correlation between the reaction rate constant and the influence of the substituent at the phenolic substrate.[177]

Figure 38: Hammett correlation plot for the hydroxylation of sodium 4-X-phenolates (X = OMe, Me, CO2Me) with 2 2 [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1).

The Hammett correlation plot of the phenolate hydroxylation with P1 is linearly fitted (red line in figure 38). From the slope, this linear regression reveals the Hammett’s constant  with a value of -1.2. The negative sign of  confirms the electrophilic character of this hydroxylation reaction, which was already mentioned above. The peroxide dicopper(II) complexes P2 and P3 show Hammett’s constants  in the same range (P2:  = -0.5, P3:  = -1.0)[42,43] and thus exhibit the same reaction mechanism. Hammett correlations are also described with other tyrosinase model systems, where calculated  values are reported between -1.84 and -2.2.[20,40,174,175] The hydroxylation of para-substituted phenolates with tyrosinase itself was first performed in presence of borate and hydroxylamine to stabilise the catechol product, with the result of  = -2.4.[60] In 2012, hydroxylation reactions were reported at physiological pH and without additives. Here, a  value of -1.75 was found.[61] Thus, the enzyme tyrosinase displays a negative Hammett’s constant as well, which validates the electrophilic aromatic substitution mechanism found for the model systems.

Tyrosinase Models

Analysis of Reaction Products

Attempts were made to analyse reaction products of the hydroxylation of sodium 4-X-phenolates (X = OMe, Me) with the peroxide dicopper(II) complex P1. For P2, NMR spectroscopic analyses are reported, which display catechol product, phenol reactant, ligand L2 and acetophenone as internal standard.[43]

To analyse reaction products via NMR, P1 is synthesised as described in section 3.3.1. by using a centrifuge tube. The formation is not monitored via UV/Vis spectroscopy but through the colour change of the solution. After complete P1 formation, excess oxygen is removed and 5 eq. of sodium 4-X-phenolate (X = OMe or Me) are added as THF solution. The hydroxylation reaction is stopped by the addition of aqueous HCl after exact 5, 20 or 30 min and a NMR spectrum of the extracted organic compounds is measured in acetone-d6. In the NMR spectrum, catecholic signals are found for both 4-methylcatechol and 4-methoxycatechol besides many undefined other signals. When calculating the amount of catechol product via the internal standard acetophenone, the yield is in both cases lower than 40 %. One possible explanation for these low phenolate conversions is the solvent THF. The complex P1 is sensitive towards other solvents than dichloromethane, especially in greater amounts. P1 was proven to tolerate 250 L of methanol in a 1 mmol L-1 (10 mL) solution (vide supra), at least in the first few minutes after addition. For the NMR spectroscopic analyses, P1 is formed in a centrifuge tube in 5 mL solution (1 mmol L-1). The phenolate is added in a 475 L THF solution and thus the amount of other solvents than dichloromethane is four-fold higher than in hydroxylation reactions described earlier in this section. Moreover, P1 is much less tolerant towards THF than methanol, which substantiate a fast decay of P1 upon phenolate addition. Hence, amounts of P1 decay prior to the desired phenolate hydroxylation, which leads to the small yield of catechol observed.

3.4.2. Catalytic Conversions

Hydroxylation Catalysis of 8-Hydroxyquinoline

Numerous structural tyrosinase model systems are known, which form a -2:2-peroxide dicopper(II) complex. Many of them show furthermore hydroxylation ability towards phenolic substrates, but only a few systems are able to hydroxylate in a catalytic manner.[37–39] Efforts are made to develop functional tyrosinase models that display high turnover numbers (TONs) and a large variety of substrates.

Tyrosinase Models

Scheme 20: Hydroxylation catalysis of 8-hydroxyquinoline with a peroxide dicopper(II) species.

Catalytic hydroxylation reactions of 8-hydroxyquinoline with the complex [Cu2{HC(3-tBuPz)2-

2 2 (4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) are performed related to literature-known protocols.[41,48,49,178] P1 is synthesised as described in section 3.3.1. in an UV/Vis measurement cell at -78 °C. After complete formation of P1, excess oxygen is kept and a substrate solution, containing 25 eq. of 8-hydroxyquinoline (per Cu2O2 core) and 50 eq. of triethylamine in dichloromethane, is added. Triethylamine is used as auxiliary base to maintain the catalytic cycle by deprotonating the substrate and providing protons for the release of the quinone product. Immediately after substrate addition, the cooling bath is removed and the solution is allowed to warm to r.t. The reaction is monitored via UV/Vis spectroscopy by using an immersion probe (figure 39).

2 2 -1 Figure 39: UV/Vis spectra of [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) (black line, 0.93 mmol L ) and of the quinone product when catalysing the hydroxylation of 8-hydroxyquinoline (25 eq.) with P1 (coloured lines). Initial temperature: -78 °C.

In figure 39, the UV/Vis spectrum of the peroxide dicopper(II) species P1 at the beginning of the catalysis is drawn as black line. With the addition of substrate solution, the characteristic absorptions at 350 and 550 nm decrease and a new feature at 413 nm arises. This absorption is characteristic for the ortho-quinone quinoline-7,8-dione, which results from the hydroxylation and subsequent oxidation of 8-hydroxyquinoline (scheme 20) with P1.[42] The maximal product formation is reached after 7.5 min (red spectrum in figure 39). At this reaction time, the solution

Tyrosinase Models still has a temperature of -19 °C. In the next few minutes, the quinone absorption decreases until after 20 min and at r.t., a stable spectrum is reached (blue spectrum in figure 39 and cyan spectrum after 60 min). The amount of quinone product is calculated as turnovers per Cu2O2 core, expressed in the so-called turnover number (TON). For this, the quinone concentration is brought in relation to the concentration of P1 (0.93 mmol L-1). To calculate the product concentration using the Beer-Lambert law (equation 25),[179] the extinction coefficient of the absorption of quinoline-7,8-dione at 413 nm ( = 1000 L mol-1 cm-1[42]) and the optical path length (0.1 cm) are required.

A =  · c · d (25) with the absorption A, the extinction coefficient , the concentration c and the optical path length d.

Thereby, a TON of 19 is calculated using the maximal absorption after 7.5 min reaction time. Within the next 12.5 min, the absorption decreases to a stable value, where a TON of 15 is calculated. This effect was first explained by the instability of the quinone product. Subsequent oxidative coupling reactions can occur and result in C-C- or C-O-coupled quinone dimers or trimers.[55,57,58,67,69] However, a deeper view into the UV/Vis spectrum displays a shift of the baseline. For this immersion probe measurement setup, it is known that upon temperature variation the baseline does not remain constant. It is obvious that this baseline shift does not behave linearly throughout all wavelengths. Hence, no shift is observed at 800 nm, but a large shift appears around 500 or 400 nm. Starting from -78 °C, the baseline shifts to a certain degree and shifts back till it reaches the initial point at 20 °C. Thus, it should be considered that the correct value for the TON is 15 (blue spectrum in figure 39, r.t.) instead of 19 (red spectrum in figure 39, -19 °C).

Duplicating the amount of substrate from 25 eq. to 50 eq. 8-hydroxyquinoline increases the TON from 19 to 20 (red spectrum in figure 40). Considering the above-mentioned baseline shift with increasing temperature, the amount of quinone product should be determined from the spectrum at r.t. (blue spectrum in figure 40). The TON is thus calculated to 15 and hence comparable to the conversion described with 25 eq. substrate.

When applying self-assembly conditions, the catalytic conversion yields quinone product as well. For this, substrate solution (containing 50 eq. of 8-hydroxyquinoline and 100 eq. of triethylamine) is added to oxygen-saturated dichloromethane at -78 °C. After addition of copper(I) precursor complex solution, the cooling bath is removed and the solution is allowed to warm to r.t. The reaction is monitored via UV/Vis spectroscopy.

Tyrosinase Models

2 2 -1 Figure 40: UV/Vis spectra of [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) (black line, 0.95 mmol L ) and of the quinone product when catalysing the hydroxylation of 8-hydroxyquinoline (50 eq.) with P1 (coloured lines). Initial temperature: -78 °C.

Figure 41: UV/Vis spectra of the quinone product when catalysing the hydroxylation of 8-hydroxyquinoline (50 eq.) 2 2 with [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1). Reaction performed under self-assembly conditions. Initial temperature: -78 °C.

In figure 41, the absorption of the quinone product at 413 nm is observed. Assuming that P1 is formed in >95 % yield, a TON of 17 (red spectrum) is calculated, or 13 (blue spectrum) when including the baseline shift, respectively. Considering that these self-assembly conditions are not ideal for the formation of P1, the calculated TONs are comparable to the quinone product formation when applying no self-assembly conditions (20 and 15, respectively). Furthermore, the formation of P1 is described to proceed also at r.t. (section 3.3.1.). Thus it is of interest, if

Tyrosinase Models the catalytic hydroxylation of 8-hydroxyquinoline can be performed under self-assembly conditions at r.t. as well. Figure 42 shows the quinone absorption at 413 nm, which appears immediately after the addition of copper(I) precursor complex solution. As the highest time resolution of the UV/Vis spectroscopic device is to measure one spectrum every 10 seconds and the maximal quinone product absorption is observed already in the first spectrum, the formation of quinoline-7,8-dione is at r.t. faster than 10 seconds. The TON is calculated to 13, as observed above, when starting the self-assembly catalysis at -78 °C and considering the baseline shift.

Figure 42: UV/Vis spectrum of the quinone product when catalysing the hydroxylation of 8-hydroxyquinoline 2 2 (50 eq.) with [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1). Reaction performed under self-assembly conditions. Initial temperature: r.t.

The hydroxylation catalysis of 8-hydroxyquinoline by using P1 as catalyst, is comparable to the reaction with other bis(pyrazolyl)methane peroxide dicopper(II) species regarding turnover

2 2 numbers. Whereas P2 ([Cu2{HC(3-tBuPz)2(1-MeIm)}2(- : -O2)](SbF6)2) displays a TON of

[43] 2 2 14, the catalysis with P3 ([Cu2{HC(3-tBuPz)2(Py)}2(- : -O2)](SbF6)2) results only in 8 eq. of quinoline-7,8-dione.[42] The more important fact is, however, the reaction time. P3 is reported to reach the maximum TON within 16 h, the maximal amount of quinone product with P2 is observed after 20 min. With the improved catalyst P1, the hydroxylation reaction reveals 15 eq. (considering the baseline shift) of quinoline-7,8-dione after a reaction time of 7.5 min and even 13 eq. after 10 seconds, when performing the catalysis at r.t. In the literature, most of the catalytic hydroxylation reactions are reported with the substrate 2,4-di-tert-butylphenol (DTBP-H). Turnover numbers are in the same range as the 8-hydroxyquinoline conversion with P1. Tetradentate pyridinylimine ligand systems show TONs of 16 or 18 within a reaction time of 1 h or 4.5 h, respectively.[48,53] Comparable or even higher product formation (TONs:

Tyrosinase Models

11-29) is reached with bidentate N-donor ligand systems, exhibiting pyridinyl, imine, benzimidazolyl, pyrazolyl or triazolyl moieties.[41,51,52,57,65] The highest turnover is reported with a bidentate benzimidazolyl-imine ligand, where the TON is calculated to 31.[50] But all these bidentate systems collectively display long reaction times with catalytic conversions within several hours. Thus, the tyrosinase model system P1 shows comparable amounts of quinone product but it is tremendously faster than literature-known model systems.

Analysis of Reaction Products

The catalytic conversion products of the reaction of 8-hydroxyquinoline with P1 are determined not only by UV/Vis, but also via NMR spectroscopy. Therefore, the catalysis is stopped after 7.5 min or 1 h, the organic compounds are extracted and measured NMR spectroscopically in acetone-d6. Regardless of the substrate concentration, neither quinone product nor the substrate 8-hydroxyquinoline were found in the spectra. Moreover, acidification or basification of the aqueous residue and subsequent extraction with organic solvent did not result in quinoline signals in the NMR spectrum. By analysing the reaction mixture with mass spectrometry, neither product nor substrate are observed. With the bis(pyrazolyl)methane peroxide dicopper(II) species P3, quinoline-7,8-dione is detected via NMR spectroscopy and mass spectrometry.[42] Tuczek et al. performed the catalysis with 8-hydroxyquinoline as well and observed the quinoline absorption at 412 nm.[55] But they could not detect the quinone product in the NMR spectrum, nor in the mass spectrum. Instead, they observed the complex

[Cu(II)(8-quinolinol)2] in the mass spectrum, where hydroxyquinoline serves as ligand for the copper centre. This complex was also verified via powder diffraction analysis.

It is obvious that the analysis of quinoline-7,8-dione from the catalytic mixture is difficult. Thus, future studies should continue to develop procedures for the detection of the quinone product in the catalytic hydroxylation reaction of 8-hydroxyquinoline. This is important, especially when considering the high catalytic velocity with P1. The turnover number cannot be calculated exactly from the UV/Vis spectrum due to the baseline shift upon increasing temperature. Thus, another quantitative analysation method has to be found.

Tyrosinase Models

3.5. Conclusion

In this chapter, the synthesis and characterisation of the new bis(pyrazolyl)methane

HC(3-tBuPz)2(4-CO2MePy) (L1) is described. L1 is an extension of the pyridinyl ligand L3

(HC(3-tBuPz)2(Py)), by possessing an ester substituent at the pyridinyl moiety. The synthesis was performed according to syntheses of related bis(pyrazolyl)methanes.[128] L1 exhibits a higher isomerisation tendency of the tert-butyl groups and thus the synthesis is costlier than in

[43] [128] the case of the related ligands L2 (HC(3-tBuPz)2(1-MeIm)) or L3. By purifying L1 via column chromatography instead of distillation, the yield can be increased from 59 to 87 %. Copper(I) bromide and copper(II) chloride complexes of L1 display the tridentate coordination of this ligand. Both pyrazolyl and the pyridinyl N-donor moieties coordinate the copper centre. The copper(II) chloride complex C1 shows a strong distorted square-pyramidal coordination sphere. The copper(I) bromide complex C2 crystallises with two molecules in the asymmetric unit, which display a highly distorted tetrahedral geometry. The copper(I) chloride complex C3 and the copper(I) iodide complex C4 bear the imidazolyl ligand L2. Both complexes show a high symmetric geometry with a mirror plane through the imidazolyl moiety, the copper centre and the anion. Regarding bond lengths, it is not possible to make statements about the influence of ester substitution on N-donor strengths.

With the ligand L1, the -2:2-peroxide dicopper(II) species P1 was synthesised as new tyrosinase model system. The formation of P1 is complete within 40 min at -78 °C and could be also performed at r.t. (3.5 min, 70 % yield). However, it is exclusively possible in dichloromethane. For P1, as well as the L2-bearing complex P2 and P3 (containing L3), formation kinetics were determined. The electron-withdrawing ester substituent leads to a weaker pyridinyl N-donor and thus, the formation of P3 is faster than that of P1. The slowest formation kinetics were obtained with the complex P2, where the calculated activation enthalpy is the highest. With P1, one of the most stable tyrosinase model system was developed (at r.t.: t1/2 = 44 min). P1 was characterised via UV/Vis spectroscopy, displaying two characteristic absorptions at 350 and 550 nm in an intensity ratio of 13:1. In UHR CSI-MS measurements

2 2 + the monocationic complex ([Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)]SbF6) was detected. XAS analysis proved copper to be in oxidation state +II and oxygen to be bound in a -2:2-peroxide fashion. The resonance Raman spectrum revealed the O-O vibration at 757 cm-1.

As a functional tyrosinase model system, P1 is able to hydroxylate phenolic substrates, even in catalytic reactions. Sodium 4-X-phenolates with X = OMe, Me or CO2Me are hydroxylated

Tyrosinase Models selectively in ortho-position in Michaelis-Menten-like substrate binding kinetics. The faster hydroxylation of electron-richer substrates proves an electrophilic aromatic substitution mechanism with a Hammett’s constant  of -1.2. The catalytic conversion of 8-hydroxyquinoline yielded the quinone product, monitored via the characteristic absorption of quinoline-7,8-dione at 413 nm. The turnover number was calculated to 20, when using 50 eq. of substrate. Considering the baseline shift with increasing temperature (that is due to the measurement setup), the TON must be corrected to 15. The catalysis occurs also under self- assembly conditions and even at r.t., although with slightly smaller TONs. With the maximal amount of quinone product reached after 7.5 min (low temperature) or even after less than 10 seconds (r.t.), this catalytic reaction is the fastest for tyrosinase model systems developed so far.

Biohybrid Conjugates

4. Biohybrid Conjugates

The combination of biological protein scaffolds with chemical catalysts leads to artificial metalloenzymes, which display new, enhanced or more selective reactivity towards external substrates.[87] Bis(pyrazolyl)methane copper complexes were shown to serve as excellent model systems of the enzyme tyrosinase as they catalyse hydroxylation reactions of phenolic substrates (see chapter 3).[42,43,56] The oxygenation of a copper(I) precursor complex results in the formation of a -2:2-peroxide dicopper(II) complex, but the stabilisation of an intermediate mononuclear superoxide species cannot be achieved. By introducing the bis(pyrazolyl)methane copper complex into a protein environment to obtain a biohybrid catalyst, this stability problem should be avoided. With a superoxide species, the substrate variety for hydroxylation reactions could be enhanced.[39]

This chapter describes the synthesis of two new maleimide-bearing bis(pyrazolyl)methanes, providing an anchoring-site for a protein scaffold. The hydroxylation ability is examined by using these ligands in tyrosinase model systems. Afterwards, the conjugation of the maleimide- bearing ligands with an apoprotein is explained. The incorporation of a metal centre and the subsequent characterisation of the artificial metalloenzyme is described, followed by oxidation reactions with different substrates.

4.1. Bis(pyrazolyl)methanes

The conjugation of proteins with bis(pyrazolyl)methane ligands can be performed by the addition reaction of the SH-group of a cysteine residue in the protein to a maleimide moiety at the ligand molecule. The successful incorporation of Grubbs-Hoveyda-type catalysts in variants of the protein FhuA is reported making use of such a maleimide anchoring.[100,180]

For the preparation of the maleimido-bis(pyrazolyl)methanes L5 and L6 (figure 43), the OH-bearing ligand L4 has to be synthesised (figure 43). This hydroxyl group can be reacted in an esterification to L5 or in a Mitsunobu reaction to L6.

Biohybrid Conjugates

Figure 43: Bis(pyrazolyl)methane ligands with a hydroxyl group (L4) and with maleimide moieties (L5 and L6). N-donor atoms are marked in blue.

4.1.1. Synthesis of 2-(4-Hydroxymethylenepyridinyl)bis(3-tert-butylpyrazolyl)methane (L4)

The synthesis of the bis(pyrazolyl)methane HC(3-tBuPz)2(4-CH2OHPy) (L4) starts with the related compound L1, where the pyridinyl moiety possesses an ester substituent instead of a hydroxyl function at C4. A schematic description of the synthesis is given in scheme 21. The

[181] reduction of the ester substituent with LiAlH4 leads to the formation of gaseous hydrogen. Aqueous, alkaline workup results in the desired bis(pyrazolyl)methane L4.

Scheme 21: Synthesis of HC(3-tBuPz)2(4-CH2OHPy) (L4).

As already mentioned in section 3.1.1., bis(pyrazolyl)methanes with substituents at the pyrazolyl moieties tend to isomerise to the thermodynamically more stable 5-tBuPz substituent. This isomerisation was also observed during the synthesis of L4 by obtaining the ligand

[141,142] HC(3-tBuPz)(5-tBuPz)(4-CH2OHPy). An acid-catalysed re-isomerisation of the tert-butylpyrazolyl unit and subsequent purification via column chromatography results in L4 as an orange, very viscous oil with an overall yield of 57 %.

Biohybrid Conjugates

4.1.2. Synthesis of 2-(4-Maleimidoethylenecarboxymethylenepyridinyl)bis- (3-tert-butylpyrazolyl)methane (L5)

The maleimide-bearing ligand HC(3-tBuPz)2(4-Mal1Py) (L5) is synthesised according to literature procedures[100,180] and the optimised synthesis is shown in scheme 23. Basically, the alcoholic ligand L4 is reacted with 3-maleimidopropionyl chloride in an esterification to give the maleimido-bis(pyrazolyl)methane ligand L5, where an ester spacer group connects the N-donor ligand with the maleimide moiety. For this synthesis, 3-maleimidopropionyl chloride is prepared starting from maleic anhydride and -alanine (scheme 22). The condensation results in 3-maleimidopropionic acid,[182] which is activated with thionyl chloride to give the acyl chloride 3-maleimidopropionyl chloride.[183]

Scheme 22: Synthesis of 3-maleimidopropionyl chloride starting from maleic anhydride and -alanine.

Scheme 23: Synthesis of HC(3-tBuPz)2(4-Mal1Py) (L5).

The synthesis of L5 is challenging, as reported reaction conditions for the esterification do not work in the present case.[100,180] Okuda et al. prepared maleimide-linked ruthenium complexes for Grubbs-Hoveyda-type catalysts by using tetrahydrofuran as solvent and sodium bicarbonate as base. The reaction is performed at r.t. and complete within 18-24 h. Sodium bicarbonate is barely soluble in THF, but sufficient for the reaction in the case of ruthenium complexes. For the deprotonation of L4, the solubility is insufficient and thus the reaction yielded only unreacted L4 (table 12, entry 1). Neither increasing the temperature (entry 2), nor using diethyl ether as solvent (entry 3) or purifying the maleimide derivative via distillation (entry 4) result in the desired maleimide ligand L5. Applying sodium hydride (entry 5) instead

Biohybrid Conjugates of sodium bicarbonate leads to the degradation of the bis(pyrazolyl)methane. Another base, which is more soluble in organic solvents and can still be removed after the reaction is triethylamine. This base is able to absorb HCl, which is formed during the reaction. As NEt3·HCl is insoluble in THF or Et2O, it can be filtered off. But approaches with NEt3 in THF (entry 6) or

Et2O (entry 7) only yield the alcoholic ligand L4. Finally, the only reaction conditions that gave the maleimido-bis(pyrazolyl)methane ligand L5 are depicted in entry 8. With the base NEt3 in THF at 25 °C and with the re-distilled maleimide derivative, L5 is obtained from the reaction mixture as yellow solid in 94 % yield (scheme 23).

Table 12: Reaction conditions for the esterification of HC(3-tBuPz)2(4-CH2OHPy) (L4) with 3-maleimidopropionyl chloride.

entry base solvent temperature maleimide desired derivative product?*

1 NaHCO3 THF r.t. washed no

2 NaHCO3 THF 50 °C washed no

3 NaHCO3 Et2O r.t. washed no

4 NaHCO3 THF r.t. distilled no 5 NaH THF r.t. washed no

6 NEt3 THF r.t. washed no

7 NEt3 Et2O r.t. washed no

8 NEt3 THF r.t. distilled yes

*The desired product is the maleimido-bis(pyrazolyl)methane HC(3-tBuPz)2(4-Mal1Py) (L5).

4.1.3. Synthesis of 2-(4-Maleimidomethylenepyridinyl)bis(3-tert-butylpyrazolyl)methane (L6)

In contrast to L5, the maleimide-bearing ligand HC(3-tBuPz)2(4-Mal2Py) (L6) exhibits a short methylene linker between the N-donor ligand and the maleimide moiety. The synthesis of L6 is performed by applying a Mitsunobu reaction[184,185] and is shown in scheme 24. In the first step, triphenylphosphane is reacted with diethyl azodicarboxylate (DEAD) and the alcoholic ligand L4. The addition of maleimide and neopentyl alcohol yields the amine compound

DEAD-H2, triphenylphosphane oxide and the maleimide-bound ligand L6. In some Mitsunobu reactions neopentyl alcohol is added with the maleimide to activate it.[185] This facilitates the reaction of maleimide with the alcoholic compound.

Biohybrid Conjugates

Scheme 24: Synthesis of HC(3-tBuPz)2(4-Mal2Py) (L6).

The described purification method is column chromatography. In the case of L6, DEAD-H2 appeared to have the same Rf value as the product. Thus, further purification by recrystallisation from ethyl acetate is required. DEAD-H2 crystallises from the cold solution within two days. L6 is obtained from the supernatant as colourless oil in 49 % yield.

4.1.4. Characterisation of the Bis(pyrazolyl)methanes L4, L5 and L6

The bis(pyrazolyl)methane ligands L4, L5 and L6 are characterised via NMR, IR spectroscopy and mass spectrometry (EI). The NMR spectra of the alcoholic ligand L4, the maleimide ligand with a longer spacer L5 and the maleimide ligand with a shorter spacer L6 are compared to the ester-substituted ligand L1 and the unsubstituted ligand L3. It is observed that most of the NMR signals are insignificantly shifted when changing the substituent at the pyridinyl moiety. The 1H-NMR signals of the protons, which are in neighbourhood to the substituent, are denoted with 3-py(H) and 5-py(H) (table 13). A comparison of the different ligand systems shows that both protons are influenced by the ester substituent (L1) but are unimpressed of alkyl substituents that bear further functional groups (L4, L5, L6). The methylene protons, which are located at the C-atom connected to the 4-py-position are abbreviated with CH2(H). Regarding L4 and L6, the chemical shift of these protons does not change significantly, although L4 bears a hydroxyl group and L6 a maleimide unit. A small shift (~0.4 ppm) is observed in the ligand L5, where an ester group with further alkyl units and a maleimide moiety binds to the methylene C-atom. In the 13C-NMR spectra, the 2-py(C) is slightly shifted with the ester substituent (L1) but insignificantly shifted upon alkyl substitution (L4, L5, L6) as it is described for the pyridinyl protons above. The pyridinyl carbon atom, where the substituent is located, is weakly influenced by the ester group (L1). This influence is much higher with alkyl-ester (L5) or alkyl- amide (L6) groups (8.8 or 9.0 ppm, respectively). The largest shift of 14.6 ppm is observed with the alkyl-alcohol substituent in L4. The signal of the methylene C-atom is similar for

Biohybrid Conjugates oxygen neighbour atoms (L4, L5), but the chemical shift is about 24 ppm lower when nitrogen is bound (L6).

1 13 Table 13: Selected H- and C-NMR signals of HC(3-tBuPz)2(4-CO2MePy) (L1), HC(3-tBuPz)2(Py) (L3), HC(3-tBuPz)2(4-CH2OHPy) (L4), HC(3-tBuPz)2(4-Mal1Py) (L5) and HC(3-tBuPz)2(4-Mal2Py) (L6).

1H-NMR 13C-NMR

3-py(H) 5-py(H) CH2(H) 2-py(C) 4-py(C) CH2(C) L1 7.45 7.81 - 157.4 138.6 - L3 6.86 7.25 - 156.0 136.9 - L4 6.91 7.26 4.65 155.9 151.5 63.4 L5 6.82 7.21 5.06 156.5 145.7 64.7 L6 6.78 7.15 4.61 156.6 145.9 40.3

The IR spectra of the ligands L4, L5 and L6 show the characteristic vibrations of a bis(pyrazolyl)methane.[128] As described for L1 above, features in the range of 1700-1750 cm-1 in the spectra of L5 and L6 are assigned to C=O vibrations,[143] which are found in the maleimide unit. Moreover, L5 exhibits an ester group in the spacer between the N-donor ligand and the maleimide moiety. In the spectra of L4, a feature at 3258 cm-1 is observed. This is characteristic for the O-H vibration of hydroxylic functions, as it is found in L4.[143]

The EI mass spectra display peaks of different degradation products of L4, L5 and L6. In every spectrum the mass peak for the respective ligand is found, although fragment peaks are of much higher intensity than mass peaks.

4.2. Tyrosinase Reactions

The bis(pyrazolyl)methane ligands L5 and L6 were designed for the conjugation into a protein scaffold. Thereby, mononuclear copper oxygen species should be stabilised to perform hydroxylation or oxidation reactions. To analyse the hydroxylation ability of copper(I) precursor complexes with L5 and L6 in advance to the preparation of the biohybrid conjugates, oxygen activating and tyrosinase modelling reactions are performed.

Biohybrid Conjugates

4.2.1. Oxygen Activation with L5 and L6

For the formation of peroxide dicopper(II) complexes with the bis(pyrazolyl)methanes L5 and L6, copper(I) precursor complexes with both ligands are prepared according to the description in section 3.3.1. These complexes, [CuL5]SbF6 and [CuL6]SbF6, are injected into oxygen- saturated dichloromethane (scheme 25). The formation of the peroxide dicopper(II) species

2 2 P5 ([Cu2{HC(3-tBuPz)2(4-Mal1Py)}2(- : -O2)](SbF6)2) and P6 ([Cu2{HC(3-tBuPz)2-

2 2 (4-Mal2Py)}2(- : -O2)](SbF6)2) is monitored via UV/Vis spectroscopy by using an immersion probe setup (figures 44 and 45).

2 2 Scheme 25: Synthesis of [Cu2{HC(3-tBuPz)2(4-Mal1Py)}2(- : -O2)](SbF6)2 (P5) and [Cu2{HC(3-tBuPz)2- 2 2 (4-Mal2Py)}2(- : -O2)](SbF6)2 (P6).

The oxygenation of [Cu{HC(3-tBuPz)2(4-Mal1Py)}]SbF6 and [Cu{HC(3-tBuPz)2(4-Mal2Py)}]-

2 2 [15] SbF6 results in characteristic absorption spectra of - : -peroxide dicopper(II) species. However, the intensity of the high-energy band of P5 is rather low. The spectrum of P5 shows absorptions at 348 (~12500 L mol-1 cm-1)[186,187] and 555 nm (~800 L mol-1 cm-1).[186,187] The reason for the rather weak 348 nm-band is not fully clarified yet. On one hand, the weak absorption might result from a small extinction coefficient, which would be calculated to 12500 L mol-1 cm-1. Side-on peroxide dicopper(II) species with bis(pyrazolyl)methane ligands, however, show extinction coefficients around 20000 L mol-1 cm-1.[42] Thus, on the other hand, P5 might be not fully formed. In this case, a hypothetical extinction coefficient of 20000 L mol-1 cm-1 would lead to the yield of 63 %. First attempts to determine the extinction coefficient via titration experiments, however, indicate P5 to be not fully formed.[187] With P6, slightly shifted absorptions at 350 (~21000 L mol-1 cm-1) and 550 nm (~1200 L mol-1 cm-1) are observed, which are comparable to the spectrum of P1 ([Cu2{HC(3-tBuPz)2(4-CO2MePy)}2-

2 2 (- : -O2)](SbF6)2). Both peroxide dicopper(II) species P5 and P6 are completely formed

Biohybrid Conjugates within 30 min at -78 °C. The decay kinetics are determined neither at -78 °C nor at r.t., but first attempts of stability experiments indicate complexes that are comparably stable as P1.

It could be demonstrated that the maleimide modification does not interfere with the formation of the peroxide dicopper(II) complex.

Figure 44: UV/Vis spectra of the reaction of [Cu{HC(3-tBuPz)2(4-Mal1Py)}]SbF6 with oxygen in dichloromethane at 2 2 -78 °C. Final concentration of the peroxide dicopper(II) complex [Cu2{HC(3-tBuPz)2(4-Mal1Py)}2(- : -O2)]- -1 (SbF6)2 (P5): 1 mmol L .

Figure 45: UV/Vis spectra of the reaction of [Cu{HC(3-tBuPz)2(4-Mal2Py)}]SbF6 with oxygen in dichloromethane at 2 2 -78 °C. Final concentration of the peroxide dicopper(II) complex [Cu2{HC(3-tBuPz)2(4-Mal2Py)}2(- : -O2)]- -1 (SbF6)2 (P6): 1 mmol L .

Biohybrid Conjugates

4.2.2. Catalytic Hydroxylation Reactions

The peroxide dicopper(II) species P5 and P6 are analysed concerning hydroxylation ability towards phenolic substrates. Catalytic reactions are performed as described in section 3.4.2. In the presence of triethylamine, 8-hydroxyquinoline and 4-methoxyphenol are converted to the corresponding quinones (schemes 20 and 26). Quinones are highly reactive products. Thus, 4-methoxy-1,2-benzoquinone is known to further react with 4-methoxyphenol in a C-O coupling reaction to result in 4-methoxy-5-(4-methoxyphenoxy)-1,2-benzoquinone (scheme 26).[57]

Scheme 26: Hydroxylation catalysis of 4-methoxyphenol with a peroxide dicopper(II) species and subsequent C-O coupling reaction.

Hydroxylation Catalysis of 8-Hydroxyquinoline

The catalytic conversion of 8-hydroxyquinoline (50 eq.) was performed with the peroxide dicopper(II) species P5 and P6. The catalytic reactions are monitored via UV/Vis spectroscopy, as quinoline-7,8-dione exhibits a characteristic absorption at 413 nm[42] (figures 46 (with P5) and 47 (with P6)).

2 2 -1 Figure 46: UV/Vis spectra of [Cu2{HC(3-tBuPz)2(4-Mal1Py)}2(- : -O2)](SbF6)2 (P5) (black line, 1 mmol L ) and of the quinone product when catalysing the hydroxylation of 8-hydroxyquinoline (50 eq.) with P5 (coloured lines). Initial temperature: -78 °C.

Biohybrid Conjugates

2 2 -1 Figure 47: UV/Vis spectra of [Cu2{HC(3-tBuPz)2(4-Mal2Py)}2(- : -O2)](SbF6)2 (P6) (black line, 1 mmol L ) and of the quinone product when catalysing the hydroxylation of 8-hydroxyquinoline (50 eq.) with P6 (coloured lines). Initial temperature: -78 °C.

The conversions of 8-hydroxyquinoline with P5 and P6 display fast quinone formation. Within 6.5 min (or 6 min in the case of P6), the maximal amount of quinoline-7,8-dione is observed. This is comparable to the reaction with P1 (7.5 min). The turnover number is calculated to 17 with P5 and 11 with P6 (based on the Cu2O2 core). With a hypothetically not fully formed P5 species, the TON would be calculated higher. As already described in section 3.4.2., during the observed short reaction time, the temperature does not reach r.t. after removal of the cooling bath. This leads to the above-mentioned baseline shift within the spectra containing the maximal turnover (red spectra in figures 46 and 47). The blue spectra in both figures show the absorption of the quinone product when r.t. is reached (after 20 min). Then, TONs are calculated to 14 with P5 (or higher with not fully formed P5) and 9 with P6, respectively. These catalytic conversions of 8-hydroxyquinoline are comparable to the reaction with P1 (TON = 15) and to published bis(pyrazolyl)methane systems (P2: TON = 14, P3: TON = 8).[42,43]

Hydroxylation Catalysis of 4-Methoxyphenol

The hydroxylation ability of P5 and P6 is furthermore studied with the substrate 4-methoxyphenol. In figures 48 and 49, the UV/Vis spectroscopic monitoring of the catalyses is depicted.

Biohybrid Conjugates

2 2 -1 Figure 48: UV/Vis spectra of [Cu2{HC(3-tBuPz)2(4-Mal1Py)}2(- : -O2)](SbF6)2 (P5) (black line, 1 mmol L ) and of the quinone product when catalysing the hydroxylation of 4-methoxyphenol (50 eq.) with P5 (coloured lines). Initial temperature: -78 °C.

2 2 -1 Figure 49: UV/Vis spectra of [Cu2{HC(3-tBuPz)2(4-Mal2Py)}2(- : -O2)](SbF6)2 (P6) (black line, 1 mmol L ) and of the quinone product when catalysing the hydroxylation of 4-methoxyphenol (50 eq.) with P6 (coloured lines). Initial temperature: -78 °C.

The absorption spectra of the conversion of 4-methoxyphenol show an arising feature at 420 nm, which is characteristic for 4-methoxy-1,2-benzoquinone ( = 1700 L mol-1 cm-1).[188] In contrast to the catalytic conversion of 8-hydroxyquinoline, the hydroxylation of 4-methoxyphenol is slower and thus the reaction already reached r.t., when analysing the quinone product. Thus, no baseline shift falsifies the calculation of the turnover number. With P5, the maximal amount of formed quinone product after 16 min correlates to a TON of 7 (or higher

Biohybrid Conjugates with hypothetically not fully formed P5), whereas with P6, the yield is even smaller (TON = 3). Considering the high reactivity of 4-methoxy-1,2-benzoquinone, further C-O coupling reactions occur to form 4-methoxy-5-(4-methoxyphenoxy)-1,2-benzoquinone (scheme 26).[68,69] Tuczek et al. reported that this coupled product exhibits a characteristic absorption at 418 nm (524 L mol-1 cm-1).[57] Since the quinone product and the coupled species show almost the same absorption wavelength, it is not clear which product is observed in the catalysis spectra of P5 and P6. With regard to the high reactivity of the uncoupled quinone, the possibility of the coupled product is obvious. Thus, the TONs are calculated to 23 for P5 (or higher with hypothetically not fully formed P5) (after 16 min) and to 10 for P6 (after 16 min), respectively. To make a definite statement about the species belonging to this 420 nm absorption, NMR spectroscopic analyses are required.

When performing this catalytic reaction with the sodium phenolate (50 eq.) instead of the phenol substrate, the phenolate coordination to the copper centre and the subsequent hydroxylation is observed (figure 50). Addition of sodium 4-methoxyphenolate to a solution of the peroxide dicopper(II) species P5 results in the decrease of the characteristic absorption at 348 nm. Simultaneously, a new absorption at around 300 nm arises, which might on one hand stem from phenolate binding to the copper centre. On the other hand, the continuing increase of this 300 nm-band can be correlated to the hydroxylation of the phenolate to the catecholate. After a while, the quinone product (or the C-O coupled product) is also observed at 420 nm due to maintaining excess of oxygen.

2 2 Figure 50: UV/Vis spectra of the reaction of [Cu2{HC(3-tBuPz)2(4-Mal1Py)}2(- : -O2)](SbF6)2 (P5) (black line, 1 mmol L-1) with sodium 4-methoxyphenolate (50 eq.) (red lines: arising quinone product). Inset: high-energy wavelength region. Initial temperature: -78 °C.

Biohybrid Conjugates

4.3. Nitrobindin Conjugates

Variants of the -barrel protein nitrobindin, which were designed by site-directed mutagenesis, are known to serve as protein scaffolds for biohybrid catalysts.[96,98,101–103] For the conjugation of bis(pyrazolyl)methane copper complexes, NB4 and its expanded variant NB4exp are used. Due to solubility reasons, copper complex conjugation is performed successively. First, the maleimide ligand is conjugated to the protein, then the conjugated protein is treated with copper nitrate. The resulting bis(pyrazolyl)methane copper complex NB4(exp) biohybrid catalyst is analysed with regard to potential substrate conversion. As oxygenation reactions are rather challenging, additional one-electron oxidation substrates are also applied.

4.3.1. Conjugation of L5 and L6 to NB4 and NB4exp

Synthesis

Prior to the conjugation, dithiothreitol (DTT) is added to the proteins to prevent disulfide bridge formation of the cysteine residues. Disulfide bridges lead to the formation of protein oligomers, which precipitate in the solution.

The conjugation of the proteins NB4 and NB4exp with the maleimide ligands L5 and L6 is performed in MOPS (3-(N-morpholino)propanesulfonic acid) buffer in a 20 mol L-1 solution.[189] 5 equivalents of the maleimide ligand are added in a DMSO solution, assuring that the organic solvent does not exceed 1 % of the total volume. The protein solution is incubated at 4 °C for 1 h to ensure ligand conjugation. Afterwards, excess ligand is removed by a desalting column. The synthesis of conjugated nitrobindin proteins is shown in scheme 27.

Scheme 27: Synthesis of conjugates of HC(3-tBuPz)2(4-Mal1Py) (L5) or HC(3-tBuPz)2(4-Mal2Py) (L6) with the proteins NB4 or NB4exp.

Biohybrid Conjugates

Characterisation

The bis(pyrazolyl)methane-conjugated nitrobindin proteins are characterised via ESI, LC-ESI and MALDI-TOF mass spectrometry. In the ESI mass spectra of NB4-L5, NB4-L6 and NB4exp-L6 (figures 51, 52 and 53), the isotopic distribution of the molecular ion peak is found. The calculated values of the conjugates deviate rather high (up to 3.88 m/z) from the experimentally found ones (table 14). LC-ESI mass spectra show similar distribution patterns and m/z values are in the same range (table 14).

Table 14: Found (ESI-, LC-ESI- and MALDI-TOF-MS) and calculated m/z values for the conjugates NB4-L5, NB4-L6, NB4exp-L5 and NB4exp-L6.

ESI-MS LC-ESI-MS MALDI-TOF-MS calculated m/z m/z m/z m/z NB4-L5 19934.040 19932.490 19936.361 19935.523 NB4-L6 19860.213 19861.647 19863.114 19863.461 NB4exp-L5 - - 23097.906 23097.094 NB4exp-L6 23021.151 23022.194 23022.903 23025.031

Figure 51: ESI mass spectrum of NB4-L5.

Biohybrid Conjugates

Figure 52: ESI mass spectrum of NB4-L6.

Figure 53: ESI mass spectrum of NB4exp-L6.

MALDI-TOF mass spectra display the molecular ion peaks with a deviation in most cases not higher than 1 m/z from the calculated values (table 14). This is within the error range for such biomacromolecules. Furthermore, a second peak around 10000 m/z (11500 m/z for NB4exp) is ascribed to the twofold positive molecular ion. In figures 54, 55, 56 and 57, the spectra of the biohybrid conjugates NB4-L5, NB4-L6, NB4exp-L5 and NB4exp-L6 are depicted.

Although the discrepancies to the calculated values are rather high, especially in the case of ESI-MS, both ESI and MALDI-TOF mass spectra confirm the conjugation of the bis(pyrazolyl)methane ligand into the protein scaffold.

Biohybrid Conjugates

Figure 54: MALDI-TOF mass spectrum of NB4-L5.

Figure 55: MALDI-TOF mass spectrum of NB4-L6.

Figure 56: MALDI-TOF mass spectrum of NB4exp-L5.

Biohybrid Conjugates

Figure 57: MALDI-TOF mass spectrum of NB4exp-L6.

4.3.2. Copper Complexation in the Conjugated Protein

Synthesis

The incorporation of copper into the maleimide ligand-conjugated proteins NB4-L5 and NB4exp-L5 is performed by the addition of an aqueous copper nitrate solution (scheme 28; the depicted structure of the copper complex in this and following schemes is only of illustrative purpose. It does not represent the actual constitution). The concentration of the copper solution is in the range of 10-15 mmol L-1, whereas the protein concentration does not exceed 350 mol L-1. Incubation at 4 °C for 30 min ensures copper coordination in the active site. Subsequently, excess copper is removed by a desalting column.

Scheme 28: Synthesis of the copper-coordinated biohybrid conjugates NB4-/NB4exp-L5-Cu. L might be a H2O ligand.

Biohybrid Conjugates

Characterisation

The incorporation of copper into NB4-L5 and NB4exp-L5 is analysed via UV/Vis and EPR spectroscopy. In figures 58 and 59, the UV/Vis spectra of NB4-L5-Cu and NB4exp-L5-Cu are depicted. Both protein spectra (black lines) show high absorptions at 280 nm. These are characteristic for proteins and stem from aromatic side chain residues (Phe, Tyr, Trp).[190]

Figure 58: UV/Vis spectra of the biohybrid conjugate NB4-L5-Cu (178 mol L-1) (black), NB4-L5-Cu with NB4-L5 as baseline (red) and the bis(pyrazolyl)methane copper complex without the protein (blue). L5-Cu is prepared as 6 mmol L-1 solution in 30:6:64 water : DMSO : acetone.

Figure 59: UV/Vis spectra of the biohybrid conjugate NB4exp-L5-Cu (153 mol L-1) (black), NB4exp-L5-Cu with NB4exp-L5 as baseline (red) and the bis(pyrazolyl)methane copper complex without the protein (blue). L5-Cu is prepared as 6 mmol L-1 solution in 30:6:64 water : DMSO : acetone.

Biohybrid Conjugates

When using NB4-L5 (or NB4exp-L5 respectively) as baseline (red lines), a weak absorption is observed at 300 nm, which is more pronounced in NB4 than in NB4exp. This absorption is ascribed to the L5-copper complex in the nitrobindin proteins. A UV/Vis measurement of the bis(pyrazolyl)methane copper complex without any protein (blue lines) shows an absorption at 320 nm. As L5-Cu is not soluble in water or buffer solution, acetone is added until the complex is completely dissolved (64 % acetone of total volume). The difference in the solvents leads to the absorption shift from 320 to 300 nm in the protein.

EPR spectra of NB4-L5-Cu and NB4exp-L5-Cu (figures 60 and 61) show signals that indicate the presence of copper(II) in the protein solution. In literature, EPR signals of copper proteins are reported in the same magnetic field range and with hyperfine structures, at least at low temperature.[6,191] In the case of nitrobindin bis(pyrazolyl)methane copper biohybrid conjugates, a hyperfine structure is hardly observed. Furthermore, with these EPR spectra it is not ensured that the copper ion is coordinated by the bis(pyrazolyl)methane ligand. Copper coordination can also be achieved with protein side chain residues of amino acids like histidine, tyrosine or glutamate. Thus, further studies (including theoretical calculations) on copper coordination in NB4-L5 and NB4exp-L5 are required.

Figure 60: EPR spectrum of NB4-L5-Cu in MOPS buffer at -173 °C. Concentration: 334 mol L-1.

Biohybrid Conjugates

Figure 61: EPR spectrum of NB4exp-L5-Cu in MOPS buffer at -173 °C. Concentration: 283 mol L-1.

The amount of copper in the protein solution was analysed via ICP mass spectrometry. The obtained values are summarised in table 15. For the protein solution NB4exp-L5-Cu, a copper loading of 81 % is determined. Additionally, the ICP-MS experiment was performed with the oxygenated protein NB4exp-L5-Cu-O2. Here, the copper loading is calculated to 66 %. For details about the oxygenation procedure see section 4.3.4. As with EPR, ICP-MS cannot evaluate where the copper is positioned in the protein. The possibility of copper coordination by other ligands than L5 is still given.

Table 15: Found (ICP-MS) and calculated amounts of copper in the protein samples NB4exp-L5-Cu and NB4exp-L5-Cu-O2. The copper loading is determined from the ppm values.

ICP-MS calculated copper loading Cu [ppm] Cu [ppm] [%] NB4exp-L5-Cu 1.18 1.45 81

NB4exp-L5-Cu-O2 3.83 5.80 66

Copper Titration of NB4-L5 and NB4exp-L5

To obtain deeper insights into the copper coordination within the biohybrid conjugates, titration experiments with different amounts of copper nitrate are performed. Figure 62 shows the intensity change of the absorption at 300 nm with the successive addition of copper nitrate to

Biohybrid Conjugates

NB4-L5 and NB4exp-L5. For both proteins, an increase in the intensity is observed with higher amounts of copper ions. The expected behaviour would be a saturation curve until 1 equivalent per biohybrid conjugate is reached. Afterwards, more equivalents of copper should not result in a further increase of the 300 nm absorption. This behaviour is not observed. Up to an addition of 2 equivalents of copper to NB4-L5 the intensity increases and reaches an area of lower increase (inset in figure 62). This reveals that after the coordination of copper by the bis(pyrazolyl)methane ligand, one additional coordination site absorbs copper ions. From 3 equivalents on, precipitation leads to an intensity increase right up to the limit of the measurement setup (Abs ~3.5). Thus, NB4-L5 does not tolerate copper concentrations higher than 3 equivalents per biohybrid conjugate. In the case of NB4exp-L5, the curve is almost similar but shifted to higher copper concentrations. This means that the biohybrid conjugate with the expanded nitrobindin variant tolerates up to 4 or 5 equivalents of copper nitrate. The reason could be another copper coordination site in the doubled region of NB4exp in contrast to NB4. Moreover, NB4exp provides more space for additional copper coordination because of the larger -barrel.

Figure 62: UV/Vis spectroscopic monitoring of the absorption at 300 nm when adding different amounts of copper nitrate to NB4-L5 and NB4exp-L5. Biohybrid conjugate concentration: 50 mol L-1. Inset: region of low copper concentration.

Although UV/Vis and EPR spectroscopy, as well as ICP-MS and the titration experiment indicate the presence of copper inside the protein, the absolute structure could not be determined reliably. Single crystal X-ray diffraction is a sufficient method for structure elucidation of proteins but was not performed yet.

Biohybrid Conjugates

4.3.3. Reduction of Copper

Synthesis

For oxygen activating reactions, the copper centre in the biohybrid catalyst must be in oxidation state +I. Prior to the reduction of copper(II), oxygen is removed from the protein solution. This is performed by bubbling N2 through the solution. A second possibility is to transfer the protein solution into the glovebox, change the buffer to degassed buffer via a desalting column and combine the elution fractions, which contain protein. Where the second method is labour- intensive, the disadvantage of N2-bubbling is the protein precipitation at the bubbles. Afterwards, the reductant is added to the degassed protein solution (scheme 29). Four different reductants (sodium dithionite, dithiothreitol, sodium ascorbate, tris(2-carboxyethyl)phosphane hydrochloride) are analysed regarding their ability to reduce copper(II) to copper(I) in the conjugated protein.

Scheme 29: Reduction of copper(II) in the biohybrid conjugates NB4-/NB4exp-L5-Cu. L might be a H2O ligand.

Characterisation

The four different reductants sodium dithionite (DT), dithiothreitol (DTT), sodium ascorbate (Asc) and tris(2-carboxyethyl)phosphane hydrochloride (TCEP) are analysed towards their ability to reduce copper(II) in the protein. The reduction was monitored via UV/Vis spectroscopy (figure 63). The signal-to-noise ratio in these absorption spectra is rather low, due to the chosen measurement setup. As mentioned above, the copper-incorporated biohybrid conjugate shows an absorption at 300 nm (black line). The addition of DT (not depicted) or DTT (blue line) to the protein solution results in an intensive absorption of the reductant itself. Thus, the copper absorption is overlaid and successful reduction cannot be evaluated. With DT, the reductant is removed after the reduction by a desalting column and thus the interfering

Biohybrid Conjugates absorption as well. However, the concentration of the protein solution is diluted to a value that is too low for UV/Vis measurements. The reduction with TCEP (green line) shows a partial decrease of the 300 nm absorption, but the best reduction result is obtained with ascorbate (red line). A new absorption at 254 nm is ascribed to the oxidation product of ascorbate.[192]

Figure 63: UV/Vis spectra of NB4-L5-Cu (100 mol L-1) (black) and of the biohybrid conjugate solution after the addition of different reductants (coloured lines).

4.3.4. Oxygen Activation

Synthesis

Scheme 30: Oxygenation of the biohybrid conjugates NB4-/NB4exp-L5-Cu(I). L might be a H2O ligand.

Biohybrid Conjugates

The copper(I)-conjugated proteins NB4-L5-Cu and NB4exp-L5-Cu are oxygenated by bubbling

O2 through the solution or by the addition of O2-saturated buffer to the protein solution. The reaction is performed in MOPS or phosphate buffer at 4-10 °C (scheme 30).

Characterisation

The oxygenated biohybrid conjugate NB4-L5-Cu-O2 is analysed via UV/Vis spectroscopy by using stopped-flow techniques. When mixing equal volumes of the protein solution and oxygen-saturated MOPS buffer, the UV/Vis spectra shown in figure 64 are obtained within 500 ms. Although the first measurement is taken after 1 ms, no change in the absorption spectrum is observed. The spectra show a tiny shoulder at about 390 nm, which might indicate a mononuclear superoxide copper(II) species. Since this absorption is only a first evidence, it should be treated with caution. Reported end-on-superoxide copper species exhibit a high- intensity absorption around 380-450 nm (2000-8000 L mol-1 cm-1) and weaker absorptions around 580-600 nm (~1000 L mol-1 cm-1) and 740-760 nm (~1000 L mol-1 cm-1).[15,39] The problem of analysing the oxygenation of biohybrid conjugates is the high protein absorption. This is, however, found at 280 nm but influences the absorption spectrum up to higher wavelengths. Another problem is the solubility of oxygen in water. O2 is much less soluble in

-1 [193] -1[147] water than in organic solvents (H2O: 1.245 mmol L , DCM: 4.3 mmol L ) and thus, the limited amount of oxygen cannot be neglected.

Figure 64: UV/Vis spectroscopic monitoring of the oxygenation of NB4-L5-Cu(I) with oxygen-saturated MOPS buffer in a stopped-flow setup at 10 °C. Final protein concentration: 50 mol L-1. Resolution: 1 spectrum per ms.

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Biohybrid Conjugates

4.3.5. Reaction with Hydrogen Peroxide

Synthesis

The reaction of copper-incorporated biohybrid conjugates with hydrogen peroxide is performed with the copper(II) protein. With a stopped-flow measurement setup (10 °C), equal volumes of

NB4-L5-Cu(II) and an aqueous hydrogen peroxide solution are mixed (scheme 31). H2O2 is provided as 100 eq. per protein molecule.

Scheme 31: Reaction of NB4-L5-Cu(II) with H2O2. L might be a H2O ligand.

Characterisation

The reaction of NB4-L5-Cu(II) with H2O2 is monitored via UV/Vis spectroscopy. Even by using a stopped-flow setup with a time resolution of 1 ms intervals, no evidence of a hydroperoxide copper species is observed. The spectrum shows only the broad protein absorption at 280 nm.

4.3.6. Substrate Oxidation Reactions

Oxidation of 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)

The reaction of ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) with one-

·+ electron oxidants results in the radical cation ABTS . NB4exp-L5-Cu-O2 is used as oxidant in an ABTS assay with the apoenzyme NB4exp, the biohybrid conjugates NB4exp-L5, NB4exp-L5-Cu, NB4exp-L5-Cu-Asc and MOPS buffer as negative controls. The enzyme laccase is used as positive control due to its oxidation ability. The oxidation of ABTS is described in scheme 32.

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Biohybrid Conjugates

Scheme 32: One-electron oxidation of ABTS with NB4exp-L5-Cu-O2.

Figure 65: UV/Vis spectroscopic monitoring of the ABTS oxidation with NB4exp-L5-Cu-O2 (red) and MOPS buffer (negative control, black). The absorption at 420 nm is followed. NB4exp-L5-Cu-O2 was prepared the day before.

Figure 66: UV/Vis spectroscopic monitoring of the ABTS oxidation with NB4exp-L5-Cu-O2 (red) and the negative controls NB4exp (blue), NB4exp-L5 (green), NB4exp-L5-Cu (cyan), NB4exp-L5-Cu-Asc (orange) and MOPS buffer (black). Laccase (magenta) is used as positive control. The absorption at 420 nm is followed. NB4exp-L5-Cu-O2 was prepared at the same day.

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Biohybrid Conjugates

The radical cation ABTS·+ exhibits a characteristic absorption at 420 nm.[194,195] Thus, the oxidation ability of NB4exp-L5-Cu-O2 is analysed by following this absorption spectroscopically (figures 65 and 66). Regarding both experiments, only a small change in the absorption is observed. With laccase (magenta line) as positive control, the intensity increases rapidly in the first few minutes and subsequently reaches the detection limit. All other samples do not show oxidation ability. In figure 65, it seems that the biohybrid conjugate even inhibits the oxidation of ABTS in comparison with the pure buffer solution.

Reaction with 4-Aminoantipyrine (4-AAP)

The 4-AAP (4-aminoantipyrine) assay is based on the formation of a coupled product that exhibits a characteristic absorption around 500 nm.[194,196] To obtain the coupled product, the phenolic substrate must be unsubstituted in para-position to the hydroxyl group. Prior to the reaction with 4-AAP, the substrate is hydroxylated by the catalyst. When applying phenol as substrate, it would be hydroxylated to the catechol. Since both the phenol and the catechol possess an unsubstituted para-position, the assay would be positive for the substrate and the product. Thus, benzene and 4-methoxyphenol are used. The latter is indeed substituted at the para-position, but after hydroxylation, a new hydroxyl group with a free para-position is received. In scheme 33, the reaction of 4-AAP with the aromatic substrate is depicted.

Scheme 33: Reaction of 4-AAP with an aromatic substrate that is hydroxylated by NB4exp-L5-Cu-O2.

Figure 67 shows the absorption intensities of the coupled product at 509 nm. It is obvious that the reaction product with the phenol (positive control, green points) reveals high absorptions, especially with higher concentration (sample 13). The negative controls (red points), which contain no NB4exp-L5-Cu-O2, display low absorption intensities, except sample 7, which contains 10 eq. of 4-methoxyphenol. The samples containing protein solution (2-6) show also low intensities. The only exception is once again the sample with 10 eq. of 4-methoxyphenol (3). It seems that 4-methoxyphenol can react with 4-AAP regardless of whether it is

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Biohybrid Conjugates hydroxylated or not. These experiments could not confirm the hydroxylation ability of

NB4exp-L5-Cu-O2.

Figure 67: Absorption intensities of the coupled product at 509 nm after the reaction of 4-AAP with phenols. Sample numbers: 1: MOPS buffer, 2: NB4exp-L5-Cu-Asc-O2 + 1 eq. 4-OMe-phenol, 3: NB4exp-L5-Cu-Asc-O2 + 10 eq. 4-OMe-phenol, 4: NB4exp-L5-Cu-Asc-O2 + 1 eq. benzene, 5: NB4exp-L5-Cu-Asc-O2 + 10 eq. benzene, 6: NB4exp-L5-Cu-Asc-O2 + 100 eq. benzene, 7: 10 eq. 4-OMe-phenol, 8: 100 eq. benzene, 9: NB4exp + DMSO, 10: NB4exp-L5-Cu + 100 eq. benzene, 11: 1 eq. phenol, 12: 10 eq. phenol, 13: 100 eq. phenol.

Oxidation of 2-Methoxyphenol (Guaiacol)

The reaction of the substrate guaiacol (2-methoxyphenol) with an one-electron oxidant results in a radical intermediate that dimerises immediately (scheme 34).[197] The product 3,3’-dimethoxy-4,4’-biphenoquinone exhibits a characteristic absorption at 470 nm, which is monitored via UV/Vis spectroscopy.[198–200]

Scheme 34: Oxidation of guaiacol with NB4exp-L5-Cu-O2 and subsequent dimerisation of the radical.

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Biohybrid Conjugates

The results of an UV/Vis measurement that was performed when reacting NB4exp-L5-Cu-O2 with guaiacol are presented in figure 68. Here, the absorption at 470 nm is monitored. It is obvious that the biohybrid catalyst (red line) is not suitable for the oxidation of guaiacol, as the characteristic absorption of the dimeric product barely increases. Both protein negative controls show almost the same behaviour.

Figure 68: UV/Vis spectroscopic monitoring of the oxidation product of guaiacol (470 nm) when oxidising with NB4exp-L5-Cu-O2 (red). Negative controls: phosphate buffer (black), NB4exp (blue) and NB4exp-L5-Cu (cyan).

Reaction with Tetramethylpiperidin-1-ol (TEMPO-H)

Superoxide copper species are reported to abstract a hydrogen atom from TEMPO-H (tetramethylpiperidin-1-ol). The resulting persistent radical TEMPO is then analysed via EPR

[201] spectroscopy. Thus, TEMPO-H is added to a solution of NB4exp-L5-Cu-O2 (scheme 35) to analyse the hydrogen atom abstracting ability of the biohybrid conjugate.

Scheme 35: Reaction of TEMPO-H with NB4exp-L5-Cu-O2.

Immediately after the addition of TEMPO-H to the protein solution, EPR measurements are performed. The spectrum shows an EPR signal characteristic for the TEMPO radical. A similar signal is observed, when measuring a solution of only TEMPO-H in water or in phosphate buffer. Thus, it seems that TEMPO-H contains already detectable amounts of TEMPO. Another possibility would be the instability of TEMPO-H towards r.t. or air atmosphere. Due to the

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Biohybrid Conjugates impurity of TEMPO in the TEMPO-H solution, no statement can be made concerning TEMPO-H consumption.

Sulfoxidation of Thioanisole

The reaction of thioanisole with an oxidant leads to the formation of methyl phenyl

[198,199] sulfoxide. Thus, a solution of NB4exp-L5-Cu-O2 is treated with thioanisole (scheme 36) to study the oxygenation ability of the biohybrid catalyst. After a reaction time of 30 min, organic compounds are extracted from the protein solution and analysed via GC.

Scheme 36: Sulfoxidation of thioanisole with NB4exp-L5-Cu-O2.

The gas chromatogram displays a large signal of the substrate thioanisole and a smaller signal of the internal standard benzyl alcohol. A very small signal of methyl phenyl sulfoxide is found as well. This signal is calculated to a product amount of less than 1 %. When measuring a sample of pure thioanisole, this amount of sulfoxide is also found. Thus, no statement can be made concerning the oxygenation ability of NB4exp-L5-Cu-O2.

Possible Side Reactions

Due to the high reactivity of superoxide species, intramolecular side reactions are not negligible. Such reactions include the abstraction of H-atoms of nearby amino acid side chains like glycine.[202] Hereby, targeted substrate reactions might be rendered impossible.

4.4. Conclusion

In this chapter, the optimised syntheses of two new maleimide-linked bis(pyrazolyl)methane ligands capable of anchoring into a protein are described. HC(3-tBuPz)2(4-Mal1Py) (L5) exhibits a C4O-linker between the N-donor ligand and the maleimide moiety, whereas in

HC(3-tBuPz)2(4-Mal2Py) (L6) the linker consists of one carbon unit. As an intermediate, the alcohol-bearing ligand HC(3-tBuPz)2(4-CH2OHPy) (L4) was synthesised.

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Biohybrid Conjugates

Both maleimide ligands L5 and L6 were applied in oxygen activation reactions. The side-on- peroxide dicopper(II) species P5 and P6 are formed in dichloromethane at -78 °C within 30 min and display characteristic absorption bands (P5: 348 and 555 nm, P6: 350 and 550 nm). Thus, it could be demonstrated that the maleimide modification does not interfere with the formation of the peroxide dicopper(II) complex. Hydroxylation reactions were performed with the substrates 8-hydroxyquinoline and 4-methoxyphenol. With 8-hydroxyquinoline, turnover numbers comparable to other bis(pyrazolyl)methane systems were achieved (P5: 14, P6: 9). In the catalytic oxygenation of 4-methoxyphenol, the structure of the resulting product is not fully clarified yet. Besides the 4-methoxy-ortho-quinone, the formation of a C-O coupled product is possible as well. For the quinone, TONs of 7 (P5) or 3 (P6) were calculated. With consideration of the coupled product, the TONs are 23 (P5) or 10 (P6). Assuming a not fully formed Cu2O2 species P5, higher turnover numbers would be calculated for both catalytic reactions. Peroxide dicopper(II) species of L5 and L6 display remarkable stabilities and activities comparable to the parental system P1.

The maleimide-bearing ligands L5 and L6 were conjugated into the nitrobindin variants NB4 and NB4exp. The conjugation was proven via ESI and MALDI-TOF mass spectrometry. Addition of copper nitrate to the biohybrid conjugates results in copper incorporation. In the UV/Vis spectra of NB4-L5-Cu and NB4exp-L5-Cu an absorption at 300 nm is found and ascribed to the bis(pyrazolyl)methane copper complex in the protein. EPR spectroscopic and ICP-MS analyses indicated the presence of copper in the protein solution. Afterwards, copper(II) should be reduced to copper(I). Hence, different reductants (sodium dithionite, dithiothreitol, sodium ascorbate and tris(2-carboxyethyl)phosphane hydrochloride) were evaluated and sodium ascorbate provided the best results. Subsequently, the copper(I) protein was oxygenated and UV/Vis spectra revealed a small feature, which might be ascribed to a superoxide species. Finally, substrate reactions with ABTS, 4-AAP, guaiacol, TEMPO-H and thioanisole were performed and could not confirm the oxidation ability of the biohybrid conjugate NB4exp-L5-Cu-O2, until now. Further studies on oxygen activating and substrate converting reactions are required.

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Conclusion and Outlook

5. Conclusion and Outlook

5.1. Conclusion

This doctoral thesis describes the application of bis(pyrazolyl)methane ligands in the field of enzyme modelling. On one hand, bis(pyrazolyl)methane copper complexes are structural and functional tyrosinase models, enabling a dinuclear catalytic centre (chapter 3). On the other hand, biohybrid conjugates with the protein nitrobindin are the first step towards mimicking mononuclear copper enzymes like particulate methane monooxygenase with this ligand class (chapter 4).

Inspired by the excellent tyrosinase models with bis(pyrazolyl)(imidazolyl)- or bis(pyrazolyl)- (pyridinyl)methanes (L2 or L3),[42,43] a new model system was developed. The new bis(pyrazolyl)methane HC(3-tBuPz)2(4-CO2MePy) (L1) possesses an ester substituent at the pyridinyl moiety in para-position to the N-donor atom. L1 was synthesised according to the general protocol for bis(pyrazolyl)methanes.[128] Due to the high isomerisation tendency of the 3-tert-butylpyrazolyl moiety in L1, the reported purification method was not applicable and an intermediate re-isomerisation step was required. Furthermore, the yield was increased from 59 to 87 % by exchanging distillation to column chromatography. The combination of L1 with copper(I) and copper(II) salts resulted in single crystals, suitable for X-ray diffraction. The molecular structures of the copper(II) chloride complex C1 and the copper(I) bromide complex C2 display that the substituted pyridinyl and both pyrazolyl moieties coordinate the copper ion. In both complexes, halogenido ligands complete the coordination sphere to result in a penta- or tetracoordinated complex, respectively. The square-pyramidal coordination geometry in the copper(II) chloride complex C1 is highly distorted. C2 crystallises with two almost identical molecules in the asymmetric unit. Thus, the degree of distortion in the tetrahedral geometry is almost the same. Regarding bond lengths in C1 and C2, no statement about the donor strength of the ester-substituted pyridinyl moiety of L1 could be made. Thus, the influence of the ester substitution is not reflected in solid state structures of copper(I) or copper(II) complexes. Besides the complexes with L1, molecular structures of copper(I) complexes with the imidazolyl ligand L2 (HC(3-tBuPz)2(1-MeIm)) were determined via X-ray diffraction analysis.

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Conclusion and Outlook

Both the copper(I) chloride complex C3 and the copper(I) iodide complex C4 show the tridenticity of the ligand L2. C3 and C4 display a high symmetry within the complex molecule, which is also reported for the copper(I) chloride complex CC3 with a quinolinyl ligand.[54] The imidazolyl moiety, the copper centre and the halogenido ligand are located on a mirror plane through the molecule.

The application of L1 in oxygen activation chemistry displayed the formation of the -2:2-peroxide dicopper(II) species P1. With a half-life of 44 min at r.t., P1 is one of the most stable tyrosinase model systems. The complete formation of P1 is obtained at -78 °C within 40 min. P1 could also be formed at r.t. (3.5 min), however the yield was only 70 %. Interesting but not yet fully understood is the fact that the formation of P1 is exclusively possible in dichloromethane. To analyse the reaction of the precursor complex [CuL]SbF6 with oxygen, formation kinetics were determined for P1, as well as the peroxide dicopper(II) species P2

(imidazolyl system) and P3 (unsubstituted pyridinyl system). With kobs values of 0.1-3.7 · 10-2 s-1, the pseudo-first-order formations of all P species are in the same range, however P2 is the slowest. The ester substituent as electron-withdrawing functional group influences the pyridinyl moiety by weakening the N-donor. Consequently, the formation of P3 is faster than that of P1. Characterisation of P1 via UV/Vis spectroscopy displays the two absorptions of side-on peroxide dicopper(II) species at 350 and 550 nm in an intensity ratio of

13:1. With UHR CSI-MS measurements, the monocationic complex ([Cu2{HC(3-tBuPz)2-

2 2 + (4-CO2MePy)}2(- : -O2)]SbF6) was found. Low-temperature resonance Raman spectra

-1 18 show a vibration feature at 757 cm , which shifts when oxygenating with O2. This is characteristic for the O-O vibration of -2:2-peroxide dicopper(II) complexes. The side-on peroxide dicopper(II) species could be proven via XAS analysis revealing characteristic atom distances and an oxidation state of +II for copper.

P1 was shown to serve as excellent tyrosinase model system, as it hydroxylates phenolic substrates, even in catalytic reactions. The hydroxylation of sodium 4-X-phenolates with

X = OMe, Me or CO2Me occurred selectively in ortho-position. The velocity of these conversions is comparable to reported systems.[62] The reactions with different substrate concentrations revealed Michaelis-Menten-like substrate binding kinetics. It was shown that electron-richer phenolates are hydroxylated faster than electron-poor substrates. Thus, the mechanism of this reaction is an electrophilic aromatic substitution with a Hammett’s constant  of -1.2. This is the same reaction mechanism as reported for the enzyme tyrosinase itself.[61] The catalytic conversion of 8-hydroxyquinoline yielded the quinone product, monitored via the characteristic absorption of quinoline-7,8-dione at 413 nm. The use of 50 eq. of substrate led

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Conclusion and Outlook to the calculated turnover number of 20. Due to the measurement setup, a baseline shift occurred upon increasing the temperature. Thus, the TON was re-calculated to 15 per Cu2O2 species. The catalysis was also performed under self-assembly conditions, and even at r.t. quinone product was observed, however with smaller TONs. The maximal amount of quinoline-7,8-dione was reached after 7.5 min (low temperature) or even after less than 10 seconds (r.t.). Thus, this tyrosinase model with the ester-substituted bis(pyrazolyl)- (pyridinyl)methane ligand L1, is the fastest system in the catalytic conversion of phenolic substrates to date. Moreover, P1 is the most stable catalytically active tyrosinase model system.

Figure 69: Overview of the synthesised and characterised compounds of chapter 3. The bis(pyrazolyl)methane ligand L1 and the copper bis(pyrazolyl)methane complexes C1-C4.

With the aim of stabilising artificial superoxide species within a protein scaffold, two new bis(pyrazolyl)methane ligands were designed. Both ligands possess a maleimide moiety, capable of the covalent attachment to a cysteine residue of the protein. In

HC(3-tBuPz)2(4-Mal1Py) (L5), the maleimide moiety is connected via a C4O-linker to the

N-donor ligand. HC(3-tBuPz)2(4-Mal2Py) (L6) exhibits a shorter linker, which consists of a single methylene group. As an intermediate, the alcohol-bearing ligand

HC(3-tBuPz)2(4-CH2OHPy) (L4) was synthesised.

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Conclusion and Outlook

The oxygen activation ability of copper complexes was analysed for both maleimide ligands L5 and L6. Although these ligands possess a maleimide modification, the formation of the side- on peroxide dicopper(II) species P5 and P6 was still observed. Both peroxide species were formed in dichloromethane at -78 °C within 30 min. P5 shows characteristic absorptions at 348 and 555 nm, whereas with P6, these absorptions are at 350 and 550 nm. P5 and P6 were also capable of transferring oxygen to phenolic substrates like 8-hydroxyquinoline and 4-methoxyphenol. The catalysis of 8-hydroxyquinoline displayed comparable turnover numbers to other bis(pyrazolyl)methane systems (P5: 14, P6: 9). The catalytic conversion of 4-methoxyphenol, however, is more difficult to analyse. The highly reactive 4-methoxy-ortho-benzoquinone can subsequently form a C-O coupled product, which reveals an absorption at the same wavelength as the quinone. Turnover numbers were hence calculated for both possibilities. Regarding the quinone, TONs of 7 (P5) or 3 (P6) were calculated; with the coupled product, the TONs are 23 (P5) or 10 (P6). P5 displayed less intense absorption bands in the UV/Vis spectrum, which might indicate a not fully formed peroxide dicopper(II) species. Under this assumption, the turnover numbers for both catalytic conversions with P5 are higher.

For analysing the ability to stabilise a superoxide species, the maleimide-bearing ligands L5 and L6 were conjugated into the nitrobindin variants NB4 and NB4exp. ESI and MALDI-TOF mass spectrometry revealed the successful conjugation. The biohybrid conjugates were treated with copper nitrate to incorporate copper ions. Complexation of the copper ions by the bis(pyrazolyl)methane ligand L5 is indicated by the rise of an absorption at 300 nm upon copper addition. Further analyses by EPR spectroscopy and ICP-MS measurements proved the presence of copper in the protein solution of NB4-L5-Cu and NB4exp-L5-Cu. For the reduction of incorporated copper(II) to copper(I), a series of reductants (sodium dithionite, dithiothreitol, sodium ascorbate and tris(2-carboxyethyl)phosphane hydrochloride) was tested. Due to the decrease of the absorption at 300 nm, sodium ascorbate was found to be the best reductant for reducing copper within the protein. The copper(I) biohybrid conjugate was subsequently oxygenated and analysed via UV/Vis spectroscopy. The spectra revealed a small feature, which might be ascribed to a superoxide absorption, but further studies are still required. Moreover, substrate reactions with ABTS (2,2’-azino-bis(3-ethylbenzo-thiazoline- 6-sulphonic acid)), 4-AAP (4-aminoantipyrine), guaiacol (2-methoxyphenol), TEMPO-H (tetramethylpiperidin-1-ol) and thioanisole were performed to analyse the oxygenation, oxidation or H-atom transfer ability of NB4exp-L5-Cu-O2. To date, no product could be detected with different analysation methods.

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Conclusion and Outlook

The conjugation of L5 into the nitrobindin variants NB4 and NB4exp is the first step towards biohybrid catalysts with bis(pyrazolyl)methane ligands. This opens the possibility of the stabilisation of a superoxide species by means of a proteinogenic second coordination sphere.

Figure 70: Overview of the synthesised and characterised compounds of chapter 4. The bis(pyrazolyl)methane ligands L4-L6 and the biohybrid conjugate NB4/NB4exp-L5-Cu.

5.2. Outlook

The bis(pyrazolyl)methane ligand HC(3-tBuPz)2(4-CO2MePy) (L1) shows high catalytic activity with phenolic substrates. However, it is still difficult to analyse catalytic products. On one hand, attempts should be made to find other methods than UV/Vis spectroscopy to characterise the quinone product. On the other hand, the reason why the catalytic reaction stops after several turnovers is still not fully clarified. A recent report describes the formation of a fluorido-bridged species as an irreversible degradation product.[57] Another possible degradation pathway is the reaction of the catalytically active species with the upcoming water as catalytic product. Thus, analysation and characterisation of other irreversible degradation products would be helpful for a better understanding of the catalytic reaction. Furthermore, hydroxylation reactions of phenolic substrates should be performed at lower temperatures than -78 °C. In recent studies, an intermediate bis(-oxide) dicopper(III) species was observed upon addition of the phenolate

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Conclusion and Outlook at -145 °C.[64] This would be a further step for the clarification of the real catalytically active species.

In the field of modelling mononuclear copper enzymes with biohybrid catalysts, first steps were made with maleimide-linked bis(pyrazolyl)methane ligands and different variants of the protein nitrobindin. The analysis of the biohybrid conjugates via UV/Vis and EPR spectroscopy, as well as ICP-MS indicates the successful incorporation of copper ions into the conjugated protein. However, for the absolute verification of the coordination by the bis(pyrazolyl)methane ligand, X-ray crystallographic data are needed. Moreover, theoretical calculations are required to obtain insights into the orientation of the bis(pyrazolyl)methane ligand and the oxygenated copper complex. Theoretical calculations would also answer the question whether the protein cavity is too large or too small for these types of reactions. Furthermore, studies on the characterisation of the oxygenated biohybrid conjugates should be continued and further studies on the conversion of substrates are required.

In the past, stabilisation of mononuclear superoxide species remained rather challenging, since copper oxygen species tend to form dinuclear complexes. With the help of a controlled proteinogenic environment, this might become possible. Thus, the way to the conversion of sophisticated substrates by using biohybrid catalysts is opened. This establishes a new and challenging project in the research field of enzyme modelling.

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Experimental Part

6. Experimental Part

6.1. General Procedures

If not stated otherwise, all ligand and complex syntheses were performed in an inert atmosphere (nitrogen) with Schlenk technique. Solutions and solvents for oxygen activation and transfer reactions were handled with gas-tight Hamilton syringes. Solvents were dried

[203] according to literature, distilled and degassed by three cycles of vacuum/N2 prior to usage. All protein reactions were conducted in the laboratory of Prof. Dr. Takashi Hayashi (Osaka University, Japan) or Prof. Dr. Ulrich Schwaneberg (RWTH Aachen University). For all biochemical reactions, ultrapure ddH2O (Milli-Q) was used.

6.2. Chemicals

If not mentioned otherwise, all chemicals were used as purchased with no further purification.

Acetone-d6 (99.96 %) Euriso-top Acetophenone (p.a.) Merck 4-Aminoantipyrine (≥98.0 %) Fluka Ammonium acetate (min. 97.0 %) Wako 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (BioChemica) AppliChem Benzene (puriss, absolute) Fluka Benzyl alcohol (99 %) Grüssing

Chloroform-d1 (99.96 %) Euriso-top Citric acid monohydrate (99.5 %) Roth Cobalt(II) chloride (p.a.) Sigma-Aldrich Copper(I) bromide (99 %) Fluka Copper(II) chloride (>97 %) Fluka Copper(I) iodide (98 %) Grüssing Copper(II) nitrate trihydrate (99.0 %) Wako Copper(II) nitrate hemipentahydrate (p.a.) Riedel-de-Haёn

Dichloromethane-d2 (99.96 %) Euriso-top Diethyl azodicarboxylate (>97 %) Fluka Dimethyl sulfoxide (for biochemical reactions, min. 99.0 %) Wako Disodium phosphate (p.a., mind. 99.5 %) AppliChem (+-)-threo-Dithiothreitol (min. 97.0 %) Wako

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Experimental Part

Formic acid (98.0 %) ChameleonReagent Hydrochloric acid (≥37 %, p.a.) Sigma-Aldrich

Hydrogen peroxide (30.0-35.5 % in H2O) Wako 8-Hydroxyquinoline (>99 %) Merck Lithium aluminium hydride (≥97.0 %) Sigma-Aldrich Maleimide (>98 %) Merck 2-Methoxyphenol (Guaiacol) (oxidation indicator, ≥98 %) Sigma 4-Methoxyphenol (≥98.0 %) Sigma-Aldrich Methyl phenyl sulfoxide (98+ %) Alfa Aesar 3-(N-Morpholino)propanesulfonic acid (99.0 %) DOJINDO Neopentyl alcohol (>99 %) ABCR Oxygen (2.5, technical grade) Westfalen Potassium persulfate (≥98.0 %) Fluka Silica gel (Geduran Si 60, 40-63 m) Merck Silver hexafluoridoantimonate (98 %) Sigma-Aldrich Sinapic acid (>99.0 %) Sigma-Aldrich Sodium L-ascorbate (>98.0 %) TCI Sodium chloride (protease and nuclease tested) nacalai tesque Sodium diphosphate (≥99.0 %) Sigma-Aldrich Sodium dithionite (>85 %) TCI Sodium hydride (60 %, oily dispersion) Aldrich Tetramethylpiperidin-1-ol (95 %) ABCR Thioanisole (99 %) Alfa Aesar Thionyl chloride (>99 %) Sigma-Aldrich p-Toluenesulfonic acid monohydrate (≥98 %) TCI Triethylamine (>99 %) Sigma-Aldrich Triphenylphosphane (99 %) ABCR Tris(2-carboxyethyl)phosphane hydrochloride (90+ %) Wako Urea (molecular biology grade) AppliChem

Solvents (analytical grade) were purchased from Fisher Chemicals or VWR Chemicals, standard chemicals from Grüssing.

Sodium hydride was washed with n-pentane for several times, dried in HV and stored under inert atmosphere. Cobalt(II) chloride was absolutised with SOCl2 and stored in a glovebox.

The syntheses of the following chemicals and starting materials were performed according to

[204] [174] literature: copper(I) chloride, sodium 4-X-phenolates (X = OMe, Me, CO2Me), 3-tert- butylpyrazole,[205] Dess-Martin periodinane,[140] methyl 2-formylisonicotinate,[139] 3-maleimidopropionic acid,[182] 3-maleimidopropionyl chloride.[183]

Supplements to the synthesis of 3-maleimidopropionyl chloride: in contrast to the literature procedure, dichloromethane and excess of thionyl chloride were removed via distillation (90 mbar, 50 °C). Furthermore, residual thionyl chloride was removed by the addition of

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Experimental Part dichloromethane (30 mL) and again distillation. 3-Maleimidopropionyl chloride was additionally purified via distillation (10-1 mbar, 120 °C oil bath) with a not-cooled u-bend. For this distillation, the crude product was heated under HV and in a receiver flask, which was cooled with liquid nitrogen, condensation of the purified product (pale yellow solid) was observed.

[43] [128] The ligands L2 (HC(3-tBuPz)2(1-MeIm)) and L3 (HC(3-tBuPz)2(Py)) were synthesised following literature procedures.

The proteins NB4 and NB4exp were expressed and purified by students either in the laboratory of Prof. Dr. Takashi Hayashi (Osaka University, Japan) or Prof. Dr. Ulrich Schwaneberg (RWTH Aachen University) and provided to use.[103]

6.3. Methods

NMR spectroscopy 1H- and 13C-NMR spectra were measured on a Bruker Avance II 400 or Bruker Avance III HD 400 nuclear resonance spectrometer. The signals were calibrated to the residual signals of the deuterated solvent. 2D-NMR spectra (COSY, HSQC, HMBC) were used for the correct assignment of the signals.

IR spectroscopy IR spectroscopic measurements were performed either on a ThermoFisher AvatarTM 360 spectrometer with KBr pellets (resolution: 2 cm-1) or a Shimadzu IRTracer 100 with CsI beamsplitter in combination with a Specac Quest ATR unit (resolution: 2 cm-1).

Resonance Raman spectroscopy Resonance Raman measurements were carried out at the Center for Free-Electron Laser Science (CFEL, group of Prof. Dr. Rübhausen) (Hamburg, Germany). A Tsunami Ti:Sapphire laser system model 3960C-15HP (Spectra Physics Laser Inc., California) in conjunction with a flexible harmonic generation unit, model GWU2 23-PS (GWU-Lasertechnik Vetriebsges.mbH, Erftstadt) providing the frequency doubled wavelength of 360 nm was applied. To determine the pulse width of the laser, a small part of the Tsunami fundamental was mirrored out using a glass plate and the reflex then coupled into an autocorrelator (AC) (APE GmbH, Berlin, Germany). The laser beam was widened with a spatial filter and then focused on the cuvette inside the cryostat. The focus spot size was around 20 m in diameter. With a micrometer screw a focal depth of around 50 m inside the cuvette was adjusted. Raman scattered light

117

Experimental Part was then captured with the entrance optics of the UT-3 triple monochromator spectrometer.[206,207] The cryostat was a slightly modified version of the one reported in literature[207] with a standard cuvette with septum instead of PEEK tubes for oxygenation. Equipped with a different Peltier element (QuickCool QC-127-1.4-6.0MS) and a new copper block which encloses three sides of the Suprasil cuvette with a sample volume of 1.4 mL (Hellma Analytics, Müllheim, Germany) temperatures below -90 °C inside the solution were reached. The used laser power in front of the entrance optics was ~3 mW. The pulse width was 1 ps. The experiments were conducted in a clean room with constant temperature (20.0 °C ± 0.5 °C) and humidity (45 % ± 3 %).

All complex preparations and the filling of the cuvette were performed in a MBraun glovebox.

Mass spectrometry High-resolution ESI mass spectra were recorded with a ThermoFisher Scientific LTQ-Orbitrap XL spectrometer (IOC, Aachen). The source voltage was 4.49 kV and the capillary temperature was 299.54 °C. The tube lens voltage was between 110 and 130 V.

EI mass spectra were measured on a ThermoFisher Finnigan MAT95 with a source voltage of 5 kV and an electron energy of 70 eV.

FAB mass spectra were obtained with a ThermoFisher Finnigan MAT95 or a Joel MStation sector field spectrometer (Munich, Germany). Ionisation was achieved with 8 kV xenon atoms on a copper target and 2-nitrobenzyl alcohol or glycerol as matrix.

CSI-MS measurements were performed on a UHR-TOF Bruker Daltonik maXis plus, an ESI- quadrupole-time-of-flight (qTOF) mass spectrometer (group of Prof. Dr. Ivanović-Burmazović, Erlangen, Germany) capable of a resolution of at least 60000 full-width at half-maximum (FWHM), which was coupled to a Bruker Daltonik Cryospray unit. Detection was in positive- ion mode and the source voltage was 4.5 kV. The flow rates were 250 L h-1. The drying gas

(N2), to aid solvent removal, was held at 198 K and the spray gas was held at 193 K. The machine was calibrated prior to every experiment by direct infusion of the Agilent ESI-TOF low concentration tuning mixture, which provided a m/z range of singly charged peaks up to 2700 Da in both ion modes.

MALDI-TOF mass spectra were measured with a Bruker Autoflex III spectrometer (Osaka, Japan) where sinapic acid served as matrix. Prior to the measurements the protein samples were treated with concentrated sinapic acid in a mixture of water : acetonitrile = 50:50, containing 0.1 % trifluoroacetic acid to precipitate.

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Experimental Part

High-resolution ESI mass spectra of conjugated proteins were recorded on a Bruker micrOTOF II-HE spectrometer (Osaka, Japan). For LC-MS measurements a Hitachi device with an UV detector L-2400 and a pump L-2100 were used. The eluent was 20:80 = acetonitrile : 0.1 % formic acid in water. These measurements required an acetate buffer. For this, MOPS buffer

-1 was replaced by ammonium acetate buffer (50 mmol L NH4OAc, pH 5) via centrifugation with an Amicon (addition of new buffer for three times).

ICP-MS analysis ICP-MS measurements of protein solutions were conducted on an Agilent 8800 ICP Triple Quad (ICP-QQQ) device (ITMC, Aachen). The aqueous protein solution was diluted with aqueous HCl prior to the measurement. The limit of quantitation is on the ppb level.

UV/Vis spectroscopy UV/Vis spectroscopic measurements of peroxide complexes or substrate conversions were performed on an Agilent Technologies Cary 60 UV/Vis spectrophotometer. The spectra were obtained with a quartz glass immersion probe (Hellma, 1 mm) connected via a Cary 50 fibre optic coupler. The reactions were performed in a commercial Schlenk measurement cell.

UV/Vis spectroscopic determinations of protein concentrations were performed with a Shimadzu Biotech BioSpec-nano Micro-volume UV/Vis spectrophotometer (Osaka, Japan) or a ThermoFisher Scientific NanoDropTM 1000 UV/Vis spectrophotometer (ABBt, Aachen).

Copper coordination in conjugated proteins was analysed with a Jasco V-670 spectrophotometer (Osaka, Japan) in a 10 mm quartz glass cuvette (70 L, 100 mol L-1).

All air-sensitive protein solutions were measured in a glovebox (humid atmosphere, ~3 ppm

H2, 0 ppm O2) with an Ocean Optics (UV/Vis Sampling System) Flame Miniature spectrometer (Osaka, Japan) with a 10 mm quartz glass cuvette (70 L, 100 mol L-1).

Products of biochemical oxidation reactions were analysed in a 96-well microtiter plate (MTP) and the absorption was monitored with a TECAN Sunrise Plate Reader (ABBt, Aachen).

Stopped-Flow UV/Vis spectroscopy Low-temperature stopped-flow UV/Vis measurements were performed with a HI-TECH Scientific SF-61SX2 device with a charge-coupled device (CCD) photodiode array detector. The optical path length of the quartz glass cuvette was 10 mm and the mixing time amounts to 2 ms.

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Experimental Part

Protein solutions were measured on a RSP-1000 Unisoku stopped-flow system (Osaka, Japan) with a photodiode array detector. The optical path length of the quartz glass cuvette was 10 mm.

Both systems used a xenon arc lamp as light source.

EPR spectroscopy EPR spectra of conjugated proteins were recorded with a Bruker EMXmicro at 100.0 K (Osaka, Japan). The centre field was 3000.000 G and the sweep width 2000.000 G. The microwave frequency was 9.661-9.668 GHz and the power 0.632 mW. The modulation frequency was 100.00 kHz and the amplitude 10.00 G. Protein solutions were in the range of 250-350 mol L-1. Products of biochemical oxidation reactions were analysed with a Magnettech MiniScope MS400.

Gas chromatography GC measurements were performed on a Shimadzu GC2010plus device with a FS-Supreme 5ms capillary column (5 % phenyl methylpolysiloxane, length: 30 m, diameter: 0.32 mm, film thickness: 0.25 m) and a flame ionisation detector.

X-ray absorption spectroscopy Measurements at the Cu K-edge were carried out at beamline BM23 of the European Synchrotron Radiation Facility ESRF (Grenoble, France) by the group of Prof. Dr. Bauer in fluorescence mode by making use of a hyperpure solid-state Ge detector. The sample was measured as a frozen solution at liquid nitrogen temperature (77 K) by using dichloromethane as matrix. Due to the low concentration of 0.01 mol L-1, several scans were conducted and averaged to increase the signal-to-noise ratio. Data reduction followed standard procedures given in the literature.[164] Due to the high residual noise, Fourier filtering was applied in the range r = 3-11.5 Å.

X-ray diffraction analysis The single crystal X-ray diffraction data were collected on a Bruker D8 goniometer with APEX CCD detector. An Incoatec microsource with Mo-K radiation ( = 0.71073 Å) was used and temperature control was achieved with an Oxford Cryostream 700. Data were collected at 100 or 110 K in -scan mode. Data were integrated with SAINT[208] and corrected for absorption by multiscan methods with SADABS.[208] The structures were solved by direct and conventional Fourier methods and all non-hydrogen atoms were refined anisotropically with full-matrix least- squares based on F2 (XPREP,[209] SHELXS[210] and ShelXle[211]). Hydrogen atoms were derived from difference Fourier maps and placed at idealised positions, riding on their parent C-atoms,

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Experimental Part

with isotropic displacement parameters Uiso(H) = 1.2Ueq(C) and 1.5Ueq(Cmethyl). All methyl H-atoms were allowed to rotate but not to tip.

The crystallographic data for the complexes C1-C4 are listed in tables 19 and 20.

6.4. Ligand Syntheses

6.4.1. 2-(4-Methoxycarbonylpyridinyl)bis(3-tert-butylpyrazolyl)methane (L1)

Figure 71: HC(3-tBuPz)2(4-CO2MePy) (L1).

The synthesis was performed according to a slightly modified literature procedure.[128]

NaH (1.46 g, 60.8 mmol, 2.2 eq.) was stirred in freshly distilled, dry THF (100 mL) at 0 °C. 3-tert-Butylpyrazole (7.20 g, 58.0 mmol, 2.1 eq.) was added in small portions under vigorous stirring and the solution was stirred until gas evolution was no longer observed. After dropwise addition of SOCl2 (2.00 mL, 27.6 mmol, 1.0 eq.), the yellow solution was stirred for 40 min at 0 °C and another 45 min at r.t. By adding methyl 2-formylisonicotinate (5.00 g, 30.3 mmol,

1.1 eq.) and catalytic amounts of CoCl2 a colour change from yellow to dark violet occurred. Subsequently, the mixture was heated to reflux for 43 h during which the colour turned to dark brown. After cooling to r.t., water (85 mL) and diethyl ether (85 mL) were added and the reaction mixture was stirred for 1 h at r.t. Afterwards, the product was extracted with diethyl ether (4 × 70 mL) and washed with water (2 × 70 mL) and with saturated aqueous NaCl

(2 × 70 mL). The combined organic layers were dried over MgSO4 and the solvent was removed in vacuo. The crude product was purified by column chromatography (silica gel, dichloromethane : ethyl acetate = 2:1 → ethyl acetate) and the resulting orange, wax-like oil was dissolved in warm iso-hexane. Cooling to -32 °C yielded the product and its isomer (see below) as an amorphous solid (combined yield: 9.47 g, 23.9 mmol, 87 %).

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Experimental Part

An alternative, but less efficient purification way is already published in literature.[56] Here, the crude product was distilled (7 × 10-1 mbar, second fraction at 180 °C) and the viscous, red oil was dissolved in warm iso-hexane. By cooling the solution to -32 °C, the product and its isomer (see below) were obtained as a powder (combined yield: 59 %).

As the pyrazole unit of this ligand tends to isomerise and hence formed the ligand

HC(3-tBuPz)(5-tBuPz)(4-CO2MePy), a further isomerisation step was required. This isomerisation was performed according to literature.[141,142] The ligand

HC(3-tBuPz)(5-tBuPz)(4-CO2MePy) (8.20 g, 20.7 mmol) was dissolved in toluene (75 mL) under air atmosphere. p-Toluenesulfonic acid monohydrate (0.82 g, 10 w%) was added to the yellow solution at r.t. under stirring. The reaction mixture was heated to reflux for 19 h during which the solution became dark brown. After cooling to r.t., the acidic solution was neutralised by the addition of a half concentrated aqueous solution of Na2CO3 (80 mL). Subsequently, the organic layer was separated, washed with water (3 × 50 mL), dried over MgSO4 and the solvent was removed in vacuo. The crude product was purified by column chromatography (silica gel, dichloromethane : ethyl acetate = 2:1 → ethyl acetate). The resulting orange, wax- like oil was dissolved in warm iso-hexane and cooling to -32 °C yielded the pure product (without isomer) as an amorphous solid (4.90 g, 12.4 mmol, 60 %).

Appearance: Pale yellow amorphous solid.

Rf (silica gel, dichloromethane : ethyl acetate = 2:1): 0.8

1 H-NMR (400 MHz, CDCl3, 25 °C):  = 1.29 (s, 18H, CH3, 6), 3.91 (s, 3H, CH3, 13), 6.18 (d,

3 3 4 JH,H = 2.4 Hz, 2H, CH, 3), 7.43 (d, JH,H = 2.4 Hz, 2H, CH, 2), 7.45 (d, JH,H = 0.8 Hz, 1H, CH,

3 4 8), 7.69 (s, 1H, CH, 1), 7.81 (dd, JH,H = 4.8 Hz, JH,H = 0.8 Hz, 1H, CH, 10), 8.75 (d,

3 JH,H = 4.8 Hz, 1H, CH, 11) ppm.

13 C-NMR (101 MHz, CDCl3, 25 °C):  = 30.6 (CH3, 6), 32.4 (C, 5), 52.9 (CH3, 13), 78.1 (CH, 1), 103.3 (CH, 3), 121.8 (CH, 8), 122.9 (CH, 10), 130.3 (CH, 2), 138.6 (C, 9), 150.3 (CH, 11), 157.4 (C, 7), 163.4 (C, 4), 165.3 (C, 12) ppm.

IR (KBr): ṽ = 3132 (w,  (C-Harom.)), 3111 (m,  (C-Harom.)), 3072 (w,  (C-Harom.)), 3018 (w, 

(C-Haliph.)), 2956 (s,  (C-Haliph.)), 2931 (m,  (C-Haliph.)), 2901 (m,  (C-Haliph.)), 2866 (m, 

(C-Haliph.)), 1736 (vs,  (C=O)), 1714 (m), 1605 (m), 1570 (m), 1529 (m), 1514 (m), 1483 (m), 1458 (m), 1433 (m), 1402 (m), 1387 (w), 1360 (m), 1335 (s), 1300 (s), 1259 (s), 1248 (vs), 1219 (m), 1209 (m), 1186 (m), 1159 (m), 1115 (m), 1093 (m), 1065 (m), 1053 (m), 1026 (w),

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Experimental Part

997 (m), 974 (m), 914 (m), 872 (w), 845 (w), 812 (m), 791 (s), 760 (s), 725 (m), 702 (s), 677 (m) cm-1.

+ 13 + 13 + + MS (EI ): m/z (%): 397 (1) [C20 C2H29N5O2] , 396 (4) [C21 CH29N5O2] , 395 (18) [C22H29N5O2] ,

13 + 13 + + 274 (2) [C13 C2H18N3O2] , 273 (18) [C14 CH18N3O2] , 272 (100) [C15H18N3O2] , 137 (36)

+ + [C7H7NO2] , 136 (23) [C7H6NO2] .

+ + HRMS (ESI , methanol, acid.): m/z calcd. for C22H30N5O2 [M+H] : 396.2398; found: 396.2373.

6.4.2. 2-(4-Hydroxymethylenepyridinyl)bis(3-tert-butylpyrazolyl)methane (L4)

Figure 72: HC(3-tBuPz)2(4-CH2OHPy) (L4).

The synthesis was performed according to a slightly modified literature procedure.[181]

LiAlH4 (0.96 g, 25.3 mmol, 1.5 eq.) was stirred in freshly distilled, dry diethyl ether (80 mL) at

0 °C. HC(3-tBuPz)2(4-CO2MePy) (L1) (6.64 g, 16.8 mmol, 1.0 eq.) was dissolved in freshly distilled, dry diethyl ether (180 mL) and added via a dropping funnel under vigorous stirring. The greenish grey suspension was warmed to r.t. and stirred for 17 h. After the addition of water (0.8 mL), aqueous NaOH (0.8 mL, 10 % solution) and another portion of water (0.8 mL) were added. The reaction mixture was stirred for 4 h at r.t. until gas evolution was no longer observed and the solution turned yellow. Subsequently, the precipitate was filtered off and the filtrate was dried over MgSO4. After removing the solvent in vacuo, the isomerised ligand

HC(3-tBuPz)(5-tBuPz)(4-CH2OHPy) was obtained.

The isomerisation was reversed according to literature.[141,142] The ligand

HC(3-tBuPz)(5-tBuPz)(4-CH2OHPy) (5.81 g, 15.8 mmol) was dissolved in toluene (57 mL) under air atmosphere. p-Toluenesulfonic acid monohydrate (0.58 g, 10 w%) was added to the yellow solution at r.t. under stirring. The reaction mixture was heated to reflux for 18 h during which the solution became dark brown. After cooling to r.t., the acidic solution was neutralised

123

Experimental Part

by the addition of a half concentrated aqueous solution of Na2CO3 (60 mL). Subsequently, the organic layer was separated, washed with water (3 × 40 mL), dried over MgSO4 and the solvent was removed in vacuo. The crude product was purified by column chromatography (silica gel, n-hexane : ethyl acetate = 1:4 → ethyl acetate) and the product was obtained as an oil (3.28 g, 8.9 mmol, 57 %).

Appearance: Orange wax-like oil.

Rf (silica gel, n-hexane : ethyl acetate = 1:4): 0.6

1 H-NMR (400 MHz, CDCl3, 25 °C):  = 1.27 (s, 18H, CH3, 6), 1.88 (s, 1H, OH, 13), 4.65 (s, 2H,

3 CH2, 12), 6.15 (d, JH,H = 2.4 Hz, 2H, CH, 3), 6.91 (s, 1H, CH, 8), 7.26 (s, 1H, CH, 10), 7.37 (d,

3 3 JH,H = 2.4 Hz, 2H, CH, 2), 7.60 (s, 1H, CH, 1), 8.56 (d, JH,H = 4.8 Hz, 1H, CH, 11) ppm.

13 C-NMR (101 MHz, CDCl3, 25 °C):  = 30.6 (CH3, 6), 32.3 (C, 5), 63.4 (CH2, 12), 78.3 (CH, 1), 103.1 (CH, 3), 119.9 (CH, 8), 121.1 (CH, 10), 130.0 (CH, 2), 149.6 (CH, 11), 151.5 (C, 9), 155.9 (C, 7), 163.2 (C, 4) ppm.

IR (ATR): ṽ = 3258 (w,  (O-H)), 2961 (m,  (C-Haliph.)), 2926 (w,  (C-Haliph.)), 2903 (w, 

(C-Haliph.)), 2868 (w,  (C-Haliph.)), 1711 (vw), 1608 (w), 1564 (w), 1521 (m), 1481 (w), 1460 (m), 1416 (w), 1403 (m), 1362 (m), 1326 (m), 1294 (w), 1247 (s), 1206 (m), 1159 (m), 1099 (m), 1053 (vs), 1026 (m), 994 (m), 984 (w), 928 (vw), 861 (w), 804 (vs), 759 (vs), 725 (m), 686 (m), 609 (w), 592 (w), 534 (w), 500 (w), 442 (m) cm-1.

+ + + + MS (EI ): m/z (%): 367 (1) [C21H29N5O] , 337 (1) [C20H27N5] , 260 (1) [C15H24N4] , 244 (1)

+ 13 + + [C14H18N3O] , 110 (3) [C5 CH7NO] , 109 (100) [C6H7NO] .

+ + HRMS (EI ): m/z calcd. for C21H29N5O [M] : 367.2373; found: 367.2366.

6.4.3. 2-(4-Maleimidoethylenecarboxymethylenepyridinyl)bis(3-tert-butylpyrazolyl)methane (L5)

The synthesis was performed according to a modified literature procedure.[100]

HC(3-tBuPz)2(4-CH2OHPy) (L4) (0.29 g, 0.8 mmol, 1.0 eq.) was stirred in freshly distilled, dry

THF (10 mL) at r.t. NEt3 (0.89 mL, 6.4 mmol, 8.0 eq.) was added to the yellow solution. 3-Maleimidopropionyl chloride (0.16 g, 0.9 mmol, 1.1 eq.) was dissolved in freshly distilled, dry THF (2 mL) and added dropwise with a syringe under vigorous stirring. The reaction mixture turned cloudy flesh-coloured, orange and was stirred at r.t. for 19 h. Afterwards, the precipitate

124

Experimental Part was filtered off and the filtrate was treated with diethyl ether (250 mL). The organic layer was washed with water (2 × 200 mL) and with saturated aqueous NaCl (1 × 200 mL). The combined organic layers were dried over MgSO4 and the solvent was removed in vacuo. The product was obtained as an amorphous solid (0.39 g, 0.8 mmol, 94 %).

Figure 73: HC(3-tBuPz)2(4-Mal1Py) (L5).

Appearance: Yellow amorphous solid.

1 3 H-NMR (400 MHz, CDCl3, 25 °C):  = 1.27 (s, 18H, CH3, 6), 2.69 (t, JH,H = 7.0 Hz, 2H, CH2,

3 3 14), 3.84 (t, JH,H = 7.0 Hz, 2H, CH2, 15), 5.06 (s, 2H, CH2, 12), 6.16 (d, JH,H = 2.4 Hz, 2H, CH,

4 3 3), 6.68 (s, 2H, CH, 17), 6.82 (t, JH,H = 0.6 Hz, 1H, CH, 8), 7.21 (dd, JH,H = 5.0 Hz,

4 3 JH,H = 1.0 Hz, 1H, CH, 10), 7.39 (d, JH,H = 2.0 Hz, 2H, CH, 2), 7.58 (s, 1H, CH, 1), 8.59 (dd,

3 4 JH,H = 4.8 Hz, JH,H = 0.4 Hz, 1H, CH, 11) ppm.

13 C-NMR (101 MHz, CDCl3, 25 °C):  = 30.6 (CH3, 6), 32.3 (C, 5), 32.9 (CH2, 14), 33.7 (CH2,

15), 64.7 (CH2, 12), 78.4 (CH, 1), 103.1 (CH, 3), 120.7 (CH, 8), 122.0 (CH, 10), 130.0 (CH, 2), 134.4 (CH, 17), 145.7 (C, 9), 149.9 (CH, 11), 156.5 (C, 7), 163.2 (C, 4), 170.3 (C, 13), 170.4 (C, 16) ppm.

IR (ATR): ṽ = 2964 (w,  (C-Haliph.)), 2904 (w,  (C-Haliph.)), 2871 (w,  (C-Haliph.)), 1743 (m,  (C=O)), 1708 (vs,  (C=O)), 1609 (w), 1566 (w), 1521 (w), 1481 (w), 1458 (w), 1446 (w), 1406 (m), 1383 (w), 1363 (m), 1322 (w), 1248 (m), 1206 (w), 1175 (m), 1144 (m), 1055 (m), 1026 (w), 998 (w), 956 (w), 931 (w), 910 (w), 827 (m), 803 (m), 763 (m), 725 (w), 695 (s), 612 (vw), 535 (vw), 484 (vw), 446 (w), 440 (w), 420 (w) cm-1.

+ 13 + 13 + + MS (EI ): m/z (%): 520 (1) [C26 C2H34N6O4] , 519 (1) [C27 CH34N6O4] , 518 (3) [C28H34N6O4] ,

13 + 13 + + 397 (1) [C19 C2H23N4O4] , 396 (4) [C20 CH23N4O4] , 395 (14) [C21H23N4O4] , 369 (1)

13 + 13 + + 13 + [C19 C2H29N5O] , 368 (4) [C20 CH29N5O] , 367 (11) [C21H29N5O] , 245 (12) [C13 CH18N3O] ,

+ 13 + + 13 + 244 (80) [C14H18N3O] , 153 (12) [C6 CH6NO3] , 152 (100) [C7H6NO3] , 125 (24) [C5 C2H11N2] ,

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Experimental Part

13 + + 13 + + 124 (39) [C6 CH11N2] , 123 (83) [C7H11N2] , 111 (11) [C5 CH8NO] , 110 (41) [C6H8NO] , 109

+ (15) [C6H7NO] .

+ + HRMS (EI ): m/z calcd. for C28H34N6O4 [M] : 518.2642; found: 518.2634.

6.4.4. 2-(4-Maleimidomethylenepyridinyl)bis(3-tert-butylpyrazolyl)methane (L6)

Figure 74: HC(3-tBuPz)2(4-Mal2Py) (L6).

Caution: the ligand HC(3-tBuPz)2(4-Mal2Py) (L6) was synthesised via a Mitsunobu reaction. Within this reaction the explosive and light, shock and heat sensitive diethyl azodicarboxylate (DEAD) was used. For safety reasons, this reaction was carried out behind an explosion prevention pane and with Kevlar gloves.

The synthesis was performed according to a slightly modified literature procedure.[185]

PPh3 (0.33 g, 1.3 mmol, 1.0 eq.) was stirred in freshly distilled, dry THF (6.5 mL) and cooled to -78 °C. DEAD (0.20 mL, 1.3 mmol, 1.0 eq.) was added dropwise with a syringe under vigorous stirring within 3 min. The yellow solution was stirred at -78 °C for 5 min.

HC(3-tBuPz)2(4-CH2OHPy) (L4) (0.52 g, 1.4 mmol, 1.1 eq.) was dissolved in freshly distilled, dry THF (15 mL) and added dropwise with a syringe within 1 min. The reaction mixture was stirred at -78 °C for another 5 min. Afterwards, first neopentyl alcohol (0.06 g, 0.7 mmol, 0.5 eq.), then maleimide (0.12 g, 1.3 mmol, 1.0 eq.) was added to the solution. After another 5 min of stirring at low temperature, the reaction mixture was warmed to r.t. and stirred for 19 h. Subsequently, the solvent was removed in vacuo and the crude product was purified by column chromatography (silica gel, n-hexane : ethyl acetate = 5:1 → ethyl acetate). The fraction containing the product was dissolved in ethyl acetate (5 mL) and storage at -25 °C yielded colourless crystals of DEAD-H2 within two days. The supernatant was taken off and reduced to dryness. The product was obtained as a colourless oil (0.27 g, 0.6 mmol, 49 %).

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Experimental Part

Appearance: Colourless oil.

Rf (silica gel, n-hexane : ethyl acetate = 1:1): 0.5

1 H-NMR (400 MHz, CDCl3, 25 °C):  = 1.26 (s, 18H, CH3, 6), 4.61 (s, 2H, CH2, 12), 6.14 (d,

3 3 JH,H = 2.8 Hz, 2H, CH, 3), 6.72 (s, 2H, CH, 14), 6.78 (s, 1H, CH, 8), 7.15 (dd, JH,H = 5.0 Hz,

4 3 JH,H = 1.4 Hz, 1H, CH, 10), 7.35 (d, JH,H = 2.4 Hz, 2H, CH, 2), 7.55 (s, 1H, CH, 1), 8.53 (dd,

3 4 JH,H = 5.0 Hz, JH,H = 0.6 Hz, 1H, CH, 11) ppm.

13 C-NMR (101 MHz, CDCl3, 25 °C):  = 30.6 (CH3, 6), 32.2 (C, 5), 40.3 (CH2, 12), 78.2 (CH, 1), 103.0 (CH, 3), 121.2 (CH, 8), 122.7 (CH, 10), 129.9 (CH, 2), 134.4 (CH, 14), 145.9 (C, 9), 149.9 (CH, 11), 156.6 (C, 7), 163.1 (C, 4), 169.9 (C, 13) ppm.

IR (ATR): ṽ = 3044 (w,  (C-Harom.)), 2991 (w,  (C-Harom.)), 2960 (w,  (C-Haliph.)), 2903 (vw, 

(C-Haliph.)), 2871 (vw,  (C-Haliph.)), 1744 (m,  (C=O)), 1709 (vs,  (C=O)), 1695 (s), 1607 (w), 1529 (s), 1481 (m), 1457 (w), 1449 (m), 1438 (w), 1429 (w), 1402 (m), 1389 (w), 1367 (m), 1315 (w), 1275 (m), 1234 (vs), 1157 (m), 1113 (w), 1060 (s), 1020 (m), 1007 (m), 986 (w), 900 (w), 885 (w), 828 (w), 796 (m), 783 (m), 759 (m), 725 (w), 695 (m), 651 (m), 617 (m), 592 (s), 444 (w), 419 (vw) cm-1.

+ 13 + 13 + + MS (EI ): m/z (%): 448 (1) [C23 C2H30N6O2] , 447 (1) [C24 CH30N6O2] , 446 (2) [C25H30N6O2] ,

13 + 13 + + 325 (1) [C16 C2H19N4O2] , 324 (3) [C17 CH19N4O2] , 323 (10) [C18H19N4O2] , 260 (2)

13 + + 13 + + [C14 CH23N4] , 259 (9) [C15H23N4] , 110 (8) [C5 CH7NO] , 109 (100) [C6H7NO] .

+ + HRMS (EI ): m/z calcd. for C25H30N6O2 [M] : 446.2426; found: 446.2425.

6.5. Complex Syntheses

6.5.1. [Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1)

Figure 75: [Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1).

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Experimental Part

A yellow solution of HC(3-tBuPz)2(4-CO2MePy) (L1) (49 mg, 0.13 mmol, 1.0 eq.) in THF

(0.5 mL) was added dropwise to a green solution of CuCl2 (17 mg, 0.13 mmol, 1.0 eq.) in THF (0.5 mL). Immediately, green solid precipitated, which was filtered and redissolved in warm acetonitrile (2.0 mL). The solution was overlaid first with diethyl ether (1.0 mL), then with n-pentane (2.0 mL). Storage at -25 °C resulted after several weeks in small green crystals suitable for XRD analysis (41 mg, 0.08 mmol, 62 %).

Appearance: Green crystalline solid.

IR (KBr): ṽ = 3130 (m,  (C-Harom.)), 3111 (m,  (C-Harom.)), 3082 (m,  (C-Harom.)), 3051 (m, 

(C-Harom.)), 2964 (m,  (C-Haliph.)), 2951 (m,  (C-Haliph.)), 2904 (m,  (C-Haliph.)), 2864 (m, 

(C-Haliph.)), 1747 (vs,  (C=O)), 1720 (m), 1680 (w), 1618 (m), 1566 (m), 1522 (m), 1516 (m), 1487 (m), 1460 (m), 1431 (m), 1400 (m), 1358 (m), 1331 (m), 1304 (s), 1290 (s), 1261 (vs), 1230 (s), 1205 (m), 1192 (m), 1121 (m), 1109 (m), 1072 (s), 1016 (m), 970 (m), 914 (m), 851 (w), 835 (w), 810 (m), 795 (m), 768 (s), 727 (m), 704 (m), 617 (w) cm-1.

+ 37 65 + 13 35 65 + MS (FAB ): m/z (%): 497 (1) [C22H29 Cl CuN5O2] , 496 (2) [C21 CH29 Cl CuN5O2] and

13 37 63 + 35 65 + 37 63 + [C21 CH29 Cl CuN5O2] , 495 (4) [C22H29 Cl CuN5O2] and [C22H29 Cl CuN5O2] , 494 (3)

13 35 63 + 35 63 + 13 65 + [C21 CH29 Cl CuN5O2] , 493 (5) [C22H29 Cl CuN5O2] , 461 (5) [C21 CH29 CuN5O2] , 460

65 + 13 63 + 63 + (18) [C22H29 CuN5O2] , 459 (11) [C21 CH29 CuN5O2] , 458 (42) [C22H29 CuN5O2] .

+ + HRMS (ESI , methanol, acid.): m/z calcd. for C22H29ClCuN5O2 [M-Cl] : 493.1304; found: 493.1282.

6.5.2. [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2)

Figure 76: [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2).

A warm solution of CuBr (27 mg, 0.19 mmol, 1.5 eq.) in acetone (0.5 mL) was added dropwise to a yellow solution of HC(3-tBuPz)2(4-CO2MePy) (L1) (49 mg, 0.13 mmol, 1.0 eq.) in THF (0.5 mL). The solution turned into red brown and after stirring for 30 min and cooling to r.t. solid precipitated. Acetonitrile (1.0 mL) was added to redissolve the solid and the red solution was

128

Experimental Part overlaid with diethyl ether (1.0 mL). After several weeks, red brown crystals suitable for XRD analysis were obtained (17 mg, 0.03 mmol, 24 %).

Appearance: Red brown crystalline solid.

IR (KBr): ṽ = 3134 (w,  (C-Harom.)), 3107 (w,  (C-Harom.)), 3070 (w,  (C-Harom.)), 3049 (m, 

(C-Harom.)), 3026 (w,  (C-Harom.)), 2956 (s,  (C-Haliph.)), 2902 (m,  (C-Haliph.)), 2862 (m, 

(C-Haliph.)), 1738 (vs,  (C=O)), 1632 (w), 1605 (w), 1564 (m), 1518 (s), 1483 (m), 1458 (m), 1435 (m), 1400 (m), 1362 (m), 1335 (m), 1302 (m), 1286 (m), 1263 (m), 1234 (s), 1200 (m), 1155 (m), 1115 (m), 1101 (m), 1057 (m), 1030 (w), 1007 (w), 970 (w), 937 (vw), 914 (w), 870 (vw), 852 (w), 808 (m), 771 (m), 764 (s), 729 (m), 723 (m), 700 (m), 677 (w), 611 (vw) cm-1.

+ 13 81 65 + 81 65 + MS (FAB ): m/z (%): 542 (7) [C21 CH29 Br CuN5O2] , 541 (25) [C22H29 Br CuN5O2] , 540

13 81 63 + 13 79 65 + 81 63 + (21) [C21 CH29 Br CuN5O2] and [C21 CH29 Br CuN5O2] , 539 (72) [C22H29 Br CuN5O2]

79 65 + 13 79 63 + and [C22H29 Br CuN5O2] , 538 (17) [C21 CH29 Br CuN5O2] , 537 (51)

79 63 + 13 65 + 13 63 + [C22H29 Br CuN5O2] , 461 (12) [C21 CH29 CuN5O2] , 460 (46) [C20 C2H29 CuN5O2] and

65 + 13 63 + 63 + [C22H29 CuN5O2] , 459 (37) [C21 CH29 CuN5O2] , 458 (100) [C22H29 CuN5O2] .

+ + HRMS (ESI , methanol, acid.): m/z calcd. for C22H29CuN5O2 [M-Br] : 458.1614; found: 458.1583.

6.5.3. [Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3)

Figure 77: [Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3).

An orange solution of HC(3-tBuPz)2(1-MeIm) (L2) (172 mg, 0.51 mmol, 1.0 eq.) in acetonitrile (1.0 mL) was added dropwise to a warm suspension of CuCl (56 mg, 0.57 mmol, 1.1 eq.) in methanol (1.0 mL). The beige solution was stirred for 30 min at 60 °C and subsequently cooled to r.t. After 3 months, colourless crystals suitable for XRD analysis were obtained (32 mg, 0.07 mmol, 15 %).

Appearance: Colourless crystalline solid.

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Experimental Part

IR (KBr): ṽ = 3142 (w,  (C-Harom.)), 3116 (m,  (C-Harom.)), 2967 (m,  (C-Haliph.)), 2950 (m, 

(C-Haliph.)), 2931 (m,  (C-Haliph.)), 2910 (m,  (C-Haliph.)), 2862 (m,  (C-Haliph.)), 1729 (w), 1637 (w), 1611 (w), 1549 (w), 1519 (s), 1501 (m), 1482 (m), 1467 (m), 1456 (m), 1439 (m), 1410 (m), 1361 (m), 1348 (m), 1335 (m), 1287 (m), 1267 (w), 1238 (vs), 1176 (w), 1158 (m), 1144 (w), 1087 (w), 1058 (m), 1035 (w), 1001 (w), 951 (m), 857 (m), 836 (m), 820 (m), 808 (s), 774 (vs), 758 (m), 730 (m), 704 (m), 667 (m), 632 (w), 608 (w), 491 (w), 458 (w) cm-1.

+ 13 65 + 13 65 + MS (ESI , acetonitrile): m/z (%): 747 (4) [C36 C2H56 CuN12] , 746 (21) [C37 CH56 CuN12] ,

13 63 + 65 + 13 63 + 745 (55) [C36 C2H56 CuN12] and [C38H56 CuN12] , 744 (45) [C37 CH56 CuN12] and

63 15 + 63 + 13 65 + [C38H56 CuN11 N] , 743 (100) [C38H56 CuN12] , 406 (3) [C18 CH28 CuN6] , 405 (18)

13 63 + 65 + 13 63 + 63 15 + [C17 C2H28 CuN6] and [C19H28 CuN6] , 404 (7) [C18 CH28 CuN6] and [C19H28 CuN5 N] ,

63 + 403 (38) [C19H28 CuN6] .

+ + HRMS (ESI , acetonitrile): m/z calcd. for C19H28CuN6 [M-Cl] : 403.1669; found: 403.1676.

6.5.4. [Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4)

Figure 78: [Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4).

An orange solution of HC(3-tBuPz)2(1-MeIm) (L2) (170 mg, 0.50 mmol, 1.0 eq.) in THF (2.0 mL) was added dropwise to a warm, white suspension of CuI (95 mg, 0.50 mmol, 1.0 eq.) in acetonitrile (2.0 mL). After stirring at r.t., the solvent was reduced to its half volume. The red solution was overlaid first with diethyl ether (1.0 mL), then with n-pentane (2.0 mL). Storage at r.t. for several weeks resulted in red brown crystals suitable for XRD analysis (86 mg, 0.16 mmol, 33 %).

Appearance: Red brown crystalline solid.

IR (KBr): ṽ = 3140 (w,  (C-Harom.)), 3120 (m,  (C-Harom.)), 3105 (s,  (C-Harom.)), 3009 (w, 

(C-Haliph.)), 2964 (s,  (C-Haliph.)), 2949 (s,  (C-Haliph.)), 2904 (s,  (C-Haliph.)), 2866 (m, 

(C-Haliph.)), 1728 (w), 1703 (w), 1635 (w), 1608 (m), 1547 (m), 1518 (vs), 1500 (s), 1481 (m),

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Experimental Part

1460 (s), 1431 (s), 1408 (m), 1362 (s), 1346 (s), 1333 (m), 1284 (s), 1236 (vs), 1207 (m), 1174 (m), 1155 (s), 1142 (m), 1084 (m), 1059 (s), 1026 (m), 1001 (m), 951 (m), 928 (m), 858 (s), 833 (s), 818 (s), 806 (vs), 773 (vs), 758 (s), 729 (s), 704 (s), 665 (m), 629 (m), 606 (m) cm-1.

+ 13 65 + 13 63 + MS (FAB ): m/z (%): 406 (24) [C18 CH28 CuN6] , 405 (100) [C17 C2H28 CuN6] and

65 + 13 63 + 63 15 + [C19H28 CuN6] , 404 (58) [C18 CH28 CuN6] and [C19H28 CuN5 N] , 403 (100)

63 + [C19H28 CuN6] .

- 13 65 - 13 63 - MS (FAB ): m/z (%): 532 (3) [C18 CH27 CuIN6] , 531 (5) [C17 C2H27 CuIN6] and

65 - 13 63 - 63 15 - 63 - [C19H27 CuIN6] , 530 (3) [C18 CH27 CuIN6] and [C19H27 CuIN5 N] , 529 (8) [C19H27 CuIN6] .

+ + HRMS (ESI , methanol, acid.): m/z calcd. for C19H28CuN6 [M-I] : 403.1669; found: 403.1642.

6.6. Formation of Peroxide Dicopper(II) Complexes

The synthesis was performed according to a slightly modified literature procedure.[42]

A solution of the respective ligand L (0.16 mmol, 1.0 eq.) in dichloromethane (5 mL) was added to CuCl (0.17 mmol, 1.1 eq.) under stirring. The solution was stirred for 1 h at r.t. AgSbF6 (0.18 mmol, 1.1 eq.) was dissolved in THF (300 L) and added dropwise to the copper(I) complex solution under vigorous shaking. AgCl precipitated to give the precursor complex

-1 [CuL]SbF6 (30 mmol L ).

For UV/Vis spectroscopic measurements with an immersion probe, dichloromethane (9.4 mL) was transferred into a measurement cell, cooled to -78 °C and saturated with oxygen by bubbling through the solvent (10 min). A baseline was measured. After starting the UV/Vis spectroscopic measurement, 650 L of the precursor complex ([CuL]SbF6) solution were added. The formation of P could be followed due to the characteristic peroxide dicopper(II) absorptions (final concentration: 1 mmol L-1).

The kinetic analysis of the formation of P was investigated by the use of stopped-flow UV/Vis spectroscopic measurements. Two gas-tight Luer Lock syringes were used to handle air- and moisture-sensitive solutions. Dichloromethane was saturated with oxygen by bubbling through the solvent at r.t. (10 min) and subsequently transferred into the first syringe. A 4 mmol L-1 precursor complex solution was prepared by adding [CuL]SbF6 stock solution (1.3 mL) to dichloromethane (9.0 mL). This solution was transferred into the second syringe. During the stopped-flow measurements equal amounts of both syringes were mixed so that the final concentration of P in the measurement cell was 1 mmol L-1. The measurements were

131

Experimental Part performed at different temperatures and prior to every measurement temperature stability was awaited.

For resonance Raman measurements, the precursor complex [CuL]SbF6 was prepared in

-1 CD2Cl2 instead of CH2Cl2 inside a MBraun glovebox. A 10 mmol L precursor complex solution was obtained by adding [CuL]SbF6 stock solution (1.0 mL) to CD2Cl2 (2.0 mL). This solution was transferred into a septum-sealed screw-cap cuvette. After a precursor complex measurement, the cuvette was cooled to -80 °C and oxygen was added by bubbling through the solution (10 min). After 30 min, the measurement was started. The final concentration of P

-1 18 16 in the cuvette was 5 mmol L . The measurements were repeated with O2 instead of O2.

2 2 6.6.1. [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1)

2 2 Figure 79: [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1).

The synthesis was performed according to a slightly modified literature procedure.[42]

For the synthesis of P1, HC(3-tBuPz)2(4-CO2MePy) (L1) (64 mg, 0.16 mmol, 1.0 eq.), CuCl

(17 mg, 0.17 mmol, 1.1 eq.) and AgSbF6 (61 mg, 0.18 mmol, 1.1 eq.) were used. The copper(I) chloride complex solution was dark red and the precursor complex

-1 ([Cu{HC(3-tBuPz)2(4-CO2MePy)}]SbF6) was red in solution (30 mmol L ).

Besides -78 °C, the formation of the peroxide dicopper(II) complex P1 was performed at r.t. as well. Dichloromethane (9.4 mL) was saturated with oxygen at r.t. and 650 L of the precursor complex [Cu{HC(3-tBuPz)2(4-CO2MePy)}]SbF6 were also added at r.t.

Appearance: Dark violet in solution.

UV/Vis: 350 nm (23800 L mol-1 cm-1, LMCT) and 550 nm (1900 L mol-1 cm-1, LMCT).

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Experimental Part

16 16 Resonance Raman (360 nm, CD2Cl2, -80 °C): 757 ( ( O- O)), 566 (undefined), ~330 (

-1 (Cu-Neq)), ~280 ( (Cu-Nax) / Cu-Cu motion) cm .

2 2 6.6.2. [Cu2{HC(3-tBuPz)2(1-MeIm)}2(- : -O2)](SbF6)2 (P2)

2 2 Figure 80: [Cu2{HC(3-tBuPz)2(1-MeIm)}2(- : -O2)](SbF6)2 (P2).

The synthesis was performed according to a slightly modified literature procedure.[43]

For the resynthesis of P2, HC(3-tBuPz)2(1-MeIm) (L2) (55 mg, 0.16 mmol, 1.0 eq.), CuCl

(17 mg, 0.17 mmol, 1.1 eq.) and AgSbF6 (63 mg, 0.18 mmol, 1.1 eq.) were used. The copper(I) chloride complex solution was flesh-coloured and the precursor complex

-1 ([Cu{HC(3-tBuPz)2(1-MeIm)}]SbF6) was colourless in solution (30 mmol L ).

Appearance: Dark purple in solution.

UV/Vis: 334 nm (~20000 L mol-1 cm-1, LMCT) and 534 nm (~1000 L mol-1 cm-1, LMCT).

2 2 6.6.3. [Cu2{HC(3-tBuPz)2(Py)}2(- : -O2)](SbF6)2 (P3)

The synthesis was performed according to a slightly modified literature procedure.[42]

For the resynthesis of P3, HC(3-tBuPz)2(Py) (L3) (54 mg, 0.16 mmol, 1.0 eq.), CuCl (17 mg,

0.17 mmol, 1.1 eq.) and AgSbF6 (63 mg, 0.18 mmol, 1.1 eq.) were used. The copper(I) chloride complex solution was yellow and the precursor complex

-1 ([Cu{HC(3-tBuPz)2(Py)}]SbF6) was greenish yellow in solution (30 mmol L ).

Appearance: Dark violet in solution.

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Experimental Part

UV/Vis: 350 nm (20000 L mol-1 cm-1, LMCT) and 550 nm (1000 L mol-1 cm-1, LMCT) in accordance with literature.[42]

2 2 Figure 81: [Cu2{HC(3-tBuPz)2(Py)}2(- : -O2)](SbF6)2 (P3).

2 2 6.6.4. [Cu2{HC(3-tBuPz)2(4-Mal1Py)}2(- : -O2)](SbF6)2 (P5)

2 2 Figure 82: [Cu2{HC(3-tBuPz)2(4-Mal1Py)}2(- : -O2)](SbF6)2 (P5).

For the synthesis of P5, HC(3-tBuPz)2(4-Mal1Py) (L5) (42 mg, 0.08 mmol, 1.0 eq.), CuCl

(9 mg, 0.09 mmol, 1.1 eq.) and AgSbF6 (32 mg, 0.09 mmol, 1.2 eq.) were used. The copper(I) chloride complex solution was yellow and the precursor complex

-1 ([Cu{HC(3-tBuPz)2(4-Mal1Py)}]SbF6) was yellow in solution (30 mmol L ).

Appearance: Pale violet in solution.

UV/Vis: 348 nm (~12500 L mol-1 cm-1, LMCT) and 555 nm (~800 L mol-1 cm-1, LMCT). The formation of P5 is assumed to be incomplete. Extinction coefficients might be higher.[186,187]

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Experimental Part

2 2 6.6.5. [Cu2{HC(3-tBuPz)2(4-Mal2Py)}2(- : -O2)](SbF6)2 (P6)

2 2 Figure 83: [Cu2{HC(3-tBuPz)2(4-Mal2Py)}2(- : -O2)](SbF6)2 (P6).

For the synthesis of P6, HC(3-tBuPz)2(4-Mal2Py) (L6) (37 mg, 0.08 mmol, 1.0 eq.), CuCl

(9 mg, 0.09 mmol, 1.1 eq.) and AgSbF6 (32 mg, 0.09 mmol, 1.2 eq.) were used. The copper(I) chloride complex solution was pale yellow and the precursor complex

-1 ([Cu{HC(3-tBuPz)2(4-Mal2Py)}]SbF6) was pale brown in solution (30 mmol L ).

Appearance: Dark violet in solution.

UV/Vis: 350 nm (~21000 L mol-1 cm-1, LMCT) and 550 nm (~1200 L mol-1 cm-1, LMCT).

6.7. Hydroxylation Reactions with Peroxide Dicopper(II) Species

6.7.1. Stoichiometric Hydroxylation of Sodium Phenolates

For reactions of peroxide dicopper(II) species with sodium phenolates, P was generated in situ in an UV/Vis measurement cell (1 mmol L-1) as described in section 6.6. After complete formation, excess oxygen was removed by cycles of vacuum/N2, followed by bubbling N2 through the solution (5 min). Sodium 4-X-phenolate (X = OMe, Me, CO2Me) was dissolved in methanol according to tables 16, 17 and 18. After starting the UV/Vis spectroscopic kinetic measurement at 350 nm, 250 L (2, 5, 10, 15, 20 or 30 eq.) of the phenolate solution were added in one portion. The hydroxylation kinetics were observed indirectly due to the decrease of the peroxide absorption. The measurement was stopped when the decay curve reached a stable value.

135

Experimental Part

Table 16: Weighed portions of sodium 4-OMe-phenolate for stock solutions for hydroxylation reactions.

-1 eq. (phenolate) m [mg] n [mmol] VMeOH [mL] c [mol L ] 2 23.5 0.16 2.0 0.08 5 29.3 0.20 1.0 0.20 10 59.1 0.40 1.0 0.40 15 88.0 0.60 1.0 0.60 20 117.6 0.80 1.0 0.80 30 350.6 2.40 2.0 1.20

Table 17: Weighed portions of sodium 4-Me-phenolate for stock solutions for hydroxylation reactions.

-1 eq. (phenolate) m [mg] n [mmol] VMeOH [mL] c [mol L ] 2 20.8 0.16 2.0 0.08 5 52.1 0.40 2.0 0.20 10 104.7 0.80 2.0 0.40 15 156.7 1.20 2.0 0.60 20 208.8 1.60 2.0 0.80 30 312.1 2.40 2.0 1.20

Table 18: Weighed portions of sodium 4-CO2Me-phenolate for stock solutions for hydroxylation reactions.

-1 eq. (phenolate) m [mg] n [mmol] VMeOH [mL] c [mol L ] 2 27.8 0.16 2.0 0.08 5 70.0 0.40 2.0 0.20 10 140.5 0.81 2.0 0.40 15 209.1 1.20 2.0 0.60 20 279.1 1.60 2.0 0.80 30 209.2 1.20 1.0 1.20

To analyse the reaction products, NMR spectroscopic studies were performed. For this, dichloromethane (5 mL) was transferred into a septum-sealed centrifuge tube, cooled down to -78 °C and was saturated with oxygen by bubbling through the solvent (10 min). 250 L of the

-1 precursor complex [CuL]SbF6 (30 mmol L , section 6.6.) were added under stirring and the formation of the peroxide species P (0.7 mmol L-1, 3.8 nmol, 1 eq.) was observed by a colour

136

Experimental Part

change. After 40 min at -78 °C, excess oxygen was removed by three cycles of vacuum/N2 and subsequently bubbling of N2 (5 min). Sodium 4-X-phenolate (X = OMe, Me) (29.4 mg (OMe) or 25.9 mg (Me), 0.2 mmol) was dissolved in THF (5 mL) to give a 40 mmol L-1 solution. 475 L (19.0 nmol, 5 eq.) of this phenolate solution were added to the peroxide solution in one portion. After a reaction time of exact 5, 20 or 30 min, the reaction was stopped by the addition of aqueous HCl (3 mL, 0.5 mol L-1) and immediately vigorous shaking. The mixture was warmed to r.t. and dichloromethane was removed in vacuo. The residue was extracted with dichloromethane (3 × 5 mL) and the combined organic layers were dried over a pipette filled with MgSO4. Subsequently, the solvent was removed in vacuo and the residue was analysed with NMR spectroscopy in acetone-d6. As an internal standard, an acetophenone solution in dichloromethane (190 L, 0.02 mol L-1, 3.8 nmol, 1 eq.) was added prior to the extraction, after the extraction or after drying over MgSO4. Moreover, an experiment without copper(I) precursor complex solution was performed as blank sample.

6.7.2. Catalytic Conversions of Phenolic Substrates

The peroxide dicopper(II) species P was generated in situ in an UV/Vis spectroscopic measurement cell (1 mmol L-1) as described in section 6.6. 8-Hydroxyquinoline (182.2 mg, 1.26 mmol) or 4-methoxyphenol (156.7 mg, 1.26 mmol) was dissolved in dichloromethane (660 L) and triethylamine (350 L, 2.52 mmol) was added as auxiliary base. After complete formation of P, a new UV/Vis spectroscopic measurement was started, 200 L (25 eq. substrate / 50 eq. base) or 400 L (50 eq. substrate / 100 eq. base) of the substrate solution were added and the cooling bath was removed. The measurement was stopped after several hours.

Alternatively, the substrate reaction was stopped after 7.5 or 60 min by the addition of aqueous HCl (3 ml, 0.5 mol L-1). The mixture was extracted with dichloromethane (3 × 3 mL) and the combined organic layers were dried over a pipette filled with MgSO4. The solvent was removed in vacuo and the residue was analysed with NMR spectroscopy in acetone-d6.

Catalytic substrate conversions were additionally performed in a self-assembly manner. Here, dichloromethane was saturated with oxygen at -78 °C or at r.t. and substrate solution was added prior to the addition of precursor complex solution. These conversions were also monitored by UV/Vis spectroscopy.

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Experimental Part

6.8. Biohybrid Catalysts

For all biochemical reactions, the proteins NB4 and NB4exp were expressed and purified by students either in the laboratory of Prof. Dr. Takashi Hayashi (Osaka University, Japan) or Prof. Dr. Ulrich Schwaneberg (RWTH Aachen University) and provided to use.[103]

6.8.1. Protein Conjugation with Bis(pyrazolyl)methane Ligands

Figure 84: L5- or L6-conjugated NB4 or NB4exp.

The proteins NB4 and NB4exp were stored in 500 L-aliquots (500 or 266 mol L-1) at -80 °C. Prior to storage or usage dithiothreitol (DTT) (2-3 mg/mL) was added to prevent the formation of disulfide bridges. For the protein reactions below, MOPS buffer or phosphate buffer was used. MOPS buffer consists of 3-(N-morpholino)propanesulfonic acid (20 mmol L-1, 4.125 g)

-1 and NaCl (150 mmol L , 8.766 g) dissolved in ddH2O (1 L) and is adjusted to a pH value of

-1 -1 7.0. Phosphate buffer consists of NaH2PO4 (50 mmol L , 6.000 g) and NaCl (100 mmol L ,

5.844 g) dissolved in ddH2O (1 L) and is adjusted to a pH value of 7.0.

After thawing the protein and/or adding DTT, the protein was purified via a desalting column (HiTrapTM 5 mL desalting column, Sephadex G-25 Superfine). The solution was diluted to 20 mol L-1 with MOPS or phosphate buffer. Ligand L5 (1.2 mg, 2.3 mol) or L6 (1.5 mg, 3.4 mol) was dissolved in DMSO (46 or 67 L) to give a 50 mmol L-1 solution. 20 L of this ligand solution (5 eq. based on protein molecules) were added to the protein solution, followed by carefully shaking of the solution and incubating for 1.5 h at 4 °C. Afterwards, the protein solution was concentrated via centrifugation (7000 rpm, 20 + 10 min, 4 °C) in an Amicon (10 kDa) and excess of ligand was removed by a desalting column. The final protein concentration was in the range of 30 mol L-1.

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Experimental Part

+ MS (MALDI-TOF, sinapic acid): m/z calcd. for C908H1411N237O264S2 [NB4-L5] : 19935.523;

+ found: 19936.361. m/z calcd. for C1045H1641N275O311S2 [NB4exp-L5] : 23097.094; found:

+ 23097.906. m/z calcd. for C905H1407N237O262S2 [NB4-L6] : 19863.461; found: 19863.114. m/z

+ calcd. for C1042H1637N275O309S2 [NB4exp-L6] : 23025.031; found: 23022.903.

+ + HRMS (ESI , acetonitrile / aqueous formic acid): m/z calcd. for C908H1411N237O264S2 [NB4-L5] :

+ 19935.523; found: 19934.040. m/z calcd. for C905H1407N237O262S2 [NB4-L6] : 19863.461; found:

+ 19860.213. m/z calcd. for C1042H1637N275O309S2 [NB4exp-L6] : 23025.031; found: 23021.151.

+ HRMS (LC-ESI , acetonitrile / aqueous formic acid): m/z calcd. for C908H1411N237O264S2

+ + [NB4-L5] : 19935.523; found: 19932.490. m/z calcd. for C905H1407N237O262S2 [NB4-L6] :

+ 19863.461; found: 19861.647. m/z calcd. for C1042H1637N275O309S2 [NB4exp-L6] : 23025.031; found: 23022.194.

6.8.2. Incorporation of Copper

Figure 85: Copper(II)-L5-conjugated NB4 or NB4exp. L might be a H2O ligand.

For the incorporation of copper into the ligand-conjugated protein (see section 6.8.1.) a 10 or

-1 15 mmol L solution of copper nitrate in ddH2O was prepared (Cu(NO3)2 · 2.5 H2O (2.5 mg,

10.7 mol) or Cu(NO3)2 · 3 H2O (2.6 mg, 10.8 mol) dissolved in ddH2O (1.075 or 0.693 mL)). Amounts of 0.5-10.0 eq. of the copper solution (based on the protein molecules) were added to the protein solution followed by up and down pipetting and incubating for 30 min at 4 °C. Afterwards, the protein solution was either directly used for analyses (UV/Vis or EPR spectroscopy) or concentrated via centrifugation (4000 rpm, 3 × 5 min, 4 °C) in an Amicon (10 kDa) and excess of copper nitrate was removed by a desalting column. The final protein concentration was in the range of 25 mol L-1.

MS (ICP): ppm of Cu calcd. for NB4exp-L5-Cu (23 mol L-1): 1.449; found: 1.175 (81 % copper loading).

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Experimental Part

ICP measurement to evaluate the copper content in a sample of an oxygenated protein (from section 6.8.4.):

-1 MS (ICP): ppm of Cu calcd. for NB4exp-L5-Cu-O2 (92 mol L ): 5.796; found: 3.833 (66 % copper loading).

6.8.3. Reduction of Copper

Figure 86: Copper(I)-L5-conjugated NB4 or NB4exp. L might be a H2O ligand.

For the reduction of copper(II) to copper(I), the conjugated protein solution (see section 6.8.2.) had to be degassed. The first method was to transfer the protein solution into a septum-sealed

Schlenk tube and to bubble N2 very slowly through the solution (15 min). The second method was to transfer the protein solution into the glovebox and change the buffer to degassed buffer via a desalting column (the buffer was degassed by bubbling N2 through the solution for 20 min). The protein was eluted in 200 L-aliquots. 8 L of each aliquot were brought out of the glovebox and the protein concentration was measured. Subsequently, the aliquots containing protein were combined.

The reduction of copper(II) to copper(I) was performed with four different reductants, which were prepared as 10 mmol L-1 solutions. Sodium dithionite (0.6 mg, 3.4 mol) was dissolved in degassed MOPS buffer (345 L), sodium L-ascorbate (1.0 mg, 5.0 mol) was dissolved in degassed MOPS buffer (505 L), (+-)-threo-dithiothreitol (0.5 mg, 3.2 mol) was dissolved in degassed ddH2O (324 L), tris(2-carboxyethyl)phosphane hydrochloride (0.5 mg, 1.7 mol) was dissolved in degassed ddH2O (174 L). Either 1.1 eq. of the reductant solution were added to the protein solution (100 mol L-1) in the glovebox (Osaka) or 1.2 eq. of the reductant solution were added to the protein solution (~25 mol L-1) in a Schlenk tube (Aachen). Subsequently, the protein solution was incubated for 20 min at 4 °C and UV/Vis spectroscopic measurements were conducted.

140

Experimental Part

6.8.4. Reaction with Oxygen

Figure 87: Superoxide-copper(II)-L5-conjugated NB4 or NB4exp.

The first method to oxygenate the Cu(I)-conjugated protein (see section 6.8.3.) made use of a stopped-flow system (in the laboratory of Prof. Dr. Takashi Hayashi (Osaka University, Japan)). Protein solution (100 mol L-1) with incorporated Cu(I) was prepared and transferred into a Hamilton syringe in the glovebox. MOPS buffer was saturated with oxygen by bubbling through the solution (20 min) at r.t. The stopped-flow system was cooled to 10 °C, flooded with argon gas and flushed with degassed ddH2O and MOPS buffer. The protein solution was brought out of the glovebox and filled into the first chamber, the oxygen-saturated buffer into the second. UV/Vis spectroscopic measurements were performed.

For the second method of oxygenation, the Cu(I)-conjugated protein, which was reduced in a Schlenk tube, was used. Either oxygen was added by bubbling very slowly through the solution (10 min) (~100 mol L-1 protein solution) or oxygen-saturated MOPS or phosphate buffer (20 %vol) was added through the septum (~30 mol L-1 protein solution).

6.8.5. Reaction with Hydrogen Peroxide

Figure 88: Hydroperoxide-copper(II)-L5-conjugated NB4.

141

Experimental Part

The Cu(II)-conjugated protein (see section 6.8.2.) was reacted with hydrogen peroxide by the use of a stopped-flow system (in the laboratory of Prof. Dr. Takashi Hayashi (Osaka University,

Japan)). The system was cooled to 10 °C, flushed with ddH2O and MOPS buffer and the first chamber was filled with protein solution (100 mol L-1). The second chamber was filled with a

-1 hydrogen peroxide solution (10 mmol L ) in ddH2O. UV/Vis spectroscopic measurements were performed.

6.8.6. Hydroxylation or Oxidation Reactions

The oxygenated copper(II)-conjugated protein NB4exp (section 6.8.4.) was reacted with different substrates that yielded either colour reactions or products, which were analysed by GC or EPR spectroscopy.

For each assay or substrate reaction, negative controls were prepared: apoprotein, Cu(II)-ligand-conjugated protein, buffer, substrate only. The negative controls were treated the same way as the samples.

ABTS (2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) Assay The following volumes were pipetted sequentially into a 96-well microtiter plate (MTP):

-1 -1 1) 50 L citric acid buffer (0.1 mol L citric acid, 0.2 mol L Na2HPO4, pH 7.0)

2) 60 L ddH2O 3) 30 L sample (20 or 100 mol L-1 protein solution) 4) 60 L ABTS (5 mmol L-1 solution)

The volumes were mixed by up and down pipetting and the absorption at 420 nm was measured with a MTP reader during the next 2 h.

4-AAP (4-Aminoantipyrine) Assay The following volumes were pipetted sequentially into a 96-well microtiter plate (MTP):

1) a) 10 L substrate solution (3 mmol L-1 benzene or 4-methoxyphenol in DMSO) or b) 9 L DMSO + 1 L substrate solution 2) 90 L sample (30 mol L-1 protein solution)

The volumes were mixed by up and down pipetting and the MTP was incubated for 30 min at r.t. under shaking (750 rpm). Afterwards, urea (25 L, 4 mol L-1 in 0.1 mol L-1 NaOH) was

142

Experimental Part

-1 added to stop the reaction. Subsequently, 4-AAP (20 L, 5 mg mL ) and K2S2O8 (20 L, 5 mg mL-1) were added and the mixture was incubated for another 30 min at r.t. under shaking (750 rpm). The absorption at 509 nm was measured with a MTP reader.

Oxidation of Guaiacol (2-Methoxyphenol) The following volumes were pipetted sequentially into a 96-well microtiter plate (MTP):

1) 90 L sample (20 mol L-1 protein solution) 2) 10 L substrate solution (0.2 or 2.0 mmol L-1 guaiacol in DMSO)

The volumes were mixed by up and down pipetting and the absorption at 470 nm was measured with a MTP reader during the next 60 min.

Oxidation of TEMPO-H (Tetramethylpiperidin-1-ol) A stock solution of TEMPO-H in DMSO (184 mmol L-1) was prepared. 10 L of this stock solution were added to the oxygenated copper(II)-conjugated protein NB4exp (600 L, 33.3 mol L-1). Immediately, EPR spectra of the reaction mixture were measured.

Sulfoxidation of Thioanisole A stock solution of thioanisole in DMSO (560 mmol L-1) was prepared. 10 L of this stock solution were added to the oxygenated copper(II)-conjugated protein NB4exp (840 L, 33.3 mol L-1). The protein solution was incubated for 30 min at r.t. Afterwards, the product was extracted with ethyl acetate (3 × 1 mL), whereby the first extraction volume contained benzyl alcohol (140 mol L-1) as an internal standard for GC measurements. The solvent was removed in vacuo and the dry residue was dissolved in ethyl acetate (1.5 mL). The product was analysed via GC analysis.

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Appendix

8. Appendix

8.1. Crystallographic Data

Figure 89: Molecular structures of [Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1) (top) and [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2) (bottom). H-atoms are omitted for clarity. Displacement ellipsoids represent the 50 % probability level.

157

Appendix

Table 19: Crystallographic data of the complexes [Cu{HC(3-tBuPz)2(4-CO2MePy)}Cl2] (C1) and [Cu{HC(3-tBuPz)2(4-CO2MePy)}Br] (C2).

Complex C1 C2 identification code u15_46_neu w15_a33sadm_c

empirical formula C22H29Cl2CuN5O2 C22H29BrCuN5O2 -1 Mr [g mol ] 529.94 538.95 crystal size [mm] 0.25 × 0.18 × 0.14 0.22 × 0.20 × 0.16 T [K] 110(2) 100(2) crystal system monoclinic monoclinic

space group Cc P21/c a [Å] 17.739(3) 13.8076(13) b [Å] 7.8560(14) 31.120(3) c [Å] 18.578(3) 11.6548(11)  [°] 90 90  [°] 110.299(4) 95.621(2)  [°] 90 90 V [Å3] 2428.2(7) 4983.9(8) Z 4 8

-3 calcd [g cm ] 1.450 1.437  [mm-1] 1.148 2.506  [Å] 0.71073 0.71073 F(000) 1100 2208 hkl range -21/21, -9/9, -22/22 -16/16, -37/37, -14/14 reflections collected 13139 56016 reflections unique 4613 9226

Rint. 0.0898 0.1105 number of parameters 296 573

R1 [l ≥ 2(l)] 0.0438 0.0402

wR2 (all data) 0.0958 0.0896 goodness-of-fit 0.942 1.020 absolute structure parameter 0.016(13) - largest diff. peak, hole [e Å-3] 0.425, -0.820 0.831, -0.452

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Figure 90: Molecular structures of [Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3) (top) and [Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4) (bottom). H-atoms are omitted for clarity. Displacement ellipsoids represent the 50 % probability level.

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Table 20: Crystallographic data of the complexes [Cu{HC(3-tBuPz)2(1-MeIm)}Cl] (C3) and [Cu{HC(3-tBuPz)2(1-MeIm)}I] (C4).

Complex C3 C4 identification code n16_a97_pl w15_a30_alex

empirical formula C19H28ClCuN6 C19H28ICuN6 -1 Mr [g mol ] 439.46 530.91 crystal size [mm] 0.24 × 0.23 × 0.22 0.25 × 0.22 × 0.12 T [K] 100(2) 110(2) crystal system orthorhombic orthorhombic space group Pnma Pnma a [Å] 12.5529(8) 12.947(3) b [Å] 16.6333(11) 16.486(4) c [Å] 10.1981(7) 10.427(3)  [°] 90 90  [°] 90 90  [°] 90 90 V [Å3] 2129.3(2) 2225.6(10) Z 4 4

-3 calcd [g cm ] 1.371 1.584  [mm-1] 1.167 2.384  [Å] 0.71073 0.71073 F(000) 920 1064 hkl range -16/14, -21/8, -13/12 -15/15, -20/20, -12/12 reflections collected 9729 23848 reflections unique 2634 2161

Rint. 0.0410 0.1460 number of parameters 140 140

R1 [l ≥ 2(l)] 0.0360 0.0363

wR2 (all data) 0.0882 0.0769 goodness-of-fit 1.026 1.041 absolute structure parameter - - largest diff. peak, hole [e Å-3] 0.426, -0.368 0.503, -1.246

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8.2. Extinction Coefficient of P1

Figure 91: UV/Vis spectroscopic determination of the extinction coefficient of the absorption of 2 2 [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) at 350 nm (-78 °C).

Figure 92: UV/Vis spectroscopic determination of the extinction coefficient of the absorption of 2 2 [Cu2{HC(3-tBuPz)2(4-CO2MePy)}2(- : -O2)](SbF6)2 (P1) at 550 nm (-78 °C).

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8.3. Protein Structure

The following data of the proteins NB4 and NB4exp are given in the literature.[103]

In both protein structures, the yellow marked Cys96 is the residue, where the maleimide coupling reactions take place. In the structure of NB4exp the duplicated sequence is underlined.

Protein primary structure of NB4:

MWSHPQFEKNQLQQLQNPGESPPVHPFVAPLSYLLGTWRGQGEGEYPTIPSFRYGEEIRF SHSGKPVIAYTQKTWKLESGAPLLAESGYFRPRPDGSIEVVIACSTGLVEVQKGTYNVDEQS IKLKSDLVGNASKVKEISREFELVDGKLSYVVRLSTTTNPLQPLLKAILDKL

Molecular weight: 19.417 kDa

Cavity size: 855 Å

Protein primary structure of NB4exp:

MWSHPQFEKNQLQQLQNPGESPPVHPFVAPLSYLLGTWRGQGEGEYPTIPSFRYGEEIRF SHSGKPVIAYTQKTWKLESGAPLLAESGYFRPRPDGSIEVVIACSTGLVEVQKGTYNVDEQS IKLKSDLVGNASKVKEISQKGTYNVDEQSIKLKSDLVGNASKVKEISREFELVDGKLSYVVRL STTTNPLQPLLKAILDKL

Molecular weight: 22.578 kDa

Cavity size: 1389 Å

Mutations in the nitrobindin variant NB4 compared to the wildtype (NB):[212]

M75L/H76L/Q96C/M148L/H158L

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