Self-assembly followed by oxidation of a new cobalt supramolecular cage

Beau Noafshar5M Research ProjectLusby Group

Submitted Friday 3/5/15

Abstract

1

A new ligand system has been synthesised and self-assembled with a cobalt(II) precursor into

tetrahedral supramolecular cage C2. The 1,4-bis(6-(1-methyl-1,3-benzimidazolyl)pyridin-3-yl)benzene

ligand (L2) is devoid of the acidic proton which was thought to make similar cage CA (assembled from LA

=1,4-bis(2-(1-(2-(2-methoxyethoxy)ethyl)-1H-1,2,3-triazol-4-yl)pyridin-3-yl)benzene) unstable at neutral

pH. Stability to body-like conditions is essential for supramolecular function as a potential drug delivery

vehicle. Even in the absence of this proton, decomposition occurred over the course of 10 days.

Comparison with a similar system CC (assembled from LC = 1,4-bis(2,2’-bipyridyl)benzene) suggests

strained geometry at the cobalt metal centres makes the C2 structure susceptible to attack from base.

C2 was confirmed structurally by mass spectrometry and x-ray crystallography experiments. Counter ion

metathesis gave water-soluble species C2∙12NO3. C2∙12NO3 is able to encapsulate triisopropylsilanol in

slow-exchange (Ka = 280 M-1), as well as camphor, chromone, 2-adamantanone and nitrobenzene in fast-

exchange. Scrambling experiments with CA∙12NO3 showed C2∙12NO3 to exhibit some substitutional

lability at 70°C, a result comparable with similar systems.

Whilst C2∙12NO3 still has potential as a catalyst, it’s instability to basic conditions makes it unsuitable as

a drug delivery vehicle. The result indicates structures with ideal geometry may be better suited to give

stable and therefore functional supramolecular assemblies.

Table of Contents

2

Abstract 2Abbreviations 5

1. Introduction 7 1.1 Overview 7 1.2 Functional systems: catalysis 12 1.3 Functional systems: drug delivery 19 1.4 Lusby group previous work 24 1.5 Project aims 31

2. Experimental 33 2.1 General information 33 2.2 Attempted L1 synthesis 34

2.3 L2 synthesis 38 2.4 C2 synthesis 41 2.5 C2 experiments 44

3. Results 45 3.1 NMR experiments 45 3.3 Mass spectrometry 46 3.4 Scrambling Experiments 49 3.5 Buffer solution experiments 50 3.6 X-ray crystallography 51 3.7 Guest binding experiments 52

4. Discussion 55 4.1 Synthesis of L1 design 55

4.2 Synthesis of L2 design 574.3Evidence for C2 structure 59

4.4 C2 stability 64 4.5 C2 host-guest chemistry 71 4.6 Summary and future work 76

5. Conclusion …77

3

Acknowledgements78References 79

Abbreviations

Five supramolecular cages (C), five supramolecular helicates (H) and five corresponding ligand systems

(L) which have been synthesised in Lusby group labs are discussed in this report. Those made by other

workers are labelled alphabetically whilst those made or hypothesized as part of this writer’s project are

4

labelled numerically. The single exception to this is helicate H2 which was synthesized by another

member of the group. Fragments (F) have either been bought or synthesized.

FA - 1-azido-2-(2-methoxyethoxy)ethane

LA - 1,4-bis(2-(1-(2-(2-methoxyethoxy)ethyl)-1H-1,2,3-triazol-4-yl)pyridin-3-yl)benzene

CA - [Co4(1,4-bis(2-(1-(2-(2-methoxyethoxy)ethyl)-1H-1,2,3-triazol-4-yl)pyridin-3-yl)benzene )6]12+

HA - [Co2(1,4-bis(2-(1-(2-(2-methoxyethoxy)ethyl)-1H-1,2,3-triazol-4-yl)pyridin-3-yl)benzene )3]6+

FB - 1-adamantanemethylazide

LB - 1,4-bis(1-adamantanemethyl)-1H-1,2,3-triazol-4-yl)pyridin-3-yl)benzene

CB - [Co4(1,4-bis(1-adamantanemethyl)-1H-1,2,3-triazol-4-yl)pyridin-3-yl)benzene)6]12+

HB - [Co2(1,4-bis(1-adamantanemethyl)-1H-1,2,3-triazol-4-yl)pyridin-3-yl)benzene)3]6+

Fc - 4-bromo-2,2’-bypyridine

LC - 1,4-bis(2,2’-bipyridyl)benzene

CC - [Co4(1,4-bis(2,2’-bipyridyl)benzene)]12+

F1a – 5-bromo-2-pyridinecarbonitrile

F1b - 2-[6-(5-bromopyridin-2-yl)-1,2,4,5-tetrazin-3-yl]ethan-1-ol

F1c - 1,4-bis(6-ethynylpyridin-3yl)benzene

L1 - 1,4-bis(2-[6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl]ethan-1-ol)benzene (proposed structure)

C1 - [Co4(1,4-bis(2-[6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl]ethan-1-ol)benzene)6]12+ (proposed structure)

F2a - 5-bromo-2-pyridinecarboxylic acid

5

F2b - 2-(5-bromopyridin-2-yl)-1-methyl-1,3-benzimidazole

L2 - 1,4-bis(6-(1-methyl-1,3-benzimidazolyl)pyridin-3-yl)benzene

C2 - [Co4(1,4-bis(6-(1-methyl-1,3-benzimidazolyl)pyridin-3-yl)benzene)6]12+

H2 - [Co2(1,4-bis(6-(1-methyl-1,3-benzimidazolyl)pyridin-3-yl)benzene)3]6+

C∙12NO3 – supramolecular cage surrounded by 12 NO3- counter ions

C∙12PF6 – supramolecular cage surrounded by 12 PF6- counter ions

Other abbreviations

CAN – (NH4)2Ce(NO3)6 - cerium ammonium nitrate

n-ESI-MS – nano-electrospray ionization- mass spectroscopy

TBAF – tetrabutylammonium flouride

1. Introduction

1.1 Overview

Supramolecular chemistry is the logical advancement from our now strong understanding of molecular

covalent bonding. Using intermolecular interactions, researchers have been able to consider discrete

compounds as a fresh set of Lego blocks. As the field’s collective grasp of the way the blocks interact,

6

cooperate and self-assemble has progressed, the number of new structures created has grown

exponentially. Advancements have allowed for the synthesis of a variety (fig. I1) 1 of large, elaborate and

functional assemblies. Initial applicable examples prove promising but as with the beginnings of covalent

chemistry we trail behind the natural world.1, 2 Biological systems already highlight the potential of

supramolecular structures for applications such as catalysis and photosynthesis.1, 2, 3 The current aim of

the field is to mimic and thereby understand these arrangements with the hope of someday surpassing

them. Just as covalent chemistry has branched extensively, it is probable that successful utilization of

supramolecular species will soon permeate many aspects of society.

Fig. I1: Possible shapes of 2D and 3D supramolecular assemblies. Reproduced from reference 1.

7

Fig. I2: Fujita’s [Pd4(4,4’-bipyridine)4(en)4]8+ supramolecular square (1).11

1.1.1 Metallomacrocycles

Very often supramolecular structures incorporate metal centres into these systems.1This is because we

can comprehensively predict the strength and directionality of a given centres bonding. Early examples

of supramolecular chemistry used simple metal geometries to create 2D architectures. There are a large

number of macrocyclic triangles4,5 and squares6,7 reported using either linear metal units as edges or

linear ligands and metal vertices. Stang in particular has reported numerous examples of supramolecular

squares which use cis-protected Pd(II) and Pt(II) vertices8,9,10. Fujita reported the first example of this

type in 199011; as with many examples in the field Fujita’s supramolecular square (1) was self-assembled.

Favourable M-L interactions between the 4,4’-bipyridine ligand and Pd(II) metal centres ((en)Pd(NO)2

precursor) resulted in the formation of 1 in ethanol. The formation of 1 is thermodynamically driven;

4,4’-bipyridine is a better ligand for Pd than the NO precursor ligand and fully satisfied geometries

between 4,4’-bipyridine and cis-protected Pd are only possible through the formation of the

supramolecular square 1. The relative weakness of the M-L bonding (when compared with average

covalent bond strength) aids this process. The bonds weak enough to break if formed in an incorrect

position during the self-assembly and then reform in a manner that builds towards the correct overall

8

structure. As a result 1 was self-assembled in 10 minutes. Whereas covalent synthesis of similarly sized

structures would be a lengthy process if at all feasible.

Fig. I3: Fujita’s Pd squares 2 a/b/c in equilibrium with corresponding Pd triangles 2 a/b/c.12

Later Fujita noted that supramolecular assemblies can still form in the absence of fully satisfied

geometry.12 When longer ligands were used by varying the X position with linkers a, b and c the

corresponding squares 2a, 2b and 2c still formed but they were found to be in equilibrium with

corresponding supramolecular triangle (3a, 3b and 3c). It was thought that increasing the length and

therefore the flexibility of the ligand allowed for deviation from linearity. This in turn reduces the strain

on the Pd centre on forming the triangle by retention of near 90° bond angles. This way the equilibrium

was seen to shift towards 3 as the length of X or flexibility was increased on going from 2a to 2b to 2c.

Whilst 2 is thermodynamically favoured, 3 is a smaller more numerous species than 1 and is therefore

entropically favoured. Multiple supramolecular structures of varying sizes are a common feature in

9

these types of system. It is the flexibility of the ligand that creates this slight distortion of the geometry

rules and the resulting new species.

Fig. I4: Saalfrank’s tetrahedral [Mg4(tetraethyl-2,3-dioxobutane-1,1,4,4-tetracarboxylato)6]4- cage (4).13

The rest of the ligands have been omitted for clarity, this is the case for all following tetrahedral cages.

1.1.2 3D coordination complexes

Flexible ligands gave rise to the serendipity that aided syntheses of the first 3D coordination based

supramolecular structures. Saalfrank and co-workers synthesized the first tetrahedral

metallosupramolecular cage 4 from a MgCl2 precursor and tetradentate ligand tetraethyl-2,3-

dioxobutane-1,1,4,4-tetracarboxylate.13

Fig. I5: Saalfrank’s tetrahedral [Fe4(tetraethyl-2,3-dioxobutane-1,1,4,4-tetracarboxylato)6] cage (5).14

10

The same workers later used a similar ligand and a Fe(II)Cl2 precursor to synthesise coordination

complex 5.14 5 was able to encapsulate small cations (such as NH4+, Na+, K+ and others) within the cavity

of the neutral cage. Recognition and encapsulation of the cations listed stems from the large size and

framework nature of 5. As the field contains many examples which also fit this description, selective

recognition and encapsulation are common functions of macrocycles and coordination complexes. Often

research groups have reported a new stable supramolecular species, then in the months or years after,

published the function of the cage as its activity is discovered.

11

1.2 Functional systems: catalysis

As seen with Saalfrank’s cage it is the cavity which gives rise to host-guest functionality. When

encapsulated by the respective cage small molecules can exhibit unusual chemistry. The reactivity of the

guest can be increased or decreased depending on the host-guest interactions, this feature has been

utilised for applications in catalysis.

1.2.1 Catalysis of the Nazarov cyclisation

Fig. I6: Raymond’s tetrahedral gallium cage [Ga4(N,N -bis(2,3-dihydroxybenzoyl)-1,5-′

diaminonaphthalene)6]12- (6).15

Raymond and co-workers synthesised a tetrahedral gallium cage 6 which was able to catalyse the

reaction of the dienol (7) to the respective pentadiene (8) (fig. I7).15This reaction, known as the Nazarov

cyclisation is commonly used in organic chemistry. The cage pre-organised the flexible substrate (8) e.g

the shape of the cavity effectively held it in a more reactive conformation. As the rearrangement

proceeded to the intermediate, the molecule was even better held by the cage. The shape of the

intermediate was more stable within the cavity, but more importantly the intermediate was a diallylic

carbocation. This positively charged species experienced an electrostatic attraction with the polyanionic

cage. Stabilisation of the intermediate rapidly accelerated the rate of the reaction up to 2.1x10 6 times

the uncatalysed reaction.

12

Fig. I7: Nazarov cyclisation catalyzed by 6 and subsequent Diels-Alder reaction to form 9.15

The product of the reaction (8) was bound more strongly than substrate 7 as the flexibility of the guest is

removed by formation of the cyclopentane ring system. However catalytic turnover cannot occur

without a vacant cavity, binding of the product caused inhibition of the cages function. This is common

where substrate and product are similar in structure. Raymond dealt with this by performing a second

reaction, addition of maleimide produced the Diels-Alder adduct of the product (fig. I7). This second

product (9) was now too large to act as a guest, the cavity was thereby vacated and catalytic turnover

resumed. Formation of the Diels-Alder adduct could however be viewed as a negative as the

cyclopentadiene was the desired product, and inhibition of the catalytic cycle was caused by trapping of

this product.

13

1.2.2 Fujita’s Pd bowl and cage

Fig I8: Fujitas Pd octahedron [Pd6(2,4,6-tris(pyridin-3-yl)-1,3,5-triazine)4]12+ (9) and Pd bowl [Pd6(2,4,6-

tris(pyridin-4-yl)-1,3,5-triazine)4]12+ (10). Reproduced from ref. 16.

Fujita and co-workers managed to resolve a somewhat similar problem with a different catalytic system.

The group self-assembled a Pd6L4 octahedron (9) as well as the Pd6L4 bowl shaped assembly from a

slightly different ligand.16 The cage was able to bind Diels-Alder reactants 11 (9-

hydroxymethylanthracene) and 12 (N-cyclohexylmaleimide) (fig. I9). In the absence of cage reaction

between 11 and 12 has been widely reported to produce the 9,10-bridged Diels-Alder adduct. When the

reaction took place within the cage the cavity pre-organised the molecules in a manner which changed

the conventional selectivity. In this arrangement 12 was close in space to the terminus of 11, this lead to

bridging at the end of 11 rather than at the middle. The product of the reaction; the unusual 1,4-adduct

(14) has conjugated π ring systems originating from the starting material (11). 14 experienced even

stronger π-π stacking interactions with the 3-tpt ligand that makes up the cage structure than 11.

Binding of two substrates within 9 causes an entropic destabilisation of the structure meaning

14

replacement of product 14 in cavity by one of each reactant (11 and 12) was very unlikely. Again due to

product inhibition catalytic turnover was not achieved.

Fig. I9: 9 catalysed Diels-Alder reaction between 11 and 12 forming unusual 1,4-adduct 14. Reproduced

from ref. 16.

With the bowl (10) this was not the case. A slightly different dienophile substrate was used ( 15, fig. I10)

and without the pre-organisation afforded as with the cage, the conventional 9,10-adduct (16) was

formed from reaction with 11. The rate of this reaction however was greatly increased, in absence of 10

the yield of the reaction (fig. I10) was 3%, but with 10 near-complete conversion (99% yield) was

achieved in the same time. As with the octahedron (9), 11 was stabilised as a substrate via π-π stacking.

However bridging of 15 across the centre of the molecule removed linearity from the product (16) and

diminished the stacking interaction. This way the affinity between product and bowl was reduced, new

substrate molecules could replace it thereby achieving catalytic turnover.

15

Fig. I10: 10 catalysed Diels-Alder reaction between 11 and 15 forming 9,10-adduct 16. Reproduced from

ref. 16.

Fig. I11: Catalytic cycle of 9 during the reaction between 17 and 18 to form 19.

Octahedron 9 was found instead to achieve catalysis of a different reaction.17 2-naphthaldehyde (17, fig.

I11) was found to bind in the hydrophobic pocket of 9. 17 could then undergo nuceophilic addition from

Meldrum’s acid (18, fig. I11) followed by dehydration to form the respective conjugated enone (19, fig.

I11). Stabilisation of the anionic intermediate was achieved through strong electrostatic interactions

with 3 Pd2+ centres around each portal. The Knoevenagel product (19) was found to be too large to fit in

the cavity and was thus dissociated. This way, this workers observed high yields for various derivatives

using only a catalytic amount of 9. When bowl 10 was used instead, the reaction proceeded with a 17%

yield as the lack of a portal surrounded Pd2+ centres meant 10 was unable to afford the same

stabilisation of the intermediate. This goes to show that the interactions necessary for successful

16

catalysis are very specific. Stabilisation of the intermediate has been seen to be most promising through

electrostatic or π-stacking interactions. The shape of the molecules must then change sufficiently that

product inhibition is avoided. The difficulty of finding substrates which perform this binding-

stabilisation-alteration sequence is the main reason examples of supramolecular catalysis are not so

numerous. The incredible range of enzymes which have been tailored to each perform this sequence on

a specific molecule is the reason we trail nature in this kind of catalysis.

1.2.3 stabilisation of P4

Fig I12: Nitschke’s tetrahedral Fe cage [Fe4(L)6]4- (20), tetrahedral P4 (22) and host-guest complex (21).18

Supramolecular cages have also been reported to produce effects opposite to catalysis, where reactive

compounds have been indefinitely stabilised. This was the case when Nitschke and co-workers stabilised

molecules of the extremely air-sensitive and pyrophoric compound P4.18 Tetrahedral cage 20 was self-

assembled and was able to bind 22 within the cavity forming host-guest complex 21 (fig. I12). Despite,

the bonding within 22 being significantly weaker than when the compound is oxidised, the molecule

remained stable indefinitely. The portals of 20 were calculated as 1Å in radius, too small to allow 22 or

even diatomic oxygen through. Although Nitschke suggests thermal fluctuations create transient

widening of the portals of 20 thus allowing O2 into the cavity of the cage. This then rules out protection

17

of 22 (as part of 21) from oxidation source as the mechanism of stabilisation. Instead it is proposed that

the transition state of any oxidative addition to the molecule creates a species too large to fit inside the

cavity, the resulting high energy barrier produces indefinite stabilization of 22. Despite this, 22 could be

easily replaced as guest within 21. Addition of benzene to the solution gave two phases. 22 was then

replaced due to its preference for the organic phase working in coordination with the competition of

benzene to pervade the cavity (fig. I13). Free white phosphorus in the organic phase regained its

sensitivity to air and reacted over time to form phosphoric acid, which then re-dissolved in the aqueous

phase due to its polarity. Though the applications are not immediately obvious the sequence

demonstrates an incredible amount of control over the reactivity of an extremely sensitive species,

afforded in part by supramolecular cage 20.

Fig. I13: Biphasic displacement of 22 from 21 by benzene and dissolution of free 22 in organic phase,

subsequent reaction of 22 to give H3PO4 which re-dissolves in aqueous phase. Reproduced from ref. 18.

18

1.3 Functional systems: drug delivery

The field’s best efforts have not yet achieved the aim of metallosupramolecular drug delivery. Examples

of successful drug vehicles have been reported in the form of surfactant based nano-particles;

micelles19,20,21 and liposomes22,23,24. Anticancer drug doxorubicin has showed improved cytotoxivity in a

breast cancer MCF-7 cell assay as part of a micellular structure made up of poly(ethylene glycol)-

poly(aspartate hydrazide) copolymers.19 Whilst the same drug as part of liposomal therapy Myocet has

been approved for treatment of metastatic breast cancer.22 Liposomal vehicles however experience

problems with in vitro stability and are rapidly cleared by the reticuloendothelial system. 25 Coordination

capsules tend to be more stable than their organic counterparts and exhibit the interlinking properties

of molecular recognition and encapsulation. These qualities suggest cages are well matched to the task

of drug delivery. Furthermore as the space within the supramolecular framework has properties defined

by the cage, organic apolar ligands often create hydrophobic cavities. Entropically driven repulsion of

water enables binding of hydrophobic molecules such drugs within this space.

1.3.1 Crowley’s Pd “lanterns”

Selective binding of drug molecules has the potential to overcome many of the problems associated

with conventional delivery methods. Workers have already reported examples of drug encapsulation

such as Crowley’s Pd2L4 “lantern” shaped cage (22, fig. I14) which was able to bind two molecules of the

anti-cancer drug cisplatin.26

19

Fig. I14: Self-assembly of Crowley’s [Pd2(2,6-bis(pyridin-3-ylethynyl)pyridine)4]4- cage (22) from precursor

[Pd(CH3CN)4](BF4)2 and IL22. Structure of anti-cancer drug cisplatin is also shown (23).26

The host guest interaction between 22 and 23 is strengthened by hydrogen- bonding interactions

between the NH3 groups of 23 and the pyridyl nitrogen atoms within the IL22 struts. This interaction can

be seen in the image of the host-guest complex (fig. I15) and is an example of function of 22 arising

from a particular feature of the ligand IL22.

Fig. I15: Encapsulation of 23 by 22 forming host-guest complex 24.

20

This system however had problems with water solubility and therefore will not have the necessary

bioavailability to function as a drug delivery vehicle. Theoretically a similar water-soluble host-guest

system could effectively protect the drug within the cavity of cage. Thereby defending it from

destruction in the gut by acid or other resistance mechanisms. This protection avoids reduction of the

effective dose noted when drugs are delivered via conventional methods. Conversely the cage can

protect healthy cells from the cytotoxicity of some drugs.26 Delivery of cisplatin would be a prime

example of this, the drug is a major component of solutions administered during chemotherapy as a

result the harsh side-effects are well known. Host-drug systems are able to target tumors, another

feature that makes them extremely well suited to the task of drug delivery. Cancerous cells exhibit poor

drainage of macromolecules, this leads to accumulation of large structures such as supramolecular cages

within tumors.27 Disassembly of the host-drug structure could then be triggered using some kind of

stimulus and the drug would be released. With Crowley’s cage competing ligands such as DMAP and

Bu4NCl were used to initiate disassembly, disassembly triggers require consideration as are cage specific.

1.3.2 Therrien’s ruthenium prisms

Bruno Therrien and co-workers have self-assembled water-soluble prismatic ruthenium cage 27 from

precursor 25 and ligand system 26 (fig. I16).28The cage displayed the novel behavior of rather than

binding a drug, 27 was able bind a scaffold within the hydrophobic cavity to which the anti-cancer drug

floxuridine (fig. I16) was tethered.

Fig. I16: Structure of Floxuridine (28).

21

Fig. I17: Self-assembly of ruthernium prismatic cage from 25 and 26, subsequent encapsulation of

pyrene gave host-guest complex 27. Reproduced from ref. 28.

Molecules floxuridine were pre-tethered to the R position of the functionalized pyrene (fig. I17). 27

showed improved solubility and cytotoxicity over floxuridine alone, thus giving a demonstration of host-

drug function. Increased cytotoxicity to ovarian cancer cells is a positive result, although a system with a

cytotoxic guest and biologically inert vehicle would be preferable. This was not the case with the

ruthenium cage reported as arene ruthenium complexes, which are similar to each metal centre in 27,

are known anti-cancer agents.29 This system shows many of the desirable features of a functional

supramolecular drug delivery vehicle although the cyctotoxicity of 27 revives the issue of side-effects, it

is also unclear whether the effectiveness of 27 is due to the host or the guest.

Molecular recognition proves a double edged sword in regards to successful drug encapsulation by a

supramolecular species. Unique host-guest interactions are necessary to ensure selective encapsulation

22

of a given drug, therefore designing a system which is sure to have the necessary interactions is not

trivial. The selectivity of cages often results in good encapsulation of a single drug rather than a range of

different molecular structures. Although, the rate at which new examples are reported is sure to

quicken as more cages are created. Whilst the field is rapidly expanding, virtually all have a common

problem of stability, the weak bonds which allow for self-assembly also lead to relatively fragile

structures.

23

1.4 Lusby group previous work

Recent work out of the Lusby group has looked to incorporate the first row transition metal- cobalt in

self-assembled supramolecular structure.30 Cobalt is smaller, cheaper and less toxic than the 2nd and 3rd

row transition metals generally employed in metallosupramolecular assemblies. Other workers have

generally looked to use the larger transition metals because of the relative structural stabilisation

afforded by substitutionally non-labile centres.31

1.4.1 Self-assembly followed by oxidation

When synthesizing the tetrahedral supramolecular cages CA and CB (fig. I18), the group used Co2+

centres but were able to stabilise the structures in an alternative manner.30CA and CB were self-

assembled from cobalt (II) precursor (CoPr, fig. I18) and linear polyaromatic ligand LA or LB (fig. I18). CA/B

was then slowly oxidised using CAN, which converted all 4 centres from Co2+to Co3+.

24

Fig. I18: Synthesis of supramolecular cobalt cages CA and CB.

This oxidation had a profound effect on the cobalt d-orbital electrons and therefore stability of the cage.

Oxidation converts the metal centres from high spin d7 to low spin d6 (fig. I19). The former has labile

bonding due to the high energy unpaired electrons whilst the latter is a filled half shell arrangement with

bonding that is substitutionally inert. Octahedral geometry at the metal centres is retained during this

change. This self-assembly followed by oxidation is a novel way of achieving a structure that was shown

via scrambling experiments to be constitutionally non-dynamic up to 70°C. Mixing of CA and CB solution

in MeCN showed no exchange of ligand between the structures, despite the similarity of LA and LB.

Fig. I19: Molecular orbital diagram for cobalt d-orbitals before (high spin d7, left) and after oxidation

(low spin d6, right).

Whilst many workers have reported formation of a mixture of supramolecular species with the same

components.1,12 The recent research out of the Lusby group demonstrates control over the dynamic

equilibrium between the cages CA/B and their respective helicate species (HA/B).

25

eg eg

t2g t2g

Fig. I20: Triple helicates HA and HB.

The group noted that formation of tetrahedron (CA/B, fig. I21) in the self-assembly reaction was

promoted by a concentrated reaction mixture followed by a slow oxidation. Conversely, selective

synthesis of HA/B (fig. I21) could also be achieved using the opposite conditions; a dilute self-assembly

reaction mixture followed by a fast oxidation gave helicates HA and HB pure. Fast addition of CAN should

effectively trap whichever species is in solution in its given state, by conversion to non-labile Co 3+. The

contrasting reaction conditions therefore suggest that before oxidation the supramolecular equilibrium

lies well towards HA/B. Conversely, slow-oxidation will give the structure time to rearrange and thereby

form the tetrahedron. Concentration has been shown in previous paper by other worker to promote

formation of larger supramolecular species over smaller structures due to Le Chatelier’s principle. As the

concentration is increased the equilibrium shifts to maintain the same number of molecules in solution,

this system does so by forming the entropically disfavoured CA/B species. The opposite is true for dilute

conditions during HA/B synthesis.

26

Fig. I21: Selective synthesis of cage (CA/B) or helicate (HA/B) species.

1.4.2 L A/B synthesis and counter ion metathesis

Synthesis of LA and LB was achieved by a copper catalysed azide-alkyne “click” reaction (fig. I22). Using

this reaction the central unit (F1c) could be readily converted to LA or LB depending on the azide fragment

used (FA/FB). The PEG group of LA gave a more water soluble cage (CA) and the bulky adamantane group

of LB aided crystallisation of CB allowing for x-ray crystallography to determine CB structure.

Fig. I22: Schemes for synthesis of LA or LB from F1c and azides FA or FB.

The workers later found however that counter ion metathesis had a greater effect on solubility than

ligand alteration. Conversion of the mixed counter ion species (MeCN:H2O, 2:1) produced by the self-

assembly to highly apolar PF6 counter ions resulted in a cage (CA∙12PF6) with good solubility in organic

solvents (MeCN, diisopropylether). Whilst conversion to all nitrate species CA∙12NO3 was found to be

make CA water soluble up to 2.5 mM. As many of the bodies uptake mechanisms rely upon aqueous

27

fluids, good water solubility and therefore bioavailability is essential to any potential function as a drug

delivery vector.

1.4.3 C A guest binding

Whilst the cavities of HA and HB were too small to encapsulate molecules, CA∙12NO3 exhibited promising

host-guest chemistry (fig. I24). The cage encapsulated triisopropylsilanol (TIPSOH) in slow-exchange (on

the NMR timescale). This strong binding interaction (Ka = 1400 M-1) was shown by the presence of 4 sets

of peaks on the NMR spectrum (fig. I23), these corresponded to free CA∙12NO3 (red peaks), bound

CA∙12NO3 (blue peaks), free TIPSOH (larger green peak) and bound TIPSOH (smaller green peaks).

Fig I23: 1H NMR spectra showing CA∙12NO3 (a), CA∙12NO3 with bound TIPSOH (b, slow exchange),

CA∙12NO3 with bound nitrobenzene (c, fast exchange) and CA∙12NO3 with bound chromone (d, fast

exchange). Reproduced from ref. 30)

A number of others bound within the cavity in fast-exchange on the NMR timescale, the most

interesting of which were 2-adamantanone (analogue of antivirals amantadine and rimantadine) and

the anticoagulant coumarin. Fast-exchange guests were confirmed by slight shifts and broadening of

both guest (green peaks in b,c and d fig. I23) and CA∙12NO3 peaks (blue peaks b,c,d fig. I23).

28

Fig. I24: Molecules that acted as guest within CA∙12NO3.

1.4.4 C A stability

29

CA∙12NO3 was acidic as a 2.5 mM D2O solution, the pH was measured at 2.5. Stability of CA∙12NO3 was

observed to be a problem when the workers moved the pH to more basic conditions using sodium

phosphate buffer solutions. When the stability was tested at pH 6 the cage was observed to decompose

into a cobalt solution and white LA precipitate over a time period of a few days (fig.I25). D2O dilution

experiments showed conversion between the triazole proton (shown in green, fig. I25) and deuterium,

indicating that the proton is weakly bound to LA and is therefore acidic. Neutrality of free LA/B suggested

that this proton was acidic partly due to the highly charged Co3+ centres of CA∙12NO3 removing electron

density from the coordinated triazole ring. The workers postulated that loss of the proton caused L A to

become weakly negatively charged. Subsequent donation of electron density onto the coordinated

cobalt centre was thought to cause a trans-effect interaction, where the opposite Co-L A bond was

labilised. Any biologically functional species cannot exhibit this kind of instability as in order to

successfully deliver drugs the structure must be stable to a wide pH range (approx. 2-8).

Fig. I25: Possible scheme for buffer mediated decomposition of CA∙12NO3.

1.5 Project aims

30

The aim of the project was to bypass the apparent instability of CA∙12NO3 to neutral conditions by

synthesising a new ligand and cage system devoid of the acidic proton. This called for a rethink of the LA

design with removal of the functionalized triazole ring from the design and subsequent substitution with

an alternate heterocycle. Crucially, one devoid of an acidic proton. However many of the successful

features of LA and LB could be retained, giving the brief for the new ligand:

- Synthetically feasible ligand system.

- Two N,N 5-membered chelate rings at the ends of the ligand system.

- Central phenyl linker unit.

- New aromatic heterocycle at triazole position, devoid of acidic proton.

- Group available at end of ligand for potential exo-functionalisation.

This brief lead to alteration of the LA design to give the motifs shown in fig. I26. The fragments 2-[6-(5-

bromopyridin-2-yl)-1,2,4,5-tetrazin-3-yl]ethan-1-ol (pyridyl-tetrazine) and 2-(5-bromopyridin-2-yl)-1-

methyl-1,3-benzimidazole (pyridyl-benzimidazole) would theoretically be di-coupled with benzene-1,4-

diboronic acid to give the full ligands (L1 and L2 fig. I26) which could then be assembled with a cobalt

precursor (using the same protocol for synthesis of CA/B) into cage systems (C1 and C2) that are stable at

neutral pH. The R positions show sites for potential functionalization. Whilst the structures of LA and LB

contained PEG and adamantane groups for solubility and to facilitate crystallization respectively,

biological or fluorescent markers could also be added for biomedical applications.

31

Fig. I26: General structures of fragments used when designing ligands L1 and L2.

32

2. Experimental

2.1 General information

Reagents were purchased from Sigma-Aldrich, Alfa-Aesar or VWR. All column chromatography was

performed using stationary phase Geduran Si60 silicagel (particle size 40-63) microns. Thin-layer

chromatography (TLC) was ran on silica 60 gel coated (0.2mm thick 60F254. Merck, Germany) aluminium

plates, and then observed using UV light (Chromato Vue Cabinet model CC-10, Upland CA91786, USA).

Solvents were obtained dry and pure from solvent purification system manufactured by Innovative

Technology, Newburyport, MA, USA. Solvents were degassed by nitrogen purging or by sonication

(ultrasonic bath) under vacuum followed by N2 backfill cycles on Schlenk apparatus.

All 1H and DOSY NMR experiments were recorded on a Bruker AV500 instrument at a constant

temperature (298 K). The data was processed using Topspin 2.1 (Bruker) and MestReNova 6.0.3

(MestreLab Research). Chemical shifts are reported in parts per million from low to high field and

are referenced against values for the residual solvent peaks. Coupling constants (J) are reported in

Hz. Standard abbreviations indicating multiplicity are used as follows: m = multiplet, t = triplet, d =

doublet, s = singlet.

33

2.2 Attempted synthesis of L1

2.2.1 General scheme (E1) for attempted synthesis of L 1

Scheme E1: Reaction scheme for synthesis of 2-[6-(5-bromopyridin-2-yl)-1,2,4,5-tetrazin-3-yl]ethan-1-ol

(F1b) from 5-bromo-2-pyridinecarbonitrile (F1a) (route (a)) plus attempted synthesis of 1,4-bis(2-[6-(5-

bromopyridin-2-yl)-1,2,4,5-tetrazin-3-yl]ethan-1-ol)benzene (L1) from 2-[6-(5-bromopyridin-2-yl)-1,2,4,5-

tetrazin-3-yl]ethan-1-ol (F1b) (route (b)) and from 1,4-bis(6-ethynylpyridin-3yl)benzene (F1c) (route (c)).

Reaction conditions and yields: (a) (i) F1a, 3-hydroxyisopropionitrile, hydrazine, EtOH, 90°C 24 hrs (ii)

sodium nitrite, AcOH, 30 mins, room temperature, 15%; (b) F1b, benzene-1,4-diboronic acid, Na2CO3,

Pd(PPh3)4, THF, EtOH, H2O, 16 hours, 80°C, no yield; (c) F1c, 3-hydroxyisopropionitrile, hydrazine, 12

hours, 90°C, no yield.

34

2.2.2 synthesis of F 1b

(a) 2-[6-(5-bromopyridin-2-yl)-1,2,4,5-tetrazin-3-yl]ethan-1-ol, F1b

(i) 5-bromo-2-pyridinecarbonitrile (499 mg, 2.73 mmol), 3-hydroxyisopropionitrile (1.86 mL,

27.3 mmol), hydrazine (3.43 mL, 109 mmol) and ethanol (5 mL) were added to a two-necked

flask and placed under nitrogen atmosphere. The flask was heated in an oil bath for 24

hours at 90°C behind a blast shield. Reaction progress was checked using TLC.

(ii) The reaction mixture was washed with DCM and water, the organic phase was then

concentrated in vacuo. (ii) The orange protonated product was oxidised by stirring with

sodium nitrite (188 mg, 2.73 mmol) in acetic acid (60 mL) for 30 minutes at room

temperature. The reaction mixture was washed with DCM and water, the organic phase was

concentrated in vacuo and purified via flash column chromatography. The title compound

was afforded as a bright pink crystalline solid. Yield=120 mg (16%) 1H NMR (500 MHz,

Chloroform-d) δ 9.03 (dd, J = 2.3, 0.6 Hz, 1H, HE), 8.58 (dd, J = 8.4, 0.7 Hz, 1H, HC), 8.16 (dd, J

= 8.4, 2.3 Hz, 1H, HD), 4.36 (q, J = 5.9 Hz, 2H, HA), 3.72 (t, J = 5.8 Hz, 2H, HB).

35

2.2.3 attempted synthesis of L 1

(b) 1,4-bis(2-[6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl]ethan-1-ol)benzene, L1 (not successfully synthesised)

F1b (25.2 mg, 88.6 µmol), benzene-1,4-diboronic acid (6.99 mg, 42.1 µmol), Na2CO3 (93.9 mg, 4.8 mmol)

and Pd(PPh3)4 (6.5 mg, 5.6 µmol) were added to a two-necked flasked and placed under nitrogen

atmosphere. THF (9 mL), EtOH (6 mL), and H2O (3 mL) were separately degassed before being added to

the two-necked flask. The light brown reaction mixture was left heating in an oil bath for 16 hours at

80°C. No product yield was observed in crude 1H NMR.

36

2.2.4 attempted synthesis of L 1 (2)

(c) 1,4-bis(2-[6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl]ethan-1-ol)benzene, L1 (not successfully synthesised)

F1c (100.1 mg, 0.3 mmol), 3-hydroxyisopropoionitrile (251.4 mg, 3.5 mmol) and hydrazine (0.4 mL, 14.1

mmol) were added to a microwave vial and placed under a nitrogen atmosphere. EtOH (2 mL) was

added and the reaction mixture was heated for 12 hours at 90°C behind a blast shield. No product yield

was observed in crude 1H NMR.

37

2.3 synthesis of L2

2.3.1 General scheme for synthesis of L 2

Scheme E2: Reaction scheme for synthesis of 2-(5-bromopyridin-2-yl)-1-methyl-1,3-benzimidazole (F 2b)

from 5-bromo-2pyridinecarboxylic acid (F2a) (route (d)) and 1,4-bis(6-(1-methyl-1,3-

benzimidazolyl)pyridin-3-yl)benzene (L2) from F2b (route (e)). Reaction conditions and yields: (d) F2a , N-

methylphenylenediamine, borane-THF, toluene, 72 hours, 110°C, 41%; (e) F2b, benzene-1,4-diboronic

acid, Pd(PPh3)4, Na2CO3, THF, EtOH, H2O, 72 hours, 70°C, 48%.

38

2.3.2 synthesis of F 1b

(d) 2-(5-bromopyridin-2-yl)-1-methyl-1,3-benzimidazole, F2b

F2a (3.27 g, 16.2 mmol) and toluene (81 mL) were added to a two-necked flask in an ice-water bath.

Borane-THF (518 µL, 5.41 mmol) was added dropwise before the mixture stirred for 30 minutes at room

temperature. N-methylphenylenediamine (615 µL, 5.41 mmol) was added and the reaction was heated

at 110°C for 72 hours. Reaction progress was checked using TLC.

Reaction mixture was concentrated in vacuo and the product was purified by silica flash column

chromatography (1:1 DCM:hexane). The title compound was afforded as a white solid. Yield = 642 mg

(41%). m.p. X°C. 1H NMR (500 MHz, Chloroform-d) δ 8.75 (d, J = 2.3 Hz, 1H H H), 8.33 (d, J = 8.5 Hz, 1H HF),

7.97 (dd, J = 8.5, 2.4 Hz, 1H HG), 7.82 (d, J = 7.5 Hz, 1H HA), 7.44 (d, J = 7.6 Hz, 1H HD), 7.36 (td, J = 7.6, 1.3

Hz, 1H HC), 7.32 (td, J = 7.6, 1.3 Hz, 1H HB), 4.26 (s, 3H HE). HR-ESI-MS (m/z): 288.014 (predicted [M+H]+ =

288.013) , 309.996 (predicted [M+Na]+ = 309.995).

39

2.3.3 synthesis of L 2

(e) 1,4-bis(6-(1-methyl-1,3-benzimidazolyl)pyridin-3-yl)benzene, L2

F2b (642 mg, 2.23 mmol), benzene-1,4-diboronic acid (175 mg, 1.06 mmol), Pd(PPh3)4 (61.3 mg, 53.1

µmol) and Na2CO3 (562 mg, 5.31 mmol) were added to a two-necked flask. THF (20 mL), H2O (10 mL) and

EtOH (16 mL) were individually degassed by purging and added to the reaction flask. The mixture was

then heated at 80°C for 72 hours. Reaction progress was checked using TLC.

The reaction formed a white precipitate which was filtered in vacuo and washed with reaction solvents.

The crude product was purified by silica flash column chromatography (2% MeOH in DCM). The title

compound was afforded as a white crystalline powder. Yield = 255 mg (48%). m.p. X°C. 1H NMR (500

MHz, Chloroform-d) δ 9.02 (dd, J = 2.3, 0.7 Hz, 2H HH), 8.54 (dd, J = 8.3, 0.7 Hz, 2H HF), 8.13 (dd, J = 8.3,

2.4 Hz, 2H HG), 7.86 (dd, J = 7.4, 1.4 Hz, 2H HA), 7.84 (s, 4H HI), 7.48 (dd, 2H HD), 7.38 (td, J = 8.0, 7.6, 1.4

Hz, 2H HC), 7.34 (td, J = 7.6, 1.4 Hz, 2H HB), 4.36 (s, 6H HE). HR-ESI-MS (m/z): 493.211 (predicted [M+H]+ =

493.214) , 515.192 (predicted [M+Na]+ = 515.195).

40

2.4 synthesis of C2

2.4.1 General scheme for synthesis of C 2

Scheme 2: General scheme for synthesis of tetrahedra and helicate; C 2∙12NO3 from L2 (route (f)(i)),

C2∙12PF6 from L2 (route (f)(ii)) and helicate species H2∙6PF6 from L2 (route (g)). Reaction conditions and

yields: (f)(i) L2 (concentrated), Co(ClO4)2∙6H2O (concentrated), MeCN, CAN dropwise, AgNO3, 24 hours,

50°C, 38%; (f)(ii) L2 (concentrated), Co(ClO4)2∙6H2O (concentrated), MeCN, CAN dropwise, NH4PF6, 24

hours, 50°C; (g) L2 (dilute), Co(ClO4)2∙6H2O (dilute), MeCN, fast addition CAN, NH4PF6, 24 hours, 50°C,

97%.

41

2.4.2 Synthesis of C 2 ∙ 12NO 3

(f)(i) [Co4(L62)]12NO3, C2∙12NO3

L2 (103 mg, 209 µmol) and Co(ClO4)2∙6H2O (51.6 mg, 139 µmol) were added to a 250 mL Schenk flask.

MeCN (16 mL) was separately degassed before being added. The reaction mixture was left to heat at 50

°C for 24 hours. The reaction was left to cool to room temperature, CAN (84.3 mg, 153 µmol) in MeCN (6

mL) was then added drop-wise via syringe pump over 2 hours. The resulting orange precipitate was

filtered onto celite and eluted with 2:1 MeCN:H2O, CG-400 anion exchange resin (1.22 g, mol) was added

to the resulting solution. This mixture was left stirring gently overnight. The resin was then removed by

vacuum filtration onto celite, the solvent mixture was removed by concentration in vacuo and replaced

with solely H2O (16 mL). To this solution AgNO3 (72.4 mg, 419 µmol) was added in an aluminium foil

covered flask, this was left for 3 hours before being filtered onto celite to remove the silver precipitate

present. Successive cycles of centrifuging followed by syringe filtering then ensured complete

elimination of silver precipitate. The resulting solution was then concentrated in vacuo to give the title

compound as an orange solid. Yield = 53.4 mg (39%). m.p. X°C. 1H NMR (500 MHz, Deuterium Oxide) δ

42

8.97 (d, J = 8.7 Hz, 1H HF), 8.82 (d, J = 8.6 Hz, 1H HG), 7.98 (d, J = 8.7 Hz, 1H HD), 7.76 (s, 1H HH), 7.63 (t, J =

7.9 Hz, 1H HC), 7.38 (s, 2H HI), 7.12 (t, J = 8.0 Hz, 1H HB), 5.52 (d, J = 8.6 Hz, 1H HA), 4.62 (s, 3H HE).

2.4.3 Synthesis of C 2 ∙ 12PF 6

(f)(ii) [Co4(L62)]12PF6, C2∙12PF6

L2 (30.1 mg, 60.9 µmol) and Co(ClO4)2∙6H2O (14.8 mg, 40.6 µmol) were added to a 250 mL Schenk flask.

MeCN (5mL) was separately degassed before being added. The reaction mixture was left to heat at 50°C

for 24 hours. The reaction was left to cool to room temperature, CAN (33.8 mg, 44.6 µmol) in MeCN (2

mL) was then added drop-wise via syringe pump over 2 hours.

The resulting orange precipitate was filtered onto celite and eluted with 2:1 MeCN:H 2O. Excess NH4PF6

(799 mg, 4.90 mmol) was then added to give a red precipitate which was removed by filtration onto

celite followed by elution with MeCN. This solution was then concentrated in vacuo to give the title

compound as a bright red crystalline solid. 1H NMR (500 MHz, Acetonitrile-d3) δ 8.86 (d, J = 8.7 Hz, 1H

43

HF), 8.79 (dd, J = 8.6, 1.8 Hz, 1H, HG), 7.93 (d, J = 8.6 Hz, 1H, HD), 7.59 (t, J = 8.2 Hz, 1H, HC), 7.56 (d, J = 1.6

Hz, 1H, HH), 7.46 (s, 2H, HI), 7.12 (t, J = 7.9 Hz, 1H, HB), 5.33 (d, J = 8.6 Hz, 1H, HA), 4.52 (s, 3H, HE).

2.5 Experiments with C2∙12NO3/ C2∙12PF6

2.5.1 Mass Spectrometry :

Mass spectrometry (n-ESI-MS) of C2∙12NO3 was carried out using Waters nanoMate and SYNAPT G2

instruments. A spectrum was produced using a 10 µM cage solution.

2.5.2 X-ray Crystallography:

X-ray crystallography quality crystals of C2∙12PF6 were grown by slow diffusion of diisopropyl ether into

an acetonitrile cage solution. This produced a sample of large dark red plate shaped crystals of C2∙12PF6.

These crystals were submitted to the University of Edinburgh crystal structure service.

2.5.3 Scrambling Experiments:

250µL of C2∙12NO3 (2.5mM) and 250 µL CA∙12NO3 (2.5 mM) were added to an NMR tube at room

temperature. The 1H NMR spectrum of this mixture was taken after 25 minutes and then again after 48

hours. The sample was then heated at 50°C then 60°C and finally 70°C over separate 24 hour periods, 1H

NMR spectra were taken at regular intervals.

2.5.4 Buffer solution experiments:

The pH of a 2.5mM D2O solution of the C2∙12NO3 was tested using pH paper. Stability tests were

performed by mixing with a NaxHxPO4 buffer solution. Buffer was added 1:1 (250 µL:250 µL) with the

2.5mM C2∙12NO3 solution.1H NMR were then taken at regular intervals. A 1:1 C2∙12NO3 (2.5 mM to D2O

sample (250 µL:250 µL) was prepared and submitted for 1H NMR spectroscopy.

44

2.5.5 Guest Binding Experiments:

0.5mL of 2.5mM C2∙12NO3 solutions were added to separate NMR tubes, 1 molar equivalent of guest

was then added to this. The tube was shaken vigorously and then submitted for 1H NMR spectroscopy.

3. Results

3.1 NMR experiments

Fig. R1: stack of 1H NMR (500 MHz, CDCl3/D2O) spectra for the pyridyl-benzimidazole fragment (green), L2

(blue) and C2∙12NO3 (red).

45

Fig. R2: DOSY NMR (500 MHz, D2O) spectrum for C2∙12NO3.

3.2 Mass Spectrometry

46

P2

P1

P4

P3

P6

P7

P5

Fig. R3: Mass spectrum of C2∙12NO3, peaks corresponding to cage are labelled according to fig R4.

Charge Mass Predicted mass Seen in spectrum

0 3932.83 - N

1+ 3870.84 3870.84 N

2+ 3808.85 1904.43 N

3+ 3746.87 1248.96 Y

4+ 3684.88 921.22 Y

5+ 3622.89 724.57 Y

6+ 3560.90 593.48 Y

7+ 3498.91 499.84 Y

8+ 3436.92 429.62 Y

9+ 3374.94 374.99 Y

47

10+ 3312.95 331.29 N

11+ 3250.96 295.54 N

12+ 3188.97 265.75 N

Table R1: Charge states of C2∙12NO3with corresponding masses.

48

[Co4L61]12+8NO3

-

P2

[Co4L61]12+7NO3

-

P3

[Co4L61]12+6NO3

-

P4

[Co4L61]12+9NO3

-

P1

Fig. R4: expansions of peaks corresponding to charge states labelled, red peaks modelled by isotopic

substitution have been superimposed, discrepancy due to calibration of n-ESI-MS.

3.3 Scrambling Experiments

49

[Co4L61]12+5NO3

-

P5

[Co4L61]12+4NO3

-

P6

[Co4L61]12+3NO3

-

P7

Fig. R5: 1H NMR spectra (500MHz, D2O) stack of CA∙12NO3, C2∙12NO3 and CA∙12NO3+ C2∙12NO3 mixture

after varying time and temperature.

3.4 Buffer solution experiments

50

CA∙12NO3

C2∙12NO3

C2∙12NO3+ CA∙12NO3 after 25 mins

Plus 48 hours

Plus 24 hours at 50°C

Plus 24 hours at 60°C

The pH of a 2.5mM D2O solution of the C2∙12NO3 was tested using pH paper. The colour suggested that

the solution was about pH 6.

Fig. R6: 1H NMR spectra (500MHz, D2O) stack of C2∙12NO3 and NaxHxPO4 + C2∙12NO3 mixture after varying

amounts of time.

3.5 X-ray Crystallography

51

C2∙12NO3

NaxHxPO4 + C2∙12NO3 after 25 minutes

Plus 24 hours

Plus 72 hours

Plus 144 hours

Crystal Data (returned from service): C250H231Co4F72N65P12, Mr = 6121.34, monoclinic, P21/c (No. 14), a =

18.6723(3) Å, b = 38.7772(8) Å, c = 38.7947(6) Å, = 92.0840(16)°, = = 90°, V = 28071.1(9) Å3, T =

120.0 K, Z = 4, Z' = 1, (CuK) = 3.447, 116463 reflections measured, 15031 unique (Rint = 0.0805) which

were used in all calculations. The final wR2 was 0.4577 (all data) and R1 was 0.1725 (I > 2(I)).

Fig. R7: Structure of C2∙12PF6 solved by X-ray crystallography.

3.6 Guest Binding Experiments

52

3.6.1 Encapsulated guest s

Fig. R8: Guest molecules for C2∙12NO3.

3.6.2 Encapsulation 1 H NMR stackplots

Triisopropylsilanol (G1)

Fig. R9: Partial 1H NMR (500MHz, D2O) spectra stack of host-guest species (red), G1 (green) and

C2∙12NO3 (blue). D2O solvent peak and portion of aliphatic region omitted for clarity.

Chromone (G2)

53

Fig. R10: Partial 1H NMR (500MHz, D2O) spectra stack of G2 (red), C2∙12NO3 (green) and host-guest

species (blue). Aliphatic region omitted for clarity.

2-adamantanone (G3)

Fig R11: Partial 1H NMR (500MHz, D2O) spectra stack of G3 (red), C2∙12NO3 (green) and host-guest

species (blue). Portion of aliphatic region and D2O solvent peak omitted for clarity.

Nitrobenzene (G4)

54

Fig. R12: Partial 1H NMR (500MHz, D2O) spectra stack of G4 (red), C2∙12NO3 (green) and host-guest

species (blue). Aliphatic region omitted for clarity.

Camphor (G5)

Fig R13: Partial 1H NMR (500MHz, D2O) spectra stack of G5 (red), C2∙12NO3 (green) and host-guest

species (blue). D2O solvent peak omitted for clarity.

4. Discussion

55

4.1 Synthesis of L1 design

4.1.1 F 1b synthesis

The two synthetic routes to obtain L1 proved unsuccessful. The first route (a→b scheme E1) involved a

tetrazine formation reaction which gave F1b from F1a and HiPN in a small yield (14%) as was a statistical

distribution of products. Homo-coupling of F1a and HiPN likely gave by-products 3,6- BPa and BPb (fig.

D1).

Fig. D1: Proposed products of tetrazine formation reaction.

4.1.2 L 1 Synthesis from F 1b

The next step used a Suzuki coupling reaction to try form L1 from F1b (fig. D2, full scheme 2.2.3).

Fig. D2: Attempted L1 synthesis from F1b.

56

This reaction was tried using Na2CO3 and TBAF as stronger and weaker sources of base. In both cases the

bright pink colour of the reaction mixture (caused by F1b) was immediately changed to a brown colour.

This was indicative of decomposition of F1b, and was likely the reason for failure of the reaction. Given

the ease of formation of the tetrazine ring, the conjugated system could well be vulnerable to attack

from the two sources of OH-. The two carbon atoms within this six membered ring are likely to be

electron deficient through bonding with two electronegative nitrogen atoms, this could leave them

susceptible to OH- in a mechanism that would break the ring system.

Fig. D3: attempted synthesis of L2 from HiPN and F1c.

4.1.3 L 1 synthesis from F 1c

The second route (fig. D3, full scheme 2.2.4) used F1c (previously synthesized by another member of the

group)34. It was thought that a reaction to that in fig. D1 would form tetrazine rings on each end of F1c

giving L1. Instead a yellow precipitate was formed which was shown by 1H NMR not to be the desired

product. As with fig. D1, carbonitrile groups are not specific in forming tetrazine rings. Therefore it is

likely that some polymerisation of F1c has occurred (fig. D4). This likely polymerization could explain the

failure of the reaction. The synthetic inaccessibility of the L1 design led to efforts being focused on L2.

Fig. D4: Polymerization of 1,4-bis(6-ethynylpyridin-3yl)benzene.

57

4.2 synthesis of L2 design

4.2.1 F 2b and L 2 synthesis

During work on the second design, F2b and L2 were synthesized cleanly and in reasonable yields. The

reaction mechanism for synthesis of F2b is shown in fig. D5. The Suzuki coupling of F2b to benzene-1,4-

diboronic acid was unaffected by the problems noted with F1b. This supports the reasoning that the

tetrazine ring system in F1b is unstable to sources of OH-.

Fig. D5: Reaction mechanism for synthesis of F2b from N-methylphenylenediamine (29) and F2a.

As with CA/B and HA/B, an equilibrium between supramolecular species is present in solution after self-

assembly. This was proven to be the case here as C2 was synthesized using a concentrated reaction

mixture followed by a slow oxidation. The helicate (H2) corresponding to C2 was selectively synthesized

from L2 by another member of the group.34 The synthesis of H2 applies the same reaction conditions as

the HA/B syntheses. The conditions for synthesis of H2 and C2 are summarized in scheme D6. Fast

oxidation is thought to freeze the equilibrium, the fact that this gives H2 suggests that after self-

assembly the solution is mostly H2. To form tetrahedra, single centres need to be oxidized which are

58

then able to rearrange into a tetrahedral structures. Le Chatelier’s principle explains the effect of

concentration, as the number of molecules in solution is increased the system seeks to correct this

change by forming the species with the lower entropy (less molecules) e.g. the cage and vice versa. This

effect is thought to be negligible though as the equilibrium before oxidation lies so far in favour of the

helicate. The two can be distinguished by characterisation using NMR experiments. Mainly DOSY, which

differentiates between size.

Fig. D6: Schemes for selective synthesis of supramolecular tetrahedron C2 or helicate H2.

59

4.3 Evidence for C2

4.3.1 1 H NMR experiments

Confirmation and characterisation of the C2 structure has been completed using several different

experimental techniques. 1H NMR spectra (fig. D7) were used to validate synthesis of F2b and L2.

Fig D7: 1H NMR spectra of F2b (bottom) and L2 (middle) and C2∙12NO3 (top).

Shifting of the assigned peaks of L2 after self-assembly was indicative of a new supramolecular species,

although was not determinant of whether the species was H2 or C2. The set of 7 aromatic peaks and 1

aliphatic peak were seen to shift either upfield or downfield, by varying degrees. Particularly strong

shifts upfield were observed for protons close to the nitrogen donor sites ( HA and HG) due to withdrawal

of electron density by the charged cobalt centres upon coordination.

60

4.3.2 DOSY NMR Experiment

The new supramolecular species was confirmed as C2 rather than H2 by diffusion-ordered NMR

spectroscopy (DOSY) (fig. D8). The spectrum indicated presence of a single species with one diffusion

constant shown by the linear plot of couplings.

Fig. D8: DOSY NMR (500 MHz, D2O) spectrum of C2∙12NO3

The corresponding diffusion constant of 2.1x105 can be used to calculate the radius of this species via

the Einstein-Stokes relationship (Eq. D1). Substituting values gives a radius value of 12.07Å.

α = kbT / 6πƞD

Eq. D1: Einstein-Stokes relationship between radius (α), diffusion constant (D), temperature (T) and

viscosity (ƞ).

61

Theoretical modelling of the cage using Spartan software gave the structure shown in fig. D9. Taking the

length of one ligand (which is an approximation of diameter) gave a value of 25.03 Å and therefore a

radius of 12.51 Å. This model agrees well with the calculated hydrodynamic radius despite the cage not

being spherical, the discrepancy of 0.44Å can be easily explained by the approximation used and

tumbling of the cage in solution. Only when leading with the ligand edge will the tetrahedron have the

maximum radius expected, other topologies will give an averaged and reduced value. Deviation from

linearity due to the slight flexibility of the ligand also contributes to reduce the radius.

Fig. D9: Theoretical modelling of C2 using Spartan software.

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25.03 Å

4.3.3 Mass spectrometry

The mass of C2∙12NO3 was determined using mass spectrometry. The masses of likely charge states,

produced by loss of subsequent counter ions, were calculated beforehand and can be seen in table R1. A

sample of 10 µM C2∙12NO3 solution in D2O was submitted giving the spectrum seen in fig. D10.

Fig. D10: Mass spectrum of C2∙12NO3.

7 Peaks from the spectrum matched the predicted masses, these peaks have been expanded and

compared with peaks calculated using isotopic distribution modelling software (fig. R4, 3.2). The

expansions match reasonably well with the isotopic distribution peaks for each respective charge state.

The slight discrepancy present is due to calibration of the n-ESI-MS instrument. Through confirmation of

the predicted charge states, the non-charged cage C2∙12NO3 was shown to be 3932.83 Da.

4.3.4 X-ray crystallography

The connectivity of the components of C2∙12NO3 was confirmed by x-ray crystallography. A service was

used for this determination, yielding the structure shown in fig. D11. Growing crystals of quality

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sufficient for x-ray crystallography was a problem with CA∙12NO3. Crystals of C2∙12PF6 were used for this

investigation rather than C2∙12NO3, as bulkier PF6 counter ions were more likely to produce high quality

crystals. The better solubility of C2∙12PF6 than C2∙12NO3 in organic solvents gave a wider range of

options for recrystallisation. The crystals which were grown by another member of the group via slow

diffusion of diisopropyl ether into MeCN were dark red, plate shaped and looked of good quality.

Fig. D11: Structure of C2∙12PF6 solved by X-ray crystallography.

4.3.5 Comparison with Spartan model

Through these experiments the size, mass, shape, and connectivity has been confirmed and shown to be

in line with the modelled structure. Establishing within reasonable doubt the formation of a new

supramolecular species. The Spartan model of C2 which has minimised steric interactions, agrees very

well with the experimental evidence. The angle created by coordination through pyridine and imidazole

rings is a good descriptor of the level of strain at the centres. Averaging all these bond angles for the real

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structure (from x-ray crystallography) gives an average coordination angle (ACA) of 81.80°. This was

0.97° closer to the ideal 90° than the model (predicted ACA 80.83°), a difference that shows the model

and the C2 structure are very similar. Electrostatics, between charged metal centres and the ligands,

which are not taken into account by the model would explain this small difference. Shortening of the Co-

L2 bonds due to electrostatic interactions would increase the coordination angle.

Fig D12: Image of modelled cage showing coordination angles at one metal centre.

4.4 C2 stability experiments

4.4.1 Scrambling experiments

Complete characterisation of system was followed by testing of its stability. In the Lusby group’s

previous work this was done by examining the stability in scrambling and buffer solution experiments.

The stack of 1H NMR spectra obtained from mixing of two cage solutions (scrambling experiment) (fig.

D13) shows very little change in the peaks (integrals or shifts) corresponding to C2∙12NO3 or CA∙12NO3

after heating at 50 °C and 60 °C.

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Fig. D13: 1H NMR spectra (500MHz, D2O) stack of CA∙12NO3, C2∙12NO3 and CA∙12NO3+ C2∙12NO3 mixture

after varying time and temperature.

In aqueous solution decomposition into components is unlikely due to the hydrophobic nature of L 2 and

LA. Lack of change in these peaks shows that there is no ligand exchange between species (at 50 °C and

60 °C). The experiment is a reflection of the metal-ligand bonding, though is dependent on both

structures. In previous work CA 12PF∙ 6 exhibited ligand exchange with CB 12PF∙ 6 in acetonitrile but only

at 70 °C. This experiment highlighted that cooperative M-L bonding played a large part is stabilising CA

and CB.30 The similarity of LA and LB means ligand exchange is feasible and would be expected to be seen

by the presence of new peaks in the 1H NMR spectrum. Though L2 has a different ligand structure, some

exchange is still expected. The lack of this suggests that all 3 structures (C2, CA and CB) have similar

constitutional dynamics due to the cooperativity of the components. A recent paper by Mike Ward and

co-workers suggests that supramolecular assembly is driven in part by the hydrophobicity of the ligand

component.33As part of the cage, half the surface area of L2 faces in towards the hydrophobic cavity

thereby achieving a lower overall amount of interaction with water. With the scrambling experiment

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performed the hydrophobicity of L2 is such that this effect may play a part, contributing towards the

relative stabilisation afforded by cooperative structures as opposed to separate components. Future

work monitoring the rate of self-assembly in water vs acetonitrile would serve to confirm or deny this.

4.4.2 Buffer solution experiments

CA∙12NO3 has a pH of 2.5. Through D2O dilution experiments this acidity was attributed to the triazole

proton of LA. The main aim of this project was to bypass this acidity with a different ligand structure. The

pH of C2∙12NO3 was shown to be about 6 using pH paper. A buffer experiment was conducted to test the

stability of the cage under constant pH 6 conditions. This experiment produced the spectra stack (fig.

D14) from which C2∙12NO3 can be seen to decompose over the course of roughly 9 days. The compound

is unstable to the buffer conditions (checked using a control experiment) and decomposes into the

ligand as a white precipitate and a cobalt solution.

Fig. D14: Scrambling experiment showing decomposition of C2∙12NO3 over approx. 12 days.

The decomposition is roughly a factor of 10X slower than that of CA∙12NO3. Puzzlingly, even though it is

devoid of the acidic proton C2∙12NO3 is still unstable to neutral pH conditions. This evidence therefore

67

suggests that the triazole proton is not involved in the mechanism of decomposition. Instead another

site in the structure must be interact with the buffer solution. A brand new (but still unpublished at the

time of writing) cage synthesised by workers in the Lusby group has again a slightly differing structure. 35

CC∙12NO3 has the same cobalt tetrahedral structure (fig. D15) but utilises ligand (LC) rather than L1 or LA.

Fig. D15: Structure of LC and CC.

Though a simpler ligand design, it appears the cage is stable to a pH 8 sodium phosphate buffer solution.

The major difference between C2, CB and CC is that in the latter the ligands coordinate through two 6-

membered rings. In the former two, coordination is through 5-membered (imidazole/triazole) and a 6-

membered (pyridine) rings. From the refined crystal structure of C2∙12PF6 (fig. R7, section 3.5) the

geometry at the 4 metal centres is considered strained. Coordination through a 5-membered rather

than 6-membered ring leads to bond angles that are far off the ideal 90° for octahedral geometry.

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The average coordination angle was 81.38° for CB∙12PF6 and 81.80° for C2∙12PF6 (example centre shown

in fig. D16). This small difference of 0.42° could explain the ten-fold difference in reactivity between

them although a larger difference is expected.

Fig. D16: Cobalt centre taken from x-ray crystallography structure of C2∙12PF6.

69

The ACA of centres in C2∙12PF6 vary somewhat with the example given being one of the more strained

centres, although the differences between centres in CB∙12PF6 are much greater. On closer inspection of

the crystal structure for CB∙12PF6 it can be seen there are two types of centre with different levels of

strain. The first (type A) shown in fig D17 has an ACA of 81.94 ° which is actually closer to the ideal 90 °

than the ACA for C2∙12PF6.

Fig. D17: Type A cobalt centre from x-ray crystallography structure of CB∙12PF6.

The second type of centre in CB∙12PF6 however is a single highly strained cobalt atom with an ACA of

79.56 ° (fig D18). With the bond angles being 10.44 ° away from the ideal this site would be energetically

unfavourable. Though this result is for CB∙12PF6 and the buffer solution experiments were conducted on

CA∙12NO3, comparisons can still be drawn as counter ion was shown not to perturb the cage structure.

The difference between LA and LB as part of CA/B is an exterior functionalization (using a 2-PEG or

adamantane group) e.g one that probably does not affect the coordination angle.

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Fig. D18: Type B centre from x-ray crystallography structure of CB∙12PF6.

Though CC has not yet been submitted for x-ray crystallography due to crystallisation issues it is a

reasonable prediction that the ACA will be 3-9 ° closer to the ideal 90 ° than for C2∙12PF6. Confirmation

of this through x-ray crystallography could corroborate that there is a linear trend between ACA (as a

measure of geometry strain) and time for decomposition in a pH 6 sodium phosphate buffer solution

(measure of stability). The proposed justification for this is that more strained geometry leads to poorer

overlap between the donating nitrogen orbital and the cobalt d-orbitals. This in turn could lead to the

highly charged cobalt metal centres being more electronically unsatisfied than the more

thermodynamically satisfied counterparts (e.g CC). A potential mechanism could possibly be attack at the

highly strained and highly charged centre by a nucleophile in solution such as OH - which is formed from

71

phosphate in the equilibrium shown in Eq. D2. However further experiments are necessary to

investigate this.

HPO42− + H2O H2PO4

− + OH

Eq. D2: HPO42- ions and water produce OH- ions and H2PO4

− ions in aqueous buffer solution.

In terms of an application in drug delivery, instability to neutral condition means C2∙12NO3 does not

meet the criteria for use within biological systems. Future workers are therefore more likely to find

success in pursuit of biologically functional systems utilising cages with less strained metal geometries.

4.5 Host-guest chemistry

4.5.1 Catalysis

In terms of catalysis, the instability of C2∙12NO3 to OH- limits its applications. Many base catalysed

reactions now become inaccessible as potentially cage catalysed syntheses. However the huge range of

organic reactions proves an advantage and as described in the introduction, cages with these shapes of

cavity have shown promising catalytic activity. Therefore testing of the catalytic activity should proceed

using substrates with favourable affinities for the cage. Reactions with anionic intermediates which may

experience a Columbic effect (with cationic cage), as well as those which are a good fit for the cavity (can

be constrictively bound) should be considered.

72

Fig. D19: Guests which displayed encapsulation by C2∙12NO3.

4.5.2 Slow-exchange guests: TIPSOH

Some initial guest binding experiments have been performed with C2∙12NO3. The guests shown in fig.

D19 were chosen according to Rebek’s suggestion that molecules which occupy 50-55% of the volume of

the cavity tend to bind well. Five of the molecules which behaved as guest for CA∙12NO3 displayed signs

of encapsulation, these were the only 5 tested with C2∙12NO3 due to time constraints. As with CA∙12NO3,

TIPSOH was seen to bind in slow-exchange on the NMR timescale. The slightly shifted cage peaks (fig.

D20) which are now in a new environment due to the guest present can be seen (red arrows) alongside

a small amount of free cage. Bound guest is also present (yellow arrows) as is the free guest in large

excess (green arrows). Such a large shift upfield is observed for the guest as it is well shielded by the

cage.

73

Fig. D20: Partial 1H NMR (500MHz, D2O) spectra stack of host-guest species, TIPSOH and C2∙12NO3.

As there are two environments for each species, the spectrum is evidence for a host-guest binding

interaction. From the molar ratios of these peaks at equilibrium the association constant of TIPSOH

binding within the cage was calculated as 280 M-1 via eq. D3.

Ka = (1-χeq)[H0] / (χeq)[H0][Geq]

Eq. D3: Relationship between association constant (Ka), % unbound cage (χeq), cage concentration ([H0])

and guest concentration ([Geq]).

This binding occurs despite TIPSOH being 220 Å3 in volume and therefore much larger than the 55% of

cavity suggested. Spartan modelling of the encapsulation of TIPSOH by C2∙12NO3 followed by energy

minimisation of steric interactions gives the image shown in fig. D21. It was found that after energy

minimisation the propyl groups of the guest aligned themselves with the portals of C2∙12NO3, this likely

necessary for the cavity to accommodate the size of TIPSOH.

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Fig. D21: Spartan model showing host-guest complex C2∙12NO3 TIPSOH.

The TIPSOH Ka for CA∙12NO3 was reported as 1400 M-1 and very recent experiments in the Lusby group

lab gave a value of 2800 M-1 for the CC∙12NO3 cage. Such stark differences in this value between the

three cages was not expected. It is thought that strong encapsulation of TIPSOH occurs through

constrictive binding. With this view, the very slight differences in cavity size between the cages are not

sufficient to explain the order of magnitude difference in association constant. Further experiments are

necessary to investigate the cause of this discrepancy, although one possible cause could be the relative

flexibility of L2 vs LC.

4.5.3 Fast-exchange guests

For the rest of the guests a different mechanism of binding was thought to occur. Whilst shape and size

are still important, molecules with electron rich functional groups were observed to bind well within

CA∙12NO3. With the 4 guests: chromone, 2-adamantanone, nitrobenzene and camphor this was

observed to also be the case for C2∙12NO3. These guests bound in fast-exchange (on the NMR

timescale), generally a weaker interaction than slow exchange. As the host-guest NMR spectrum (fig.

D22 for chromone as an example) shows a time-average of the fast equilibrium between bound and

unbound cage species, an NMR titration experiment is necessary to obtain an association constant. This

process involves tracking host-guest peaks (via 1H NMR spectroscopy) as the concentration of guest is

slowly increased from 0 to 1 equivalent (of host). This experiment was not performed due to time

constraints.

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Fig. D22: Partial 1H NMR (500MHz, D2O) spectra stack of chromone, C2∙12NO3 and host-guest species.

4.5.4 Fast-exchange guests: chromone

Spartan modelling of chromone encapsulated within C2∙12NO3 gave the energy minimised (steric

interaction minimised only) host-guest structure shown in fig. D23. This suggests the guest molecule

occupies the space as shown. There were indications with CA∙12NO3 that hydrogen bonding between

the pyridine proton facing into the cavity (HH spectrum I) and an electron rich functional group of the

guest molecule helped to strengthen binding. This may be the case here as the internal pyridine proton

(HX) of C2∙12NO3 can be seen (from x-ray crystallography data) to be in a similar orientation. The

carbonyl group of chromone points towards the vertices of the cavity, e.g. the space which the internal

pyridine proton occupies. It is therefore highly likely that a similar hydrogen-bonding interaction is

helping to stabilise the guest molecule within the cavity.

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Fig. D23: Spartan model showing host-guest complex C2∙12NO3 chromone.

4.5.5 Fast-exchange guests: 2-adamantanone nitrobenzene and camphor

A similar interaction between this internal proton and the carbonyl group of 2-adamantanone and

camphor along with the nitro group of nitrobenzene is thought to encourage binding of these guests.

More time would enable NMR titrations of these guests, determination of association constants and

quantification of the strength of binding. Comparison of Ka values with position of the internal pyridine

proton (slightly different across the 3 cages) could yield a qualitative (possibly even quantitative with

host-guest x-ray crystallography data) insight into the relationship between structure and guest-binding

ability.

4.6 Summary and future work

Successful synthesis of C2 alone proves a positive result, a result which confirms the strength of the self-

assembly followed by oxidation protocol. By the relatively simple act of oxidising the self-assembly

product, the interlinking problems of lability and stability that plague supramolecular chemistry are

somewhat overcome. This feature allows for now relatively rapid synthesis of tetrahedral cobalt cages. If

a suitable ligand system can be designed and synthesised it is likely that, via the self-assembly followed

by oxidation protocol, a cage can be obtained pure. This is shown by the rate at which the Lusby group is

producing new cages. Other workers have commonly reported mixtures of supramolecular species, self-

assembly followed by oxidation allows for selective synthesis of either cage (CA, CB, CC, C2) or the

77

corresponding helicate (HA, HB, HC, H2) by variance of concentration and speed of oxidation. Since these

factors are now understood it would be interesting to examine the effect of a third variable. The

hydrophobic effect has been reported to aid supramolecular synthesis of larger species (e.g. tetrahedra)

over smaller species (e.g. helicate), it would therefore be interesting to examine the self-assembly

reaction of C2∙12NO3 (or CA/CB) in aqueous solution.33 An increase in the rate vs self-assembly in

acetonitrile would provide additional evidence for this theory. As would an increase in the relative

amount of cage vs helicate, due to the reduction of hydrophobic interaction afforded by the larger

species.

Future work on C2∙12NO3 would therefore include: NMR titrations of confirmed guests with the aim of

assessing the structure-function relationship, testing with substrates similar in structure to the Nazarov

substrate and other unimolecular reagents with the aim of catalysis, and examination of the rate of self-

assembly when performed in water to assess whether the reaction is hydrophobically driven. This work

can continue hand-in-hand with the now relatively easy synthesis of new more stable cages which

currently include alternate linker units to the central benzene as well as 6-membered coordinating units.

Conclusion

During this project a new cobalt tetrahedral supramolecular cage (C2) was synthesized from a cobalt (II)

precursor and ligand system L2. The structure has been confirmed using NMR, X-ray crystallography and

n-ESI-MS experiments. Initial guest binding shows behavior similar to a previously synthesized cage (CA),

encapsulation of TIPSOH occurred in slow-exchange whilst camphor, 2-adamantanone, nitrobenzene

and chromone were bound in fast-exchange. Stability studies showed that though decomposition of

C2∙12NO3 (in a pH 6 sodium phosphate buffer solution) is slower than for CA∙12NO3, though the

78

structure is still unstable to near-neutral conditions. Comparison with a more stable newly synthesized

cage (CA) suggests that strained geometry (around cobalt centres in C2∙12PF6) due to coordination

through 5- membered imidazole rings is the cause of this instability. Though this feature limits the use of

the system as a drug delivery vector, the cage’s ability to catalyse unimolecular reactions is still to be

tested. The result demonstrates the strength of the self-assembly followed by oxidation protocol when

synthesizing these types of cage, but also shows the necessity for ideal cobalt geometry when designing

for functionally stable assemblies.

Acknowledgements

I would like to thank P.J. Lusby for use of his laboratory and chemicals and for answering all questions

during the course of the project, however basic they may have been.

I would like to thank fellow project students R. Stewart and U. Mitreviciute for their company around

the lab and for their sharing of glassware.

79

Finally I would like to thank the PhD students in the group T. Sooksawat, D. August, M. Edwards and M.

Burke without whom completion of the project would have been impossible.

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