1

Investigation into Metal Carbonyl and Gold

Nanoparticle Conjugates as Bio-Imaging

Contrast Agents

Chemistry 4MX Research Report

2015/2016

Written by

Aaron King s1123432

Supervisor

Prof. Leong Weng Kee

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Table of Contents

Table of Contents 2

Abstract 5

1. Introduction 6

2. Objectives 17

3. Results & Discussion 18

4. Conclusion 33

5. Future Work 35

6. Acknowledgements 36

7. Experimental 37

8. References 42

9. Appendix 44

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i. List of Figures

List of Figures

Figure 1.1. Metal-Carbonyl bonding diagram

Figure 1.2. Averaged Raman spectrum of the maximum [Mn(tpm)(CO)3]Cl signal

inside a HT29 human colon cancer cell and B) a typical averaged

Raman spectrum from a region within the cell lacking any metal

complex.

Figure 1.3. Simplified diagrammatic comparison between Raman and SERS

scattering

Figure 1.4. A diagrammatic representation of the electromagnetic and chemical

enhancement mechanisms

Figure 1.5. An image of the rat brain vasculature produced with in vivo imaging

with the Osmium salt

Figure 1.6. The structure of the diiron dithiolate photoCORM

Figure 1.7. The structure of of N-acteylneuramic acid (Neu5Ac).

Figure 1.8. Structure of the Neu5ac/PAPBA boronic acid-diol complex

Figure 2.1. Structure of planned phenylboronic photoCORM to be used for SERS

imaging

Figure 3.1. Dose-Response curve for the osmium boronic acid cluster 2c.

Figure 3.2. Structure of phenylboronic acid 2b and the thiolate-triosmium cluster

2d.

Figure 3.3. Structure of triosmium-benzoic acid cluster 2e.

Figure 3.4. Comparison of the cell viability when treated with 50 µM of

compounds 2a - f

Figure 3.5. Structure of triosmium-mercaptophenol cluster 2f.

Figure 3.6. TEM images of 12 nm gold nanoseeds (A) and 60 nm gold nanostars

(B).

Figure 3.7. UV/vis spectrum for 60 nm gold nanostars.

Figure 3.8. Structure of diphenyl-butadiyne (3a), phenylacetylene (3b),

diphenylactylene(3c), and propiolic acid (3d).

Figure 3.9. Raman spectra of alkynes 3a - d conjugated to 60 nm gold nanostars.

Figure 3.10. Structure of planned acidic internal diyne.

Figure 3.11. Structure of 4 alkynes; acetylene (5a), acetylenedioc acid (5b),

phenylacetylene (3b), and propiolic acid (3d).

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Figure 3.12. Raman spectra of alkynes 3a, 3d, 5a, 5b, and 4b conjugated to 60

nm gold nanospheres.

Figure 3.13. Structure of the two phenylactyelene derivatives; p-

fluorophenylacetylene (6a) and 4-ethynylanisole (6b).

Figure 9.1. IR spectrum for osmium cluster 2c.

Figure 9.2. 1H NMR spectrum for osmium cluster 2c.

Figure 9.3. IR spectrum for osmium cluster 2e.

Figure 9.4. 1H spectrum for osmium cluster 2e.

Figure 9.5. 13C NMR spectrum for osmium cluster 2e.

Figure 9.6. Dose-Response curve for the osmium acid cluster 2e.

Figure 9.7. IR spectrum for osmium cluster 2f.

Figure 9.8. 1H spectrum for osmium cluster 2f.

Figure 9.9. 13C NMR spectrum for osmium cluster 2f.

Figure 9.10. Dose-Response curve for the osmium cluster 2e.

Figure 9.11. ORTEP plot of the molecular structure of 2e. Thermal ellipsoids are

drawn at the 50% probability level.

Figure 9.12. ORTEP plot of the molecular structure of 2f. Thermal ellipsoids are

drawn at the 50% probability level.

List of Schemes

Scheme 3.1. Synthesis of the diiron dithiolate boronic acid complex 1a.

Scheme 3.2. Synthesis of Os3(CO)10 (ACN)2.

Scheme 3.3. Synthesis of triosmium boronic acid cluster 2c.

Scheme 3.4. Mechanism showing the oxidation of phenylboronic acid to phenol.

Scheme 3.5. Glaser coupling of methyl propiolate to produce the diyne 4a.

Scheme 3.6. Planned synthesis of internal diyne on the surface of gold

nanostars.

List of Tables

Table 9.1. Crystallographic data for compounds 2e and 2f.

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Abstract

This project was divided into two separate investigations; both focussing on the

imaging of cancer cells. The first investigation aimed at using metal carbonyl

complexes contrast agents in SERS imaging, with a phenylboronic acid used to

target sialic acid on the cell surface. A diiron complex was found to be unstable to

light so a more stable triosmium cluster was investigated. This was shown to be

highly toxic, and therefore not suitable for imaging purposes. The second

investigation concentrated on the use of alkynes conjugated to gold nanostars to

be used as contrast agents for both SERS and photoacoustic imaging. Difficulties

were found in conjugating the alkyne to the nanoparticle, with only

phenylacetylene successfully conjugating to the nanostar and producing a Raman

signal.

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1. Introduction

1.1 Metal Carbonyl Chemistry

Carbon monoxide (CO) has widespread use as a ligand in organometallic

chemistry. Metal carbonyls are utilised within a number of industrial processes as

catalysts, for example, the use of cobalt carbonyl in the oxo process for the

production of aldehydes from alkenes. They are also common precursors in

organometallic synthesis. The bonding of CO to a transition metal centre has two

major components. First, a sigma bond is formed via donation of an electron pair

from the non-bonding sp-hybridized orbital on the carbon to the metal centre. This

donation increases the electron density on the metal. To compensate for this

increase, a pair of filled metal d-orbitals donate electron density back into the

vacant * orbitals[LWK(P1] on the carbonyl. This is given the name -backbonding.

It is this interaction that gives metal carbonyl complexes enhanced stability. The

bonding is summarised in Figure 1.1.

In more recent studies, metal carbonyl complexes have been shown to have great

potential in the area of bioorganometallic chemistry, in particular, as bio-imaging

agents and as a means of delivering therapeutic quantities of CO.

1.2 Metal Carbonyls as Bio-Imaging Agents

Bio-imaging refers to the visualisation of biological processes and tissues. It is

becoming an ever more powerful tool in the diagnosis and treatment of many

diseases. Developments in bio-imaging now allow for the in vivo analysis of a

π

π

π π*

σ

Figure 6.1. Metal-Carbonyl bonding diagram

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number of biological processes, including receptor kinetics, molecular/cellular

signalling, and the detection of disease biomarkers. An ideal bio-imaging modality

is one that can produce highly resolved images and is sensitive at clinically

relevant depths within tissues.

An important addition to the current medical imaging methods is optical imaging,

which uses the interaction of light with biological molecules to produce images.

The clinical imaging modalities commonly used include magnetic resonance

imaging (MRI), X-ray, and positron emission tomography (PET). Both X-ray and

PET use ionizing radiation, optical imaging however uses non-ionizing radiation

and therefore is safer for both the operator and the subject. Furthermore, valuable

physical and chemical information can be obtained with the use of different

wavelengths of light, based on the absorption and scattering of different

molecules. Examples of optical imaging include fluorescence, infrared (IR) and

Raman imaging.

What makes metal carbonyl complexes excellent candidates for use as bio-

imaging agents is the strong CO stretching vibration in the mid-IR region (1800 –

2200 cm-1). This so happens to be within a spectral window for living organisms,

and is free from interference by other functional groups.

1.2.1 IR Imaging

Mid-IR imaging is a light absorption technique which requires a change in dipole

moment during a molecular vibration. Transition metal carbonyl complexes are of

relevance here as the CO vibrations are very strong and can be easily tuned via

the ligand sphere on the metal. Its advantages as a bio-imaging technique include

narrower line-widths, allowing for multiplexed applications, as well as a lower

energy content. Metal carbonyl complexes were first shown to be feasible

biomarkers by Jaouen et al.1 A modified estradiol-chromium tricarbonyl complex

was synthesized and incubated with lamb uterine cytosol. With the use of FT-IR

spectroscopy, the characteristic CO peaks could be detected within the

spectroscopically silent region of ~2000cm-1.

Leong et al., demonstrated that it was possible to use water-soluble

organometallic carbonyl compounds for bio-imaging in the mid-infrared region.2

Osmium clusters were selected as they are relatively inert in the presence of air

and water and have multiple sites to attach various ligands and thus the properties

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of the clusters can be adjusted as necessary. The tested compounds, at the

relevant doses, showed only a small effect on the viability of the cells. The group

was able to achieve a spatial resolution of approximately 6 µm, and therefore not

suitable for intracellular imaging. IR microscopy has a limited spatial resolution

due to the diffraction limits given by λ/2. Therefore at 2000 cm-1 a maximum

spatial resolution of 2.5 µm can be obtained.

1.2.2 Raman Imaging

Raman spectroscopy does not suffer from the same fundamental spatial resolution

problem of IR spectroscopy due to the use of shorter wavelengths, mostly within

the visible region. Furthermore, Raman imaging can circumvent any problems with

interference from water in IR spectroscopy. A metal carbonyl complex was first

used for imaging of a live cell by 3D Raman imaging by Meister et al.3 Living HT29

human colon cancer cells were incubated with the stable manganese carbonyl

complex, [Mn(tpm)(CO)3]+. This compound can also act as a photo-inducible

CORM (CORMs are discussed in section 1.3.1 of this introduction). Raman

spectroscopy was used to study both the uptake and intracellular fate of the

carbonyl complex. It was the C=O stretching vibrations of the organometallic

complex that acted as intrinsic labels. Figure 1.2 shows the averaged Raman

spectrum of the [Mn(tpm)(CO)3]Cl signal inside a HT29 human colon cancer cell

Figure 1.7. Averaged Raman spectrum of the maximum [Mn(tpm)(CO)3]Cl signal inside a HT29 human colon cancer cell and B) a typical averaged Raman spectrum from a region within the cell lacking any metal complex.3

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(A) and a typical averaged Raman spectrum from a region within the cell lacking

any metal complex (B).

When comparing the two spectra a significant difference between the two spectra

is seen in the range 1900 to 2100 cm-1. A peak at 1963 cm-1 is clearly visible in A

but not in B, and can be assigned to the C=O stretching vibration of

[Mn(tpm)(CO)3]Cl within the cell. Raman spectroscopy gives a much improved

spatial resolution and with minimal interference from water,4 as well as a lower

scattering cross section in comparison to IR spectroscopy.5 However a higher

concentration of the metal carbonyl biotag was required than what was used in

the previous IR experiments. This gives rise to the problem of unwanted toxicity,

thus limiting the potential for use within clinical applications.

1.2.3 SERS Imaging

The requirement for higher concentrations of metal carbonyl is due to the relative

weak Raman signal produced by the C=O bond stretch. However, Jeanmaire and

Van Duyne were able to show how the Raman signals of molecules on colloidal

gold or silver nanoparticles could be enhanced through the phenomenon of

surface-enhanced Raman scattering (SERS).6 Silver and gold nanoparticles are

capable of allowing for single molecule detection, with enhancements as great as

1014.7 The enhancement is attributed to two mechanisms: the chemical and

electromagnetic effects.

Figure 1.8. Simplified diagrammatic comparison between Raman and SERS scattering.

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The electromagnetic (EM) effect gives the greatest enhancement (up to 12 orders

of magnitude). It is caused by the excitation of localized surface plasmons, the

collective oscillation of conduction band electrons. The effect is greatest when the

plasmon frequency, ωp, is equal to the frequency of incident radiation, ωL. For

most nanoparticles, this resonance occurs within the near-IR region. The

enhancement of the field magnifies the intensity of the incident light, which in turn

excites the Raman modes of the molecule in question, thus increasing Raman

scattering. The Raman signal is enhanced further by the same mechanism that

enhances the incoming radiation. A simplified visualization of the effect is given

on the left hand side of Figure 1.4.

The chemical (CM) effect, unlike the electromagnetic effect, is not applicable to all

cases. Molecules containing a lone pair of electrons, once adsorbed onto the

surface, form a charge transfer state with the metal. The HOMO to LUMO transition

for many molecules requires energy greater than those involved in Raman

spectroscopy, where IR or visible light are most commonly used. If the HOMO and

LUMO of the species in question falls symmetrically on either side of the Fermi

level of the metal surface, the metal can act as a charge-transfer intermediate.8

Therefore light at half the wavelength can be used to effect the transition, making

it possible for the excited state to be reached with visible light.

The use of a metal carbonyl complex as an effective SERS imaging agent was first

demonstrated by the Leong group.9 A metal carbonyl biotag was prepared by

combining an osmium carbonyl cluster with gold nanoparticles, forming an

Figure 1.9. A diagrammatic representation of the electromagnetic and chemical enhancement mechanisms.

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organometallic-gold conjugate (OM-NP). The enhancement of the CO Raman

signal was demonstrated by comparing the Raman spectrum of the osmium

carbonyl cluster and the OM-NP. No CO signal was detected at a concentration of

10 mM, and was only just detectable at 50 mM. The CO signals for the OM-NP

conjugates however were significantly enhanced, the intensity increased by a

factor of ~15000.

1.2.4 Photoacoustic Imaging

There is one other imaging modality which metal carbonyl complexes have been

utilised for, that being photoacoustic imaging (also known as optoacoustic

imaging). Unlike the imaging methods mentioned previously, photoacoustic

imaging (PAI) is a hybrid modality which combines the strengths of both optical

and ultrasound imaging. Photoacoustic imaging has garnered much attention over

recent years due to it having both the excellent contrast of optical imaging and

the high resolution that is afforded by ultrasound.

PAI is based upon the photoacoustic effect, whereby electromagnetic energy is

absorbed and subsequently converted into acoustic sound waves. In photoacoustic

imaging the sample is irradiated with a light source (typically in the NIR region

due to the biological window); a fraction of the incident light energy is absorbed.

This energy is then rapidly converted into heat within picoseconds. This rise in

temperature leads to thermoelastic expansion, the sudden pressure increase

propagated as a sound wave. The low diffusion of the acoustic wave allows for

greater coherence in comparison to reflected optical signals which are more easily

scattered within tissue. The pressure wave is detected on the surface using

acoustic transducers. The use of algorithms and an array of transducers allows for

an image to be rendered.

Endogenous contrast agents, namely oxy- and deoxyhaemoglobin, can be used in

PA imaging and are sufficient to provide functional information. However, the

imaging capabilities of this modality can be improved greatly with the introduction

of an exogenous contrast agent.

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The only example of a metal carbonyl complex used as a photoacoustic contrast

agent was demonstrated by the Leong group. The water-soluble osmium carbonyl

cluster [Os3(CO)10(µ-H)(µ-S(CH2)2COO-]-Na+ was synthesised, the salt was then

used for the in vivo photoacoustic imaging of a rat’s cerebral vasculature.10 An

osmium carbonyl was selected due to it exhibiting a high photoacoustic signal as

well as excellent chemical stability and minimal cytotoxicity. The carbonyl cluster

contrast agent afforded high spatial resolution and good sensitivity.

1.3 Metal Carbonyls in Medicine

Carbon monoxide has been shown to be an important low concentration cell

signalling molecule within mammalian systems. CO is produced naturally in every

mammalian cell by heme catabolism. The process is catalysed by the enzyme

heme oxygenase (HO).11 CO, Fe2+, and biliverdin are the resultant products of the

catabolism of heme by HO. The green biliverdin is subsequently reduced to yellow

bilirubin. The action of HO is commonly observed in the development of bruises,

as the colour changes from red/purple through to yellow.

Much research has been carried out on the biological actions of CO. It has been

found that the molecule is anti-inflammatory, anti-apoptotic and anti-proliferative,

it protects tissues against hypoxia or ischemia reperfusion injuries and causes

vasodilation.12 Use of CO gas has already been shown to increase survival rates in

rat cardiac transplants, reverse pulmonary hypertension and suppress

Figure 1.10. An image of the rat brain vasculature produced with in vivo imaging with the Osmium salt.10

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arteriosclerotic lesions due to graft rejection.13 The main problem faced when

developing medical uses for CO is its toxicity. Carbon monoxide is often referred

to as the “silent killer” as the gas is both colourless and odourless. CO causes toxic

effects within the body by combining with hemoglobin to form carboxyhemoglobin.

The affinity of hemoglobin is approximately 210 greater for CO than O2, thus

preventing the hemoglobin from carrying oxygen to tissues. On average, exposure

at 100ppm or above can have damaging effects on human health. 14

This characteristic toxicity of CO in higher concentrations has led to research

towards methods in which the quantities of CO, and the manner in which it is

released, can be easily controlled and monitored. A potential solution is the use of

organometallic carbonyl compounds which can be triggered to release CO.

1.3.1 Carbon Monoxide Releasing Molecules

Carbon monoxide releasing molecules or CORMs, are typically transition metal

carbonyl complexes with which the release of the CO ligands can be triggered by

environmental factors. Triggers include chemical interactions, thermal triggers,

and the interaction with light. Examples of early CORMs include Mn2(CO)10,

[Ru(CO)3Cl2]2, and Ru(CO)3Cl(glycinate).15–17 Ru(CO)3Cl(glycinate) (CORM-3) was

shown to have low toxicity, to be a vasodilator, and greatly increased the survival

rates of mice following heart transplants.17There are numerous challenges to the

development of metal carbonyl complexes as CORMs. Many metal carbonyls are

toxic due to their lack of stability. This can then lead to the uncontrolled release

of CO as well as the release of heavy metals into the blood. Much research has

focussed on finding metal carbonyls that have sufficient stability as well as CO

releasing properties.

Certain CORMs release CO when irradiated with light, these are given the name

photoCORMs. The use of light as the trigger allows for greater control and precision

when administering CO within tissues. There have been many metal carbonyl

complexes reported that release CO once irradiated with UV, including the

manganese carbonyl complex mentioned previously, [Mn(tpm)(CO)3]+.3 This

photoCORM was shown to have photo-initiated cytotoxicity against the human

colon cancer cell line HT29.18 More recently, however, photoCORMs which are

triggered by visible light have also been described. These include

tricarbonylmanganese,19 and iron carbonyls containing cysteamine ligands.20 A

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diiron dithiolate complex which was both water soluble and stable has also been

described; the complex was shown to release all six CO ligands within 30 minutes

of irradiation with broad-band visible light.21

1.3.2 Targeted Applications of Metal Carbonyls

One advantage of using metal complexes for clinical treatment is the potential to

co-ordinate ligands with differing functions. For biological applications, it is often

required to target particular tissue types or areas within the body. This calls for

the production of therapeutic/imaging agents that are able to target specific

biomarkers within the body. For bioimaging applications, targeting is often

required to insure that there is sufficient accumulation of the agent within the

targeted tissue to allow for high contrast images to be produced. Numerous

biomarkers can act as potential targets, including specific cells, molecules, genes,

gene products, enzymes, or hormones.

For this project the biomarker selected was sialic acids. Sialic acid is the term

given to a family of carbohydrates that are derived from the nine carbon sugar

neuramic acid. There are over 50 carbohydrates that belong to this family, with

the most commonly found being N-

acteylneuramic acid (Neu5Ac). Sialic acids are

synthesised in, and expressed by, essentially

every vertebrate cell. They play a role in a

multitude of physiological processes, for

example, differentiation, proliferation, immune

response, cell-cell communication, and

microbial infection. They very rarely exist freely

and are mostly found at the terminal position

Figure 1.7. The structure of of N-acteylneuramic acid (Neu5Ac).

Figure 1.6. The structure of the diiron dithiolate photoCORM

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of oligosaccharide chains (such as glycoproteins and glycolipids) located on the

surface of cells.

Increased levels of sialic acids were observed in patients suffering from a number

of different cancers, including lung cancer,22 cervical cancer,23 and oral cancer.24

It is this overexpression on the surface of cancer cells that make sialic acids

excellent biomarkers for both imaging and therapeutic agents.

One method for labelling this group of carbohydrates is by reacting them with

phenylboronic acid (PBA) to form five- and six-membered cyclic complexes. PBA

was combined with quantum dots to produce a biotag which effectively targeted

sialic acid on living cells.25 The PBA allows for efficient tagging of sialic acids (and

other saccharides) due to its ability to react reversibly with 1,2- and 1,3-diols. The

selectivity of the method derives from the interaction requiring the correct

stereochemistry; formation of a stable boronic acid-diol complex requires the

presence of syn-periplanar diol groups.26 The structure of PAPBA (3-

(propionamido)phenylboronic acid) complexed to Neu5Ac is given in figure 1.8.

1.4 Gold Nanoparticles in Bio-Imaging

The ability of gold (and silver) nanoparticles to enhance the Raman signals of

metal carbonyls bound to the surface has been mentioned previously. This is the

basis of SERS imaging. As well as being utilised for SERS, gold nanoparticles

(AuNP’s) are also able to act as excellent contrast agents for photoacoustic

imaging. This is due to their strong optical absorption as a result of the surface

plasmon resonance. It is possible to tune the absorption of the AuNP’s by altering

their size and shape; absorption in the near-IR region is desired for imaging as

attenuation by blood and tissue is minimised. There have been a number of gold

Figure 1.8. Structure of the Neu5ac/PAPBA boronic acid-diol complex

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nanostructures developed alongside the original colloidal nanospheres, including

nanorods, nanoshells, nanocages and nanostars.

Gold nanoparticles as photoacoustic imaging contrast agents was first

demonstrated by Copland et al.27 The group was able to successfully demonstrate

the feasibility of using gold nanoparticles for the detection of deeply-seated

tumours via PAI. Antibodies were conjugated to the AuNP’s to target human breast

cancer cells. The cells were detected by PAI in a gelatin phantom at concentrations

as low as 109 NP’s per ml and at depths of up to 6 cm. The peak absorption of the

nanospheres was at 532 nm, just outside of the desired near-IR window.

Gold nanorods (AuNR’s) were demonstrated to be more suitable contrast agents

by Eghtedari et al.28 The advantage of nanorods is that their dimensions are more

easily tuned, and hence peak absorption within the NIR region can be obtained. A

PA signal was detectable at a depth of 12 mm with a 25 µl aliquot of AuNR’s, at a

concentration of 1.25 pM, injected into live mice, demonstrating that it was

possible to detect and localize AuNR’s at very low concentrations deep within living

tissue. The ability to adjust the dimension of nanorods also allows for the potential

of multiplexing. Multiplexing is the targeting of multiple targets simultaneously.

This is necessary for the detection of cellular content that is typical in cancers.

This multiplexed approach to PA imaging with the use of gold nanorods was

demonstrated by Li et al.29 Different antibodies were conjugated to AuNR’s with

different aspect ratios in order to target two unique cancer cell types, OECM1 and

Cal27. By switching the wavelength of the laser, the corresponding cell type could

be identified and distinguished from the other. In vitro and in vivo experiments

showed contrast enhancements of up to 10 and 3.5 dB, respectively.

The ability to conjugate certain functionalized molecules onto the surface of gold

nanoparticles allows for the possibility of contrast agents with dual functionality.

For example gold nanorods functionalized with Raman active dyes were utilised

for both PA and SERS imaging of ovarian cancer within live mice.30 The

photoacoustic modality allowed for imaging at depths of around 3 – 4 cm and the

SERS overcomes the lack of sensitivity afforded by PA imaging. This then allowed

for the operative resection of the tumour, and successfully demonstrated the

utility of a nanogold-based contrast agent with dual PA and SERS imaging

functionality.

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2. Objectives

My work whilst at NTU is split between two projects. The aim of the first is to

synthesise a diiron carbonyl complex that can act as both a SERS imaging agent

and a photoCORM. The complex is based on the diiron photoCORM designed by

the Leong group,21 with the carboxylic acid replaced with a phenylboronic acid

group. It is hoped that the phenylboronic acid will target the sialic acids that are

over-expressed on the surface of cancer cells and the CO Raman signals will be

detected with SERS within the biological window; the ability to construct a cellular

image from the SERS signal of CO ligands has been demonstrated previously.9 For

the enhancement, a phenylboronic acid gold nanoparticle will have to be co-

administered with the complex. The compound should show low cytotoxicity, high

sensitivity and targeting for cancer cells.

The second branch of my project is research into alkyne-gold nanoparticle

conjugates for dual modal SERS/PA imaging in vivo. It has already been shown

that it is possible to create a contrast agent that has dual functionality by

conjugating gold nanoparticles with Raman active molecules.30 The greater

symmetry of the C≡C bond in an alkyne conjugate is expected to afford a more

intense Raman signal than the carbonyl ligand, and they also appear within the

previously mentioned biological window.31 By varying the alkyne conjugated, due

to the narrow signals produced, it is possible to target more than one biomarker

simultaneously. Various alkynes will be screened in order to find those that give

the strongest Raman signals and which do not overlap. Once the alkynes have

been selected, the nanoparticle conjugate will be combined with a targeting

biomolecule to target different cancer cells.

Figure 2.1. Structure of planned phenylboronic photoCORM to be used for SERS imaging

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3. Results & Discussion

3.1 Diiron-mercaptophenylboronic acid complexes as dual imaging/therapeutic

agents of cancer cells

As mentioned in the introduction the aim of this section of the project is on the

use of a diiron carbonyl complex as both a SERS imaging agent and a photoCORM.

The structure of the desired complex is as shown in Figure 2.1, and its synthesis

in Scheme 3.1.

The diiron thiolate bridged boronic acid complex was synthesised by reacting

Fe3(CO)12 with two molar equivalents of 4-mercaptophenylboronic acid. The

complex was unstable to light; the colour changed from orange to brown on

exposure to ambient light. Due to the relative instability of this diiron complex, it

was desirable to have a complex that could better withstand irradiation with a

laser, as would be required for a functional imaging agent.

A more stable family of compounds are the osmium clusters. The use of a boronic

acid functionalised osmium cluster has been previously utilised in a SERS-based

assay for glucose.[1] The improved stability of the osmium in comparison to the

diiron complex, however, may result in a reduction in the CO releasing

functionality. The osmium cluster synthesis has been previously documented; the

precusor is produced by reacting triosmium dodecacarbonyl, Os3(CO)12, with

Scheme 3.1. Synthesis of the diiron dithiolate boronic acid complex 1a.

1a

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trimethyl N-oxide (3 molar equivalents) and acetonitrile to give Os3(CO)10 (ACN)2

as shown in Scheme 3.2

Once the reaction was deemed complete, as monitored by IR spectroscopy, the

product was isolated and reacted with 4-mercaptophenylboronic acid. The identity

of the product was confirmed by NMR spectroscopy.

The NMR spectra is given in Figure 9.2. The peak at -16.9 ppm indicates the

bridging hydride. The large upfield shift is due to the extensive shielding effects

of the electron-rich osmium atoms donating e- density to the hydrogen atom. The

other peaks of note are those in the aromatic region (7 – 8 ppm); the doublets at

7.8 ppm and 7.4 ppm can be attributed to the aromatic hydrogens at positions

Scheme 3.2. Synthesis of Os3(CO)10 (ACN)2.

2a

2a 2b 2c

Scheme 3.3. Synthesis of triosmium boronic acid cluster 2c.

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(2,3) and (4,5) respectively. The presence of the hydride bridging peak, in

particular, gives sufficient evidence that the synthesis was successful.

The cytotoxicity of the osmium cluster 2c was evaluated on Hela cancer cells, as

per the technique described in section 7.2.5. Hela cells are derived from cervical

cancer and were selected due their excellent resilience making them easy to

handle. A dose response curve was generated using a sigmoidal dose response

(variable slope) equation. The IC50 was found to be 23 ± 2 µM.

The high toxicity of the compound was surprising, given its relative stability, and

warranted further investigation. A toxic compound has little use as an imaging

agent as the cells are required to survive the treatment so that an image can then

be produced. The cytotoxicity test was repeated alongside the ligand, 2b and a

simple thiolato-triosmium cluster, 2d

Figure 3.1. Dose-Response curve for the osmium boronic acid cluster 2c.

21

The IC50 of the osmium cluster 2c was determined this time to be 27 ± 3 µM. It

was also found that both 2b and 2d had little effect on the cell viability, even at a

high concentration of 75 µM. These results suggest that it is not the ligand which

is inducing the toxic effects, and therefore it must either be the metal carbonyl or

their complex that is the source of toxicity. However, although cluster 2d shows

little effect on the cell viability, it may have to do with its low solubility and hence

low cellular uptake. To test this last hypothesis, an analogue which is soluble in

aqueous conditions was required.

The cluster 2e was synthesised using the same method that was used to produce

both clusters 2c and 2d, using the appropriate ligand (thiophenol for 2d and 4-

mercaptobenzoic acid for 2e). The x-ray crystal structure of 2e was also

determined, and is given in Figure 9.11.

2b 2d

Figure 3.2. Structure of phenylboronic acid 2b and the thiolate-triosmium cluster 2d.

22

An attempt at the determination of the IC50 value for this complex was not possible

as the data gave a poor fit to the expected sigmoidal curve. Nevertheless, the

data acquired suggest that 50 % cell viability was shown at approximately 50 µM.

Though it is not possible to make direct comparisons between the IC50 values of

2c and 2b, it can be observed that the toxicity of 2e lies somewhere in between

the two. This is further demonstrated by comparing the cell viability of all

complexes at doses of 50 µM, as shown in Figure 3.5.

One of the characteristic features of cancer cells is the accumulation of high

concentrations of hydrogen peroxide.[2] Phenylboronic acid can undergo cleavage

in the presence of an oxidant such as hydrogen peroxide. The mechanism for such

an oxidation is given in Scheme 3.4. To better understand the toxic mechanism of

Os-BA, the toxicity of a phenol functionalised osmium cluster (2f) was also

investigated, and also given in Figure 3.5.

2e

Figure 3.3. Structure of triosmium-benzoic acid cluster 2e.

Scheme 3.4. Mechanism showing the oxidation of phenylboronic acid to phenol.

23

The phenol cluster 2f was found to be cytotoxic, even more so than 2c; the IC50

was determined to be 12.7 ± 0.7 µM. This may give an indication as to what

causes the toxicity of the 2c. The slightly greater toxicity of 2f than 2c could be

due to the incomplete conversion of one to the other.

Though this data appears to show that the toxicity is indeed a result of cluster 2c

being oxidised by increased levels of peroxide produced by the cancer cells, more

information is required before a conclusion can be drawn. Although phenylboronic

acid can be oxidised by hydrogen peroxide, whether there is a sufficient

concentration at the cellular level has yet to be determined. The general cellular

environment must also be considered; the cellular target has not been

determined.

Figure 3.4. Comparison of the cell viability when treated with 50 µM of compounds 2a - f

24

Figure 3.5. Structure of triosmium-mercaptophenol cluster 2f.

25

3. 2 Alkyne-Gold NP conjugate for dual modal SERS/PAT imaging of cancer

cells

The first step in designing a contrast agent that could be applied to both SERS and

photoacoustic imaging was to determine which alkyne when functionalized to the

gold nanoparticles would give sharp, strong C≡C Raman absorptions. The need for

strong, well defined Raman peaks is give a great enough contrast so that a highly

resolved image can be produced. Narrow peaks also allow for the possibility of

multiplexing (targeting two distinct biomarkers simultaneously), as it would be

possible to differentiate the signals of two or more different alkynes.

The gold nanostructure to be functionalized with the alkynes was chosen to be

gold nanostars (AuNS’s). Nanostars were selected rather than other

nanostructures as AuNS’s have high photothermal conversion efficiency, due to

their strong absorption of near-infrared radiation. AuNS’s have been demonstrated

to produce greater photoacoustic signals than gold nanorods at equivalent

concentrations.[3] The synthesis of the AuNS’s was adapted from that reported by

Vo-Dinh et al.[4] This method allows for the simple synthesis of the AuNS’s without

the use of the toxic surfactant cetylammonium bromide (CTAB), thus facilitating

the clinical use of nanostars.

In this method, altering the concentration of AgNO3 allows for the adjustment of

the size of the resultant nanostars. As mentioned in the introduction of this report,

altering the dimensions of a nanostructure will change its optical properties. With

the aim of the nanostars to absorb light within the near-IR region, a concentration

of 2.0 mM was chosen. The structures of both the nanoseeds and nanostars were

confirmed with the use of transmission electron microscopy (TEM). The images

produced are given in in Figure 3.6; the sizes of the nanoseeds and nanostars are

approximately 12 and 60 nm, respectively.

26

The UV-Vis spectrum of the gold nanostars show an absorption peak at

approximately 728 nm (Figure 3.6). This falls well within the near-IR region of the

electromagnetic spectrum, as is preferable for bio-imaging.

Figure 3.7. UV/vis spectrum for 60 nm gold nanostars.

Figure 3.6. TEM images of 12 nm gold nanoseeds (A) and 60 nm gold nanostars (B).

27

Of the first alkynes tested (Figure 3.8), 3a and 3c produced no Raman signals in

the desired region of 1800 – 2200 cm-1. The reason for this was attributed to the

lack of solubility of the two compounds; a white precipitate was observed when

DMSO solutions of these were added into the aqueous suspension of gold

nanostars. Both alkynes 3b and 3d produced identifiable signals within the target

region. The normalized spectra of the four gold nanostar conjugates are given in

Figure 3.9; the peaks for phenylacetylene and propiolic acid are highlighted.

3a

3b

3c

3d

3a

3d 3c

3b

Figure 3.8. Structure of diphenyl-butadiyne (3a), phenylacetylene (3b), diphenylactylene (3c), and propiolic acid (3d).

Figure 3.9. Raman spectra of alkynes 3a - d conjugated to 60 nm gold nanostars.

28

Although both gave recognisable peaks it is clear that phenylacetylene gave the

stronger signal. It is desirable to be able to detect different targets simultaneously

– termed multiplexing. In order for this to be realised, two or more alkynes are

required, each with strong, narrow absorption peaks which are distinguishable. A

possible candidate to complement phenylacetylene is a water soluble internal

diyne, as diynes have been shown to give greater Raman intensities than

alkynes.[5] The target molecule was decided to be the diyne di-acid shown in Figure

3.10.

The synthesis of this compound has not been reported, so a modification of the

Glaser coupling reaction using CuI as a catalyst was devised.[6] Since the coupling

reaction requires basic conditions, the acid groups were protected as the methyl

propiolates. The planned reaction scheme is given below.

Scheme 3.5. Glaser coupling of methyl propiolate to produce the diyne 4a.

4a

Figure 3.10. Structure of planned acidic internal diyne.

29

After multiple attempts at synthesising the diyne, a yellow oil was isolated and

then purified by column chromatography. The expected mass of the product was

166, however a mass of 684.05 m/z was recorded. The NMR spectrum also did

not agree with that expected. The reason as to why the synthesis was unsuccessful

is unclear. However, as there are no reported examples of this coupling reaction

being successfully carried out on alkynes with ester groups, it is likely that it is

this functionality that is interfering with the reaction.

A more radical approach to the synthesis was explored; the idea was to carry out

the coupling of two terminal alkynes on the surface of gold nanoparticles. The

planned scheme for this coupling is given in Scheme 3.6.

Scheme 3.6. Planned synthesis of internal diyne on the surface of gold nanostars.

The use of a TMS protecting group allows for a water soluble alkyne to be

conjugate with the gold nanostars, as the TMS functional group can easily be

removed under basic conditions (this is desirable so as to not reduce the stability

of the gold nanostars). Once the TMS protecting group has been removed, it would

allow for the coupling of a second terminal alkyne via a Glaser coupling. This

method would also allow for variation of the –R group, and avoid the use of the

acidic/ester groups which are believed to have interfered with the Glaser coupling.

As the coupling reaction and the deprotection both require basic conditions, the

synthesis has the potential to be streamlined, combing steps one and two.

Unfortunately, problems arose during the attempt to conjugate the TMS-acetylene

onto the gold nanostars. First 40 µl of the alkyne was added at a concentration of

4b

30

10 mM as per the experimental described in 7.3.3. The Raman spectra was taken,

shown in Figure 3.12, but no signal was detected in the 1800 – 2200 cm-1 region.

The experiment was repeated with the concentration of the TMS-acetylene

increased by a factor of 5. Still no Raman peaks were observed.

It[LWK(P2] was decided to no longer investigate the potential of TMS-alkyne

nanostars in the synthesis of diyne functionalized nanostars, as the conjugation

was not possible. The reason for why it had been unsuccessful could be due to the

non-planar structure of the TMS group. All other successful conjugations had been

carried out with planar molecules (propiolic acid and phenylactylene). This may

indicate that alkyne molecules conjugate to the nanoparticles side-on and the

relevant bulk of the TMS group inhibited the conjugation. Analysing the

conjugating mechanism of alkynes to gold nanoparticles is beyond the scope of

this investigation.

Rather than trying to find a way to utilise diynes as conjugates, the focus was

shifted to finding potential functionalization candidates have distinct Raman

spectra that allows for multiplexing. Meaning that strong, narrow Raman signals

had to be produced for a compound to warrant further investigation. Four planar

alkynes were tested; acetylene (produced via reacting calcium carbide and water),

propiolic acid, phenylacetylene, and acetylene dicarboxylic acid. The reason why

phenylacetylene and propiolic acid was tested once more was that due to the

alkynes being tested under different conditions. 10 µl of 5 mM stock solutions of

each alkyne (excluding acetylene) were added to 1 ml solutions of 60 nm gold

nanospheres (which had been stockpiled from previous experiments carried out

by the group). The acetylene was produced by slowly adding water to a large

excess of calcium carbide within a sealed vessel and bubbling the gas produced

via a capillary tube through 1ml of gold nanospheres. After 2 hours the SERS

spectra were recorded.

31

Figure 3.11. Structure of 4 alkynes; acetylene (5a), acetylenedioc acid (5b), phenylacetylene (3b), and propiolic acid (3d).

5a

5b

3d

3b

3b

3d

5b

5a

4b

Figure 3.12. Raman spectra of alkynes 3a, 3d, 5a, 5b, and 4b conjugated to 60 nm gold nanospheres

32

The strongest Raman signal was produced by phenylactetylene, followed by the

propiolic acid. It was theorised that the aromaticity could play a role in the greater

enhancement of the Raman signal, to put this to the test and to also investigate

two more potential conjugates for multiplexing, two phenylacetylene derivatives

were tried. One being p-fluorophenylacetylene, with the fluorine acting as an

electron withdrawing group. The second derivative was 4-ethynylanisole, with the

methoxy group acting as an electron donating group. By comparing how different

electrophilic directing groups alter the strength and/or positon if the alkyne signal,

more information can be gained on the importance of the aromatic ring in giving

the strong signals that have been observed previously.

As with the previous tests, stock solutions of 5 mM were made up. 10 µl of each

of the two solutions was added to 1 ml of 60 nm nanospheres. The samples were

refrigerated until they were able to be tested. Upon removal of the samples if was

apparent that the nanoparticles had aggregated (were no longer a free

suspension). This occurs due to a drop in the stability of the nanoparticles; can be

caused by a number of factors. This aggregation meant that no spectra could be

obtained.

Figure 3.13. Structure of the two phenylactyelene derivatives; p-fluorophenylacetylene (6a) and 4-ethynylanisole (6b).

6a 6b

33

4. Conclusion

4.1 Diiron-mercaptophenylboronic acid complexes as dual imaging/therapeutic

agents of cancer cells

A diiron complex with functionalised with a phenylboronic acid ligand was

successfully synthesised and characterised, with the purpose to use the complex

as both a contrast SERS imaging of cancer cells and a photoCORM. Once the

complex had been produced it was found to be light sensitive and would

decompose upon exposure. This instability makes the compound unsuitable for

use as an imaging agent as would not be able to withstand irradiation with a laser

pulse.

As an alternative a more stable triosmium complex, which had already been

demonstrated to be a feasible probe for the SERS-based assay for glucose,

became the focus of investigation. The stability of the complex meant for the loss

of photoCORM functionality. Once synthesised, the cytotoxicity of the complex was

tested on Hela cells and was found to be highly toxic. The IC50 was calculated to

be 23 ± 2 µM. The toxicity is surprising due to the relative stability of the osmium

cluster.

An investigation into the mechanism behind the toxicity was carried out. It was

theorised that that toxicity was due to the phenylboronic acid being oxidised by

high concentrations of hydrogen peroxide to form phenol. The IC50 of the phenol

functionalised cluster was found to be 12.7 ± 0.7 µM. In comparison, a cluster

with benzoic acid rather than boronic acid was found far less toxic. An IC50 could

not be calculated but cell viability was much greater at comparable concentrations

than either the boronic acid or phenol clusters. This is not sufficient evidence to

determine the mechanism of toxicity and further research is required.

In reflection upon the initial aims of this investigation it is very apparent that these

aims were not reached. The reasons being due to the unforeseen instability of the

diiron complex and the toxicity of the triosmium cluster. The toxicity however lead

to research into the use of the triomsium cluster as an anti-cancer drug.

34

4.2 Alkyne-Gold NP conjugate for dual modal SERS/PAT imaging of cancer

cells

60 nm gold nanostars were synthesised from citrate stabilised nanoseeds, the

structure of the nanoparticles was confirmed with TEM and UV/vis. In an attempt

to produce a contrast agent for both SERS and photoacoustic imaging 4 different

alkynes; propiolic acid, diphenlyactylene, phenylactylene, and diphenyl-butadiyne

were added to 1 ml samples of the gold nanostars in an attempt to conjugate

them.

Raman spectra were recorded for the four samples, the only alkynes that produced

a signal was propiolic acid and phenylacetylene. The reason for this was believed

to be a matter of solubility. For the purpose of creating an internal diyne and thus

an identifiable spectra in comparison to the two external alkynes, the synthesis of

a diyne di-acid was attempted. This was unsuccessful.

A more radical approach to forming an internal diyne was taken; the aim of which

was to first conjugate TMS-alkyne to the nano-surface. Once conjugated, the TMS

protecting group would be removed and the Glaser coupling would be attempted.

This too was unsuccessful, due to an inability to conjugate the TMS-alkyne to the

nanoparticle.

Focus of the investigation was shifted away from finding a suitable diyne, but

instead on other alkynes that produced strong, narrow Raman signals and were

distinguishable from that of phenylacetylene. The alkynes tested were acetylene

gas, 2-butynedioic acid, p-fluoroacetylene and 4-ethynylanisole. None of these

were found to be suitable; due to time constraints, a lack of progress and greater

success with the other project efforts were shifted away from this investigation.

The aim of this investigation was to create a contrast agent for both SERS and PA

imaging based on alkyne conjugated to gold nanostars. . Difficulties were found

in conjugating the alkyne to the nanoparticle, with only phenylacetylene

successfully conjugating to the nanostar and producing a Raman signal.

35

5. Future Work

5.1 Diiron-mercaptophenylboronic acid complexes as dual imaging/therapeutic

agents of cancer cells

This investigation could be taken in two different directions, either continue

research into the use of the Os-BA complex as an anti-cancer drug or attempt to

adjust the complex as to reduce cytotoxicity and allow for use as an SERS imaging

contrast agent. For either of these two possibilities it is important to determine

what cause the toxicity of the complex.

If it is shown that the phenylboronic acid functionality is at the root of the effects

then by selecting a different target on the cancer cell and replacing the

mercaptophenylboronic acid ligand for one that will bind to the new target.

Assuming that the new ligand doesn’t induce toxicity, this would produce a viable

contrast agent for SERS imaging.

To research the usefulness of the product as an anti-cancer drug, an investigation

into the selectivity of the cluster would be necessary. It is still yet to be determined

whether the boronic acid gives a great enough selectivity for cancer cells. It would

also be worth investigating any difference cytotoxicity towards cancer cells other

than Hela cells.

5.2 Alkyne-Gold NP conjugate for dual modal SERS/PAT imaging of cancer

cells

Throughout this investigation the main obstacle proved to be conjugating the

alkynes to the gold nanoparticles. The only real success was found with the use of

phenylacetylene. Therefore, in continuing this investigation the best avenue of

research would be focus solely on phenylacetylene, and no longer focus on

multiplexing capabilities.

Attempting to produce both SERS and photoacoustic images in vitro and if proven

viable in vivo, would act as a proof of concept. Once the phenylactetylene-gold

nanostar conjugates have been shown to been viable contrast agents for both

SERS and photoacoustic imaging, trialling other phenylacetylene derivatives for

multiplexing could be carried out.

36

6. Acknowledgements

First I wish to thank my placement supervisor at Nanyang Technological

University, Associate Professor Leong Weng Kee, for providing me with the

opportunity to be a member of his research group for the duration of my exchange.

His insight and advice was invaluable for my work.

Particular thanks must go to my mentor in the lab, Mr Lam Zhiyong, without whom

I certainly would not have been able to complete this project. His knowledge of

the field and willingness to assist me with my project whenever necessary allowed

me to learn a great deal about a topic which I knew little about prior to joining the

group.

I also would like to acknowledge the other members of the research team who

welcomed me into the group and also gave me advice and assistance with my

project.

Finally thanks those at The University of Edinburgh that made this exchange to

NTU possible, including Dr Michael Cowley, Dr Peter Kirsop and Dr Simon Daff.

37

7. Experimental

7.1 General Experimental

All reactions and manipulations were carried out under an argon atmosphere using

standard Schlenk techniques unless stated otherwise. Solvents that were used for

reaction were distilled over the appropriate drying agents under argon before use.

Compounds were typically purified by column chromatography on silica gel, or by

preparative thin-layer chromatography (TLC) using 20 cm × 20 cm plates coated

with silica gel 60 F254 with the appropriate mobile phase.

Infrared (IR) spectra were recorded on a Bruker Alpha FT-IR spectrometer.

Solution spectra were recorded in DCM, unless otherwise stated, in a solution IR

cell with NaCl windows and a path length of 0.1 mm, at a resolution of 2 cm-1.

1H NMR spectra were recorded on a JEOL ECA 400 or ECA 400SL at 400 MHz,

referenced to the residual proton resonance of acetone-d6, unless otherwise

stated. 13C NMR spectra were recorded on the same instruments at the

corresponding frequency. Chemical shifts, δ, are in ppm and coupling constants,

J, are in Hz.

Mass spectra (MS) were recorded on a Thermo Deca Max (LCMS) mass

spectrometer with an ion-trap mass detector at 15 eV, 40 °C in the Electrospray

Ionization (ESI) mode. Copper specimen grids (300 mesh) with carbon support

film were purchased from Beijing XXBR Technology Co. Ltd. TEM images were

collected on a JEM-1400 transmission electron microscope operated at 100 kV.

The Raman spectral measurements were carried out using a Reinshaw InVia

Raman (UK) microscope with a Peltier cooled CCD detector and an excitation

wavelength of 633 nm, where the laser beam was directed to the sample through

a 50× objective lens, which was used to excite the sample and to also collect the

return Raman signal. All Raman spectra were processed with the WiRE 3.0

software. The maximum laser power at the sample was measured to be 6.1 mW

and the exposure time was set at 10 seconds unless stated otherwise. Prior to

each measurement, the instrument was calibrated against a silicon standard with

a Raman peak centred at 520 cm-1.

38

7.2 Diiron-mercapthenylboronic acid complexes as dual imaging/therapeutic

agents of cancer cells

7.2.1 Synthesis of diiron-mercaptophenylboronic acid complex

Dry THF (5 ml) was added to Fe3(CO)12 (48.3 mg, 0.096 mmol) and 4-

mercaptophenylboronic acid (32.5 mg, 0.211 mmol). The mixture was refluxed

for 40 mins. Over which time the colour changed from green to orange/brown.

The THF was then removed under pressure. A recrystallization was attempted in

order to purify the product. The product was dissolved in a minimal amount of

DCM and heated. The undissolved residue was removed via filtration and then

dissolved in acetone. Removal of the acetone yielded an orange product.

7.2.2 Synthesis of Os3(CO)10(NCCH3)2

Os3(CO)12 (200 mg, 0.2205 mmol) was stirred in dichloromethane (80 ml) with

bubbling of argon. TMNO (58 mg, 0.7455 mmol) dissolved in acetonitrile (40 ml)

was added dropwise over 1 hour, with stirring at room temperature. Once all

TMNO had been added the reaction mixture was stirred for a further 15 minutes.

The reaction was monitored with use of IR spectroscopy, with the bis ACN stretch

identifying the desired product.

The yellow solution was filtered through a short silica column. The solvent was

removed under reduced pressure, though not to the point of complete dryness.

The product was dried fully by blowing N2 over the sample.

7.2.3 Synthesis of Os-BA

Os3(CO)10(NCCH3)2 (39.2 mg, 0.042 mmol) and HSC6H4B(OH)2 (7.8 mg, 0.050

mmol) was dissolved in THF (7 ml) and left to stir overnight under argon at room

temperature. The progress of the reaction was monitored with IR spectroscopy.

The residue was purified by TLC using ethyl acetate and hexane (1:2, v/v) as the

eluent. This yielded two yellow bands, with the second band being the desired

product, as confirmed by the IR. The Rf value = 0.19. Extraction of this yellow

band gave a yellow solid.

Experimental yield = 57.1 %.

39

7.2.4 Synthesis of Os-COOH

Os3(CO)10(NCCH3)2 (55.5 mg, 0.042 mmol) and HSC6H4COOH (12.5 mg, 0.084

mmol) was dissolved in THF (8 ml) and left to stir overnight under argon at room

temperature. The progress of the reaction was monitored with IR spectroscopy.

The residue was purified by TLC using ethyl acetate and hexane (1:1, v/v) as the

eluent. This yielded two yellow bands, with the second band being the desired

product, as confirmed by the IR. The Rf value = 0.24. Extraction of this yellow

band gave a yellow solid. The purity of which was confirmed by 1H NMR.

7.2.5 Cell culture and cell viability assay

Experimental cultures of Hela cells were obtained from the American Type Culture

Collection (ATCC) and cultured in tissue culture dishes (Nunc Inc., IL). The cells

were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Biowest, France)

supplemented with 10 % fetal bovine serum (FBS; Gibco®, NY), 1 % L-glutamine (PAA

Laboratories, Austria), and 1 % penicillin/streptomycin (HyClone, UT) at 37 ᵒC in

5 % CO2 atmosphere. Phosphate buffered saline (PBS) was obtained from PAA

Laboratories. Stock solutions of clusters 2a, 2c – 2f, and ligand 2b in sterile-filtered

dimethyl sulfoxide (DMSO) were prepared and serial diluted to lower

concentrations. For treatment with the compounds, cells were seeded in wells

within 96 well plate in growth medium at the same initial density of 10 000 cells

per well, and allowed to adhere and grow for 24 hours. This was followed by

treatment with the indicated concentrations of compounds in DMEM (0.5 % DMSO)

for 24 hours. Control cells were treated with vehicle (0.5 % DMSO). To each well,

10 µl of MTS reagent (MTS Cell Proliferation Assay Kit, BioVision, CA) was added

and then left to incubate in a 37 °C incubator with 5 % CO2 for 2 hours. The

absorbance intensities at 490 nm were then measured and the cell proliferation

relative to the control sample was calculated. Each sample was analyzed in

triplicates and was corrected with corresponding background intensities from the

same incubation conditions with the cells. The curves were generated using a

sigmoidal dose response (variable slope) equation, using Graph Pad Prism 5

software, with the IC50 determined.

40

7.3 Alkyne-Gold NP conjugate for dual modal SERS/PAT imaging of cancer

cells

7.3.1 Synthesis of alkyne conjugated gold nanostars

In a typical reaction, stock gold nano-seed solution (100 µl) was added to HAuCl4

(10 µl, 0.25 mM) and HCl (10 µl, 1M). The mixture was stirred whilst AgNO3 (100

µl, 2.0mM) and ascorbic acid (50 µl, 100mM) were simultaneously. There was an

immediate colour change from pale red to dark blue. The mixture was stirred for

a further 2 mins before addition of the alkyne, (40 µl, 20mM). After 10 mins of

stirring, polyacrylic acid (2 ml, 4 mg/ml) and NaOH (3 ml, 0.1M) was added. The

reaction mixture was stirred for 2 h after which the product was separated using

centrifuge.

7.3.2 Glaser coupling of methyl propiolate

Copper iodide (100.94 mg, 0.55 mmol), sodium carbonate (112.15 mg, 1.07

mmol) and iodine (67.26 mg, 0.57 mmol) were added to a stirred solution of

methyl propiolate (44.7 mg, 0.53 mmol) in DMF (1 ml). The mixture was heated

at 80ᵒC for 3 hours. The reaction was monitored with spot TLC. Once the reaction

had been deemed complete, the mixture was cooled and filtered. The filtrate was

washed with saturated Na2S2O3 and dried with MgSO4. Solvent was removed under

reduced pressure leaving behind a yellow oil. The oil was then purified using

preparative TLC with an eluent mixture of hexane and acetone (1:1 v/v). Mass

spectroscopy was used to confirm the presence of the product.

7.3.3 Synthesis of TMS-acetylene conjugated gold nanostars

A stock solution of citrate-stabilized gold nanostars was prepared by the addition

of citrate solution (1 %) to boiling HAuCl4 (1 mM) under vigorous stirring. After

stirring for 15 mins, the solution was cooled and filtered, and then kept at 4ᵒC.

This solution is used as a seed solution for subsequent syntheses.

An aliquot (100 µl) of gold nanocluster seed solution was added to HAuCl4 (10 µl,

0.25 mM) and HCl (10 µl, 1M). The mixture was stirred whilst AgNO3 (100 µl,

2.0mM) and ascorbic acid (50 µl, 100mM) were added simultaneously. There was

an immediate colour change from pale red to dark blue. The mixture was stirred

for a further 2 min before addition of TMS-acetylene alkyne (40 µl, 20 mM/100

41

mM). After 10 min of stirring, polyacrylic acid (2 ml, 4 mg/ml) and NaOH (3 ml,

0.1M) were added. The reaction mixture was stirred for 2 h, after which the

product was separated using centrifuge.

42

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9. Appendix

Figure 9.1. IR spectrum for osmium cluster 2c.

Figure 9.2. 1H NMR spectrum for osmium cluster 2c.

45

Figure 9.3. IR spectrum for osmium cluster 2e.

Figure 9.4. 1H spectrum for osmium cluster 2e.

46

Figure 9.5. 13C NMR spectrum for osmium cluster 2e.

Figure 9.6. Dose-Response curve for the osmium acid cluster 2e.

47

Figure 9.7. IR spectrum for osmium cluster 2f.

Figure 9.8. 1H spectrum for osmium cluster 2f.

48

Figure 9.9. 13C NMR spectrum for osmium cluster 2f.

Figure 9.10. Dose-Response curve for the osmium cluster 2e.

49

Figure 9.11. ORTEP plot of the molecular structure of 2e. Thermal ellipsoids are drawn at the 50% probability level.

Figure 9.12. ORTEP plot of the molecular structure of 2f. Thermal ellipsoids are drawn at the 50% probability level.

50

Table 9.1. Crystallographic data for compounds 2e and 2f.

Compound 1 2

Empirical formula C17H6O12Os3S • CH2Cl2 C64H24O44Os12S4 •

2.5CH2Cl2

Formula weight 1089.80 4119.79

Temperature (K) 153(2) 103(2)

Wavelength 0.71073 0.71073

Crystal system Triclinic Triclinic

Space group P -1 P1

a (Å) 8.4854(5) 8.4267(6)

b (Å) 12.0178(6) 14.1473(9)

c (Å) 12.8840(7) 21.5478(13)

81.639(3) 89.327(3)

77.714(3) 87.798(4)

76.102(3) 75.631(4)

V (Å3) 1240.01(12) 2486.6(3)

Z value 2 1

calc (g/cm3) 2.919 2.751

a) (mm-1) 15.692 15.561

F(000) 980 1841

Crystal size (mm3) 0.280 x 0.380 x 0.420 0.120 x 0.100 x 0.060

Theta range for data

collection

2.27 to 26.37° 2.40 to 26.37°

Reflections collected 5058 10175

Independent reflections 5058 10175 [R(int) = 0.1147]

Completeness(%) 99.9 (26.37°) 99.9 (25.24°)

Absorption correction Multi-Scan Semi-empirical from

equivalents

Max. and min. transmission 0.0960 and 0.0580 0.46 and 0.16

Refinement method Full-matrix least-squares

on F2

Full-matrix least-squares

on F2

Data / restraints /

parameters

5058 / 0 / 325 10175 / 9 / 611

Goodness-of-fit on F2 1.081 1.075

Final R indices [I>2sigma(I)] R1 = 0.0392

wR2 = 0.1021

R1 = 0.0557

wR2 = 0.1430

R indices (all data) R1 = 0.0442

wR2 = 0.1054

R1 = 0.0753

wR2 = 0.1590

Largest diff. peak and hole

(e•Å-3)

3.037 and -1.795 3.779 and -3.544