Radboud Honours Academy

Faculty of Science

Research Proposal

Towards Artificial Leaves:Exploring the Effects of Surrounding Ligands on

a Synthetic Oxygen Evolving Center

Jasper van der Kolk, Judith Schaart, Laurens Sluyterman,Mike Smeenk, Iris Zahn

With supervision ofDr. Kim Bonger

2015-2016

Contents

1 Details of the Proposal 11.1 Research Proposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Applicants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Supervisor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Summaries 22.1 Scientific Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2 Summary for the General Public . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

3 Scientific Background 33.1 Artificial Photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33.2 Thermodynamic and Kinetic Conditions for Artificial Photosynthesis . . . . . . . . . . . . . . . . . . . 43.3 Oxygen Evolving Center in Photosystem II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.4 Artificial Oxygen Evolving Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.5 Housing of Artificial Catalysts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4 Aims and Objectives of the Research 104.1 Social Relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.2 Aim and Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

5 Research Question and Goals 11

6 Plan of Work 126.1 G1: Create a nanosphere which is able to host the artificial OEC, as reported by Zhang et al. [1] . . . . 12

6.1.1 Verification Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136.2 G2: Create a functioning system, containing the encapsulated artificial OEC, a sensitizer and an

electrode, and analyse this system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146.3 G3: Use a plug-and-play approach to systematically alternate the functionalisation of the nanosphere,

thereby changing the environment of the OEC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

7 Timetable 16

8 Statements by the Applicants 17

9 Acknowledgements 17

10 Appendix 18

11 References 20

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1 Details of the Proposal

1.1 Research Proposal

Title: Towards Artificial Leaves: Exploring the Effects of Surrounding Ligands on a Synthetic Oxygen EvolvingCenterArea: Chemistry/Biochemistry

1.2 Applicants

Name: Jasper van der KolkGender: Male

Name: Judith SchaartGender: Female

Name: Laurens SluytermanGender: Male

Name: Mike SmeenkGender: Male

Name: Iris ZahnGender: Female

1.3 Supervisor

Name: Dr. Kim BongerTelephone number: +31(0)24 3616753 / 52535Email address: K.M.Bonger@science.ru.nlInstitute and Department: Institute for Molecules and Materials & Radboud Institute for Molecular Life Sciences,Biomolecular Chemistry

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2 Summaries

2.1 Scientific Summary

Because of the upcoming deficiency of fossil fuels and the growing climate problem much research is done towardsrenewable energy sources. Producing fuels via artificial photosynthesis is a promising concept. However, moreknowledge is needed before an efficiently working system can be developed. In nature the Oxygen Evolving Center(OEC) is part of photosystem II (PSII) in the photosynthetic reaction chain. This Mn4Ca-cluster is coordinatedbetween two protein subdomains of PSII and splits water into oxygen, protons and electrons. Recently, a syntheticvariant of the natural OEC was reported. Although the electrochemical properties resemble those of the naturalvariant, actual water splitting was not yet accomplished due to incorrect orientation of the surrounding coordinationligands. In this proposal we describe experiments in which the effect of the surrounding ligands on the water splittingpotential of a synthetic variant of the natural OEC is studied. By encapsulating the synthetic OEC in nanosphereswhich are endohedrally coated with single amino acids or oligopeptides, we will attempt to gain a better understandingof the workings of the OEC. A basic system will be produced in which all functionalisations end with a carboxylicacid. To test its functionality, this nanosphere-OEC complex is dissolved in a solution with a Ru(bpy)3 sensitizerand an electrode to stimulate an electron flow. By changing the sequence or length of the coating oligopeptidesvia a systematic plug-and-play approach, the influence of the environment on the functionality of the OEC can bestudied by comparing the characteristics of the complexes. The information gained from this research can be usedas a starting point for designing useful, highly efficient water splitting complexes in the future.

2.2 Summary for the General Public

De invloed van de omgeving op het functioneren van een synthetisch Oxygen Evolving Center

Het klimaatprobleem groeit en fossiele brandstoffen raken op. Daarom zijn er andere, schonere brandstoffen nodig,bijvoorbeeld geproduceerd via kunstmatige fotosynthese. Een van de onderdelen van dit proces is watersplitsing.Tijdens natuurlijke fotosynthese gebeurt dit in het Oxygen Evolving Center (OEC). In dit voorstel worden exper-imenten beschreven waarbij een synthetisch OEC wordt ingepakt in een nanokooi die bekleed is met verschillendefunctionele groepen die het OEC kunnen coordineren. Door varianten te maken en te bestuderen kan onderzochtworden welke invloed de omgeving uitoefent op het functioneren van het OEC. Hiermee willen wij een basis leggenvoor de ontwikkeling van kunstmatige, efficiente watersplitsende complexen in de toekomst.

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3 Scientific Background

3.1 Artificial Photosynthesis

Because of the upcoming deficiency of fossil fuels and the growing climate problem, much research has been donetowards renewable and clean energy sources. One of the main interests in this field is artificial photosynthesis. Thisconcept is based on the natural principles of photosynthesis, by attempting to produce fuels out of sunlight and wa-ter. The designed systems, based on this principle, are called “artificial leaves” [2]. They consist of a light harvestingsensitizer (also called a dye), a water oxidizing catalyst (WOC) and a fuel producing component, linked to electrodes.A sensitizer, like the Ru(bpy)3 complex, can absorb photons from sunlight, leading to an energized electron thatcan be transferred to, for example, a TiO2-anode. The sensitizer is left in the oxidized state. It can be reduced bywithdrawing electrons from the WOC and thereby intitiating a water splitting reaction as shown in equation (1).

2H2O(l) −→ 4H+ +O2(g) + 4e− (1)

An electron cycle starts, in which electrons are gained via water oxidation and eventually are given to the anode [3].At the cathode of the system fuel can be produced, for example hydrogen or carbohydrates This can be achieved bya redox reaction which takes place directly on the cathode or by using catalysts, which use the electrons provided bythe system. A schematic representation of a system for artificial photosynthesis is shown in figure 1.

Figure 1: Schematic representation of thebuilding of a system for artificial pho-tosynthesis, containing a photosensitizer(PS), WOC and hydrogen evolving com-plex (HEC) as fuel producing component.The PS is excited by light the and startsthe electron cycle by donating an electronto the electrode. Water is oxidized by theWOC and hydrogen gas is produced by theHEC [4].

At the moment, the main problems of artificial photosynthesis are the efficiency and stability of the systems. Theseproblems are mainly caused by photobleaching (damage of the sensitizer caused by photons with high energy), inef-ficient charge separation (movement of excited electrons between molecules), charge recombination (falling back ofexcited electrons to the ground state) and inefficient light absorption by the sensitizer. Charge separation is neces-sary to maintain the electron cycle, but in artificial systems this process is often very slow, so the system does notfunction properly. Charge recombination is in most current systems for artificial photosynthesis a very fast process.However, it lowers the efficiency of the system dramatically, because electrons cannot be passed to the electrode, sono electron cycle is generated and thus no fuel can be produced. Inefficient light absorption by the sensitizer may becaused by the fact that the complex can only absorb a small range of wavelengths. As a result of this, the quantumefficiency (ratio of used photons to the total photon incidence) becomes very low. A lot of research is done to solvethese problems. For commercial applications of artificial photosynthesis, a robust system has to be used and the cur-rent systems are not stable enough. Next to that, more efficient linkage of the components of the system is required [5].

Some examples to overcome these hurdles in artificial photosynthesis have been explored. For instance Yamamoto etal. [6] produced a system in which a sensitizer and a WOC were covalently linked with a pyridylmethyl-substituteddialkoxybiphenyl bridge. According to Yamamoto et al. the bridge would suppress charge recombination and thussolve one of the problems of designing an artificial system for water splitting. A schematic representation of thesystem produced by Yamamoto et al. is shown in figure 2.

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Figure 2: Schematic figure of visible light-driven water oxidation using wide-bandgap TiO2 functionalised with aruthenium water oxidation catalyst–porphyrin linked dyad as the photoanode [6].

3.2 Thermodynamic and Kinetic Conditions for Artificial Photosynthesis

To use artificial systems for studying specific processes of water splitting, it is necessary to make sure every electrontransfer occurs readily and quickly. Thus the thermodynamics and kinetics of such a system are key, as is preventingcharge recombination. To achieve a favourable thermodynamic system, there must be a nett loss of Gibbs free energyin the system, as stated by the second law of thermodynamics. For reactions involving electron transfers this can betranslated to the rule that the potential E, described by the Nernst equation (2), should be positive [7].

E = EΘ − RT

νFln(Q) (2)

In this equation, E stands for the reaction potential of the reaction in Volts, EΘ for the standard potential for thereaction in Volts, R for the gas constant, T for the absolute temperature in Kelvin, ν for the amount of transferredelectrons, F for the Faraday constant and Q for the reaction quotient. The standard potential for a reaction isdefined in equation (3) [7].

EΘ = −∆GΘr

νF(3)

In this equation ∆GΘr is the difference in Gibbs free reaction energy for the two states of the reaction. A larger

reaction potential equals a more thermodynamically favourable reaction.

For water splitting reactions, the standard electron potential is 1.23 Volts [7]. To transfer the electrons that areproduced during this reaction, a sensitizer should be chosen that has a potential higher than 1.23 Volts. This wouldresult in a positive reaction for the total reaction in standard conditions. To increase the efficiency of the system,the right balance between thermodynamic and kinetic characteristics is needed. Thus, the potential should not betoo high, as that would cause kinetic problems due to the fact that the kinetics and thermodynamics are entwinedin electron transfer as stated by the Marcus theory [8]. Another problem with a low potential of the sensitizer is thatthe sensitizer may not be able to lose its electrons after excitation, as the excited potential is also lower. This wouldcause fast charge recombination and thus a lower efficiency.

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3.3 Oxygen Evolving Center in Photosystem II

Artificial photosynthesis is based on natural photosynthesis, the process in which plants, algae and cyanobacteriause sunlight to create sugars and oxygen from water and carbon dioxide. The water splitting process is part of thelight-dependent reactions, in which eventually the energy-carrying molecules NADPH (from NADP+) and ATP areformed, as showed in figure 3.

Figure 3: Schematic representation of the light-dependent reactions in photosynthesis [9].

A central process in these reactions is the transfer of electronsand energy through a protein cascade in the thylakoid membranes.A catalytic site in photosystem II (PSII) delivers the electrons.This catalytic site, called the Oxygen Evolving Center (OEC),splits water into oxygen, protons and electrons by using light en-ergy. Oxygen and protons are released into the thylakoid lu-men, while the electrons are brought to P680, a chlorophyl-basedreaction centre of PSII, via a tyrosine residue. The oxidizedform of P680 acts as the primary oxidant and accepts an elec-tron originating from the water molecule. The electron is thenexcited by light energy, but this higher energy level is unstable,so the electron is released and P680 gets oxidized again. Thefreed electron enters then a transport chain towards photosystemI. [10].

Figure 4: S-state cycle of photosynthetic water oxidation, showingthe optimized geometry, protonation pattern, and Mn oxidationstates of the inorganic core.Two forms exist in the S2 state, anopen cubane (S2A) and a closed cubane (S2B) structure [10].

As stated above, the OEC provides the elec-trons for this transport chain. It is a manganese-calcium cluster, consisting of four manganeseatoms, one calcium atom and five oxygenatoms. The calcium atom and three man-ganese atoms form a cube, while the fourthmanganese dangles outside this structure. Theoxygen atoms form bridges between the fivemetal atoms. The whole structure of the clusteris asymmetrical, because the interatomic bondlengths vary. During the water splitting pro-cess, the OEC undergoes structural changes,as is shown in figure 4. The cluster goesthrough five different transition states, calledSi states with i = 0-4 for the number of elec-trons released during the cycle. In each tran-sition from S0 to S4 an electron is transferredto the tyrosine after which the OEC changesback into the S0 state to complete the cy-cle [10].

Since complex is part of PSII several amino acids of PSII are directly ligated to the cluster, which are shown in figure5. These ligands stabilize the oxidation states of the OEC. Each manganese atom is linked with six ligands, while thecalcium atom is ligated by seven ligands in the first coordination sphere. These are two monodentate ligands (D1-His332 and D1-Glu189) and five carboxylate bidentate ligands (D1- Asp170, D1-Glu333, D1-Asp342, D1-Ala344 andCP43-Glu354). In this nomenclature D1 stands for the reaction subunit of PSII in which these ligands are presentand CP43 is one of the core antenna subunits of PSII [11]. To release electrons from water, four water moleculesare ligated to the OEC. Next to that oxo-bridges contribute to the stability of the oxidation states as well. Whichligands are linked to which atom exactly, is shown in table 2 in the appendix, as well as the interactomic distancesin the OEC itself. In the second coordination sphere some ligands are indirectly linked with the OEC. The ligandsD1-Asp61, D1-His337 and CP43-Arg357 form hydrogen bonds with the oxo-bridges of the first coordination sphere,influencing the surroundings of the OEC [12].

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Figure 5: The structure of the OEC and its surroundings obtained by using an X-ray free electron laser (XFEL) andsynchroton radiation (SR). (a) Structure of the OEC with the ligated water molecules (W), (b) Natural OEC withits ligand environment [12].

3.4 Artificial Oxygen Evolving Center

Recently, Zhang et al. reported a synthetic Mn4Ca-cluster mimicking the natural OEC in metal-oxygen core struc-ture and in binding ligands [1]. They produced a complex from inexpensive commercial chemicals. The team usedseveral techniques, including Cyclic Voltammetry (CV) and Electron Paramagnetic Resonance (EPR) spectroscopyto determine the properties of their synthetic cluster. The cyclic voltammogram shows similarties with that of thenative OEC. The most important result is that five redox-states can be accessed. This means that the five S-statesdescribed before can be accessed, just as in the natural OEC [1]. This is essential for the four-electron process withwhich water can be split. The fact that these states can be accessed by the OEC, without the full PSII surroundingit, is an important aspect.

Although the two complexes are very similar, there are a few notable structural differences (see figure (6)). Firstly,in the artificial complex O4 is missing, it is replaced by a carboxylate group. Secondly, the terminally coordinatedwater molecules (outside the core) are replaced by more complex groups. And finally, but most importantly, thecoordination of O5 to Mn3/4 is much shorter in the artificial OEC than in the natural OEC. In the native complexO5 is located far from the core, which creates the possibility to form five bonds instead of four. This allows a fifthcoordination site to be open which, in the natural OEC, is used to bind a substrate water molecule. This differenceprobably emerges from the fact that in the native OEC O5 is held in place by several amino acids, whereas in theartificial version only one carboxylate group holds the oxygen in place. This makes it possible for O5 to move closerto the center, thereby blocking its fifth coordination spot [1].

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Figure 6: Crystal structure of the native OEC and the synthetic Mn4Ca complex in S1 state. (A) Natural catalystmetal-oxygen core, (B) Artificial complex metal-oxygen core, (C) Natural catalyst, including ligating protein side-chains and water molecules, (D) Artificial complex including all ligand groups, (E) Synthesis of the artificial complex.All distances are given in A [1].

Even though this artificial OEC is similar to the natural complex in many ways, it deviates in several importantaspects. Nevertheless, the artificial OEC has a very high synthetic flexibility in the surrounding ligands that has notbeen explored previously [1]. By alternating the surrounding ligands it would be possible to allow water binding tooccur and to create a useful water splitting catalyst based on the synthetic Mn4Ca-cluster.

3.5 Housing of Artificial Catalysts

Artificial catalysts on their own are not capable of functioning as well as their natural counterparts, as the environ-ment in which they need to work is not optimal. Improving the environment of an artificial catalyst and creatingthe perfect surroundings is therefore crucial for optimizing the catalytic activity and understanding the workings ofthe natural counterpart.

In this study, we aim to use MnL2n nanospheres to study the influence of ligand orientation on the working mechanismof the synthetic Mn4Ca-cluster [1]. These nanospheres are self-assembling complexes built of metal ions (M) whichare coordinated with multidentate ligands (L). These ligands are able to bind two or more metal ions and thus areable to form a network between metal ions. The n in the formula is 6, 12, 24, 30 or 60 and represents the number ofmolecules. The size and shape of these complexes is dependent on n and the schematic forms of the complexes arevisualized in figure 7 [13].

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Figure 7: Schematic representation of the different forms and sizes the nanospheres can have. Below each structurethe nummer of metal ions (M) and ligands (L) is stated [14].

Platinum ions (Pt2+) and palladium ions (Pd2+) are often used for formation of a nanosphere, because they bothhave a square planar coordination chemistry. This means that the metal atom is surrounded by four ligands, whichare in a plane. The ligands which are used are bent, so that they are able to form a spherical structure by coordi-nating to different metal ions. The angle of the bent ligand influences the size and shape of the sphere [13].

It is possible to synthesize complexes which consist of two different ligands [13]. Sun et al. [14] found that the size ofthese mixed-ligand spheres depends on the ratio of the used ligands. Either only M12L24 complexes or only M24L48

complexes were formed during their experiments. By using mixed-ligand spheres asymmetric complexes can beformed. This is interesting for our research, as the synthetic OEC is asymmetric as well. Thus the complex mightfit better in an asymmetric sphere. However, a statistical mixture of complexes with different ligand-ratios will beformed, so structural information is lost.

An interesting feature of nanospheres is the possibility to functionalise them. This can be done either on the outside(exohedral) or on the inside (endohedral) by linking functional groups to the bending ring of the ligand, as showedin figure 8. For example, oligopeptides, porphyrins, oligosaccharides and oligonucleotides can be attached [13] [15].The functionalised cores can be used to encapsulate molecules. Because the linked molecules are close together ina nanosphere, the effect of weak interactions on the substrate is enhanced by cooperativity. Most research towardsfunctionalising nanospheres is done with M12L24 complexes, which have a diameter between 3.5 and 5.2 nanome-ters [13]. Suzuki et al. [15] already managed to decorate these kind of complexes with 24 to 96 amino acid residues in2007. Since the synthetic OEC has only a size of some Angstroms, we expect that it can fit within a cage which isfunctionalised with amino acids. Suzuki et al. also state that peptide-linked complexes can be used as a housing forartificial enzymes [15]. Next to that, Wang et al. [16] have reported a way to encapsulate catalysts and substrates ina M12L24 nanosphere.

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Figure 8: In figure (a) and (b) a schematic representation of the addition of functional groups to ligands is shown,at the left site of the arrow. In figure (a) exohedral functionalisation is visualized. Figure (b) shows endohedralfunctionalisation. The nanospheres which are formed from these ligands are shown at the right sight of the arrow.In figure (c) different examples of endohedral functionalisation possibilities are shown [13].

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4 Aims and Objectives of the Research

4.1 Social Relevance

Growing CO2 emissions and decreasing availability of fossil fuels are growing problems in the modern world. Becauseof this, the quest for cheap, efficient and renewable energy sources has been a main topic of research in the lastcouple of years. One of the biggest possibilities to replace fossil fuels lies in molecular fuels, especially molecularhydrogen, produced by using solar energy. System designs range from aqueous photocatalyst colloids to commercialphotovoltaic modules connected to electrolysers. A whole scale of integrated photo-electrochemical (PEC) devices liesbetween these extremes [17]. We aim to contribute to this field of research with our research, as the gained knowledgecan be used as a starting point for designing useful, highly efficient water splitting complexes in the future.

4.2 Aim and Objectives

In this research we aim to understand the effects of the surrounding ligands on the synthetic OEC, reported byZhang et al. [1]. We will study the effects on the structural and electrochemical properties, influencing the potentialof the complex to split water. By studying this, we endeavour to gain a better understanding of its workings andcontribute to finding scalable and efficient sources of molecular fuels using solar energy in the future. We proposeto do this by modifying the ligands of the complex by using an amino acid functionalised MnL2n nanosphere. In asystematic plug-and-play approach we will alternate the surrounding ligands of the sphere in their nature and length.To carry out the research, our first objective is to synthesize a nanosphere containing the artificial OEC. Secondly,we need to create a functional system, consisting of the encapsulated OEC, a sensitizer and an electrode. Our thirdobjective is to alternate the surrounding ligands of the OEC and study the effects of the variations on the workingsof the complex.

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5 Research Question and Goals

Much research has been done towards the synthesis of biofuels making use of artificial photosynthetic componentswhich are derived from natural systems. One of the essential components of natural photosynthesis is the OECand understanding its functions will allow for a better design of artificial systems for photosynthesis. We proposeto study the functioning of the OEC by using the artificial complex as reported by Zhang et al. [1]. In a personalcorrespondance, Zhang stated that his artificial OEC needs a housing which creates the right environment for it tobe an efficient-working WOC.

Our primary research question is:

What are the effects of the orientation of the surrounding ligands on the water splitting potential ofthe artificial OEC, reported by Zhang et al. [1]?

To answer this primary question, we set out several goals:

G1: Create a nanosphere which is able to host the artificial OEC, as reported by Zhang et al. [1].

We suggest to use a M12L24 cuboctahedral nanosphere as a basis to create tha housing for the synthetic OEC. Thisnanosphere can be endohedrally functionalised by linking amino acids with acidic residue groups (like aspartic acid(Asp) or glutamic acid (Glu)) or oligopeptides ending with these amino acids to the ligands which form the sphere.We initially prepare the synthetic OEC which we will add to the solution in which the nanosphere will be formed.We expect the carboxylic acid groups surrounding the synthetic OEC to be replaced by the carboxylic acid residuesof the amino acids functionalising the sphere, as a result of cooperative interactions. EPR will be used to comparethe synthesized OEC with the artificial OEC as reported by Zhang et al. [1]. To verify the encapsulation of the OECin the nanosphere we will use Cold-Spray Ionisation Mass Spectrometry (CSI-MS), Nuclear Magnetic Resonance(NMR) spectroscopy and X-ray diffraction.The electrochemical properties are explored by CV.

G2: Create a functioning system, containing the encapsulated artificial OEC, a sensitizer and an electrode, andanalyse this system.

In order to study the influence of the environment on the functioning of the OEC, we must create a basic, functionalsystem first. This system should at least contain a WOC, a sensitizer and an electrode. We propose to use theencapsulated OEC as WOC, a Ru(bpy)3 complex as sensitizer and a TiO2 electrode, as the function and propertiesof these are well known as a result of studies in the past [18]. The functioning of the system can be tested by using CV todetermine the redox potentials of the system and by measuring the turnover number of oxygen production. We will useNMR spectroscopy, Diffusion Ordered Spectroscopy (DOSY-) NMR, Ultraviolet-Visible (UV-Vis) spectroscopy, Cold-Spray Ionisation Mass Spectrometry (CSI-MS) and X-ray diffraction for the structural analysis of the nanospherecomplex, after its functioning has been shown.

G3: Use a plug-and-play approach to systematically alternate the functionalisation of the nanosphere, therebychanging the environment of the OEC.

To determine the influence of the environment on the water splitting potential of the OEC, several ways of changingthe functionalisation of the nanosphere are possible. Firstly, we suggest to change the amino acids which are directlycoordinating the OEC. Secondly, we will alter the size of the cavity in which the OEC is encapsulated. Thirdly,if oligopeptides are used, we will also change the amino acids further away from the OEC. Finally, we can use amixture of differently functionalised ligands. We propose to use a plug-and-play method to explore the optimal ligandenvironment in a fast and efficient manner. Using software that is able to optimize packaging of metal-complexes inprotein cavities might provide an additional tool to find optimal ligand coordination. For this purpose the RosettaVIPsoftware could be useful. We will test all synthesized complexes for functionality and characteristics similar to theanalysis of the basic system.

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6 Plan of Work

6.1 G1: Create a nanosphere which is able to host the artificial OEC, as reported byZhang et al. [1]

The first step of our research is creating the nanospheres, including the functionalisations, which host the artifi-cial OEC. Firstly, we synthesize the OEC, as described by Zhang et al. [1]. The produced OEC is surrounded bycarboxylic acid ligands and a pyridine group. Secondly, we create the cages. The M12L24 nanospheres consist of2,6-bis(4-pyridylethynyl)-aniline ligands, which constitute the edges of the polyhedra, and palladium ions (Pd2+),which constitute the vertices of the structure. The synthesis of this structure is worked out in the appendix (figure12). We will functionalise the edge ligands endohedrally by attaching amino acids to the benzene core via an ethanoicacid linker, which is shown in the appendix (figure 13). Condensation reactions will allow for the N-terminus to forma peptide bond with the ligand. An example of how such a cage looks like can be found in figure 9a.

For the basic system, we propose to use only one type of amino acid, which will ultimately form a symmetricnanosphere. We will use an amino acid with a side chain containing a carboxylic acid, Asp and Glu are thereforeboth likely candidates (figure 9b). We have two reasons for this decision. Firstly, the natural OEC is linked to severalAsp and Glu amino acids of PSII. Secondly, the manganese core of the synthetic OEC is coordinated with carboxylicacids.

We add the synthesized OEC to the reaction mixture before the ligands are added, so that it can be taken up duringformation of the nanosphere. This formation will take place in acetonitril (CH3CN), the same solvent as used in theproduction process of the artificial OEC [1].

Figure 9: In figure (a) a schematic representation of a nanosphere with 1,3-di(4-pyridyl)benzene ligands and Pd2+ isshown [19]. The cage proposed for encapsulation of the artificial OEC also contains alkyn linkers between the pyridyl-and the benzene rings which will lengthen the edges of the cage. These are not shown in the figure. In figure (b) achemical representation of the amino acids Asp and Glu, which have carboxylic acid residues (in blue). These canbe used for endohedral functionalisation of the “start cage” and will be linked to the ligands via the amine-group(indicated with the red circle)

One potential issue is the coordination of the artificial OEC in the nanosphere. If, after analysis with MS, IRspectroscopy and X-ray diffraction, we find the OEC not to be encapsulated by the nanosphere, we will follow adifferent approach. We will attach the pyridine ligand of the OEC to 1

24 of the nanosphere ligands (see figure 10).

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Because of the large size of the OEC, we expect only one of these ligands to be incorporated into each nanosphere [20].

Figure 10: Schematic representation of the production of the nanospheres, where one out of 24 ligands is functionalisedwith the artificial OEC. Metal ions (M), in this case Pd2+, are added to the functionalised ligands to create the cages.

6.1.1 Verification Methods

We will analyse the synthesized OEC and nanosphere by a variety of techniques and compare it with reporteddata. We verify the synthesis of the OEC by using Electron Paramagnetic Resonance (EPR) spectroscopy, elementalanalysis, X-ray diffraction, Cyclic Voltammetry (CV), UV-Vis spectroscopy and Fourier Transform Infrared (FTIR)spectroscopy. The data obtained by EPR and CV function as ‘fingerprints’, while the other data provide directinformation about the structural characteristics of the synthesized complex. To verify the functionalisations of thenanosphere ligands, we will use mass spectrometry (MS), infrared (IR) spectroscopy and Nuclear Magnetic Reso-nance (NMR) spectroscopy. We will use Cold Spray Ionisation (CSI-) MS, NMR spectroscopy and X-ray diffractionto verify the encapsulation of the OEC in the nanosphere. The techniques used are listed below and explained inmore detail for clarity.

• EPR is a spectroscopy technique. It is used to study complexes which contain d-block elements, like man-ganese [21]. The results from the EPR-measurement give a fingerprint of the complex. By comparing the EPRdata of the complex we have synthesized with the results of Zhang et al. [1], we will be able to verify if we havesynthesized the same complex.

• CV provides information about the redox potentials of the synthesized complex. This technique is used withcompounds that have electron transfer processes that are reversible, for example the artificial OEC. Using thistechnique, we can determine if the synthesized complex is redox active and how many electrons are involved inits electron transfer process. CV involves three electrodes: a working, counter and reference electrode. Theseare submerged in an acetonitril electrolyte solution containing the synthesized compound. A voltage is appliedto the working electrode (with respect to the reference electrode). This voltage is then linearly increased, andthen decreased with a rate of 100 mV/s. When it is increased, the compounds around the working electrodewill be oxidized and current response is passed from the working electrode to the counter electrode. Severaloxidation steps might be accessed. Decreasing the potential will reverse the process. The current response withrespect to the applied voltage can be plotted and gives information about the redox states of the compound [22].To verify synthesis of the OEC we suggest to use this technique, as the results can be compared with those ofZhang et al. [1].

• Elemental analysis is a method of determining the relative proportions of the elements present in a com-pound [23]. We will use this technique to test if the right synthetic OEC is formed by comparing the results tothose of Zhang et al. [1].

• X-ray diffraction can be used in different ways. By using Serial Femtosecond Crystallography (SFX) highresolution structures can be determined [24]. X-ray Free-Electron Laser (XFEL) techniques can be used to studycrystallographic characteristics of the produced complexes at room temperature. A great advantage of using

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XFEL is that this technique is able to measure radiation-sensitive complexes, like the OEC, while they are notdamaged yet by the X-ray, which may induce structural changes. This way of acting is called the “probe beforedestroy” approach. This approach is possible because the X-ray pulses have a very high intensity, but are veryshort (femtoseconds) [25]. Single crystal synchrotron X-ray diffraction can be used to determine the nanospherestructure [13], as this technique provides information about bondlenghts and coordination of the atoms of themeasured crystal [26].

• UV-Vis spectroscopy will be used to determine the absorption pattern and absorption intensity of the OECand nanosphere. Photons at specific wavelengths are absorbed by the sample and cause electronic transitions inthe compound, which are specific for a compound. The non-absorbed light will be detected and compared witha control sample to make a dataset [27].The absorption intensity later gives the possibility to tell if the OECabsorbs photons more effectively when used in this cage than in another, when compared to both compounds(cage and OEC) separately [15] [27].

• IR spectroscopy gives insight in the bonds which are present in the molecule. The sample is bombarded withIR light of different frequencies. When a bond is excited with exactly the frequency of its own vibration, theIR light is absorbed [13]. IR will be used to determine the vibrational properties of the functionalised ligands,which will be used for nanosphere formation. FTIR spectroscopy is an improved version of the IR technique. Itscans all frequencies simultaneously, which gives it a higher sensibility [23]. FTIR helps to determine the shapeof the solid OEC by measuring vibrational properties of the complex in KBr pellets. The spectra acquired byIR measurements give information about the chemical bonds which are present in the synthetic OEC [21].

• MS allows for determination of the mass and structure of a complex. For MS the sample is vaporized andionized. After ionization the charged fragments are accelerated between to negatively charged plates. Afterthat the fragments are brought into the analyzer tube, in which a magnetic field is present. This field in-fluences the path of the fragment, depending on the mass-to-charge ratio of the fragment. We will use ColdSpray Ionization (CSI-) MS, a special form of MS. CSI-MS is very suited for measuring the properties of rel-atively labile organometallic species. [28] It uses a special method for ionisation of the sample. According toYamaguchi [28] CSI-MS allows “simple and precise characterization of labile non-covalent complexes that aredifficult or impossible to observe by conventional MS techniques”. Since the nanospheres we propose to use arebuilt via non-covalent interactions, we think CSI-MS is the best way to gain structural information about thenanosphere [13].

• NMR spectroscopy is a technique which helps to identify the structure of organic compounds. 1H-NMRand 13C-NMR are both suitable for verifying the linkage of the functionalisations to the ligands. We will useDOSY-NMR spectroscopy to determine the size of the nanosphere [13].

6.2 G2: Create a functioning system, containing the encapsulated artificial OEC, asensitizer and an electrode, and analyse this system.

After we have produced nanospheres containing the artificial OEC, we will create a functioning system for watersplitting. To do this, we add the sensitizers, nanospheres and an electrode to an acetonitril/water solution, as theOEC needs water to function and is relatively stable in acetonitril. As mentioned before, we use Ru(bpy)3 as asensitizer. The reason for this choice is that this ruthenium complex has a oxidation potential that is high enoughto oxidize the OEC. As for this purpose a potential of 1.23 Volts is needed [7]. The Ru(bpy)3 has a potential of1.29 Volts and thus is capable of doing this [29]. This sensitizer is commercially available. For the anode we usea TiO2 electrode. For our purpose, what happens to the electrons is not interesting, but because there should bea closed circuit, they do need to go somewhere. For this we direct the electron flow to ground. Once the entiresystem has been assembled, we will determine its functionality. We do this by measuring the the turnover number,by determining the amount of water molecules which is converted. This can be derived from the evolution of oxygenmolecules, by using an oxygen electrode.

If the built system is not functional, we must make changes to the basic system. There are three reasons whya system may not be functional. Firstly, the OEC does not split water. In this case, we have to change the

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nanosphere endohedrally to influence the workings of the OEC, in order to make a functional system. Secondly, thechosen sensitizer does not accept electrons from the OEC. This may be caused by thermodynamic or kinetic reasons.To solve this, we choose another sensitizer to substitute the Ru(bpy)3. Lastly, the TiO2 cannot accept the electronsfrom the excited sensitizer. We can solve this problem by changing the type of electrode or using an electrolyteinstead.

6.3 G3: Use a plug-and-play approach to systematically alternate the functionalisa-tion of the nanosphere, thereby changing the environment of the OEC.

Once we have managed to develop a working basic system, we can alter the cage to find a surrounding that gives abetter result. Several ways of changing the functionalisation of the nanosphere are possible.

Firstly, we suggest to change the amino acids which are closest to the OEC. For example, Asp and Glu can be ex-changed to study the effect of different acidic amino acids. Another possibility is to use amino acids which containedalcohol groups, like serine, threonine and tyrosine, as this functional groups may influence the functionality of theOEC. The natural OEC is, apart from acidic groups, also surrounded by histidines, so introducing this amino acidis also an option.

Secondly, the size of the cavity in which the OEC is encapsulated can be altered. This can be done by changing thelength of the functionalising oligopeptides, for example by linking glycine (Gly) to the chain or by adding linkersto the ligands. We suggest to use Gly because this is a flexible amino acid which has no functional groups in itsresidue. About one hundred amino acid residues can fit in the cage when using the ligands we will use [15]. Thisof course does depend on which amino acid residue is used. A possible advantage of a longer peptide is the factthat this will give the peptide more flexibility (a short stick is less flexible than a long, bendable string). This inturn allows the cavity to be more asymmetric, something we believe will be beneficial for the, also asymmetric,OEC. In the other method spacer molecules can also be added to the ligands, effectively enlarging (or shrinking)the entire cage by lengthening its edges. This allows the diameter of the cage to vary from around 3.5 nm to 5.2 nm. [13]

Thirdly, if oligopeptides are used, the amino acids further away from the OEC can be varied too. These might notcoordinate directly to the OEC, but can influence the cavity in beneficial ways, for example by changing the polarity.

Finally, a mixture of differently functionalised ligands can be used. In this case a statistical mixture of nanosphereswill be synthesized. We expect that using a mixture of differently functionalised ligands will give asymmetric spheres,which is most likely favourable, as the OEC itself is also asymmetrical. Because of the statistical mix no structuralinformation can be gathered from these systems, there are simply too many different types of spheres. However,qualitative information might be gathered from this approach, because something can be said about the effects ofcombining different oligopeptides, even if it is not possible to find beneficial ratios and distributions in a single cage.

We propose to use a plug-and-play method for modifying the cages. Using the RosettaVIP software could be useful,as this software is able to optimize packaging of metal-complexes in protein cavities. To be able to use this program,the data derived from X-ray diffraction of the starting nanosphere have to be saved in a PDB coordinate entry file.The program runs an algorithm, which can predict mutations which improve the packaging of the core. It is expectedthat it also works for improving the peptide groups linked to the cage [30]. We think this program may extend thepossibilities for optimalisation of the nanosphere for the OEC. All synthesized complexes will have to be tested forfunctionality and characteristics similar to analysis of the basic system.

To determine the structural and functional properties of all the systems, we will use the same techniques as used inthe first goal. DOSY-NMR, NMR, CSI-MS, UV-Vis and single crystal synchrotron X-ray diffraction can be used todetermine the structure of the complexes. We will use CV to determine the redox properties of the systems. We willuse the turnover number to determine the functionality of the system. To do this final measurement all complexesneed to be part of the complete system, including the sensitizer and anode. By comparing the properties in the newsystem with the basic system and looking at the functionality of the new system, the properties which are importantfor the functionality can be found.

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7 Timetable

In order to answer the research question, three goals were formulated. The planning of these three goals is given inthe folowing timetable. The total duration is four years which have been split into 16 periods of three months.

Table 1: This is timetable for the project. Each period is three months long.

Figure 11: This is a colour-wise representation of the mentioned timetable

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8 Statements by the Applicants

By submitting this document we declare that we satisfy the nationally and internationally accepted standards forscientific conduct as stated in the Netherlands Code of Conduct for Scientific Practice 2012 (Association of Universitiesin the Netherlands (VSNU)).

9 Acknowledgements

We would like to give our thanks to Dr. Chunxi Zhang for his email correspondence which helped us to furtherdevelop this idea. Our words of thanks also go to the Durrant group (Imperial College London), in particular Dr.Laia Francas Forcada and Madeleine Morris, for allowing us to have a look in their labs and discussing our researchproposal. Furthermore, we would like to thank Prof. Bill Rutherford, Dr. Andrea Fantuzzi, Prof. Peter Nixon,Dr. James Murray and their groups for their guidance and insights on our idea. Their remarks and out-of-the-boxthinking during our visit to their groups at Imperial College inspired us while making this proposal. We also giveour gratitude to Dr. Martin Feiters, Dr. Jan Keltjens and Dr. Gert Vriend for filling in the gaps in our knowledgeregarding encapsulation of metal complexes, artificial water splitting and bioinformatics respectively. Last, but notleast, we would like to thank our supervisor Dr. Kim Bonger for her guidance, enthusiasm and assistance the pastyear and the Radboud Honours Academy for giving us the opportunity to challenge ourselves with this excellenceprogramme.

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10 Appendix

Table 2: Interatomic distances in the natural OEC obtained using XFEL and SR techniques [12]

Figure 12: Reaction mechanism for the synthesis of the ligand, based on the synthesis of Suzuki et al. [15]

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Figure 13: Reaction mechanism for the coupling of the aminoacids to the ligand. The spheres represent the aminoacids coupled via the principle shown

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