Using Dynamic Combinatorial Chemistry to Construct Novel Thioester and Disulfide Lipids

Using dynamic combinatorial chemistry to construct novel thioester and disulfide lipids

A dissertation submitted to the University of Manchester for the degree of MSc by Research Chemistry In the Faculty of Science and Engineering

2017

Jack A. Yung

School of Chemistry

Table of Contents

List of figures, tables and equations 5 Symbols and abbreviations 10 Abstract 12 Declaration 13 Copyright statement 13 Acknowledgements 14 The author 14 Chapter 1. Introduction 15 1.1 The cell membrane 16 1.1.1 Function and composition 16 1.2 Membrane lipids 16 1.2.1 Lipid classification 16 1.2.2 Amphiphiles 17 1.2.3 Glycolipids 17 1.2.4 Sterols 18 1.2.5 Phospholipids 19 1.3 Lipid vesicles 20 1.3.1 Supramolecular self-assembly 20 1.3.2 Interaction free energies 21 1.3.3 Framework for the theory of self-assembly 22 1.3.4 Micelles 23 1.3.5 Lipid bilayers 24 1.3.6 Vesicles 25 1.3.7 Phase-transition temperature 27 1.4 Amphiphilic building blocks 27 1.5 Thioesters 29 1.5.1 Thioester reactivity 29 1.5.2 Trans-thioesterification 30 1.5.3 Thioester exchange reactions in DCC 31 1.6 Disulfides 32 2

1.6.1 Disulfide reactivity 32 1.6.2 Thiol-disulfide interchange reactions 32 1.6.3 Disulfide exchange reactions in DCC 33 1.7 Pre-biotic lipids 34 1.7.1 Sources of pre-biotic organic compounds 34 1.7.2 The first pre-biotic membrane structure 36 1.7.3 The role of sulfur in pre-biotic chemistry 36 1.8 Artificially designed vesicles 37 1.8.1 Applications of artificially designed vesicles 37 1.8.2 Zeta-potential 37 1.8.3 Vesicle design 38 1.9 Targets 39 1.9.1 Aims 39 Chapter 2. Lipid synthesis and characterisation 43 2.1 Synthesis of 2-nitro-4-(palmitoylthio)benzoic acid 44 2.2 Synthesis of 2-nitro-5-(stearoylthio)benzoic acid 45 2.3 Trans-thioesterification of (2,3)-dimercapto-1-propanol 46 2.3.1 Synthesis of (S,S’)-(3-hydroxypropane-1,2-diyl)dihexadecanethiolate 46 2.3.2 Synthesis of (S.S’)-(3-hydroxypropane-1,2-diyl)dioctadecanethiolate 52 2.4 Hydroxyl-group functionalisation 53 2.4.1 Attempted synthesis of (2,3)-bis(palmitoylthio)propyl phosphate 53 2.4.2 Attempted synthesis of (2,3)-bis(palmitoylthio)propyl sulfate 57 2.5 Trans-thioesterification of (2,3)-dimercapto-1-propanesulfonic acid 59 2.5.1 Synthesis of (2,3)-bis(heptadecanoylthio)propane-1-sulfonate 60 2.5.2 Synthesis of (2,3)-bis(pentadecanoylthio)propane-1-sulfonate 64 2.6 Thiol-disulfide interchange of 1d 65 2.6.1 Attempted synthesis of 5-(hexadecyldisulfaneyl)-2-nitro-benzoic acid 65 Chapter 3. Vesicle, kinetics and DCC studies 72 3.1 Vesicle studies 73 3.1.1 DLS and zeta-potential studies of 3a and 3b 73 3.2.1 Encapsulation of 5(6)-carboxyfluorescein into vesicles of 3a and 3b 77 3.1.3 Release of 5(6)-carboxyfluorescein from vesicles composed of 3a and 3b 78 3.2 Dynamic combinatorial chemistry studies 81

3.2.1 Introduction 81 3.2.2 Generated dynamic combinatorial libraries from 2a and 2b 83 3.3 Kinetic studies 86 3.3.1 Introduction 86 3.3.2 Stability of 1a 87 3.3.3 Stability of 1a towards methanlyosis 87 3.3.4 Stability of 1a towards hydrolysis 88 3.3.5 Technical issues 88 3.3.6 Trans-thioesterification of 1a with 1d and 1e 89 3.3.7 Trans-thioesterification of 1a with 1i and 1j 93 Chapter 4. Experimental 96 4.1 General materials, instrumentation and other notes 97 4.2 Synthetic methods 99 4.3 DLS and zeta-potential measurements 107 4.4 Encapsulation of 5(6)-carboxyfluorescein 107 4.5 Release of 5(6)-carboxyfluorescein 108 4.6 Kinetic Studies 109 4.6.1 Preparation of buffer and 1a stock solution 109 4.6.2 Trans-thioesterification of 1a with di-thiol sources 110 4.6.3 Trans-thioesterification of 1a with thiol sources 110 Chapter 5. Conclusions and further work 112 5.1 General conclusions 113 5.2 Future work 114 Chapter 6. Annex 118 6.1 1H and 13C spectra 119 References 130

Final word count for dissertation: 26,688

List of figures, tables and equations

Figure 1.1 Cell membrane. 16 Table 1.1 Lipid classification. 17 Figure 1.2 Chemical structure of glycolipid. 18 Figure 1.3 Chemical structure of cholesterol. 18 Figure 1.4 Chemical structure of glycerol. 19 Figure 1.5 General chemical structure of phospholipid. 19 Figure 1.6 Phospholipid functionalities. 20 Figure 1.7 Hydrophobic effect. 22 Equation 1.1 Packing parameter. 23 Figure 1.8 Micelle structure. 24 Figure 1.9 Rod-like micelle structure. 24 Figure 1.10 Lipid bilayer structure. 25 Figure 1.11 Lipid mobility. 25 Figure 1.12 Vesicle structure. 26 Table 1.2 Vesicle classification. 26 Table 1.3 Lipid bilayer phase states. 27 Figure 1.13 Chemical structures of 1a and TNB2-. 28 Figure 1.14 Reaction mechanism for Ellman’s test. 28 Figure 1.15 Thioester and ester bonding descriptions. 29 Figure 1.16 General reaction scheme for trans-thioesterification. 30 Figure 1.17 General reaction mechanism for trans-thioesterification. 30 Figure 1.18 Thioester hydrolysis reaction mechanism. 30 Figure 1.19 General thioester DCL generation. 31 Figure 1.20 General thioester DCL generation reaction scheme. 31 Figure 1.21 General thioester DCL generation reaction mechanism. 32 Figure 1.22 General thiol-disulfide interchange reaction mechanism. 33 Figure 1.23 General disulfide DCL generation reaction scheme. 33 Figure 1.24 General disulfide DCL generation reaction mechanism. 34 Figure 1.25 General reaction scheme of FTT. 34 Figure 1.26 Generation and delivery of extra-terrestrial molecules. 35

Figure 1.27 Chemical structure of 6-methyldecandoic acid. 36 Figure 1.28 Reaction scheme for In-vivo enzymatic vesicle triggers. 39 Figure 1.29 Chemical structure of thioester lipid 1a. 39 Figure 1.30 Chemical structure of 1d, 1e and glycerol. 40 Figure 1.31 Trans-thioesterification reaction scheme to synthesize lipids 2a-3c. 40 Figure 1.32 General reaction scheme for hydroxyl phosphorylation and sulfation. 41 Figure 1.33 Thiol-disulfide interchange reaction scheme to synthesis lipids 2f and 3f. 41 Figure 1.34 Chemical structures of disulfide lipids 2f and 3f. 41 Figure 1.35 Reaction scheme for DCL generation via disulfide and thioester exchange reactions. 42 Figure 2.1 Reaction scheme for the synthesis of thioester lipid 1a. 44 Figure 2.2 Mechanism for the conversion of palmitoyl chloride into 1a. 44 Figure 2.3 Reaction scheme for the synthesis of thioester lipid 1b. 45 Figure 2.4 Reaction scheme for the trans-thioesterification of 1d. 46 Figure 2.5 Reaction mechanism for the trans-thioesterification of 1d. 47 Figure 2.6 Chemical structure of (TNB2-) (DIPEA+) salt. 49 Figure 2.7 1H NMR spectra of resulting crude on reaction of 1a with 1d. 49 Figure 2.8 Chemical structures of 2a and 1d with corresponding molecular ion-peaks. 50 Figure 2.9 Overlaid 1H NMR spectra of purified 2a and dimerized by-product. 51 Figure 2.10 Comparison of 1H NMR splitting patterns of 2a and dimerized by-product. 51 Figure 2.11 Chemical structures of possible dimerized by-products of 2a. 52 Figure 2.12 Reaction scheme for the phosphorylation of lipid 2a. 53 Figure 2.13 Reaction mechanism for the generation of a chlorophosphite group. 54 Figure 2.14 Reaction mechanism for the hydrolysis and or E2 elimination of chlorophosphite. 54 Figure 2.15 1H NMR phosphorylation time course studies. 55

Figure 2.16 Overlaid 1H NMR spectra of phosphorylated crude and 2a. 55 Figure 2.17 COSY spectra of phosphorylated product. 56 Figure 2.18 Reaction mechanism for intra-molecular acyl-migration of 2a on phosphorylation. 57 Figure 2.19 Chemical structure of acyl-migrated product on phosphorylation. 58 Figure 2.20 Reaction mechanism for intra-molecular acyl-migration of 2a on sulfation. 59 Figure 2.21 Reaction scheme for the trans-thioesterification of 1e. 59 Figure 2.22 Reaction mechanism for the trans-thioesterification of 1e. 60 Figure 2.23 Chemical structure of (3b)DIPEA salt. 62 Figure 2.24 COSY spectra of 3b, showing hidden DIPEA environments. 63 Figure 2.25 Enlarged 1H NMR spectra of thioester lipid 3b. 63 Figure 2.26 Overlaid 1H NMR spectrum of 1a/3b/3a. 64 Figure 2.27 Reaction scheme for the generation of disulfide lipid 1f 65 Equation 2.1 Thiol-disulfide interchange rate law. 66 Figure 2.28 Reaction scheme for competing thiol-disulfide interchange reactions. 66 Figure 2.29 Enlarged 1H NMR spectra of resulting 1f crude. 67 Figure 2.30 Overlaid 1H NMR spectra of 1g and disulfide lipid 1f. 68 Figure 2.31 Reaction scheme for the synthesis of disulfide lipid 2f. 68 Figure 2.32 LCMS spectrum of resulting crude on reaction of 1f with 1d. 70 Figure 3.1 Chemical structure of thioester lipids 3a and 3b. 70 Figure 3.2 Intensity size-distribution graphs of 3a and 3b on DLS analysis. 71 Figure 3.3 Intensity zeta-potential distribution graphs of 3a and 3b 75 Table 3.1 DLS and zeta-potential data. 76 Figure 3.4 Chemical structure of 5(6)-CF. 77 Equation 3.1 Encapsulation efficiency. 78 Equation 3.2 (%) Release of 5(6)-CF. 79 Figure 3.5 Graph of rate of release (%) of 5(6)-CF from vesicles 3a and 3b. 80 Figure 3.6 Chemical structure of 3a and DPPC. 81

Figure 3.7 Reaction scheme for thioester DCC generation from 2a and 2b. 81

Figure 3.8 Reaction mechanism for thioester DCC generation from 2a and 2b. 82 Figure 3.9 Reaction mechanism for the generation of mixed lipids 2ab/2ba. 82 Figure 3.10 DCC 1H NMR time-course studies. 83 Figure 3.11 Reaction scheme for DCL water contamination. 84 Figure 3.12 LCMS spectrum of DCC crude. 85 Figure 3.13 Acyl-migration mechanism on DCC formation. 85 Figure 3.14 Chemical structures of thiol species 1i and 1j. 86 Table 3.2 Kinetic studies; hydrolysis of 1a in bulk conditions. 88 Figure 3.15 Reaction scheme for the hydrolysis and trans-thioesterification of 1a. 89 Equation 3.3 Absorbance intensity increase. 90 Figure 3.16 Time-dependent absorbance intensity change of 1a on trans-thioesterification with di-thiol substituents. 90 Equation 3.4 Extent of trans-thioesterification. 91 Equation 3.5 Beer lambert law. 91 Table 3.3 Kinetic data and conditions for 1a and 1d. 92 Table 3.4 Kinetic data and conditions for 1a and 1e. 92 Figure 3.17 Reaction scheme for the trans-thioesterification of 1a with either 1i or 1j. 93 Figure 3.18 Time-dependent absorbance intensity change of 1a on trans-thioesterification with thiol substituents. 93 Table 3.5 Kinetic data and conditions 1a with 1i. 94 Table 3.6 Kinetic data and conditions 1a with 1j. 94 Figure 4.1 Schematic of GPC procedure. 108 Figure 4.2 Schematic for release of 5(6)-CF procedure. 109 Table 4.1 Conditions for the trans-thioesterification of 1a with 1d. 110 Table 4.2 Conditions for the trans-thioesterification of 1a with 1e. 110 Table 4.3 Conditions for the trans-thioesterification of 1a with 1i. 110 Table 4.4 Conditions for the trans-thioesterification of 1a with 1j. 111

Figure 5.1 Reaction scheme for the generation of thioester lipid 2c. 115 Figure 5.2 Chemical structure of disulfide lipid 3f. 116 Figure 5.3 Reaction scheme for acyl-migration blocking 116 Figure 5.4 Chemical structure of mixed acyl-chained thioester lipids. 117

Symbols and abbreviations cmc Critical micellar concentration Conc. HCl Concentrated hydrochloric acid

CDCl3 Deuterated chloroform

CHCl3 Chloroform CysOMe L-Cysteine methyl ester hydrochloride DCC Dynamic combinatorial chemistry DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DLS Dynamic light scattering DOPC 1,2-Dioleoyl-sn-glycero-3-phosphocholine DPPC Dipalmitoylphosphatidylcholine DSC Differential scanning calorimetry DTNB 5,5-dithiobis-(2-nitrobenzoic acid) EDL Electrical double-layer FTIR Fourier-transform infrared spectroscopy FTT Fisher-tropsch type reaction GVs Giant vesicles H-bond Hydrogen bond HPLC High-performance liquid chromatography 1H NMR Proton nuclear-magnetic resonance ISM Inter-stellar medium LCMS Liquid chromatography mass-spectrometry LUVs Large unilamellar vesicles MP Mobile phase MS Mass-spectrometry MVs Multimellar Vesicles ppm Chemical shift

Rf Retention-factor RT Room temperature TLC Thin-layer chromatography 10

TNB 5-mercapto-2-nitrobenzoic acid TNB2- 2-nitro-5-sulfidobenzoate UV Ultra-violet Water-acyl Water-acyl-hydrocarbon-contract

ZD Intensity-mean weighted diameter ζ Zeta-potential 5(6)-CF 5(6)-Carboxyfluroescein

Using dynamic combinatorial chemistry to construct novel thioester and disulfide lipids

Jack A. Yung

2017

A dissertation submitted to The University of Manchester for the degree of MSc by Research Chemistry

In the faculty of Science and Engineering

Given the involvement of sulfur containing compounds in pre-biotic chemistry, the synthesis of sulfur containing lipids that are analogues in structure to phospholipids has been attempted.

This thesis details the potential of trans-thioesterification and thiol-disulfide interchange reactions in creating new types of sulfur-containing lipids. The resulting supramolecular structures they formed were both sized, and studied, to give possible indication of biological application.

Previous work has shown successful trans-thioesterification reactions of 2-nitro-4- (palmitoylthio)benzoic acid. Using this, several thioester lipids that are analogues in structure to phospholipids were successfully synthesised. Successful synthesis was achieved by the trans-thioesterification between di-thiol substituents (2,3)-dimercapto-1- propanol and (2,3)-dimercapto-propanesulfonic acid and thioester lipids 2-nitro-4- (palmitoyl thio)benzoic acid and 2-nitro-4-(stearoylthio)benzoic acid.

Several of the thioester lipids generated were observed to self-assemble into supramolecular structures that display unilamellar morphology, along with being in the size range of large vesicles. Analytical studies showed the resulting vesicle structures displayed an affiliation to encapsulate compounds of hydrophilic nature, along with excellent stability in aqueous conditions.

Declaration I hereby declare that no portion of the work referred in this dissertation has been submitted in support of an application for another degree or qualification of this or any other university or institute of learning.

Copyright statement i. The author of this dissertation (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given. The University of Manchester has certain rights to use such Copyright, including for administrative purposes. ii. Copies of this dissertation, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patent Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trade marks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the dissertation, for example graphs and tables (“Reproductions”), which may be described in this dissertation, may not be owned by the author and may be owned by third parties. Such intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s policy on Presentation of Thesis.

Acknowledgment and dedication I would like to thank my supervisor, Dr. Simon J. Webb for his support, guidance and scientific advice over the course of this project. I am extremely grateful to have him as my future PhD supervisor, and I am looking forward to future years of playing 5-a side football with him. I would also like to thank the group members of the SJW group, not only have you provided me with guidance, you have also provided me with friendship. Both Natasha Eccles and Thomas Fallows in particular have been incredibly helpful over the course of the year. I would also like to acknowledge the following staff members; Rehanna Sung (HPLC/LCMS) and Dr. Matthew Cliff (NMR). The Author BSc (Hons) in chemistry from the University of Sheffield. No previous research experience prior to this project.

Chapter 1

Introduction

1.1 The cell membrane

1.1.1 Function and composition

The cell membrane is a thin semi-permeable membrane that serves as a physical barrier between the cell and its external surroundings. By being selectively permeable to ions and organic molecules, the cell membrane can effectively regulate the movement of substances between the cell and its external environment.1

Fundamentally, the cell membrane consists of a mixture of lipids and proteins. The large lipid composition comprises mostly from the phospholipid bilayer of the cell membrane. The phospholipid bilayer is a structurally fundamental component of the cell membrane, and serves to act as a physical barrier between the internal and external aqueous environments of the cell. Proteins are embedded throughout the phospholipid bilayer, and act to provide a variety of specific functions, such as the selective transport of substances across the membrane (See Figure 1.1). As the proteins are non-covalently bound to the lipid constitutes, they are free to laterally diffuse within the membrane. Moreover, as a result, the cell membrane is a dynamic structure, by which its structure is best established by the fluid-mosaic model.1

Figure 1.1: Schematic illustration of a cellular membrane.

1.2 Membrane lipids

1.2.1 Lipid classification

Lipids constitute a large and diverse group of heterogeneous organic compounds that are related more by their physical properties, than chemical. As a result, lipids are classified by their physical properties, such as solubility. For instance, all lipids are typically soluble in organic solvents such as chloroform, acetone and benzene, and are therefore categorised by their differing solubilities in aqueous media and polar organic solvents.

In 1986, Small et al.2 deduced that all lipids can be categorised into one of four classes, based by their bulk solubility and structural behaviour in aqueous conditions (See Table 1.1).

Lipid Solubility in Structural Example class water behaviour in water I Insoluble Non-swelling Cholesterol amphiphiles II Insoluble Swell in water, and Phosphatidylcholine form mesomorphic phases IIIA Soluble Form isotropic Lysolecithins crystals, and micelles above cmc IIIB Soluble Form micelles above Bile salts cmc, but do not form isotropic crystals Table 1.1 Comparison of lipid classes based on their solubility and structural behaviour in water.2 (cmc) is the critical micellar concentrations.

1.2.2 Amphiphiles

An amphiphile is a term used to describe any molecule that possesses both a hydrophilic (polar) and hydrophobic (non-polar) moiety. All lipids are amphiphilic, the hydrophobic moiety of a lipid is typically a hydrocarbon chain of ten carbon atoms or more, whilst the hydrophilic moiety is a polar functional group, such as a phosphate, sulfonic acid or alcohol.3

1.2.3 Glycolipids

The basic structure of a glycolipid comprises of a hydrophilic monosaccharide or oligosaccharide, attached by a glycosidic bond to one or two hydrophobic hydrocarbon chains of varying length ‘R’ (See Figure 1.2(a)). A glycerol backbone can be introduced into the structure (glyeroglycolipids), in which one or two acyl hydrocarbon chains are acylated at the two oxygen sites of glycerol. This effectively acts to anchor the hydrophilic and hydrophobic moieties together (See Figure 1.2(b)).4

Figure 1.2: Chemical structures of a) glycolipids and b) glyeroclycolipids, where R is an alkyl substituent of varying length.

Glycolipids are located on the exterior of cellular membranes. The hydrophobic moiety buries inside the membrane, allowing the hydrophilic carbohydrate to be exposed on the surface of the membrane, where it can facilitate cell-cell interactions by acting as a recognition site.4

1.2.4 Sterols

Structurally, sterols are comprised of a hydrophobic moiety in the form of a hydrocarbon tetrameric fused ring structure, and in most cases, a short aliphatic tail.5 The hydrophilic moiety originates from the 3-hydroxyl group in a β-configuration of the fused ring structure, and contributes to sterol’s small degree of polarity. Cholesterol is the most abundant sterol in animal membranes, and contributes to 20-30% of all sterols found in nature (See Figure 1.3).6

Figure 1.3: Chemical structure of cholesterol.

Cholesterol plays an essential role in the formation of lipid rafts. As first proposed by Simons et al.7 lipid rafts comprise of regions within the membrane, known as microdomains. These consist of regions of highly ordered lipids, relative to the ‘sea’ of liquid phase lipids known as bulk lipids.8 Furthermore, cholesterol facilitates lipid raft formation, by orientation of its polar hydroxyl group towards the hydrophilic head-groups of the phospholipid bilayer. This orientation further extends both the hydrophobic fused

18 ring system and aliphatic tail into the hydrophobic core of the membrane. This mechanism of action induces further order into the hydrocarbon chains of the hydrophobic core.8

1.2.5 Phospholipids

Phosphoglyercerides, are the most abundant class of phospholipids found in nature and structurally, they consist of a glycerol backbone (See Figure 1.4).9

Figure 1.4: Chemical structure of glycerol; a triol with hydroxyl functionalities located on each of its 3 carbon-atoms.

The hydrophobic and hydrophilic functionalities are introduced into the glycerol molecule by esterification of saturated and or unsaturated acyl-hydrocarbon chains of varying length

(R=C20-C12), at both the C1 and C2 positions respectively, along with the phosphorylation of the hydroxyl group at the C3 position (See Figure 1.5).9

Figure 1.5: General chemical structure of a phosphoglyceride, in which the R substituent

is an alkyl chain of varying length (C9-C17).

The vast diversity that phospholipids display in nature arises from their propensity to react with alcohols such as serine, choline or ethanolamine. These functionalities can result in either a zwitterionic, or anionic hydrophilic head group (See Figure 1.6).9

Figure 1.6: Common functionalities of the hydrophilic head-group in phospholipids, in some cases such as R’=choline, the head-group is zwitterionic.

Dipalmitoylphosphatidylchloine (DPPC) is the most abundant phospholipid in animal cell membranes. DPPC consists of a hydrophobic moiety in the form of two saturated 10 hydrocarbon chains of R=C12H25, and a zwitterionic hydrophilic moiety (R’=choline).

In aqueous mediums, phospholipids can self-assemble into larger supramolecular structures, such as micelles, lipid-bilayers and vesicles due to their hydrophobic and hydrophilic components. The resulting self-assembled structures they form will be discussed in the following section.

1.3 Lipid vesicles

1.3.1 Supramolecular self-assembly

As stated in Section 1.2, amphiphiles possess both a hydrophobic and hydrophilic moiety. Small amphiphilic molecules such as lipids, can self-assemble into larger structures such as micelles, bilayers and vesicles, in aqueous media, with two main factors dictating their resulting structure;11

1) Chemical nature of the lipid

2) Environment of self-assembly

A general rule of thumb is that the resulting structure can be predicted by the quantity of hydrocarbon chains the lipid possesses. For example, a micelle is expected for the possession of one hydrocarbon chain, whilst a lipid-bilayer and vesicle can be predicted 20 from the presence of two hydrocarbon chains.11 These two main factors can be broken down further, into an overall fundamental theory of self-assembly, in which the self- assembly of small molecules into large structures is facilitated by;12

1) Interaction Free Energies

2) Molecular geometry

3) Correct thermodynamic treatment of the whole system

1.3.2 Interaction free energies

In 1936, Hartley et al.13 first proposed that the hydrophobic effect is the driving force behind the self-assembly of small molecules such as lipids, into defined supramolecular structures.

When a phospholipid is subjected to an aqueous environment, the dominating electrostatic interaction is the unfavourable acyl-hydrocarbon-water interaction, also known as the water-acyl-hydrocarbon (water-acyl) contact. Elimination of this unfavourable water-acyl contact is driven by the interfacial free energy of attraction that water has on itself, through hydrogen bond (H-bond) interactions. Subsequently a partly ordered cage of water molecules around the hydrophobic moiety is favoured as its replacement (See Figure 1.7).14 Furthermore, elimination of the water-acyl contact allows for the formation of favourable hydrophobic interactions on acyl-acyl contact. This brings the interaction free energy (Gibbs free energy) per lipid molecule to a minimum, and as a result, the self- assembly of phospholipids is thermodynamically favourable.15

Figure 1.7: Schematic illustration of the hydrophobic effect whereby in a) water-acyl contacts dominate, b) elimination of water-acyl contacts is driven by the interfacial free energy of attraction that waster has on itself and c) water-acyl contacts are reformed as a partially ordered cage of water molecules, along with acyl-acyl contacts. Water-head group contacts remain constant throughout, as represented by the blue.

These hydrophobic interactions are the dictating electrostatic interactions in facilitating both the aggregation of lipid molecules, and the formation of well-defined supramolecular structures such as micelles and vesicles. Favourable hydrophilic interactions (H-bond interactions) between water molecules and the polar-head group of the lipid, imposes a constant water-head group contact. This constant contact develops an interface with the aqueous medium, and facilitates the resulting size of the self-assembled structure.14 The resulting set of hydrophobic and hydrophilic interactions act as opposing forces that facilitate the self-assembly of amphiphilic lipids into supramolecular structures.

1.3.3 Framework for the theory of self-assembly

A framework of interaction free energies, molecular geometry, and entropy can be used to establish why single-chained lipids tend to form micelles, and why double-chained lipids tend to form lipid-bilayers and or vesicles.

The geometric factor, also known as the packing parameter (p) (See Equation 1.1) of a lipid is a dimensionless parameter.16

v p = aolc

Equation 1.1 Where ‘v’ is the hydrocarbon chain volume, ‘ao’ is the surface area of the

polar head group, and ‘lc’ the chain length, in which the chain behaves as a solid, rather than a liquid.16

The geometric packing rule can be used to predict the self-assembled structures of single 1 1 and double hydrocarbon-chained lipids. In the case of micelles, ( ≥ p ≥ ) is expected, 2 3 1 whereas (1 ≥ p ≥ ) is expected for lipid-bilayers and vesicles.16 However, the geometric 2 packing rule only applies to an individual amphiphilic lipid, and therefore does not consider the multiple interactions occurring between lipids on self-assembly. As a result, slight deviations from the predicted structure can occur. However, these deviations can be used to highlight the role of thermodynamic parameters such as entropy, on the resulting self-assembled structure.17

For example, geometric packing rules incorrectly predicts the self-assembly of lipids that possess either one or two hydrocarbon chains into micelle structures. However, it correctly predicts that only lipids possessing one hydrocarbon chain can self-assemble into micelles. From this deviation in geometric packing rules, you can deduce that two chained lipids cannot self-assemble into micelles due to geometric factors. Whereas, single chained lipids do not self-assemble into bilayers and vesicles due to entropic effects.16 From this, a framework for the self-assembly of lipids intro supramolecular structures is created, which takes molecular geometry, interaction free energies and entropy into consideration.

1.3.4 Micelles

Micelles are the simplest form of all self-assembled structures lipid structures, and vary in size between 2-20 nm.14 Micelles comprise of polar-head groups orientated outwards, towards the aqueous environment, whilst the hydrocarbon chains are orientated inwards, away from the aqueous environment. This orientation minimizes unfavourable hydrophobic-hydrophilic interactions (See Figure 1.8(a)).

Self-assembly into micelles is driven by the degree of hydrophobic interactions acting between the hydrocarbon-chains of the lipids. Furthermore, the parameter cmc defines the lipid concentration required for micelle formation. Below the cmc, the corresponding lipids will exist as a monolayer at the air-liquid interface. Solvent environment is also a crucial factor for micelle formation. For example; when an aqueous suspension of a corresponding

23 lipid is in an organic solvent, an inverted micelle structure can be observed (See Figure 1.8(b)).14

Figure 1.8: Schematic illustration of a) micelle structure and b) inverted micelle structure. Blue areas denote water, red-circles denote the hydrophilic moiety, yellow tails denote the hydrophobic moiety.

Micelles can also deviate in spherical morphology, by introducing a surfactant polar-head group. On increasing the concentration of the surfactant-lipid, the degree in deviation away from a spherical morphology is increased, and thus results in rod-like structures (See Figure 1.9).

Figure 1.9: Schematic illustration showing the effect of increasing the surfactant concentration on the spherical morphology of the micelle; the resulting structure is ‘rod- like’.

1.3.5 Lipid bilayers

Lipid bilayers are formed when two-chained lipids, stack in reflecting layers (head-tail-tail- head) and consists of a top and bottom plane of polar-head groups, which are in contact with the aqueous medium. The hydrocarbon acyl chains are situated inside the planes, in a tail-to-tail fashion. By having this arrangement, the two hydrocarbon acyl chains have

24 maximum protection from the aqueous medium, and thus provide stability to the lipids (See Figure 1.10).14

Figure 1.10: Schematic illustration of a lipid-bilayer structure, consisting of two-chained lipids, stacked in reflected layers (head-tail-tail-head).

Nomenclature of bilayer forming lipids is dictated by the number of bilayers formed. For example, unilamellar-lipid is for one bilayer and multilamellar-lipid is for multiple bilayers stacked upon each other.

Lipids can move within the plane of the bilayer (lateral mobility), or between the planes (flip-flop motion), with the latter being less common. Insertion of foreign molecules such as detergents can facilitate flip-flop motion (See Figure 1.11).18

Figure 1.11: Schematic illustration showing the two modes of mobility that lipids have within the two planes of the bilayer.

1.3.6 Vesicles

Vesicles are formed when two-chained lipids are dispersed in large aqueous mediums. Self-assembly results in a lipid bilayer with circular morphology, consisting of a

25 hydrophobic region of hydrocarbon chains surrounded by an aqueous core. The periphery of the circle consists of polar head groups, which interact with the aqueous medium (See Figure 1.12).14

Figure 1.12: Schematic illustration of a vesicle structure, by which the aqueous core is represented in blue.

There are 5 different vesicle classes, in which vesicles are classified by both their size, and degree of lamellarity (See Table 1.2).14 Given their resulting size range, large unilamellar vesicles (LUV) can be artificially synthesised to act as pharmaceutical drug carriers.19-23 Whereas, giant vesicles (GV) closely resemble the structural compartment of cellular membranes, and thus can be artificially synthesised to act as a cellular model for membrane investigations. An example of such application would be the investigation of biomembrane fission.24

Lipid vesicle class Size (nm) Bilayer composition Small unilamellar vesicles <50 nm Single bilayer (SUV) Large unilmellar vesicles 50-1000 nm Single bilayer (LUV) Giant vesicles 1000 nm< Single bilayer (GV) Multi-lamella vesicles 1000 nm< Multiple concentric bilayers (MLV) Multi-vesicular vesicles 1000 nm< Non-concentric vesicles (MVV) inside a large vesicle Table 1.2: Classification of vesicles with respect to size, and bilayer composition. On sizes >1000nm, vesicles are solely classified by their bilayer composition.14

1.3.7 Phase-transition temperature

Lipid bilayers can be classified into three different phase-states, based on the relative degree of interactions between membrane components, and their membrane mobility (See Table 1.3).25

Phase state Magnitude of interactions Membrane mobility between neighbouring lipids

Solid ordered (so) Strong Low

Liquid disordered (ld) Weak High

Liquid ordered (lo) Strong Moderate Table 1.3. Showing the three phases of a lipid-bilayer, along with their corresponding interaction strength between neighbouring lipids, and membrane mobility.

At a certain temperature called the phase-transition temperature (Tm), the phase state of the lipid-bilayer depends solely on the relative degree of interactions between the hydrocarbon chains. On reaching the Tm, the lipid bilayer undergoes a phase transition from a solid 25 ordered phase to a liquid disordered phase. The Tm is a characteristic parameter associated with all lipids, and can give a relative indication of the strength of interactions and or relative degree of packing between the hydrocarbon chains. Tm can be directly measured using differential scanning calorimetry (DSC).

1.4 Amphiphilic building blocks

This project has extended on the work of Lopez et al.26 in which the potential of thiol- thioester exchange reactions (trans-thioesterification) to create magnetoresponsive materials was explored. Several thioester derivatives were successfully synthesised, including the thioester lipid; 2-nitro-4-(palmitoylthio)benzoic acid (1a), designed with the intention of possessing spectrophotometric properties on nucleophilic attack, by the displacement of 2-nitro-5-thiobenzoate (TNB2-) (See Figure 1.13).26 TNB2- absorbs visible light at 412 nm, and displays strong leaving-group capabilities, an effect of its low pKa (~4.6)26. This is due to the stabilization of negative charge over the aromatic ring.

Figure 1.13: Chemical structures of both 1a and TNB2-.26

The basis of 1a’s spectrophotometric design was influenced by the works of Ellman, 27 who in 1958, developed a technique to quantify the concentration of thiol groups in a sample (Ellman’s-Test). 27 The technique proceeds by dissolving the sample of interest, along with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) in buffer (pH 8). In basic conditions, a thiolate anion is generated, and thus cleaves the disulfide bond of DTNB, generating both TNB2- and a new disulfide equivalent. On generation of TNB2-, the absorbance intensity at 412 nm increases, this can be used to quantify the concentration of thiol-substituents in the sample (See Figure 1.14).27

Figure 1.14: Reaction mechanism for Ellman’s test, by which DTNB is cleaved.27

Furthermore, Lopez et al.26 displayed the suitability of thioester lipid 1a as a thioester source for trans-thioesterification reactions. With use of ultraviolet-visible (UV) spectroscopy, the extent of trans-thioesterification of 1a with a series of thiol-derivatives, 26 ranging in pKa values (6.8, 7.5, and 10) was measured. The studies were performed in bulk aqueous conditions and the increase in absorbance intensity at 410 nm on generation of TNB2- was measured. The studies were successful and showed high extents of trans- thioesterification in the range of 70-80% being achieved on reaction of thioester lipid 1a with a series of thiol substituents. The results obtained highlighted the strong electrophilic nature of 1a, with high extents of trans-thioesterification being recorded for thiol- 26 derivatives in the lower pKa range. Studies performed by Lopez et al. have shown 1a to

28 be a potential building block, by which the thioester lipid can have its hydrophilic moiety modified, by using trans-thioesterification reactions.

1.5 Thioesters 1.5.1 Thioester reactivity

Thioesters are active acylation intermediates, and are involved in numerous biochemical reactions, both in nature and in the pharmaceutical industry. Examples include; peptide and RNA synthesis,28,29 along with having pharmaceutical application in antibiotic synthesis.30 Furthermore, thioesters have also been proposed as pre-cursors of life, given their significant role in pre-biotic chemistry.31In comparison to their corresponding oxygen analogues (esters), thioesters display a higher degree in reactivity, with many factors contributing to this resulting higher reactivity. One being, that the ‘C-S’ bond is less able to participate in electron-delocalisation through the acyl-group, in comparison to that of a ‘C-O’ bond. This is a result of a poorer orbital overlap between the 3p orbital of sulphur and the 2p orbital of carbon, thus the ‘C-S’ bond is less stable, and resultantly weaker (See Figure 1.15).32

Figure 1.15: General chemical structures of an ester and thioester, along with the resulting p-orbital bonding descriptions.

- Another factor is that on nucleophilic acylation, the resulting thiolate anions (RS ) (pKa 13) - 33 acts as a stronger leaving group in comparison to alkoxides (RO ) (pKa 17). This is a - result of RS being more acidic in nature (lower pKa), and is thus more stabilised in its anionic form.

1.5.2 Trans-thioesterification Thioester-thiol exchange (trans-thioesterification) reactions involve the exchange of an ‘R’ substituent of a thiol, with the ‘R’ substituent of a thioester (See Figure 1.16).

Figure 1.16: General reaction scheme for a trans-thioesterification reaction between a thioester and a thiol. The mechanism of action proceeds by the deprotonation of the thiol substituent, to generate the corresponding thiolate anion. Given that thiolate anions in aqueous conditions possess both a high degree of polarizability, and low degree of solvation, they behave as strong nucleophiles.34

Nucleophilic acyl-substitution (SNAc) then occurs, by which the active nucleophilic species (RS-) attacks at the carbonyl centre of the thioester. The resulting position of the - - equilibrium is dictated by the relative pKa’s of both SR’ and SR’’ . With the thiolate anion 35 of lower pKa being the favoured leaving group (See Figure 1.17).

Figure 1.17: General reaction mechanism of trans-thioesterification.

Given that the active nucleophilic species is a thiolate anion, trans-thioesterification can be base catalysed. However, given the relative electrophilicity of thioesters, a competing thioester hydrolysis reaction will occur in aqueous conditions. Therefore, the addition of base can induce further hydrolysis via base mediated hydrolysis (See Figure 1.18).

Figure 1.18: Reaction scheme for the hydrolysis of thioesters in aqueous conditions. a) either acidic or basic conditions.

1.5.3 Thioester exchange reactions in DCC

As first reported by Giuseppone et.al.36 DCC is widely used as a laboratory technique to generate vast libraries of molecules that can be screened for biologically active substances and new materials.37

Library generation is based on using reversible reactions under thermodynamic control, with exchange reactions occurring via covalent or non-covalent bonds. Members that make 30 up the dynamic combinatorial library (DCL) will consist of a set of different building blocks which, can reversibly interchange by connections, between specifically chosen functional groups (See Figure 1.19).

Figure 1.19: Schematic showing the reversible nature by which building blocks (as denoted by shapes) can interchange between thioester functionalities, to generate new members that constitute the DCL.

Prior tailoring of the functional groups will affect both the conditions required for exchange activation, and the kinetics of library member generation. To date, many well- known covalent exchange reactions have been used to generate DCL members, with hydrazones, disulfides, and thioesters functionalities, being regarded as the most effective functional groups in generating vast DCLs in short periods of time.37,38

Larsson et al.39 report successful thioester DCL formation though thioester exchange. Library generation occurred rapidly through a series of trans-thioesterification between a set of thioester species and a catalytic thiolate anion (1% mol/mol) (See Figure 1.20).40

Figure 1.20: Reaction scheme showing the formation of a DCL of interchanging thioester species in a statistical disruption where a) thiolate anion (1%mol/mol).

The mechanism of action proceeds by a series of trans-thioesterifications between the catalytic thiolate anion and thioester species, until a near statistical distribution of thioester members is met (See Figure 1.21).

Figure 1.21: Reaction mechanism showing the subsequent steps (1 to 3), to generate a DCL of thioester members by a series of trans-thioesterification reactions.

As the thioester-exchange process occurs through a series of trans-thioesterification reactions, the rate of exchange can be increased by selectively tailoring the catalytical 40 thiolate to possess a lower pKa.

1.6 Disulfides

1.6.1 Disulfide reactivity

Disulfides are typically formed by the oxidation of two thiol groups, this oxidative process results in the formation of a disulfide bond (RS-SR). Disulfide bonds are relatively strong (~251kJmol-1),41 and as a result, play a significant role in both the tertiary and quaternary structures of proteins; with significant disulfide cleavage resulting in complete loss of protein activity.41

1.6.2 Thiol-disulfide interchange reactions

Thiol-disulfide interchange reactions proceed by nucleophilic substitution, in which a new disulphide is formed, along with a thiolate anion derived from the original disulfide. The mechanism of action proceeds by the nucleophilic backside attack of the thiolate anion, along the ‘S-S’ bond axis of the disulfide; this in turn displaces a thiolate anion (See Figure 1.22). Given that the active nucleophile is a thiolate anion, thiol-disulfide interchange reactions can be base catalysed.

Figure 1.22: Reaction mechanism for a thiol-disulfide interchange reaction. In this case, - thiolate anion SR2, has the lower pKa and thus is the favoured leaving group.

As with trans-thioesterification reactions, the resulting position of the equilibrium is dictated by the relative pKa’s of the resulting thiolate anions. Given, that thiol-disulfide interchange reactions involve the cleavage and formation of a strong ‘S-S’ bond, the reaction can still proceed reversibly in basic conditions at room temperature (RT).41

1.6.3 Disulfide exchange reactions in DCC

DCLs that consist of interchanging disulfides were first reported by Otto et al.42 and Hioko et al.43. In a similar manner to trans-thioesterification reactions, thiol-disulfide interchange reactions serve as an efficient way to form vast DCLs of mixed disulfides through disulfide exchange reactions. The exchange process occurs by mixing disulfides in the presence of a catalytic amount of thiolate anion (1%mol/mol), affording a near statistical distribution of mixed disulfides (See Figure 1.23).40 The mechanism of action proceeds by a series of thiol-disulfide interchange reactions, between the catalytic thiolate anion and the disulfide species, until a near statistical distribution of disulfide members is met (See Figure 1.24).

Figure 1.23: Reaction scheme showing the formation of a DCL through disulfide exchange reactions, where a) catalytic amount of thiolate anion (1% mol/mol).40

Figure 1.24: Reaction mechanism showing the subsequent steps (1 to 3), to form a DCL of disulfide members by a series of thiol-disulfide interchange reactions.

As the disulfide-exchange process occurs through a series of thiol-disulfide interchange reactions, the rate of exchange can be increased by selectively tailoring the catalytical thiolate to possess a low pKa. An increase in pH can also increase the rate of exchange process to a much greater extent, than in comparison to thioester-exchange, due to the lower susceptibility of disulfides to hydrolysis.40 However, in comparison to the relative rates of thioester-exchange reactions, disulfide exchange reactions occur at a slower rate, given the stronger strength of the disulfide bond.41

1.7 Pre-biotic lipids

1.7.1 Sources of pre-biotic organic compounds

Currently, there are two proposed methods for the generation and or delivery of lipids, capable of self-assembling into the first cellular life on Earth.44

The first method involves the endogenous synthesis of lipids via the Fisher-Tropsch-type (FTT) reaction.45 Today, FTT acts as a well-known industrial hydrogenation method for the synthesis of long-chained hydrocarbons, and their corresponding oxy-compounds from mixtures of carbon monoxide (CO) and hydrogen-gas (H2) over a transition metal catalyst. (See Figure 1.25).45

Figure 1.25: General reaction scheme of the FTT reaction, whereby (n=10-20). The reaction is performed over a transition-metal catalyst.45

Mccollom et al.45 was the first to propose that hydrothermal vents were a key-source of pre-biotic lipids. The proposed reaction scheme occurs, by the generation of H2. Given the significantly high thermal conditions at the site of hydrothermal vents, on the interaction of water with ferrous minerals, water is reduced into H2 gas. The generated H2 acts as a reduction source towards carbon dioxide (CO2), which is contained within migrating fluids. Subsequently, the FTT reaction of CO2 and H2 can generate homologous lipids 45 ranging in size from (>C30) onwards. This proposal was successfully tested by Mccollom et al.45 They report on the successful generation of a homologous lipid series, consisting of alkanoic acids and alcohols. Furthermore, the resulting lipids are capable of self- assembling into vesicles, in conditions mimicking that of pre-biotic hydrothermal vents.45

A second proposed source of pre-biotic lipids comes through the exogenous delivery of intact extra-terrestrial molecules. In this process, C, N, S, O, and P formed in the interior of the star, are ejected and dispersed into the interstellar medium (ISM). Chemical and physical processes, such as UV-radiation and cosmic-rays, act as a generation source of gas-phase compounds from the ejected material. On concentration of the resulting gases, 44,46 dense molecular clouds are formed. At temperatures of 10-50K ice-mantles form inside the dense molecular clouds. These ice mantles compose of H2O with mixtures of CO, CO2, methanol (CH3OH), and ammonia (NH3) On exposure to ionizing radiation, these ice- mantels act as a source of the complex-molecular series expected in pre-biotic earth, such as simple lipid molecules and amino acids (See Figure 1.26).44

Figure 1.26. Schematic illustration, showing a) the ejection and dispersion of C, N,S,O,P into the ISM, on the death of a star b) generation of gas-phased compounds from the ejected material on exposure to UV-radiation/cosmic-rays, c) at temperature conditions (10-50K), resulting ice-mantles are exposed to UV-radiation/cosmic-rays, promoting the formation of complex-molecular species d) resulting organic species are transferred to pre- biotic earth by exogenous delivery (meteorites/asteroids/comets).

This proposed source of pre-biotic organic compounds was further justified by the research of Pierazzo et al.47 who calculated that on meteorite impact with our planetary atmosphere,

35 a sufficient fraction of extra-terrestrial organics remains intact. Furthermore, research by Love et al.48 approximated that 4 billion years ago, during the late-bombardment period, exogenous delivery of organic matter would be of magnitudes higher than that is currently delivered to contemporary earth (107 kg/year).48 Based on these findings from Pierazzo et al.47 and Love et al.48 exogenous delivery is a probable, and likely source of organic matter on pre-biotic earth.

1.7.2 The first pre-biotic membrane structure

Given the relative complexity of phospholipids, it is extremely unlikely that pre-biotic membranes comprised of any membrane lipids that are currently found in nature. Moreover, given the relatively basic complexity of proposed pre-biotic organic compounds it is more likely that pre-biotic membranes comprised of structurally basic lipids. In 1978, Hargreaves et al.49 reported on the successful extraction of long-chained lipids possessing both acid and alcohol functionalities, from carbonaceous meteorites found on earth. Furthermore, these lipids were observed to self-assemble into vesicles. Hargreaves et al.49, also report that extracted 6-methyldecandoic acid (See Figure 1.27) could successfully self-assemble into vesicle structures at concentrations as low as 85mM. Given its self- assembling behaviour, and simple complexity, 6-methyldecanoic acid, or a similar derivative, is likely to have constituted one of the first pre-biotic membranes.

Figure 1.27: Chemical structure of 6-methyldecandoic acid.49

1.7.3 The role of sulfur in pre-biotic chemistry

In 1958, Miller et al.50 reported on the successful synthesis of 20 sulfur containing amino- acids, in conditions mimicking that of pre-biotic earth. This was successfully achieved by subjecting a mixture of H2O, H2, methane (CH4), ammonia (NH3) and hydrogen sulphide

(H2S) to spark discharges, with the intention to mimic lightning. The Miller-Uray experiment, not only validated the potential of amino acids to form in pre-biotic conditions, it also highlighted the essential role of thiol functionalities in the synthesis of pre-biotic organic compounds, such as amino acids.50

Given the significant roles of both thioesters and disulfides in biochemistry, Wallace and others,51,46 have explored the potential use of both thioesters and disulfides in pre-biotic chemistry. In 1966, Wallace et al.51 proposed that pre-biotic disulfide formation was 36 possible through the oxidiative monodimerization of thiols by using ferric ions, at low temperatures (33-55°C).

Given the significant role thioesters have in biochemistry, the research group of A.L Weber have extensively researched the potential role of thioesters in pre-biotic chemistry. Their research over the years has both proposed and justified that thioesters played a significant role in the pre-biotic synthesis of peptides and amino acids.51-55

1.8 Artificially designed vesicles

1.8.1 Applications of artificially designed vesicles

Since the first observation of vesicle structures by Bangham et al.56 extensive research has been carried out on the design, and application of artificially designed vesicles as pharmaceutical, diagnostic and cosmetic agents.57

Vesicles display many desirable characteristics that have made them suitable to act as drug-delivery systems. These include the ability to spontaneously self-assemble in aqueous conditions, high loading affiliation towards compounds of a hydrophilic nature (encapsulation), excellent cell uptake, and the ability to easily tailor specific properties of the vesicle, by simple chemical modifications.

1.8.2 Zeta-potential

Vesicles that display a net-neutral charge at the hydrophilic moiety, can by chemically modified by the introduction of a negatively charged functionality at the polar-head group. This resulting change in the net charge of the vesicle, will sufficiently increase the encapsulation affinity of the vesicles towards compounds of a hydrophilic nature. For example, Allen et al.58 report that the hydrophilic antiviral drug Cidofovir, displays enhanced encapsulation with positively charged vesicles in comparison to that of neutrally charged vesicles.

Subsequently, zeta-potential (ζ) measurements act as effective parameter to quantify both the resulting net-charge displayed by a vesicle, and the resulting stability of the vesicle dispersion. For example, by assuming charged spherical morphology, the resulting ζ measurements give magnitude of the resulting repulsive and attractive interactions acting between the vesicles. Destabilisation of the vesicles will occur when the attractive interactions exceed the repulsive interactions, and thus vesicles will coagulate together. ζ values ranging from 0 to ± 5 mV, indicate a high tendency of coagulation, whilst values exceeding ± 61 mV indicate a very low tendency of coagulation.59 Both the sign (±) and 37 magnitude of the ζ value acts to quantify the resulting net-charge displayed by the vesicle. With a positive value indicating an overall positive-net charge displayed and a negative value indicating an overall negative-net charge.

Furthermore, the ζ values of charged vesicles can act as an effective indication of their encapsulation efficiencies towards hydrophilic compounds, and thus can be tailored by the further introduction of charged moieties to maximise encapsulation.

1.8.3 Vesicle design

On designing a vesicle for drug delivery, three important rules are considered;16

1. Vesicle components need to display high permeability towards cellular and anatomical barriers, to effectively reach the target site.

2. Specificity towards the target cell needs to be displayed.

3. A mechanism of drug-transfer needs to be identified, either by triggered release of the encapsulated drug, or by progressive release.

The triggered released of liposomal contents has been extensively explored over the past- two decades, and originates from the desire to have both a minimal drug-leakage from the membrane, whilst also having effective drug release at the target site.58,60 One successful technique to reduce the resulting ‘leakiness’ of the vesicle is to incorporate cholesterol into the lipid bilayer. Cholesterol acts to effectively tighten up the lipid bilayer, by the formation of lipid-rafts.16

Moreover, triggered release can come through two mechanisms; remote triggers, which include heat, ultrasound and light, or ‘in-vivo’ triggers, such as enzymatic hydrolysis (See Figure 1.28).61 In this case, the phosphatidylcholine membrane components are hydrolysed by the enzyme ‘phospholipase c’. Subsequently, the membrane of the vesicle is broken down to release the entire content of encapsulated drug at the target site.

Figure 1.28: Reaction scheme for the enzymatic triggered release of encapsulated 60 compounds in a membrane of phosphatidylcholine, where a) phospholipase c and H2O.

Effective indication of a vesicle’s suitability to act as a drug-delivery system can be determined by both its ability to encapsulate the desired drug, and to also release the drug inside the target cell, at a high enough level to mediate an effective therapeutic response. These properties can be determined at the early stages of the vesicle design, by the resulting encapsulation and release studies of fluorescent hydrophilic compounds.62 An example of such studies, is the encapsulation of 5(6)-carboxyfluorescein (5(6)-CF), which given its spectrophotometric properties, can be assayed using fluorescence spectroscopy to quantify the encapsulation efficiency of a given vesicle.

1.9 Targets

1.9.1 Aims

As discussed in Section 1.4 this project is extended on work of Lopez et al.26 by which the thioester lipid 1a was successfully synthesised (See Figure 1.29). The studies performed by Lopez et al.26 have shown 1a to be a potential building block, by which the thioester- lipid can be modified in regard to its hydrophilic moieties, by using trans-thioesterification.

Figure 1.29: Chemical structure of the thioester lipid 1a.26

The primary aim of this project is to construct thioester and disulfide containing lipids, through thiol-disulfide and thioester exchange reactions, and to study the resulting supramolecular structures they form.

Initially the aim of this project will be to explore the potential of trans-thioesterification, as a means of chemically modifying the hydrophilic moiety of 1a. The di-thiols (2,3)- 39 dimercapto-1-propanol (1c) and (2,3)-dimercapto-propanesulfonic acid (1d) were identified as suitable di-thiol substituents, given their analogous nature to glycerol, and high solubility in aqueous media (See Figure 1.30). Their synthesis will be explored, and subsequent trans-thioesterification will be performed.

Figure 1.30: Chemical structures of 1d, 1e and glycerol.

If successful, this would create a trans-thioesterification framework, by which sulfur- containing analogues of phospholipids could be synthesised in one-pot, and at RT. Moreover, by using acyl-chain derivatives of 1a, a series of different thioester lipids, of varying acyl-chain lengths could be generated (See Figure 1.31).

Figure 1.31: Potential general reaction scheme, by which the thioester lipids (2a-3c) can be synthesised via trans-thioesterification.

Moreover, the hydroxyl-group functionality of 2a/2b/2c, will allow for one-step chemical modifications to the hydrophilic moiety, and thus the potential effect of the hydrophilic moiety on bilayer and vesicle formation can be studied (See Figure 1.32).

Figure 1.32: General reaction scheme, by which the hydroxyl group of the thioester lipids 2a/2b/2c can be chemically modified by a) phosphorylation (phosphorus oxychloride

(POCl3), pyridine) and b) sulfation (pyridine-sulfur trioxide complex).

Furthermore, the next aim will be to synthesise the disulfide lipid 1f. If successful, the same procedure as above will be applied to generate families of different disulfide containing lipids, by use of thiol-disulfide interchange reactions (See Figure 1.33). The proposed resulting disulfide-lipid structures are analogues to ether-linked phospholipids (See Figure 1.34). As a result, they would be expected to behave in a similar manner in aqueous media, and thus self-assemble into bilayer structures and ultimately vesicles.

Figure 1.33: Potential general reaction scheme, by which disulfide lipids 2f and 3f can be synthesised.

Figure 1.34 Chemical structures of disulfide lipids 2f and 3f to show their analogous structure to ether-linked phospholipids.

Once both types of sulfur-containing lipids have been successfully synthesised, the resulting structures they form will be sized using dynamic-light scattering (DLS). ζ measurements will also be recorded, to identify both the resulting stability the self- assembled structures display in aqueous media, along with the resulting net-charge displayed. Furthermore, if vesicle structures are present, both the encapsulation efficiency towards hydrophilic compounds of a fluorescent nature, along with the subsequent progressive release of fluorescent hydrophilic compounds will be tested. This will act as an indicator of the potential suitability the resulting vesicles could have as drug-delivery systems.

The next aim of the project will be to form DCLs consisting of families of interchanging thioester and disulfide lipids (See Figure 1.35). If successful, this will allow the effect of the bilayer, on the interchange of hydrocarbon-chains to be studied.

Figure 1.35: Reaction scheme for the formation of DCLs consisting of families of interchanging a) disulfides and b) thioesters.

The final aim of the project will be to conclude the plausibility of disulfide and thioester lipids as potential pre-biotic lipids.

Chapter 2 Lipid synthesis and characterisation

2.1 Synthesis of 2-nitro-4-(palmitoylthio)benzoic acid (1a)

Following the procedure from Lopez et al.26 DTNB was treated with tris(2-carboxy- ethyl)phosphine hydrochloride (TCEP.HCl), in a mixture of acetonitrile-water (99:1), and heated to reflux at 80oC overnight, to give thiol TNB (See Figure 2.1).

Figure 2.1: Reaction scheme for the synthesis of 1a, where a) is TCEP.HCL, and b) is palmitoyl chloride, followed by concentrated (conc.) HCl.

TCEP.HCl was used due to its strong irreversible reducing power towards disulfide bonds, in comparison to other readily available disulfide reducing agents such as dithiothreitol and 2-mercaptoethanol. The resulting solution was concentrated and dried under high vacuum (UHV) conditions to ensure complete removal of water, as palmitoyl chloride used in the subsequent step is highly moisture sensitive. Treatment of the generated thiol TNB with N,N-Diisopropylethylamine (DIPEA), generates a yellow colour change, indicative of the formation of the thiolate anion TNB2-.

The resulting thiolate anion attacks at the carbonyl of the palmitoyl chloride, generating both a chloride ion and 1a in equilibrium, which proceeds via nucleophilic acyl- substitution (SNAc). The chloride anion acts as the leaving group, and because of its low 33 pKa (-7) the equilibrium lies far to the right, and thus the formation of 1a is favoured (See Figure 2.2).

Figure 2.2 Mechanism for the conversion of palmitoyl chloride into 1a via (SNAc) nucleophilic acyl-substitution.

Thioester 1a was obtained as a pale yellow solid in 73% yield, after recrystallisation from a mixture of hexane:dichloromethane (DCM) (4:1).

In comparison to the procedure followed by Lopez et al.26 a significantly higher yield was obtained (73%>38%), even with the reaction being completed on a much larger scale (6×). As 1a is afforded as a pale-yellow powder in the reported procedure, the difference in yield is most likely attributed by the recrystallisation technique employed. As the recrystallisation technique was not described in detail, a few possibilities can be assumed in regard to the lower afforded yield. One possibility is that too much recrystallisation solvent was used, which would result in a larger amount of purified solid being lost in solution. Another possibility is that the crystal growth time was too short; this would afford smaller crystals. These smaller crystals are more likely to be lost during filtration, with some crystals being smaller in porosity than that of the filter paper, hence causing loss of purified product.

2.2 Synthesis of 2-nitro-5-(stearoylthio)benzoic acid (1b)

Following on from the successful synthesis of 1a, synthesis of the stearoyl equivalent, 1b, was attempted (See Figure 2.3). The procedure reported by Lopez et al.26 was modified by replacement of palmitoyl chloride with stearoyl chloride.

Figure 2.3: Reaction scheme for the synthesis of 1b, where a) is TCEP.HCl and b) is stearoyl chloride, followed by conc. HCl.

Mechanistically, the synthesis of 1b does not differ from that of the palmitoyl-equivalent 1a (See Figure 2.2). Successful synthesis of 1b was afforded as a pale-yellow solid in yield 68%, by recrystallisation from a mixture of hexane:DCM (1:4).

2.3 Trans-thioesterification of (2,3)-dimercapto-1-propanol (1d)

2.3.1 Synthesis of (S,S’)-(3-hydroxypropane-1,2-diyl)dihexadecanethiolate (2a)

Figure 2.4 Reaction scheme for the trans-thioesterification of 1d.

The synthetic strategy to obtain 2a (See Figure 2.4) was based on the trans- thioesterification studies of 1a in aqueous buffered conditions (pH 7.5), performed by Lopez et al.26. In these studies, the extent of trans-thioesterification between 1a and a series of different thiols were monitored spectrophotometrically, by the absorbance intensity increase at 410 nm over time, due to the generation of TNB2-. Correspondingly, the procedure was modified by use of a di-thiol, (2,3)-dimercapto-1-propanol (1d), to obtain the di-substituted trans-thioesterification product (2a).

The mechanism of action proceeds by generation of the thiolate anion of 1d (pKa: ~9.6, ~8.9)33 under basic conditions (pH 7.5), which undergoes nucleophilic acyl-substitution 2- (SNAc) at the carbonyl of 1a, generating both TNB and the mono-substituted product in equilibrium. Subsequently, the attack process occurs again through the second thiol group of 1d, to generate the di-substituted product 2a. Due to its ability to delocalise negative 2- charge over the aromatic ring, TNB has a low pKa for a thiol (4.6 vs 13 for most alkylthios)33 and thus is the favoured leaving group. As a result, the equilibrium is shifted far to the right and the reaction favours the formation of 2a (See Figure 2.5).

Figure 2.5: Mechanism for the conversion of 1a/1b into 2a/2b, via two separate 2- nucleophilic acyl-substitution reactions (SNAc). TNB is generated in each case.

On first attempt, 1d was dissolved in a mixture of aqueous buffered conditions (0.1M, pH 7.5) and methanolic aliquots of 1a, in the presence of TCEP.HCl. By using an aqueous reaction system, along with the spectrophotometric properties of the leaving group TNB2-, if self-assembled vesicles were produced, a cloudy-yellow colour would result. The cloudiness would arise from the formation of vesicles, which can scatter light. Over the course of the reaction, a yellow colour resulted, and large quantities of the starting material (1a) precipitated out of solution, which was validated by 1H NMR spectroscopic analysis of the filtered precipitate. To determine if small quantities of product 2a had been produced, as possibly indicated by the yellow colour change, the resulting aqueous solution was diluted with chloroform (CHCl3). Organic extraction was performed, and the resulting crude was analysed by both 1H NMR spectroscopy and mass-spectrometry (MS). Analysis of the resulting 1H NMR spectrum, indicated the presence of both starting materials in large quantity, with MS showing no evidence of 2a. It was thought possible that 2a could not ‘fly’, and therefore was not detectable with MS.

Given that both 1a and 2a share a palmitoyl-functionality, product identification was therefore focused on the dimercapto-region (from 3.10 to 3.80 ppm), with proton environments expected to be resolved from that of the starting material. This was expected due to the influence of carbonyl moiety of the thioester, shifting proton environments in the dimercapto region downfield due to its electron-withdrawing effects. However due to the strong intensity resonances from both starting materials, it was deemed too difficult to resolve any proton peaks in this region of the spectra.

Thin-layer chromatography (TLC) analysis was taken of the crude by using mobile phases (MP) of either DCM or ethyl acetate. Ethyl acetate proved the most suitable of the two, as

47 two spots with retention-factor (Rf) values of (~0.7) were identified upon irradiation with UV-light.

However due to poor resolution of the two spots (~0.1), a MP system of ethyl acetate/hexane (1:1) was used to evolve the two spots more effectively. This proved successful, with a resolution of (~0.5) being obtained. It was reported that column chromatography is unsuitable as a purification technique for 1a, due to its poor stability on silica columns.26 A gradient-elution of ethyl acetate/hexane starting from (1:3) to (1:1) using a small column was then attempted, with the expectation that only 2a would be eluted. This purification proved unsuccessful, as no spots from TLC analysis were obtained from any of the collected fractions. Elution with DCM/methanol (9:1) was then performed to elute all remaining compounds off the column. One streaky spot using TLC analysis was obtained from the collected fractions, giving possible indication of 2a’s instability on silica columns. On concentration of the collected fraction, a very small quantity of solid was obtained, and 1H NMR spectroscopic analysis showed only solvent.

Attention was then focused on improving the solubility of 1a in aqueous conditions by vortexmixing dilute methanolic aliquots of 1a with aqueous buffer, prior to reaction initiation. Initially this proved successful, however on addition of TCEP.HCl, 1a began to progressively precipitate out. This observation fitted with reports that at high concentrations of TCEP.HCl, the solubility of 1a is effectively further lowered in aqueous conditions.26 TCEP.HCl is an acid, and will protonate the carboxylate of 1a, this in turn reduces the hydrophilicity of the lipid, and further lowers its solubility in water.

Due to the poor solubility of 1a in aqueous media, and given the good solubility of 1b/1a in deuterated chloroform (CDCl3), it was deemed appropriate to use an organic reaction system. Given the strongly electrophilic nature of 1a, an inert solvent system along with a base of low nucleophilicity (DIPEA) seemed appropriate. However, reactions had to be performed without the disulfide reducing agent TCEP.HCl, due to its hydrophilic nature, and resulting poor solubility in organic solvents such as CHCl3. To minimize unfavourable oxidation reactions of thiol substituents into disulfides, reactions were performed under an inert atmosphere and in the presence of argon.

On mixing of 1a and 1d in a 2:1 stoichiometric ratio, a dark-red colour resulted, rather than the expected bright-yellow. This can be accounted for by the possible formation of a TNB2-(DIPEA+) salt (See Figure 2.6).

Figure 2.6: Chemical structure of the TNB2- (DIPEA+) salt.

Dilution of the mixture with distilled water, along with several water washes, completely removed all visible amounts of TNB2- from the organic layer. Concentration of the organic layer afforded a pale-waxy solid. 1H NMR spectroscopic analysis showed significant quantities of both unreacted 1a and 1d, with minor product peaks that were unresolved due to their strong signal intensity resonances.

An excess of 1d was then employed, and 1H NMR spectroscopic analysis of the resulting crude, showed a set of two triplets in the region of 2.60-2.50 ppm. The first triplet is observed at 2.50 ppm with a slight shoulder, whilst the second triplet at 2.60 ppm is characteristic of 2.60 ppm (2H, t, SCOCH2) in 1a (See Figure 2.7). On using correlation spectroscopy (COSY) analysis, the shoulder is identified as another possible product peak, as both overlaying peaks at 2.50 ppm are coupled to the multiplet at 1.60 ppm (2H, m,

CH2(CH2)15), As the relative intensities of the two overlaying signals at 2.50 ppm are significantly different, it was deduced that a mixture of products had been formed, rather than the shoulder being identification of the disubstituted 2a.

Figure 2.7. 1H NMR spectra; Zoom in (2.45 ppm to 2.63 ppm), of the resulting crude on reaction of 1a with 1d in molar excess. Traces of 1a are observed (blue), along with a triplet at 2.5 ppm that appears with a shoulder (red).

On TLC analysis, in a MP system of hexane/ethyl acetate (1:1), two spots of sufficient resolution and Rf, were identified upon irradiation with UV-light. Moreover, as the two possible product spots were UV-detectable, diluted samples of the crude dissolved in DCM were eluted through an analytical normal-phase high-performance liquid chromatography (HPLC), by use of gradient-elution hexane/ethyl-acetate (6:1 to 1:1). Hexane/ethyl acetate (4:1) afforded the best resolution of the two peaks, and the resulting fractions were collected.

As neither compounds degraded on the column, it seemed suitable that liquid- chromotography mass spectroscopy (LCMS), would be appropriate for MS analysis. LCMS analysis of the two separate fractions, afford molecular ion peaks of m/z=623, which corresponded to 2a (M+Na+), and m/z=745 (M+Na+) which corresponded to 2a plus an additional 1d substituent (m/z=122.20) (See Figure 2.8).

Figure 2.8: Chemical structures of 2a and 1d.

1H NMR spectroscopic analysis of the resulting fractions showed similar spectra, only differing in the signal integration in the dimercapto region (from 3.1 to 3.8ppm). This correlates with the LCMS data obtained, by which the two molecular ion peaks m/z=623, and m/z=745 only differ by a dimercapto substituent. For 2a, the signal at 2.50 ppm appears as a multiplet, and on COSY analysis, is resolved into two overlapping triplets, corresponding to ((SCOCH2)2). This slight difference in chemical shift can be elucidated from the asymmetric nature of 2a. Whereas the by-product affords a single triplet at 2.50 ppm, corresponding to ((SOCH2)2), which is a result of a symmetrical structure. Moreover, a multiplet, corresponding to (SSCHCH2), appears further downfield at 3.50 ppm in comparison to the multiplet at 3 ppm for the corresponding (SSCHCH2)2 environment (See Figure 2.9).

Figure 2.9: Overlaid 1H NMR spectra of the obtained a) by-product (M+Na+) (m/z=745), + and b) product (M+Na ) (m/z=623). The corresponding SCOCH2 (red) and SSCH (blue) environments have been circled.

Evidently, from the obtained LCMS data, it was concluded that by-product formation was a result of dimerzation of the di-thiol into a disulfide (See Figure 2.10).

Figure 2.10: Asymmetric chemical structure of 2a along with the symmetric chemical structure of the mono-dimerized by-product. The resulting splitting pattern at ~2.5 ppm,

corresponding to their SCOCH2 environments are shown.

Furthermore, Li et al.63 reported intramolecular acyl-migration of hydroxyl groups in phospholipids of lysophosphatidic acid. Along with the LCMS and 1H NMR spectroscopic data obtained, this suggested two possible by-product structures. (See Figure 2.11).

Figure 2.11: Chemical structures of possible by-products of 4a and 5a, where the 5a by- product would be a result of mono-dimerization and intramolecular acyl-migration, as proposed by 1H NMR spectroscopic analysis and relevant literature.63

Subsequent fourier-transform infrared spectroscopy (FTIR) and C13 NMR spectroscopic analysis supported 4a as the by-product structure, with only thioester functionalities being present on analysis, as evident from the SC=O absorption stretch at 1690 cm-1.

To prevent formation of this by-product, it seemed appropriate to add TCEP.HCl, as a strong irreversible disulfide reducing agent. Due to its poor solubility in CHCl3, a polar solvent system of acetonitrile-methanol (4:1) was used. This proved successful, and improved the overall yield of 2a from 13% to a moderate 47%, by significantly reducing the quantity of dimerised by-product (4a) obtained. 2a was thereby successfully synthesised and purified via normal-phase HPLC in a MP solvent system of hexane/ethyl acetate (4:1), affording a white-waxy solid in 47% yield.

2.3.2 Synthesis of (S,S’)-(3-hydroxypropane-1,2-diyl)dioctadecanethiolate (2b)

On successful synthesis of 2a, the same procedure was applied for the stearoyl equivalent (2b). Mechanistically the two trans-thioesterifications do not differ, and are therefore expected to proceed via the same mechanism (See Figure 2.5).

2b was successfully synthesised and purified via normal-phase HPLC in a MP solvent system of hexane/ethyl acetate (4:1), affording a white-waxy solid in 52% yield.

2.4 Hydroxyl-group functionalisation

On successful synthesis of 2a and 2b, the next step was to functionalise the hydroxyl group via either phosphorylation or sulfation reactions (See Figure 2.12). If successful, this would introduce a hydrophilic moiety in the form of a charged polar head-group. This could potentially lead to vesicle-forming lipids.

Figure 2.12: Reaction scheme for the hydroxyl-group functionalisation of 2a, via phosphorylation (6a) and sulfation (7a).

2.4.1 Attempted synthesis of (2,3)-bis(palmitoylthio)propyl phosphate (6a)

64 Following the literature from Ikemoto et al. 2a, phosphorylchloride (POCl3) and 5 pyridine-d in near equivalent stochiometric ratios were mixed in CDCl3. Over the course of 90 minutes, the solution was heated to 50°C, and monitored in-situ at 30-minute time intervals using 1H NMR spectroscopic analysis. On completion, the reaction solution was diluted with two portions of water, and the organic layer extracted, and concentrated.

The phosphorylation mechanism proceeds via attack of the hydroxyl group on the phosphorus centre of POCl3, which generates a chloride ion on nucleophilic substitution. Pyridine serves as the base, and deprotonates the acidic proton of the chlorophosphate ester group, and effectively ‘mops’ up the generated HCl. (See Figure 2.13). Further reaction of the chlorophosphate ester group with water, hydrolyses the remaining two P-Cl bonds, thus generating the phosphoric ester derivative (OPO(OH)2), and two equivalents of hydrochloric acid (HCl), which are ‘mopped’ up by the pyridine (See Figure 2.14(a)). Chlorophosphate, is an excellent leaving group, and thus excess use of pyridine can induce the E2 elimination of the hydroxyl to an alkene (See Figure 2.14(b)).

Figure 2.13: Mechanism for the generation of a chlorophosphate ester group via

nucleophilic substitution of the hydroxyl group of 3b at the phosphorus centre of POCl3.

Figure 2.14: Reaction schemes for the hydrolysis of the chlorophosphate ester group with a) near equimolar ratio of pyridine, and the E2 elimination of the chlorophosphite group into an alkene with b) molar excess of pyridine.

In-situ 1H NMR spectroscopy studies over the course of 300 minutes showed the appearance of possible product peaks 4.22 ppm (1H, m), 4.17 ppm (1H, dd), 4.05 ppm (1H, dd) and 2.25 ppm (m, 2H) with similar coupling constants, to corresponding peaks in 2a. After 300 minutes, no further change in the relative integral ratios of 2a and possible product peaks had occurred, and the reaction was judged to have reached completion (See Figure 2.15).

Figure 2.15. Overlaid 1H NMR spectra, where a) is 30 minutes, b) is 90 minutes, c) is 300 minutes. Possible product formation is identified after 30 minutes, by the appearance of the triplet at 2.25 ppm (black circle), along with the appearance of 4.22 ppm (m, 1H), 4.17 ppm (1H, dd) and 4.05 ppm (1H, dd) after 300 minutes (red circle).

Subsequently, the resulting crude was extracted, and 1H NMR spectroscopic analysis confirmed a mixture of both starting material and product (See Figure 2.16).

Figure 2.16. Overlaid 1H NMR spectra of a) the resulting crude and b) the starting material 2a. Comparison of the two spectra allows for the product peaks 4.22 ppm (m, 1H), 4.17 ppm (1H, dd), 4.05 ppm (1H, dd), 2.50 ppm (t, 2H) and 2.25 ppm (t, 2H) to be identified (blue circles), impurity peaks are denoted by (x).

On TLC analysis, hexane/ethyl acetate (4:1) was identified as the most suitable MP system to evolve the resulting three bands with substantial resolution. Preparative TLC was then employed to allow for larger quantities of crude (up to 100 mg) to be loaded onto the TLC plate. By using a MP system of hexane/ethyl acetate (4:1), the resulting three bands were scraped off the silica-plate, and analysed by both 1H NMR spectroscopy and LCMS. The 3rd band was identified as purified product (6a) by LCMS (m/z=679). On 1H NMR spectroscopic and COSY analysis, the appearance of two sets of triplets at 2.50 ppm (t, 2H) and 2.25 ppm (t, 2H) in replacement of the expected multiplet of two overlapping triplet signals, gave an indication that 6a had not formed. To identify whether the triplet at 2.25 ppm was a product peak, COSY analysis was taken, and showed both sets of triplets at 2.50 ppm and 2.25 ppm to be coupled to the multiplet at 1.60 ppm (6H, m), and thus validated the triplet at 2.25 ppm as a product peak (See Figure 2.17).

Figure 2.17: COSY spectra of the purified product by preparative TLC analysis. Coupling

of both 2.25 ppm (2H, t) and 2.50 ppm (2H, t) to 1.60 ppm (3H, m, (CH2)12CH3), is shown by the dotted lines.

It was proposed that the chemical shift (ppm) difference between the two triplets, is not an effect of an asymmetrical structure, but rather a formation of two different carbonyl functionalities. To validate this claim, FTIR spectroscopy was employed, and showed the appearance of ester (OC=O, 1737 cm-1), thioester (SC=O, 1692 cm-1) and phosphoric acid (P=O, 1165 cm-1) functionalities.

Based on these analytical findings, it was proposed that an intramolecular acyl-migration reaction between the C2 thioester and hydroxyl group had occurred on treatment with base, 56 and thus generated an ester and thiol at the C1 and C2 positions respectively. The resulting thiol at the C2 position was then subjected to phosphorylation, affording the proposed structure 8a (See Figure 2.18). This phenomenon was found to be common, as reported numerously in literature and seemed unavoidable without the prior blocking of acyl migration at the hydroxyl group.65-68

Figure 2.18. Mechanism for the intramolecular acyl-migration of 2a at the primary hydroxyl group (C1), with the thioester at the C2 position. Subsequent phosphorylation generates 8a.

Consequently, due to time constraints of the project, acyl-migration blocking was not attempted. However, a possible route for acyl migration-blocking would have been to phosphorylate and effectively ‘tie-up’ the hydroxyl group of 1d prior to thio-acylation. For this to proceed successfully, it would require prior cyclic dimerization protection of the thiols substituent. A synthetic procedure is discussed in detail in Chapter 5 (See Section 5.2).

2.4.2 Attempted synthesis of (2,3)-bis(palmitoylthio)propyl sulfate (7a)

The sulfation of 2a (See Figure 2.12), using a pyridine sulfur trioxide complex was attempted. Thioester 2a and pyridine sulfur trioxide complex were heated to reflux (~110°C) in dry toluene and in the presence of a calcium chloride drying tube, for 40 minutes. Toluene was used as a replacement to benzene to significant reduce the safety hazards affiliated with reaction, due to benzene’s highly carcinogenic nature. The drying tube was employed to prevent exposure of the moisture sensitive sulfur trioxide complex to water during heating to reflux. After 40 minutes, the reaction mixture was concentrated in vacuo, and dissolved in chloroform at 0oC, resulting in a precipitate of unreacted sulfur- trioxide complex, which was filtered off.

Mechanistically, the sulfation reaction occurs through nucleophilic substitution, via nucleophilic attack of the hydroxyl group at the sulfur atom of pyridine sulfur trioxide complex. Consequently, on generation of the sulfonic ester intermediate, pyridine acts as the leaving group and then serves as a base to deprotonate the resulting sulfonic ester (See Figure 2.19). 57

Figure 2.19: Mechanism for the sulfation of 2a into 7a, as proceeded by nucleophilic attack by the hydroxyl group of 2a on the sulfur centre of the pyridine-sulfur trioxide complex.

1H NMR spectroscopic analysis of the resulting crude product showed unreacted 2a and intramolecular acyl migrated product, as suggested by the triplet observed at 2.25 ppm corresponding to the migrated ester (CH2COO) environment. To validate this proposal, purification and full characterisation of the resulting acyl-migrated product was required. Preparative TLC was employed, with the resulting three bands scraped off and analysed by both 1H NMR spectroscopy and LCMS. Evidently from LCMS analysis, band 3 was identified as the possible acyl migrated product (m/z=679.8) (See Figure 2.19).

Figure 2.19: Chemical structure of the acyl-migrated product of 7a.

1H NMR and COSY spectroscopic analysis afforded identical spectra to that of the phosphorylated migrated product 8a. Characteristic triplet signals to that of an acyl- migrated structure are present at both 2.50 ppm and 2.25 ppm, indicative of both thioester and ester functionalities; 2.50 ppm (2H, t, SCOCH2), 2.25 ppm (2H, t, OCOCH2). FTIR spectroscopic analysis further validated this proposed structure, with thioester (SC=O, 1687 cm-1), and ester functionalities being evident (OC=O, 1733 cm-1). The proposed mechanism of intramolecular acyl migration is shown below (See Figure 2.20).

Figure 2.20: Mechanism for the intramolecular acyl-migration between the primary hydroxyl group (C1), and the thioester at the C2 position. Subsequent sulfation affords 9a.

As for compound 7a, time constraints of the project prevented blocking of acyl-migration from being attempted. However, a possible route for acyl migration-blocking, complementary to that of the phosphorylation reaction, would have been to sulfonate and effectively ‘tie-up’ the hydroxyl group of 1d, prior to thio-acylation. For this to proceed successfully, it would require prior cyclic dimerization protection of the thiol substituents. A synthetic procedure is discussed in detail in Chapter 5 (See Section 5.2).

2.5 Trans-thioesterification of (2,3)-dimercapto-1-propanesulfonic acid (1e)

Following the successful trans-thioesterification synthesis of 2a and 2b, a similar synthetic procedure was employed to synthesise the following sulfonic acid equivalents 3a and 3b (See Figure 2.21).

Figure 2.21: Reaction scheme for the trans-thioesterification of either 1a or 1b into either 3a or 3b.

Mechanistically, the two trans-thioesterifications should not differ, and the same reaction mechanism is expected (See Figure 2.22).

Figure 2.22: Mechanism for the trans-thioesterification of either 1a or 1b into either 3a or 3b, via two separate nucleophilic acyl-substitution reactions. TNB2- is generated in each case.

2.5.1 Synthesis of (2,3)-bis(heptadecanoylthio)propane-1-sulfonate (3b)

Initially 1b and 1e were mixed in a medium of acetonitrile/methanol in the presence of both DIPEA and TCEP.HCl for 5 hours. A dark-red colour change occurred and the resulting mixture was concentrated and re-suspended in a mixture of distilled water and chloroform.1H NMR spectroscopic analysis of the organic layer, showed complete absence of 1b. However, there was no evidence of 3b being present in the organic layer. Given this observation, it was proposed that 3b had higher affiliation to the aqueous layer. Subsequent acidic treatment of the aqueous layer with 5 mL of conc. HCl, followed by organic extraction was unsuccessful, with the resulting 1H NMR spectrum being very messy in appearance and showed no indication of 3b. It was proposed that acidic conditions promoted the hydrolysis of 3b.

The reaction was repeated, and the resulting aqueous layer concentrated under reduced pressure. This time on concentration, a red foam of bubbles was rapidly formed inside the rotary evaporator. This is indicative of compounds with a surfactant nature. As large quantities of the aqueous sample were lost inside the rotary evaporator, freeze-drying was deemed to the most appropriate technique to concentrate the aqueous layer.

On 1H NMR spectroscopy of the aqueous layer after freeze-drying, a possible product peak was identifiable by the multiplet at 2.50 ppm. COSY spectroscopy analysis further validated the multiplet as two overlapping triplet peaks, and consistent with the asymmetric structure of 3b. Purification was attempted by TLC analysis with a MP system 60 of chloroform/methanol/water (73:23:3). Under UV-light, one spot was observed with significant streaking, which was a possible indication of poor stability on silica. Reverse- phase HPLC was then employed with the same MP system. However, due to the significant quantity of chloroform required and its low boiling point, large quantities of bubbles were produced inside the tubing of the HPLC. Subsequently, this caused significantly poor resolution on UV-detection.

Based on the proposed amphiphilic nature of the product, it was suggested that gel- permeation chromatography (GPC) would be an acceptable purification technique if the resulting product (3b) self-assembled into vesicles in aqueous media. GPC, also known as size-exclusion chromatography, acts as a purification technique by separating molecules or molecular aggregates according to their size and shape, with larger structures such as vesicles being eluted first.70 This technique however relied on the absence of 1b, as it may self-assemble into micelles in aqueous solution, which may result in both 3b and 1b being eluted together. To ensure complete removal of 1b, the aqueous layer was washed several times with DCM. Purification of 3b was successful using GPC, with the first elution being a ‘creamy’ colour on appearance, consistent with scattering from a concentrated solution of vesicles. 3e was afforded as a white waxy solid in 61% yield by purification using GPC.

1H and 13C NMR spectroscopic analysis of 3b proved difficult, due to the compounds poor solubility in NMR solvents. At first attempt, concentrated deuterated-water (D2O) solutions of the resulting eluent afforded a low intensity spectrum. This is a possible result of 3b self-assembling into vesicles in solution. On the formation of large supramolecular structures, slow tumbling is observed. Slow tumbling occurs when there is a fast relaxation of the transverse magnetisation. For vesicle formation, enhanced spin-spin interactions occur, this leads to a faster relaxation, and thus a spectrum of weak intensity is produced.71 The overall rate of molecular tumbling, especially for thermostable vesicles, can be increased by performing 1H NMR spectroscopy at elevated temperatures. Without prior knowledge of the resulting vesicles thermostability, different NMR solvents were used.

Deuterated-dimethyl sulfoxide (DMSO-d6) and deuterated-dimethylformamide (DMF-d7) systems proved unsuccessful, along with mixtures of CDCl3 with small quantities of D2O. 1 Dilute solutions of CDCl3 were found to be the most successful solvent system for H NMR spectroscopic analysis. 1H NMR and COSY spectroscopic analysis showed that a DIPEA salt of the proposed structure had formed, given that the GPC purification technique would have excluded DIPEA (See Figure 2.23).

Figure 2.23: Chemical structure of 3b/DIPEA salt, as indicated by 1H NMR spectroscopic analysis.

Initial indications of a 3b/DIPEA salt became evident from presence of an acidic proton signal at 8.00 ppm, a sharp doublet at 1.30 ppm for the NCH(CH3)2 environment, and a multiplet at 2.50 ppm due to the two overlapping triplets of SCOCH2. Protons in the dimercapto region (3.10 to 3.80 ppm) proved the most difficult to characterise, with 4 proton environments of low intensity being present, in which two of the environments were overlapping with DIPEA signals, along with possible second-order effects being displayed at 3.10 ppm. These resulting issues could have been resolved by use of stronger magnetic field, such as a 500 MHz NMR spectrometer, however due to time-constraints and availability of the spectrometer, this was not completed. Despite this, full peak assignment was completed by comparative COSY analysis of both 1e and 3b, allowing for both hidden DIPEA environments (See Figure 2.24), and environments in the dimercapto region to be fully assigned (See Figure 2.25).

Figure 2.24: COSY spectra of 3b, by which a hidden DIPEA environment (+NCH) is found at 3.25 ppm (green circle). The coupling relationship between proton environments in the dimercapto region (3.1 to 4.1 ppm) (see dotted lines) allowed for full 1H NMR spectroscopic characterisation of 3b.

Figure 2.25: 1H NMR spectra zoom-in of the dimercapto region of 3b, showing the

assigned diastereotopic protons Ha/Hb. along with hidden DIPEA environments (blue / red circle). Possible impurity is denoted by x.

MS analysis via both LCMS and high-resolution MS (HRMS) further indicated the presence of 3b, as both showed [M-H-] (m/z=719), however there was no detection of the DIPEA cation in both the positive and negative spectrum. It was assumed that the DIPEA 63 cation did not fly through the spectrometer, as 1H NMR analysis provided sufficient evidence of its presence.

2.5.2 Synthesis of 2,3-bis(pentadecanoylthio)propane-1-sulfonate (3a)

On successful synthesis of 3b, the same synthetic procedure was applied to obtain the palmitoyl-equivalent 3a, which was afforded as a white waxy solid in 57% yield by purification using GPC.

Full NMR characterisation of 3a was not achieved due to its poorer solubility in CDCl3, a possible result of a shorter hydrocarbon moiety. 1H NMR spectroscopic analysis of 3a in dilute solutions of CDCl3 afforded a broad spectrum, indicating substantial quantities of undissolved solid (See Figure 2.26).

Figure 2.26: 1H NMR spectrum overlay of a) 1a, b) 3b and c) 3a. Matching protons environments are colour coordinated.

As observable in Figure 2.26(c), the resulting spectrum of 3a in CDCl3 is broad, however on comparison with the spectrum of 3b, expected integration constants are obtained, along with similar ppm. As 3a was purified by GPC, the only possible impurity would be 1a, if 1a formed micelles in aqueous media. On comparison of Figure 2.26(a) and Figure 2.26(c), there are no aromatic signals (7.80 to 7.5 ppm), corresponding to 1a that are observable in the 1H NMR spectrum of 3a. Furthermore, LCMS showed only the presence of ions corresponding to 3a (m/z=663.4 [M-H]-).

1 H NMR spectroscopic analysis was then performed at elevated temperatures in D2O with a 500MHz spectrometer. This proved unsuccessful with low-intensity spectrums being afforded, with peaks of even poorer resolution. Given the effectiveness of GPC in 1 purifying 3a, along with H NMR spectroscopic analysis in CDCl3 showing no observable impurity peaks, vesicles studies were performed on 3a, with the expectation that 3a was pure. Unfortunately, due to time constraints and the unavailability of an 800 MHz NMR spectrometer, in both the Chemistry Building and the Manchester Institute of Biotechnology MIB, a 1H and 13C NMR spectrum was unattainable. Any future work with 3a will require prior successful 1H NMR spectroscopic analysis, to validate its proposed structure (See Section 5.2).

2.6 Thiol-disulfide interchange of 1d

2.6.1 Attempted synthesis of 5-(hexadecyldisulfanely)-2-nitro-benzoic acid (1f)

Using reported literature on base catalysed thiol-disulfide interchange reactions between DTNB and thiol-substituents,72 the synthesis of 1f was attempted by mixing 1- octadecanethiol (1g) with a molar excess of DTNB in chloroform. The thiol-disulfide reaction was catalysed by the addition of DIPEA (See Figure 2.27).

Figure 2.27: Reaction scheme for the thiol-disulfide interchange reaction between DTNB and 1g, affording both 1f and TNB2-.

Many properties suggested DTNB as a suitable disulfide source for thiol-disulfide interchange reactions. The first being its weak S-S disulfide bond, thus reduction by most thiols should go to completion. Secondly, the spectrophotometric properties of TNB2- generated after reduction, allows for indication that the reaction is proceeding, whilst providing information on reaction-kinetics via spectrophotometric studies.

The corresponding mechanism of action proceeds via nucleophilic substitution and is base catalysed. On deprotonation (Ka), the generated thiolate anion undergoes backside nucleophilic attack along the ‘S-S’ bond axis of DTNB, which generates TNB2- as a

65 leaving group (k1). A competitive secondary reaction can also occur, given the relative concentration of 1g, whereby the alkyl thiolate anion (1g-) undergoes nucleophilic attack at 2- the disulfide bond of 1f, generating both an alkyl-disulfide (3f) and TNB (k2) (See Figure 2.28).

Figure 2.28. Reaction scheme showing the two-competing thiol-disulfide interchange reactions (b&c), along with the corresponding thiol-dissociation equilibrium (a).

Whitesides et al.72 reported on rate-law derivation for a series of thiol-interchange reactions involving monothiols and DTNB (See Equation 2.1).

훿(푁푇퐵2−) = 푘표푏푠[(푅푆−) + (푅푆퐻)(퐷푇푁퐵)] + 푘표푏푠[(푅푆−) + (푅푆퐻)(푁푇퐵 − 푅푆)] 푡 1 2

Equation 2.1: Thiol-disulfide interchange rate-law.

The obtained kinetic data also showed a 10-fold increase the in the corresponding rate 72 constant of (b), in comparison to competing reaction (c) (k1>10k2). Based on this analysis, having DTNB in molar excess, will limit the contribution of thiol-disulfide interchange (c), to negligible.

1g and excess DTNB were mixed in CHCl3 in the presence of DIPEA for 5 hours. The resulting mixture was diluted with distilled water, and the organic layer extracted and then concentrated under reduced pressure. LCMS analysis of the organic crude identified the presence of 1f; (M-H-) (m/z=482.5). However, on 1H NMR spectroscopic analysis, a mixture of compounds was evident, with two sets of impurities being present. The first, is the slight shoulder on the triplet at 2.65 ppm corresponding to SSCH2 (See Figure 2.29).

Figure 2.29: 1H NMR spectra zoom-in (2.68 to 2.59 ppm). Shows the appearance of a

shoulder (blue circle) on the triplet at 2.65 ppm corresponding to SSCH2.

On COSY spectroscopic analysis the shoulder was confirmed to be that of the SCH2CH2 environment in 1g. The second set of impurity peaks appear in the aromatic region of the spectra (7.75 to 7.25 ppm), and given their apparent spitting pattern and integration (2H, d) (2H, d) (2H, dd), it was proposed that the aromatic impurity corresponded to unreacted DTNB (See Figure 2.30).

Figure 2.30: Overlaid 1H NMR spectra of a) the starting material (1g) and b) the product crude. Comparison of the two spectra allows for the identification of impurities; DIPEA (x), DTNB (x), and 1g (x).

Purification was attempted via recrystallisation using a solvent system of hexane-DCM (4:1). However, this proved unsuccessful and afforded a mixture of both 1f and 1g. Due to the difficulty in separating 1g, and given its low relative quantity, it was deemed appropriate to perform the sequential thiol-disulfide interchange reaction between 1f and 1d, directly from the organic crude afforded from the procedure above.

2.6 Attempted synthesis of (2,3)-bis(heptadecyldisulfaneyl)propan-1-ol (2f)

As with the previous procedure, base-catalysed thiol-disulfide interchange reactions were used as a means of synthesising new disulfide equivalents 2f, by reaction of 1f with 1d (contained within a crude mixture) (See Figure 2.31).

Figure 2.31: Reaction scheme for the synthesis of 2f.

On first attempt, the crude from the previous step was dissolved in CHCl3, followed by the addition of 1d, and DIPEA under an inert atmosphere. A resulting dark red colour was generated, and the reaction was stirred for 5 hours, before dilution with distilled water and subsequent extraction. The organic layers were dried with MgSO4, filtered and the solvent removed from the filtrate under reduced pressure. However, given that the generation of 2f required disulphide bond formation, the reaction had to occur in the absence of TCEP.HCl, which is a strong disulfide reducing agent. This had a dramatic effect on the reaction, as a series of disulfides and mixed dimerized products were obtained.

1H NMR spectroscopic analysis of the crude showed a complete absence of peaks in the aromatic region, suggesting complete starting material (1f) reduction. Peaks consistent with that of an octadecanoyl region of the spectrum were also present. However, integration ratios of the peaks corresponding to the octadecanoyl region of the spectrum

2.50 ppm (4H, m, SCH2), 1.55 ppm (4H, m, SCH2CH2), and 1.20 ppm (60H, s, (CH2)15), did not match. Subsequent COSY analysis showed no underlying impurity peaks which could have possibly skewed the integration ratios. The corresponding multiplet at 2.50 ppm would be consistent with that of the proposed structure if composed of two overlapping triplets. However, LCMS analysis showed no evidence of 2f formation, as indicated by absence of the molecular ion peak (m/z=692). Evidence of mono-substituted and dimerized-mono-substituted derivatives were however present (See Figure 2.32). Therefore, the corresponding multiplet at 2.50 ppm is most likely to have been caused by the overlapping of two triplets from the dimerized and non-dimerized mono-substituted derivatives of 2f.

Figure 2.32. LCMS spectra of the resulting crude, showing only the presence of disulfides 4f and 5f, with possible fragmentation by loss of oxygen.

Conversely, due to the conditions required for thiol-disulfide interchange reactions, the formation of the dimerized disulfide is unavoidable without use of a reducing agent, such as TCEP.HCl. Also as evident from the LCMS, the mono-substituted product is clearly favoured, with complete absence of both the dimerized-disubstituted 6f (m/z=814) and disubstituted 2f (m/z=693) disulfide lipids (See Figure 2.33).

Figure 2.33: Chemical structures of 6f and 2f, along with their expected molecular-ion peaks.

Singh et al.34 reports that 1,2-di-thiols have a high tendency to polymerise at concentrations higher than 1 mM, and a high tendency of ring closure at concentrations lower than 1 mM. As the effective concentration of 1d was high (46.6 mM), a possible explanation is that the disulfide polymerization of 1d to polydisulfide (2d) had caused insufficient quantities of 1d available to undergo di-substitution, and therefore the mono-substituted product was 70 favoured (See Figure 2.34). Subsequently, reactions were then performed at lower concentrations of both 1d (5 mM), this however proved unsuccessful as on LCMS and 1H NMR spectroscopic analysis, large quantities of 1f remained unreacted. This observation is consistent to that of Singh, et al.34 in which cyclic bis(disulfide) dimer (3d) (See Figure 2.34) is the major product at low concentrations of 1d. Based on 1H NMR spectroscopic analysis, it could be plausible that 3d acts as a thermodynamic sink (very stable), and thus, thiol-disulfide interchange does not proceed.

Figure 2.34 Chemical structures of 2d and 3d.

Consequently, the formation of 1f seemed unachievable without a reducing agent, due to the high tendency of 1d to form poly-disulfides (2d) and cyclic bis(disulfide) dimers (3d).

Chapter 3 Vesicle, kinetics and DCC studies

3.1 Vesicle Studies

3.1.1 DLS and zeta-potential studies of 3a and 3b

Given the charged hydrophilic moiety of successfully synthesised lipids 3a and 3b, it seemed appropriate to both size and quantify the relative stabilities of their proposed vesicle structures (See Figure 3.1).

Figure 3.1: Chemical structures of 3a and 3b.

DLS was identified as the most appropriate analytical technique, given its ability to obtain intensity-mean weighted diameters (ZD) for diameters ranging from 1 nm to 3µm, along with quantifying their relative size distributions, as indicated by the poly-dispersity index. Poly-dispersity index can also be used to quantify the degree of lamellarity (i.e. number of bilayers), and thus categorise resulting vesicles as either unilamellar or multilamellar.73 Furthermore, the Malvern Zetasizer equipment used for DLS measurements, also has a ζ functionality. ζ values allow the relative degree of stability between charged particulates to be quantified, along with the magnitude of net charge displayed by the vesicles. These two characteristics coincide with each other. For example, a negative ζ value exceeding -61 mV, would indicate excellent vesicle stability, along with the vesicle displaying a high net- negative charge.59 This relationship between net charge and stability is a result of the electrical-double layer (EDL) theory.

The procedure of Li et al.74 was followed, and either 3a or 3b were mixed separately in 1 mL of HPLC grade water at a concentration of 4.6 mM. Prior filtration of the HPLC grade water was required through a syringe-filter, to remove any traces of large particulates such as dust. Both DLS and ζ measurements are very dust sensitive, with dust-particles contributing to the resulting poly-dispersity index. 73

The resulting solutions of either 3a or 3b were then bath-sonicated for 20 minutes at RT, which would disrupt a suspension of multimellar vesicles (MVs) from being formed, and thus would afford a lower poly-dispersity index value. After bath-sonication, the solutions were left to incubate for 1 hour at RT, before being transferred into zeta-cells, which were used for both DLS and ζ measurements.

DLS measurements were performed at 25°C and at a backscattering angle of 127°, sizing measurements for each sample were performed twelve times, this process was repeated three times for two different samples of either 3a or 3b. The afforded intensity size distributions graphs for both 3a and 3b are shown below (See Figures 3.2 a&b).

Figure 3.2: Intensity size distribution graphs taken directly from the ZetaSizer software for a) 3a and b) 3b. Six measurements were performed, with three measurements for two different samples of either 3a or 3b. The observable dust contaminant in a) is circled in green (~ 1000 nm) and a possible second structural morphology (~ 80 nm) in b) is circled in blue.

From the intensity size distributions graphs, a second possible structural morphology is observed (~80 nm) in Figure 3.2(b). Given the size of this second structural morphology, it would not be classed in the size range of a micelle (2-20 nm), and therefore a possible impurity in the form of 1b can be discounted.14 As this second morphology is only observed in one of out of the six measurements performed, it can be proposed as an anomaly.

ζ measurements were performed at 25°C and at a backscattering angle of 127°. Measurements for each sample were performed twelve times, this process was repeated three times for two different samples of either 3a or 3b. The afforded intensity ζ distributions graphs for both 3a and 3b are shown below (See Figures 3.3 c&d).

Figure 3.2: Total count ζ distribution graphs taken directly from the ZetaSizer software for d) 3a and d) 3b. Six measurements were performed, with three measurements for two different samples of either 3a or 3b.

On cumulative analysis of the resulting intensity distribution graphs, ZD and ζ values, along with an estimated width of distribution (poly-dispersity index) were obtained. Cumulative analysis is automatically performed on the resulting intensity-distribution graphs by the ZetaSizer software. The average ZD, ζ and corresponding poly-dispersity index values were taken from the six measurements of both 3a and 3b, and are shown in Table 3.1.

Thioester Average ZD Poly-dispersity Average ζ Poly-dispersity Index Index [4.6 mM] (nm) (mV)

3a 158.7 0.281 -77.9 0.422

3b 215.6 0.253 -92.1 0.567

Table 3.1: Average ZD, ζ, and poly-dispersity index values taken from the resulting 6 measurements obtained from cumulative analysis, as performed by the ZetaSizer software.

Table 3.1 shows that both ZD values for 3a and 3b lie in the size-range of ‘large’ vesicles 14 (100 nm-3 µm). Based on the average ZD data, it can be concluded that both 3a and 3b self-assemble intro supramolecular structures that lie in the size-range of vesicles, and therefore do not self-assemble into micelle structures (2-20 nm). Also evident is that the resulting poly-dispersity index values indicate that both 3a and 3b are near monodisperse in solution, and thus most vesicles in the sample are of a near similar size. Given that unilamellar vesicles of phosphatidylcholines can have a PDI values ranging from 0.10 to

0.36, it can be proposed, based on the obtained poly-dispersity index and ZD values for both 3a and 3b, that in aqueous media and on bath-sonication at RT, thioester lipids 3a and 3b self-assemble into monodispersed large unilamellar vesicles (LUVs).

As indicated by the negative ζ values (See Table 3.1), the LUVs of both 3a and 3b display a net-negative charge. This correlates with the structures of both 3a and 3b, in which the acting hydrophilic moiety is a negatively charged sulfonic acid group. The strongly negative values of ζ, indicate that at 25°C, LUVs of 3a and 3b should display high stability. In the case of 3a, a higher ζ value was expected in comparison to that of 3b, given the higher surface charge density, due to a smaller diameter. However, this is not the case, and given that the DIPEA counter-ion can lower the net negative charge displayed by the LUVs, perhaps it can be proposed that a larger proportion of LUVs consisting of 3a occur in the presence of a DIPEA counter-ion.

In conclusion, the ZD, ζ and corresponding poly-dispersity index values have indicated that both 3a and 3b self-assemble into LUVs in aqueous conditions. As both LUVs display high stability and high net negative charge at 25°C, they both have potential application in the encapsulation of compounds displaying hydrophilicity.

3.1.2 Encapsulation of 5(6)-carboxyfluorescein into vesicles of 3a and 3b

Following the procedure of Li et al.74 the encapsulation efficiency of amphiphiles 3a and 3b towards the hydrophilic (5)6-carboxyfluorescein (5(6)-CF) (See Figure 3.4) were investigated. 5(6)-CF is highly coloured, so the quantity of entrapped 5(6)-CF can be identified by measuring absorbance intensity at 488 nm using UV-visible spectroscopy.

Figure 3.4: Chemical structure of 5(6)-CF.

To obtain the absorbance intensity prior to encapsulation, 4 µL of a 2 mM 5(6)-CF stock solution was added to 2 mL of HPLC-grade water to give a 4 µM solution of 5(6)-CF. The resulting solution was then assayed using UV-visible spectroscopy, with an absorbance intensity of 0.092 being recorded for the 5(6)-CF peak at 488 nm).

Subsequently, either 3a and 3b were both separately dissolved in 2.5 mL of a 2 mM 5(6)- CF stock solution, so that the resulting concentration of the lipid was 4.6 mM. The resulting lipid/5(6)-CF mixtures were bath sonicated at RT for 15 minutes to induce vesicle formation, and then stored in the dark at RT for 2 hours, to prevent the photodestabilisation of 5(6)-CF. The non-encapsulated 5(6)-CF was then removed by GPC. On the second elution of 3.5 mL, the resulting lipid eluents were green in appearance, indicating the presence of entrapped 5(6)-CF. An aliquot of the post-GPC eluent (2 mL) was then immediately assayed using UV-visible spectroscopy (200-700 nm), which showed the absorbance peak of 5(6)-CF being approximately shifted by 4 nm. This shift may be due to interactions of 5(6)-CF with the thioester lipid membrane. Absorbance intensities of 0.136 and 0.517 were recorded at 492 nm for 3a and 3b respectively. Resulting encapsulation efficiencies were obtained using Equation 3.1.

A(5)6CF(ʎ492 nm) [(5)6퐂퐅(ʎ488 퐧퐦)] EE (%) = GPC Dilution Factor × ( ) × ( ) × 100 A(5)6CF(ʎ488 nm) [(5)6CF(ʎ492 퐧퐦)]

Equation 3.1: Where EE is encapsulation efficiency, A(5)6CF(ʎ492nm) is the absorbance

intensity of (5)6CF at wavelength 492 nm, A(5)6CF(ʎ488nm) is the absorbance intensity of

(5)6CF at wavelength 488 nm, [5(6)-CF(ʎ488nm)] is the concentration of 5(6)-CF affiliated

with the absorbance intensity at 488 nm, [(5)6CF(ʎ492nm)]0 is the concentration of the 5(6)- 2 CF encapsulation stock-solution, and the GPC dilution factor is ( ). 3

2 0.136 4×10−6M 1) 3퐚 EE (%) = ( ) × ( ) × ( ) × 100 = 0.41% 3 0.092 2×10−3M

2 0.517 4×10−6M 2) 3퐛 EE (%) = ( ) × ( ) × ( ) × 100 = 1.57% 3 0.092 2×10−3M

Encapsulation efficiencies of 0.41 and 1.57% were obtained for 3a and 3b respectively. Moreover, the encapsulation efficiencies correspond with the ζ values, whereby 3b, which has a higher average ζ value than 3a, displays a 3-fold increase on encapsulation efficiency. A possible explanation for this, is that the higher net-negative charged displayed by the vesicles of 3b increases the tendency of the hydrophilic 5(6)-CF to be encapsulated.

3.1.3 Release of 5(6)-carboxyfluorescein from vesicles composed of 3a and 3b

Following on from the encapsulation studies of 5(6)-CF with vesicles 3a and 3b, it seemed appropriate to measure the permeability of the resulting vesicles. This can be carried out by quantifying the rate of release of 5(6)-CF from the vesicle, which effectively allows quantification of the relative ‘leakiness’ of the lipid bilayers.

Following the procedure of Booth et al.41 either 3a or 3b were dissolved in 1 mL of a concentrated solution of 5(6)-CF (0.05 M) in (3-morpholinopropane-1-sulfonic acid (MOPS) buffer (20 mM), sodium chloride (NaCl) (100 mM), pH 7.4), so that the final concentration of the lipids was 20 mM. The lipid/5(6)-CF mixture was bath sonicated for 20 minutes at RT, to induce vesicle formation, and then left to incubate in the dark for 2 hours at RT. The solution was then diluted to 2.5 mL with phosphate-buffered saline (PBS), and the non-encapsulated dye was removed by GPC. After obtaining the vesicle- containing eluent, the resulting suspension was centrifuged at 2200×g for 15 minutes, resulting in the sedimentation of the vesicles out of the suspension. This procedure resulted in only released 5(6)-CF contributing to the resulting fluorescence intensity at 517 nm.

After the supernatant buffer was removed, resuspension of the green coloured vesicle mass in PBS gave a total volume of 1 mL. Purification by centrifugation was repeated, and at the 45 minute time-point, 20 µL aliquots of the resulting supernatant buffer were added to 2 mL of PBS. The fluorescence at 517 nm (excitation 492 nm) was recorded, this value was used to give an indication of the relative percentage (%) release of 5(6)-CF at the 45 minute time-point. Subsequently, the encapsulated solution was left to stand for 24 hours at RT, heated to 42°C for 15 hours and then briefly bath sonicated for 2 minutes at RT, this allowed for complete release of the entrapped 5(6)-CF. A further 20 µL aliquot of the supernatant was taken, and added to PBS, whereby the fluorescence at 517 nm (excitation 492 nm) was recorded. This final value was assumed to be 100% release of 5(6)-CF, and was used to calculate the percentage release value after 45 minutes (See Equation 3.2).

(I517 nm)45min % Release of (5)6CF = ( ) × 100 (I517nm)24hr

Equation 3.2: Where (퐈517 퐧퐦)45퐦퐢퐧 is the fluorescence intensity of 5(6)-CF at 517 nm

after the 45 minute time-point, and (퐈517퐧퐦)24퐡퐫 is the fluorescence intensity of 5(6)-CF at 517 nm after the 24 hour time-point.

182.320 1) 3a %Release of (5)6CF = ( ) × 100 =70.7 ± 0.1% 257.899

265.018 2) 3b % Release of (5)6CF = ( ) × 100 = 42.9 ± 4.2% 624.534

The data presented in Figure 3.5 is for the (%) of 5(6)-CF from the vesicles of 3a and 3b at a time-point of 45 minutes.

1 0 0

3 a F

C 8 0 3 b

)

(

f 6 0

e s

a 4 0

e R

2 0 %

a b 3 3

Figure 3.5: Average (%) release of 5(6)-CF from LUVs of 3a and 3b. Samples were incubated at RT with the fluorescent intensities being measured 45 minutes after incubation. Sample measurements were repeated three times.

As observed from Figure 3.5: vesicles composed of 3a have a higher 5(6)-CF percentage release (71%) in comparison to that from vesicles composed of 3b, which haves a percentage release (43%) at the 45 minute time-point. These values also correlate with the encapsulation studies, in which after 2 hours of incubation, vesicles composed of 3a had an encapsulation efficiency that was 3-fold lower in comparison to vesicles composed of 3b. It can therefore be concluded that the bilayer of vesicles composed of 3a are leakier in comparison to vesicles composed of 3b. This could possibly be a result of 3a’s shorter acyl chains.

LUVs of 3a and 3b are significantly loose, in comparison to that of vesicles composed of DPPC and (1,2)-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (5(6)-CF (%) release values of 11% and 8% respectively, as reported by Booth et al.41. The relative tightness of the vesicle bilayer corresponds to the degree of packing between the acyl chains. Given the acyl chains of 3a and DPPC are analogous (See Figure 3.6), a possible conclusion to be drawn would be that the zwitterionic head group in DPPC significantly increases the resulting tightness of the bilayer.

Chibowski et al.77 report that the resulting charge density on the vesicle affects the resulting tightness, with vesicles of a higher change density being packed more loosely. 80

This would correlate with the observed difference in percentage release of 5(6)-CF values between 3a and DPPC, by which DPPC approximately affords ζ and PDI values that are 1.5 times lower than that of 3a.76

Figure 3.6: Chemical structures of 3a and DPPC, in which their analogous acyl-chains can be identified.

3.2 Dynamic combinatorial chemistry studies

3.2.1 Introduction

In this section, the potential of using DCC as means of generating families of interchanging lipids through thioester exchange reactions will be discussed. The system was based on the works of Larsson et al.39 and involved the mixing 2a and 2b in equimolar amounts in CDCl3 at RT, followed by the addition of a catalytic quantity (1% mol/mol) of thiolate anion 1h (See Figure 3.7).

Figure 3.7: Reaction scheme for the formation of DCLs through thioester exchange reactions, as initiated by the addition of a catalytic quantity of 1h (1% mol/mol). The afforded libraries are expected to consist of a family of interchanging lipids, in a (1:1:1:1) ratio.

Mechanistically, the proposed thioester exchange reactions occur through a series of nucleophilic acyl-substitution (SNAc) reactions. The exchange process is catalysed by the addition of 1h, which acts as the initial nucleophile by attacking at the thioester carbonyl centres of either 2a or 2b (See Figure 3.8).

Figure 3.8: Mechanism for the nucleophilic acyl-substitution reaction (SNAc), via nucleophilic attack by 1h at the carbonyl centre of either 2a or 2b, in which either 2ma- or 2mb- acts as the leaving group.

Further nucleophilic acyl-substitution can occur, in which the thiolate anion 2ma-/2mb- attacks at the carbonyl-centre of either 2a or 2b. Subsequently, the thiolate anion 2ma- /2mb- is generated, along with a new lipid of mixed lengths ‘R’ 2ab/2ba (See Figure 3.9). This mechanism of action proceeds until a near statistical ratio (1:1:1:1) of thioester lipid products is reached.

Figure 3.9: Mechanism for the nucleophilic substitution of either 2ma- or 2mb- at the carbonyl-centre of either 2a or 2b, in which a thiolate anion of either 2ma- or 2mb- is generated along with mixed lipid of either 2ab or 2ba.

3.2.2 Generated dynamic combinatorial libraries from 2a and 2b

DCL formation was monitored over time, by 1H NMR spectroscopic analysis at pre- determined time intervals (See Figure 3.10).

Figure 3.10: Overlaid 1H NMR spectrums for the time-course studies of DCL formation from thioesters 2a and 2b, in the presence of catalytic 1h (1%mol/mol). Where a) is 30 minutes b) 60 minutes, c) 90minutes, along with a further addition (0.09 eq) of 1h, d) 120 minutes, e) 150 minutes. A water impurity is indicated by the blue circle, possible acyl-

migration corresponding to the OCOCH2 environment is indicated by the red circle, and further addition of 1h is indicated by the green circle.

The 1H NMR spectroscopic time-course studies show visible traces of water being present at 1.50 ppm (blue circle). Contamination by water is detrimental to the formation of the DCL, as the water can be deprotonated in the presence of a base, such as 1h. The generated hydroxide anion can then hydrolyse the thioesters of 2a and 2b, to form the corresponding carboxylic acids (See Figure 3.11(1&2)). As carboxylic acids are acids, it is expected that the thiolate species (2ma-/2mb-/1h) required to form the DCL will be protonated (See Figure 3.11(3)). The generated thiol species on protonation will be unable to undergo nucleophilic acyl-substitution at the thioesters of 2a and 2b due to their poor nucleophilicity. As a result, DCL formation will be stopped.

Figure 3.11: Reaction scheme for the formation of carboxylic acids, on exposure of 1h to trace quantities of water. 1) deprotonation of water by 1h results in the formation of a hydroxide ion. 2) Generated hydroxide ion is able to hydrolyse the thioester to generate the corresponding carboxylic acid. 3) Carboxylic acid will be able to protonate all thiolate species in the system.

LCMS analysis of the crude mixture after a reaction time 150 minutes, showed no evidence of mixed lipid 2ab/2ba formation (m/z=628) along with the absence of both starting lipids 2a and 2b, (m/z=600) and (m/z=656) respectively. Only ions related to 2ma- /2mb- were observed (See Figure 3.12). The most probable explanation for the LCMS data will be the exposure of the reaction system to water as identified by 1H NMR spectroscopic analysis. The proposed hydrolysis of lipids 2a and 2b, along with the protonation of thiolate anions 2ma-/2mb-, is consistent with the LCMS data. Based on 1H NMR and LCMS spectroscopic analysis, it can be proposed that the DCL formation from thioesters 2a and 2b did not occur, because of water contamination to the reaction system.

Figure 3.12: LCMS spectrum of the resulting DCL crude, in which ions possibly related to 2ma- (red) and 2mb- (blue) are observed.

Also evident from the 1H NMR spectroscopic time-course studies is the possible formation of the acyl-migrated product of either 2a or 2b, as suggested by a triplet at 2.25 ppm that indicates a OCOCH2 environment (See Figure 3.10). An explanation for this observed effect, is that the thiolate anion 1h is a base (pKa ~ 10.6) and can deprotonate the primary hydroxyl group of either 2a or 2b.33 The generated nucleophilic hydroxide ion attacks at the carbonyl of the thioester (C2), via nucleophilic acyl-substitution (SnAc), to generate the acyl-migrated product (See Figure 3.13).

Figure 3.13: Reaction mechanism for the proposed generation of the acyl-migrated product of 11k on treatment of either 3b or 4b with 1k.

In conclusion LCMS analysis has shown the observable presence of ions related to 2ma- /2mb-, and the observable absence of ions related to lipids 2a/2b and mixed acyl-lipids

2ab/2ba. A possible explanation for the observable absence of mixed acyl-lipids 2ab and 2ba in LCMS analysis is the exposure of the reaction system to water, as indicated by 1H NMR spectroscopic analysis. Consequently, due to time constraints of the project, further DCC studies were not performed. However, 1H NMR spectroscopic analysis has indicated that possible acyl-migration of thioester lipids 2a and 2b, has occurred in the conditions required for DCL formation (catalytic strong base). Given both 2a and 2b’s poor stability in the presence of a base, further DCC studies using thioester lipids 2a and 2b should be avoided. Future DCC studies should instead be focused on the formation of DCLs using their sulfonic acid equivalents 3a and 3b, See Chapter 5 (Section 5.2) for further details.

3.3 Kinetic Studies

3.3.1 Introduction

As discussed in Chapter 2, the research described in this thesis explores the use of trans- thioesterification and thiol-disulfide interchange reactions, as a potential source of forming sulphur containing lipids. Furthermore, the trans-thioesterification between 1a/1b and 1d/e proved successful. Subsequently, to further validate the kinetic details regarding optimum reaction conditions and reaction times, kinetic studies were performed on the trans- thioesterification between 1a and 1d/1e.

Moreover, following on from the work of Lopez et al.26 this current chapter will focus on discussing the corresponding relative extents (%) of each trans-thioesterification reaction between 1a and 1d/1e. To act as a comparative, the extents for the trans-thioesterification reactions of 1a with 2,3-mercapto-1-propanol (1i) and sodium 3-mercapto-1- propanesulfonate (1j) were also obtained (See Figure 3.14).

Figure 3.14 Chemical structures of 1i and 1j.

As stated in Chapter 1, lipid 1a was designed by Lopez et al.26 with its spectrophotometric properties in mind. On addition of 1a with 1d/1e/1i/1j in basic conditions, 1a undergoes a trans-thioesterification reaction, in which an aromatic thiolate (TNB2-) is released. Thiolate TNB2- is intensely yellow and absorbs in the UV-visible region at 410 nm. TNB2- also has 26 a significantly lower pKa (4.53), than the corresponding di-thiols/thiols (See Section 3.1.5), which in turn makes the species less nucleophilic, and a better leaving group, thus

86 forcing the equilibrium towards the products. The spectrophotometric properties of TNB2- allows for the trans-thioesterification processes involving a series of different di-thiols and monothiols with 1a to be monitored by UV-visible spectroscopy. Along with the subsequent extent for both 1a hydrolysis and trans-thioesterification to be established.

Prior to discussing the trans-thioesterifcation of 1a in aqueous conditions, initial attention should be drawn towards the effect of hydrolysis and methanolysis on the resulting extents of reaction.

3.3.2 Stability of 1a

Both aliphatic and aromatic thioesters are very electrophilic in nature, and are susceptible to nucleophilic acyl substitution from solvents of a nucleophilic nature, such as methanol and water. Aromatic thioesters such as 1a, are even more susceptible than their aliphatic counterparts. The extent to which hydrolysis and methanolysis contributes to the rate of 1a trans-thioesterification was investigated by Lopez et al.26

3.3.3 Stability of 1a towards methanolysis

The extent of methanolysis of 1a was followed by 1H NMR spectroscopy at RT for time points 1 hour, 24 hours, 48 hours and 1month. Over the time period of 1 month, no evidence of 1a decomposition occurred. As a result, it was concluded that 1a does not undergo methanolysis in these conditions.26

3.3.4 Stability of 1a towards hydrolysis TCEP.HCl has been reported by Tsuda et al.78 to increase the electrophilicity of thioesters like 1a, and thus facilitates hydrolysis. Given the importance of TCEP.HCl in the trans- thioesterifcation as a means of limiting disulfide formation, the extent to which TCEP.HCl contributes to the hydrolysis of 1a was also investigated by Lopez et al.26 (See Table 3.2).

Conditions Reaction time Absorbance at Extent of kh 1a [0.02 mM] (minutes) 410 nm (×10-3) hydrolysis (×10-5s-1) (%) 1a 40 18.27 ± 4.25 7.25 ± 1.7 0.82 ± 0.04

1a 40 32.05 ± 2.56 12.72 ± 1.0 1.12 ± 0.08 TCEP [0.2mM] 1a 40 40.72 ± 0.47 10.77 ± 9.3 1.50 ± 0.1 TCEP [1.0mM] Table 3.2: Hydrolysis of 1a in bulk conditions, as performed by Lopez et al.26 Extent of hydrolysis values (%) are obtained from the experimental molar coefficient value of TNB2- (12.6×103M-1cm-1).26

The extent of hydrolysis values obtained from Lopez et al.26 shows that 1a is susceptible to hydrolysis in buffered conditions (0.1 M, pH 7.5). Also evident is that on increasing concentrations of TCEP.HCl, the rate of hydrolysis is further accelerated, and thus validates the proposal by Tsuda et al.78 The proposed mechanism for TCEP.HCl facilitated hydrolysis of 1a, comes from the dramatic decrease in pH of the buffered systems when in 33 the presence of TCEP.HCl (pKa = 7.7). This abrupt change in pH promotes the hydrolysis of 1a.

3.3.5 Technical issues

As with all the experiments performed in this study, the quality of resulting UV-spectra at 410 nm was poor, with large degrees of noise being observed, along with sharp drops and sharp increases in absorbance that would occur spontaneously. On running blank samples and samples in the absence of a stirrer bar, it was identified that rate of stirrer bar revolution affected the occurrence of these fluctuations, with faster revolutions increasing their frequency. As a result, an optimum stirrer bar-revolution had to be found, at which this problem was minimised. Subsequently, for most of these experiments, this worked,

88 however in the case of the reaction between 1a with 0.5 mM of 1d, sharp spikes in absorbance increase appeared even at the optimum stirrer bar-revolution.

Another issue was the varying degree in initial absorbance values obtained on reaction initiation which meant that ΔA≠0 at t=0. Given that the same quartz cuvette was used for both the blank and sample measurements, and that the sample was measured immediately on reaction initiation with the same initial concentration of 1a. It would imply that there was a technical issue with the UV-spectrometer, regarding the blank being effectively subtracted and or measured. A number of these technical issues with the SJW-owned UV- spectrometer coincided with the UV-visible spectrometer being moved to another section of the laboratory by people outside of the group, as none of these issues were encountered prior to it being moved. Given that there were insufficient UV-visible spectrometers available with stirring capabilities in the MIB, and that reasonable error bars were obtained after averaging of data points, along with general kinetic trends expected, studies were continued using the same UV-visible spectrometer.

3.3.6 Trans-thioesterification reactions of 1a with 1d and 1e

The resulting extents (%) for the trans-thioesterifcation reaction of 1a with dimercapto- analogues 1b and 1e were obtained in bulk conditions, using an aqueous buffer medium (0.1 M phosphate buffer, pH 7.5) at RT (See Figure 3.15).

Figure 3.15: Reaction scheme for the trans-thioesterification reaction of 1a with either 1d or 1e, along with the corresponding hydrolysis of 1a.

An aqueous buffered system of pH 7.5 seemed appropriate, considering the relative pKa values of both 1d and 1e (~9.6, ~8.9, and 7.5, ~6.7 respectively), 33 along with an expected increase in the extent of hydrolysis of 1a on using a higher pH.

A series of experiments were performed, in which 1a at a constant initial concentration of 0.02 mM was reacted with di-thiols of an alcohol (1d) and sulfate (1e) functionality, at varying initial concentrations of 0.1mM and 0.5mM. TCEP.HCL was also added to the reaction system, at an initial concentration that was double the concentration of the di-thiol reactant, this was to prevent disulfide formation. The reaction conditions were based on preliminary data, which found a 0.02 mM concentration of 1a was the most effective, given the poor solubility of 1a in aqueous conditions in the presence of TCEP.HCl.26

On addition of an methanolic aliquot of 1a to the requisite di-thiol, the absorbance intensity increase (ΔA) at 410 nm was immediately monitored over a time period of 27 minutes. (ΔA) can be calculated using Equation 3.3, and is the difference between the absorbance intensity (A) at time (t) and the initial absorbance intensity (Ao) as attributed by 1a.

ΔA = A − 퐴표

Equation 3.3:

The resulting (ΔA) at 410 nm was recorded over (t), by taking absorbance intensity measurements at 100 second intervals. Each series experiment was repeated 3 times, and a curve of the average (ΔA) against (t) was plotted (See Figure 3.3 a&b).

Figure 3.16: Plots of the time-dependent absorbance intensity change at 410 nm on generation of TNB2- for the trans-thioesterification of 1a (0.02 mM) with a) 1d and b) 1e at initial concentrations of 0.1mM ( ) and 0.5mM ( ).

As observed from Figure 3b, sigmoidal kinetic plots are observed for the trans- thioesterification of 1a with 1e (0.1 mM and 0.5 mM). This is indicated by the big delay (~100 seconds), and subsequent rapid increase in the absorbance intensity at 410 nm. This observed sigmoidal behaviour indicates possible autocatalytic behaviour. Autocatalysis is the catalysis of a reaction by the resulting products.75 For example, in Figure 3.16(b), the

90 rate ate of absorbance intensity increase is initially slow, as there is little 3a present (0-100 seconds), the rate of reaction then rapidly increases (~180 seconds) as the amount of 3a increases, along with 1a and 1e still being present. The reaction then begins to slow down (~500 seconds) as the concentration of 1a is diminished.

Based off these kinetic findings, it can be proposed that the formation of 3a catalyses the trans-thioesterification reaction between 1a and 1e. Furthermore, the kinetic plots, obtained for the trans-thioesterification of 1a with 1d to generate 2a displays pseudo-first order kinetics (See Figure 3.16(a)). In comparison of resulting lipid products 3a and 2a, only lipid 3a has been observed to self-assemble into vesicle structures in aqueous conditions. It can therefore be proposed, that the formation of lipid 3a and subsequent self-assembly into vesicle structures in aqueous conditions, autocatalyses the trans-thioesterification reaction of 1a with 1e.

Moreover, from these kinetic plots, the extent of trans-thioesterification (%) of 1a can be estimated by the maximum absorbance intensity change recorded (ΔAmax), relative to the maximum absorbance expected (ΔAexmax) (See Equation 3.4).

ΔAmax Extent of trans − thioesterification (%) = ( ) × 100 ΔAexmax

Equation 3.4

ΔAexmax can be estimated using the beer lambert law, and in this case, is the maximum absorbance expected on the complete conversion of 1a into TNB2- (See Equation 3.5).

ΔAexmax = ƹ × 푐 × 푙

Equation 3.5: ΔAexmax can be estimated by multiplying the resulting molar absorption coefficient of 2b (ƹ =12.6×103 M-1cm-1) at 410 nm, the initial concentration of 3b (0.02×10-3 M) and the length of solution that UV-light passes through (l=1 cm).26

3 −1 −1 −3 ΔAexmax = 12.6 × 10 M cm × 1cm × 0.02 × 10 M = 0.252

As, ΔA ≠ 0 at t=0 for all the obtained kinetic plots, the value of ΔA at t=0 was subtracted off the ΔAmax values obtained, to account for the varying initial absorbances. The reaction conditions for the trans-thioesterification of 1a with either 1d or 1e are shown in Tables 3.3&3.4, along with their corresponding extent (%) of trans-thioesterification.

1a (mM) 1d (mM) TCEP.HCl Reaction ΔAmax Extent of (mM) time reaction (minutes) (%)

0.02 0.5 1.0 27 0.106 ± 42.1 ± 0.1 0.001

0.02 0.1 0.2 27 0.037 ± 14.7 ± 0.3 0.003

Table 3.3: Reaction conditions and corresponding extents of reactions, as calculated from

the average ΔAmax observed, for the trans-thioesterification reaction of 1a with 1d in the presence of TCEP.HCl

1a (mM) 1e (mM) TCEP.HCl Reaction ΔAmax Extent of (mM) time reaction (minutes) (%)

0.02 0.5 1.0 27 0.056 ± 22.2 ± 0.8 0.008

0.02 0.1 0.2 27 0.053 ± 21.0 ± 0.6 0.006

Table 3.4: Reaction conditions and corresponding extents of reactions, as calculated from

the average ΔAmax observed, for the trans-thioesterification reaction of 1a with 1b in the presence of TCEP.HCl.

As evident from Tables 3.3&3.4 after 27 minutes, the trans-thioesterification reaction of 1a with either 1d or 1e affords low extents of reaction. The extents are even lower when considering the effects of TCEP.HCl on the extents of hydrolysis. For the series of 1a with 1d [0.5 mM], the 4-fold increase in the extent of reaction, in comparison to the [0.1 mM] series can be accounted for by the fluctuations in absorbance on stirring. When comparing the [0.1 mM] series of the two di-thiols, 1d would be expected to afford a higher reaction 33 extent, given its higher pKa (~9.6, ~8.9) this was not the case as 1e afforded a moderately higher extent. Furthermore, the slight increase in extents of reaction on doubling the concentration of the di-thiol reactant 1e, can be accounted for by the doubling 92 in TCEP.HCl concentration. As TCEP.HCl facilitates 1a hydrolysis, the difference can be accounted for by the extent of hydrolysis of 1a.

3.3.7 Trans-thioesterification reactions of 1a with 1i and 1j

Figure 3.17 Reaction scheme for the trans-thioesterification reaction of 1a with monocapto derivatives 1i and 1j, along with 1a’s corresponding hydrolysis.

Furthermore, the resulting procedure above was repeated for the monothiol equivalents (See Figure 3.17) and the resulting (ΔA) at 410 nm was recorded over (t), by taking absorbance intensity measurements at 100 second intervals. Each series experiment was repeated 3 times, and a curve of the average (ΔA) against (t) was plotted (See Figure 3.18).

Figure 3.18 Plots of the time-dependent absorbance intensity change at 410 nm on generation of TNB2- for the trans-thioesterification of 1a (0.02 mM) with e) 1i and b) 1j at initial concentrations of 0.1mM ( ) and 0.5mM ( ).

From Figure 3.18(f), a kinetic plot displaying non-sigmoidal behaviour is observed on the generation of the monocapto equivalent of 3a (3aj). As 3aj possesses one acyl-chain, it would not be expected to self-assemble into vesicle structures in aqueous media.

Moreover, this observation corresponds with the proposal that the self-assembly of 3a into vesicles, displays autocatalytic behaviour for the trans-thioesterification of 1a with 1e.

As, ΔA ≠ 0 at t=0 for all the obtained kinetic plots, the value of ΔA at t=0 was subtracted off the ΔAmax values obtained, to account for the varying initial absorbances. The reaction conditions for the trans-thioesterification of 1a with either 1i or 1j are shown in Tables 3.5&3.6, along with their corresponding extent (%) of trans-thioesterification.

1a [mM] 1i [mM] TCEP. Reaction ΔAmax Extent of HCl time reaction [mM] (minutes) (%)

0.02 1.0 1.0 27 0.061 ± 24.2 ± 0.7 0.007

0.02 0.2 0.2 27 0.053 ± 21.03 ± 1.2 0.012

Table 3.5: Reaction conditions and corresponding extents of reactions, as calculated from

the average ΔAmax observed, for the trans-thioesterification reaction of 1a with 1i in the presence of TCEP.HCl.

1a (mM) 1j (mM) TCEP.HCl Reaction ΔAmax Extent of (mM) time reaction (minutes) (%)

0.02 1.0 1.0 27 0.083 ± 32.9 ± 1.2 0.012

0.02 0.2 0.2 27 0.092 ± 36.5 ± 0.8 0.008

Table 3.6: Reaction conditions and corresponding extents of reactions, as calculated from

the average ΔAmax observed, for the trans-thioesterification reaction of 1a with 1j in the presence of TCEP.HCl.

As evident from Tables 3.5&3.6 after 27 minutes, the trans-thioesterification reaction of 26 1a with either 1i or 1j affords low extents of reaction. Lopez, et al. reports that after 30 94 minutes, the trans-thioesterification reaction of 1a with L-cysteine methyl ester (CysOMe) (0.2 mM, pKa ~7.5) 26 affords an extent of reaction that is near 3-fold higher than that of 1i 26 in near-identical conditions. As the pKa value (~9.6) of 1i is higher than that of CysOMe, it would be expected to obtain a closer extent of reaction, given that the reaction was monitored for a shorter time period.

As evident from the obtained extents of reaction, the expected relationship between the pKa values of the di-thiol/thiol substituents and the extent of reaction was not observed. For both studies, a possible explanation for the low extents of reactions afforded would be that the pH of the reaction system was not measured before reaction initiation. As stated before, TCEP.HCl is a strong acid, and in concentrations of 0.2mM and 1.0mM, would be expected to lower the pH of the buffer (pH 7.5). For the di-thiol species, some of the thiol- substituents had pKa’s in the range of (6.7-7.5),33 as a result, if the pH was further lowered, these thiol substituents would be expected to have a higher proportion of their protonated forms.

Furthermore, in all series experiments, there was variation in the initial absorbance value measured i.e. ΔA≠0 at t=0. This will have affected the obtained extents of reaction, and is the most probable explanation for the obtained extents of reaction, not correlating with the pKa’s of the di-thiol and thiol substituents. As discussed above, this was a technical issue with the UV-spectrometer. As a result, this study should be repeated with a fully functioning UV-spectrometer, along with the pH of the buffer being measured and subsequently adjusted after TCEP.HCl addition. Unfortunately, due to time-constraints, this was not attempted.

Chapter 4 Experimental

4.1 General materials, instrumentation and other notes All reagents and solvents were obtained from commercial suppliers; (Sigma-Aldrich, Alfa Aesa, Fisher Scientific and Thermo Scientific), unless stated other-wise.

TLC analysis and preparative TLC purification was carried out on Merck silica gel 60 F254 plate. Visualisation was achieved by use of either UV-light or potassium manganate

(KMnO4). All GPC columns used for vesicle purification were PD-10 desalting columns, containing Sephadex G-25 medium (size exclusion limit Mr=7000Da). Normal-phase HPLC was performed on an Agilent 1100 series system with an ACE 5 SIL column, of dimensions (250 ×21.2 mm).

1H and 13C NMR spectrums were recorded on 400 and 500 MHz Bruker DPX spectrometers. 1H NMR shifts were references to the residual deuterated solvent peak 13 (CDCl3; 7.27), and C NMR shifts were referenced to the carbon resonance of the solvent (CDCl3; 77.0). Coupling constants (J) are reported in Hertz (Hz) with chemical shifts being recorded in parts-per-million (ppm). Multiplicities are reported with the appropriate abbreviations; singlet (s), doublet (d), triplet (t), doublet-doublet (dd), broad-singlet (bs) and multiplet (m). Mestrelabs MestReNova software package was used to assign all NMR spectra.

LCMS was performed by use of a Micromass LCT instrument, using a Waters 2790 separation module with electrospray-ionisation (ES+/-). While HRMS was performed on a Waters Q-time-of-flight (TOF) micro instrument, with an ES+/- ion-source.

Fourier transform infrared (FTIR) spectrums were obtained by use of a Bruker Alpha-P instrument with OPUS 6.5 software package. UV-visible spectroscopy (200-800 nm) was performed on a Jacso V660 instrument (1000 nm/min), with Jasco EHC-716 Peltier temperature control, along with a DIA integrating sphere attachment. Samples were measured in Sigma Spectrophotometer Silica Quartz cuvettes with a 10 mm path length. All kinetic data was plotted using GraphPad Prism software package version 6.07.

Fluorescence spectroscopy was performed on a Perkin-Elmer LS55 fluorimeter instrument, with an attached Julabo F25-HE water circulator for temperature control. All samples were measured in disposable fluorimeter cuvettes.

DLS and zeta-potential measurements were performed on a Malvern Zetasizer Nano S (He-Ne laser 633nm) instrument. Samples were measured in disposable capillary cells

(DTS1070). All subsequent data analysis was completed with use of the Malvern Zetasizer software package.

Bath-sonication was performed by use of a TranssonicTM T460 bath-type sonicator, centrifugation was performed by using a Heraeus Instruments Megafuge 1.0R, vortex mixing was performed by using a Vortext Genie 2 (600-2700 rpm), all pH measurements were recorded on a HANNA pH 212-microprocessor pH meter, and melting points were recorded using a Stuart Scientific SMP10 instrument.

4.2 Synthetic methods

2-nitro-4-(palmitoylthio)benzoic acid

2-nitro-4-(palmitoylthio)benzoic acid was synthesised by following the method of Lopez et al. 26 DTNB (1.00 g, 2.55 mmol) and TCEP.HCl (731.00 mg, 2.55 mmol) were heated to reflux in a mixture of acetonitrile-water (99:1, 50 mL) and was stirred for 15 hours. The solvent was then removed under reduced pressure, to give a bright orange solid that was re- o suspended in anhydrous-DCM, under flowing N2 and cooled to 0 C. DIPEA (1.8 mL, 10.00 mmol) was then added dropwise; with a resultant red-colour change indicating the presence of the reduced DTNB. Subsequently palmitoyl chloride (774 µL, 2.55 mmoles) was added dropwise, and a colourless colour change resulted. The reaction then stirred at RT for 12 hours. On completion, the reaction mixture was diluted with 150 mL of DCM, 500 mL of conc. HCl and brine. The organic layer was separated, dried (MgSO4), filtered and the solvent removed from the filtrate under reduced pressure to give a bright yellow solid. The titled compound was afforded as a pale-yellow solid (73%, 813.00 mg) by recrystallisation from a mixture of boiling hexane-DCM (4:1);

1 - H NMR (400 MHz, CDCl3, 25°C) δH: 7.92 (1H, d, J=1.9Hz, CarHCarCO2 ), 7.83 (1H, d,

J=8.4Hz, CarHNO2), 7.64 (1H, dd, J=8.4, 1.90Hz, CarHCarS), 2.64 (2H, t, J=7.5Hz,

CH2C=O), 1.66 (2H, m, CH2CH2(C=O)), 1.24-1.18 (24H, bs, (CH2)12CH3), 0.81 (3H, t,

J=6.8Hz, CH3). 13C NMR (100 MHz, CDCl3) δC: 137.81 (CarS), 135.43 (CarSCarH), - - 134.47 (Car(COO ), 125.81 (Car(NO2)CarH), 124.40 (Car(COO ), 44.27 (CH2(C=O)), 25.56 - - (CH2CH2(C=O)), 22.71 ((CH2)12), 14.08 (CH3). MS (ES , DCM) m/z: 436.3 [M-H] . FTIR ʋ (cm-1): 2920, 1850, 1712, 1540, 1349, 1297, 961, 833, 751.

13 C NMR peaks; 148.64 (Car(NO2)), 168.84 (Car(COO-)) and 194.79 (CH2S(C=O)) are missing from the spectra. All remaining data is in correspondence with the literature. [30]

2-nitro-4-(stearoylthio)benzoic acid

2-nitro-4-(stearoylthio)benzoic acid was synthesised by following the method of Lopez et al. 26 DTNB (1.00 g, 2.55 mmol) and TCEP.HCl (731.00 mg, 2.55 mmol) were heated to reflux in a mixture of acetonitrile-water (99:1, 50 mL). The solvent was then removed under reduced pressure, to give a bright orange solid. The resulting residue, along with stearoyl chloride (772.00 mg, 2.55 mmoles) was resuspended in anhydrous-DCM, under o flowing N2 and cooled to 0 C. DIPEA (1.8 mL, 10.00 mmol) was then added dropwise; with a resultant red-colour change indicating the presence of the reduced DTNB. The reaction then stirred at RT for 12 hours. On completion, the reaction mixture was diluted DCM (150 mL), and washed with conc. HCl (500 mL) and brine. The organic layer was separated, dried (MgSO4), filtered and the solvent removed from the filtrate under reduced pressure to give a bright yellow solid. The titled compound was afforded as a pale-yellow solid (68%, 1.18×103 mg) by re-crystallisation from a mixture of boiling hexane-DCM (4:1).;

1 H NMR (400 MHz, CDCl3, 25°C) δH: 7.83 (1H, d, J= 1.9Hz, CarHCarCO2), 7.79 (1H, d,

J=8.4, CarHNO2), 7.63 (1H, dd, J=8.4, 1.9Hz, CarHCarS), 2.65 (2H, t, J=7.5Hz, CH2C=O),

1.67 (2H, m, CH2CH2(C=O)), 1.22-1.18 (28H, bs, (CH2)14CH3), 0.81 (3H, t, J=6.8Hz,

CH3). 13C NMR (100MHz, CDCl3) δC: 137.01 (CarS), 135.16 (CarSCarH), 134.44 - - (Car(COO ), 125.86 (Car(NO2)CarH), 124.44 (Car(COO ), 44.28 (CH2(C=O)), 25.59 - - (CH2CH2(C=O)), 22.77 ((CH2)14), 14.01 (CH3). MS (ES , DCM) m/z: 464.4 [M-H] . FTIR ʋ (cm-1): 2920, 1848, 1715, 1542, 1349, 1297, 961, 833, 751.

13 C NMR peaks; 148.64 (Car(NO2)), 168.84 (Car(COO-)) and 194.79 (CH2S(C=O)) are missing from the spectra. All remaining data is in correspondence with the literature. [30]

100

(S,S’)-(3-hydroxypropane-1,2-diyl)dihexadecanethioate

2-nitro-4-(palmitoylthio)benzoic acid (60.00 mg, 0.14 mmol, 1eq) and TCEP.HCl (34.50 mg, 0.14 mmol) were dissolved in mixture of acetonitrile-methanol (4:1, 15 mL), under an argon atmosphere. (2,3)-dimercapto-1-propanol (8.60 µL, 0.69×10-1 mmol) was added to the reaction mixture dropwise, followed by the addition of DIPEA (24 µL, 0.14 mmol). On addition of DIPEA, the solution turned from colourless to dark-red. The reaction mixture was then allowed to stir at RT for 5 hours. On completion, the solvent was removed under reduced pressure, and the resulting crude was dissolved in a mixture of distilled water (50 mL) and chloroform (40 mL). The organic layer was separated, and then further washed with several-portions of distilled water, until the resulting aqueous washes were colourless. The organic layer was separated, dried (MgSO4), filtered and the solvent removed from the filtrate under reduced pressure to give a pale-brown waxy solid. The titled compound was afforded as a pale-white waxy solid (47%, 19.50 mg) from normal-phase HPLC, using a MP system of hexane-ethyl-acetate (4:1). The product was eluted after a run time of 330 seconds using an ACE 5 SIL silica column (250 × 21.2mm).

1 H NMR (400 MHz, CDCl3, 25°C) δH: 3.67 (2H, m, CH2OH), 3.53 (1H, m, SCH), 3.24 (1H, dd, J=14.3, 7.9Hz, SCH’H), 3.13 (1H, dd, J=14.3, 4.6Hz SCH’H), 2.53 (2H, t,

J=7.5Hz, (SCOCH2), 2.49 (2H, t, J=7.5Hz, SCOCH2), 1.59 (4H, m, ((CH2CH2(C=O))2), + 1.21-1.18 (48H, bs, ((CH2)12CH3)2)), 0.81 (6H, t, J=6.8Hz, (CH3)2); MS (ES , DCM) m/z: 623.7 [M-Na]+

101

(S,S’)-(3-hydroxypropane-1,2-diyl)dioctadecanethioate

2-nitro-4-(stearoylthio)benzoic acid (64.10 mg, 0.14 mmol) and TCEP.HCl (34.50 mg, 0.14 mmol) were dissolved in mixture of acetonitrile-methanol (4:1, 15mL), under an argon atmosphere. (2,3)-dimercapto-1-propanol (8.60 µL, 0.69×10-1 mmol) was added to the reaction mixture dropwise, followed by the addition of DIPEA (24.00 µL, 0.14 mmol). On addition of DIPEA, the solution turned from colourless to dark-red. The reaction mixture was then allowed to stir at RT for 5 hours. On completion, the solvent was removed under reduced pressure, and the resulting crude was dissolved in a mixture of distilled water (50 mL) and chloroform (40 mL). The organic layer was separated, and then further washed with several-portions of distilled-water, until the resulting aqueous washes were colourless The organic layer was separated, dried (MgSO4), filtered and the solvent removed from the filtrate under reduced pressure to give a pale-brown waxy solid. The titled compound was afforded as a pale-white waxy solid (52%, 16.66 mg) from normal- phase HPLC, using a MP system of hexane-ethyl-acetate (4:1). The product was eluted after a run time of 380 seconds using an ACE 5 SIL silica column (250 × 21.2mm).

1 H NMR (400 MHz, CDCl3, 25°C) δH: 3.68 (2H, m, CH2OH), 3.51 (1H, m, SCH), 3.24 (1H, dd, J=14.3, 7.9Hz, SCH’H), 3.13 (1H, dd, J=14.3, 4.6Hz, SCH’H), 2.53 (2H, t,

J=7.5Hz, SCOCH2), 2.49 (2H, t, J=7.5Hz, SCOCH2), 1.59 (4H, m, (CH2CH2(C=O))2), + 1.24-1.18 (56H, bs, ((CH2)12CH3)2)), 0.81 (6H, t, J=6.8Hz, (CH3)2); MS (ES ) m/z: 679.7 [M-Na] +

102

S-(1-(palmitoyloxy)-3-(palmitoylthio)propan-2-yl)phosphorothiolate

(S,S’)-(3-hydroxypropane-1,2-diyl)dihexadecanethioate (26.30 mg, 0.44×10-1 mmol) was 5 -1 dissolved in a mixture of pyridine-d (4.60 µL, 0.57×10 mmol) and CDCl3 (2 mL). The resulting solution was then added to a solution of phosphorus oxychloride (4.9 µL, -1 ° 0.53×10 mmol) in CDCl3 (1 mL). The mixture was heated to 50 C for 90 minutes, whilst stirring under an inert-atmosphere, and subsequently cooled to RT. On completion, the solution was then diluted with two portions of distilled water (2x 25mL), and the resulting organic layer was separated, dried (MgSO4), filtered and the solvent removed from the filtrate under reduced pressure, resulting in a white-waxy crude. The titled compound was afforded as a pale-white waxy solid (71%, 21.10 mg) from preparative TLC using, a MP system of hexane-ethyl-acetate (4:1) (Rf = 0.75).

1 H NMR (400 MHz, CDCl3, 25°C) δH: 4.22 (1H, m, SCH), 4.17 (1H, dd, J=11.3, 4.6Hz) CH’HOH), 4.05 (1H, dd, J=11.3, 5.8Hz, CH’HOH), 3.17 (1H, dd, J=14.3, 7.9Hz, SCH’H),

3.07 (1H, dd, J=14.3, 4.6Hz, SCH’H), 2.50 (2H, t, J=7.5Hz, SCOCH2), 2.27 (2H, t,

J=7.5Hz, OCOCH2), 1.51 (4H, m CH2((CH2)12)2) 1.28-1.18 (48H, bs, ((CH2)12CH3)2), 0.81

(6H, t, J=6.8Hz, (CH3)2). 13C NMR (100 MHz, CDCl3) δC :43.91 (CH2(C=O)), 34.15 - (SCH), 29.45 (SCH2), 25.58 (CH2CH2(C=O)), 22.71 ((CH2)12), 16.08 (CH3). MS (ES ) m/z: 678.4 [M-H]-. FTIR ʋ (cm-1): 2920, 1848, 1737, 1692, 1412, 1297, 1165.

2- 13C NMR (100 MHz, CDCl3) δC; ~199 (SC=O), ~180 (OC=O) and ~81 (CH2OPO3 ) are missing from the spectra.

103

(2,3)-dis(palmitoylthio)propyl sulfate

(S,S’)-(3-hydroxypropane-1,2-diyl)dihexadecanethioate (20.00 mg, 0.33×10-1 mmol) and sulfur trioxide pyridine complex (6.40 mg, 0.40×10-1 mmol) were dissolved in 1mL of anhydrous-toluene. The mixture was heated to reflux at 140°C for 30 minutes, in the presence of a calcium chloride drying-tube. On completion, the resulting solution was cooled to RT, followed by removal of the solvent under reduced pressure. The resulting crude material was then dissolved in chloroform (0.5 mL) and cooled to 0°C, with the resulting precipitate filtered off. The filtrate was then removed under reduced pressure to afford a pale-orange waxy solid. The titled compound was afforded as a pale-white waxy solid (77%, 17.40 mg) from preparative TLC, using a MP system of hexane-ethyl-acetate (4:1) (Rf = 0.60).

1 H NMR (400MHz, CDCl3, 25°C) δH: 4.29 (1H, m, SCH), 4.21 (1H, dd, J=11.3, 4.6Hz, CH’HOH), 4.14 (1H, dd, J=11.3, 5.8Hz, CH’HOH), 3.22 (1H, dd, J=14.3, 7.9Hz, SCH’H),

3.08 (1H, dd, J=14.3, 4.6Hz, SCH’H), 2.40 (2H, t, J=7.5Hz, SCOCH2), 2.27 (2H, t,

J=7.5Hz, (OCOCH2), 1.57 (4H, m, (CH2(CH2)12)2)) 1.18 (48H, s, ((CH2)12CH3)2)), 0.81 - - -1 (6H, t, J=6.8Hz, (CH3)2); MS (ES ) m/z: 679.8 [M-H] . FTIR ʋ (cm ): 2955, 1733, 1678, 1412, 1297, 961,

104

(2,3)-bis(stearoylthio)propane-1-sulfonate

2-nitro-4-(stearoylthio)benzoic acid (11.15 mg, 0.24×10-1 mmoles), TCEP.HCl (6.50 mg, 0.24×10-1 mmoles) and (2,3)-dimercapto-1-propanesulfonic acid sodium salt monohydrate (2.76 mg, 0.12×10-1 mmoles), were suspended in acetonitrile (5 mL) under an argon atmosphere. On stirring, methanol was added to the reaction mixture dropwise, until the (2,3)-dimercapto-1-propansulfonic acid was fully dissolved. DIPEA (4.22 µL, 0.24×10-1 mmol) was then added to the reaction mixture dropwise, and on addition, the colour of the solution changed from colourless to dark-red. The reaction mixture was then stirred at RT for 5 hours. On completion, the solvent was removed under-reduced pressure, followed by the addition of distilled water (15 mL) and DCM (10 mL). The aqueous layer was then separated, and freeze-dried to give a dark-red solid. This was dissolved in HPLC-grade water (2.5 mL) and purified by gel-permeation chromatography. The resulting eluted fraction was then concentrated, to afford a pale-white waxy solid of the titled product (61%, 10.54 mg).

1 + H NMR (400MHz, CDCl3, 25°C) δH: 8.02 (1H, s, NH), 4.10 (1H, m, SCH), 3.61 (1H, dd, J=14.3, 7.9Hz, SCH’H), 3.33 (1H, dd, J=14.3, 4.6Hz, SCH’H), 3.33 (2H, m, N(CH)2),

3.09 (2H, m, CH2OH), 3.08 (2H, q, CH3CH2N) 2.48 (2H, t, J=7.5Hz, SCOCH2), 2.45 (2H, t, J=7.5Hz, SCOCH2) 1.56 (4H, m, (SCOCH2CH2)2), 1.33 (12H, d, J=6.3Hz, CH3CHN),

1.22 (3H, t, CH3CH2N), 1.26-1.18 (56H, bs, ((CH2)14CH3)2)), 0.81 (6H, t, J=6.8Hz, - - - + (CH3)2); MS (ES ) m/z: 719.5 [M-H] . HRMS for [C39H75O5S3 +H] calculated 720.2000, found 719.4789 [M-H]-.

105

(2,3)-bis(palmitoylthio)propane-1-sulfonate

2-nitro-4-(palmitoyl)benzoic acid (10.47 mg, 0.24×10-1 mmoles), TCEP.HCl (6.50 mg, 0.24×10-1 mmoles) and (2,3)-dimercapto-1-propanesulfonic acid sodium salt monohydrate (2.76 mg, 0.12×10-1 mmoles), were suspended in acetonitrile (5 mL) under an argon atmosphere. On stirring, methanol was added to the reaction mixture dropwise, until the (2,3)-dimercapto-1-propansulfonic acid was fully dissolved. DIPEA (4.22 µL, 0.24×10-1 mmol) was then added to the reaction mixture dropwise, and on addition, the colour of the solution changed from colourless to dark-red. The reaction mixture was then stirred at RT for 5 hours. On completion, the solvent was removed under-reduced pressure, followed by the addition of distilled water (15 mL) and DCM (10 mL). The aqueous layer was then separated, and freeze-dried to give a dark-red solid. This was dissolved in HPLC-grade water (2.5 mL) and purified by gel-permeation chromatography. The resulting eluted fraction was then concentrated, to afford a pale-white waxy solid of the titled product (57%, 9.03 mg);

1 H NMR (400MHz, CDCl3, 25°C) δH: 8.13 (1H, m, NH+), 4.26 (1H, m, SCH), 3.38 (8H, m), 2.56 (4H, m, (SCOCH2)2), 1.64 (4H, m, (SCOCH2CH2)2), 1.42 (15H, m), 1.27 (48H, - - m, (CH2)12CH3)2), 0.90 (6H, m, (CH3)2). MS (ES ) m/z: 664.1 [M-H] .

1 Expected, based off stearoyl-equivalent: H NMR (400MHz, CDCl3, 25°C) δH: 8.02 (1H, s, +NH), 4.10 (1H, m, SCH), 3.61 (1H, dd, J=14.3, 7.9Hz, SCH’H), 3.33 (1H, dd, J=14.3,

4.6Hz, SCH’H), 3.33 (2H, m, N(CH)2), 3.09 (2H, m, CH2OH), 3.08 (2H, q, CH3CH2N)

2.48 (2H, t, J=7.5Hz, SCOCH2), 2.45 (2H, t, J=7.5Hz, SCOCH2) 1.56 (4H, m,

(SCOCH2CH2)2), 1.33 (12H, d, J=6.3Hz, CH3CHN), 1.22 (3H, t, CH3CH2N), 1.26-1.18

(56H, bs, ((CH2)12CH3)2)), 0.81 (6H, t, J=6.8Hz, (CH3)2)

106

4.3 DLS and zeta-potential measurements

The following procedure was taken from Li et al.74 Thioester lipids of either 3a or 3b were mixed separately in syringe-filtered HPLC-grade water (1 mL), so that the resulting concentration of the lipid was 4.6 mM. The resulting lipid suspensions of either 3a or 3b were then bath sonicated for 20 minutes at RT, before being left to incubate at RT for 2 hours. The Malvern Zeta-Sizer machine was then calibrated using a polystyrene latex standard, followed by the loading of each lipid solution (1 mL) of either 3a or 3b into a disposable capillary cell (DTS1070). DLS and zeta-potential measurements were ° ° performed at 25 C, with a back-scattering angle of 173 , an equilibration time of 120 seconds, and 3 cycles of measurements, with each sample measurement being repeated twice.

4.4 Encapsulation of 5(6)-carboxyfluorescein

The following procedure was taken from Li et al.74 5(6)-CF (75.30 mg, 0.02 mmol) was suspended in HPLC grade water (100 mL). To fully dissolve the 5(6)-CF, the pH of the solution was increased to pH 7 by addition of sodium phosphate dibasic whist stirring, to give a 2 mM 5(6)-CF stock solution. Subsequently, either 3a or 3b was dissolved in 2.5 mL of the 2 mM 5(6)-CF stock solution, so that the final concentration of the lipid was 4.6 mM. The resulting lipid/5(6)-CF mixture was then bath sonicated for 20 minutes at RT, before being left to incubate in the dark at RT for 2 hours. GPC was then employed to remove non-encapsulated 5(6)-CF. Firstly, the column storage solution (0.15% Kathon) was discarded, and then followed by column equilibration with HPLC grade water (30 mL). The lipid/5(6)-CF mixture (2.5 mL) was then loaded onto the column, with the first eluted fraction being discarded (2.5 mL). The column was then eluted with HPLC grade water (3.5 mL), and the resulting green-eluent was collected (3.5 mL) (See Figure 4.1).

107

Figure 4.1: Schematic illustration showing; a) equilibration of the GPC column with HPLC-grade water (30 mL), b) loading of the lipid/5(6)-CF mixture onto the column (2.5 mL), c) purification of encapsulated 5(6)-CF.

An aliquot of the post-GPC eluent (2 mL) was then immediately assayed using UV-visible spectroscopy (200-700 nm), (600 nm/min), with three cycle counts (0.6 min/cycle). The absorbance intensity at 492 nm was then measured. An aliquot of the 5(6)-CF 2 mM stock- solution (4 µL) was added to HPLC-grade water (2 mL) in a quartz cuvette (10 mm path length) and then assayed using UV-visible spectroscopy (200-700 nm), (600 nm/min), with three cycle counts (0.6 min/cycle). The resulting absorbance intensity at 488 nm was then recorded.

4.5 Release of 5(6)-carboxyfluorescein

The following procedure was taken from Booth et al.41 Either lipids 3a or 4a were dissolved in 1 mL of (5(6)-CF (0.05 M) in MOPS (20 mM), NaCl (100 mM), pH 7.4)), so that the resulting concentration of the lipid was 20 mM. The lipid/5(6)-CF mixture was then bath sonicated for 20 minutes at RT, before being incubated in the dark for 2 hours at RT. The lipid/5(6)-CF mixture was then diluted to 2.5 mL with PBS. GPC was then employed to remove non-encapsulated 5(6)-CF. Firstly, the column storage solution (0.15% Kathon) was discarded, and then followed by column equilibration with PBS (25 mL). The lipid/5(6)-CF mixture (2.5 mL) was then loaded onto the column, with the first eluted fraction being discarded (2.5 mL). The column was then eluted with PBS (3.5 mL), and the resulting green-eluent was collected (3.5 mL). After obtaining the vesicle-

108 containing eluent, the suspension was centrifuged at 2200×g for 15 minutes (See Figure 4.2).

Figure 4.2: Schematic illustration showing a) formation of the sediment lipid mass, on centrifugation at 2200×g and b) suspension of the sediment lipid mass containing encapsulated 5(6)-CF, in PBS.

The resulting sediment lipid mass was incubated at RT for 45 minutes, before an aliquot of the supernatant (20 µL) was added to PBS (2 mL) in a fluorescence cuvette. Subsequently, the resulting fluorescence intensity at 517 nm (excitation 492 nm) was measured using a fluorimeter. After 24-hours of incubation at RT, the sediment lipid mass was heated to 42°C for 15 hours, before the sample was bath sonicated for 2 minutes at RT. Furthermore, another aliquot of the supernatant (20 µL) was added to a fluorescence cuvette, containing PBS (2 mL). The resulting fluorescence intensity at 517 nm (excitation 492 nm), was then recorded. This final value was assumed to be 100% release of 5(6)-CF and was used to calculate the percentage release after 45 minutes.

4.6 Kinetic studies

4.6.1 Preparation of buffer and 1a stock-solution

Anhydrous sodium phosphate monobasic (13.90×103 mg) was dissolved HPLC-grade water (500 mL), to make a 0.20 M sodium phosphate monobasic stock solution. Anhydrous sodium phosphate monobasic (53.70×103 mg) was then dissolved in HPLC- grade water (1×103 mL), to make a 0.20 M sodium phosphate dibasic stock solution. The 0.20 M sodium phosphate monobasic stock solution (48 mL) was then combined with the 0.20 M sodium phosphate dibasic stock solution (252 mL). The resulting solution was then 109 diluted with HPLC-grade water (300 mL), to give a 0.10 M sodium phosphate buffer of pH 7.5 (600 mL).

2-nitro-4-(palmitoyl)benzoic acid (1a) (5.00 mg) was dissolved in methanol (180 mL) to give a 6.40 mM methanolic stock solution of 1a.

4.6.2 Trans-thioesterification of 1a with di-thiol sources

The following procedure was taken from Lopez et al.26 An aliquot of the 6.40 mM methanolic stock solution of 1a (1 mL) was added to a quartz cuvette of 10 mm path length, containing a stirred solution of the requisite di-thiol (2 mL) and TCEP.HCl in a 0.10 M sodium-phosphate buffer (pH 7.5) (See Table 4.1&4.2).

1a (mM) 1d (mM) TCEP.HCl (mM) 0.02 0.50 1.00 0.02 0.10 0.20 Table 4.1 Conditions for the trans-thioesterification reaction of 1a with 1d, in the presence of TCEP.HCl

1a (mM) 1e (mM) TCEP.HCl (mM) 0.02 0.50 1.00 0.02 0.10 0.20 Table 4.2 Conditions for the trans-thioesterification reaction of 1a with 1e, in the presence of TCEP.HCl.

On addition, the increase in absorbance intensity at 410 nm over 27 minutes was monitored by UV-visible spectroscopy. Absorbance measurements were taken at 100 second intervals, with each measurement being repeated 3 times.

4.6.3 Trans-thioesterification of 1a with thiol-sources

The following procedure was taken from Lopez et al.26 An aliquot of the 6.40 mM methanolic stock solution of 1a (1 mL) was added to a quartz cuvette of 10 mm path length, containing a stirred solution of the requisite thiol (2 mL) and TCEP.HCl in a 0.10 M sodium-phosphate buffer (pH 7.5) (See Tables 4.3&4.4).

1a (mM) 1i (mM) TCEP.HCl (mM) 0.02 0.50 1.00 0.02 0.10 0.20 Table 4.3. Conditions for the trans-thioesterification reaction of 1a with thiol-source 1i.

110

1a (mM) 1j (mM) TCEP.HCl (mM) 0.02 1.00 1.00 0.02 0.20 0.20 Table 4.4 Conditions for the trans-thioesterification reaction of 1a with thiol-source 1j.

111

Chapter 5 Conclusions and further work

112

5.1 General conclusions

The primary objective of this project was to explore the potential of trans-thioesterification and disulfide interchange reactions, in the construction of novel thioester and disulfide containing lipids. These sulfur-containing lipids are analogous of phospholipids, and were expected to self-assemble into lipid-bilayers and vesicles.

The initial aims proved successful, as two thioester lipids possessing sulfonic acid functionality, and differing acyl-chain lengths were successfully synthesised via trans- thioesterification reactions. Both lipids displayed self-assembling behaviour in aqueous media, with DLS analysis suggesting both resulting aggregates were in the size range of large vesicles, along with displaying unilamellar morphology. As a result, both self- assembled structures were classified as potential LUVs. Given their sulfonic acid functionality, zeta-potential measurements confirmed both vesicles to display high stability in aqueous conditions, along with being of a negatively charged nature. Furthermore, both vesicles displayed an affinity to encapsulate compounds of a hydrophilic nature. Encapsulation studies were performed and measured the relative ‘leakiness’ of the vesicle bilayer. Both vesicle bilayers displayed high permeability to compounds of a charged nature, and thus encapsulated material was released through the bilayer at a significantly fast rate, in comparison to that of pharmaceutical drug transport vesicles.

The synthesis of thioester lipids possessing an alcohol functionality, and differing acyl- chain lengths were explored. On chemical modification of the hydroxyl group via phosphorylation and sulfation, both lipids displayed poor stability when in the presence of a base, and thus the intramolecular-acyl migrated product was formed. Although unsuccessful, the synthetic results have displayed the poor suitability of thioester lipids possessing an alcohol functionality, as potential synthetic building blocks for the construction of sulfur containing lipids.

A common issue throughout this project was the dimerization, of di-thiol reactants on exposure to oxygen. Although great care was taken to ensure reactions were performed in the complete absence of oxygen, the resulting dimerization of the di-thiol reactants seemed unavoidable without use of the strong reducing agent TCEP.HCl. This prevented the formation of disulfide containing lipids, and given the time constraints of the project, this was not taken further.

Moreover, the corresponding trans-thioesterification reactions were assayed by UV-visible spectroscopy, with the intention of obtaining kinetic data in the form of trans- 113 thioesterification extents, rate constants and corresponding rate equations. Obtaining the corresponding rate constants and rate equations proved unsuccessful, given the technical faults associated with the UV-visible spectrometer. As these studies were performed in the final weeks of the project, they were not repeated. However, the trans-thioesterification of (2,3)-dimercapto-1-propan sulfonic acid with the thioester lipid 1a, afforded an interesting kinetic plot by which sigmoidal behaviour was observed. This proposed a mechanism by which vesicle formation displays autocatalysis on trans-thioesterification reaction.

Attempted DCL generation of thioester lipids possessing alcohol functionality was also attempted. This study had the intention of generating families of interchanging thioester lipids, by which thioester lipids of mixed acyl-chain lengths could be successfully synthesised. However, due to the reaction system being contaminated by water, successful DCL generation was not achieved. Although unsuccessful, this has emphasised the requirement for an anhydrous reaction system, as trace-amounts of water can have a detrimental effect to the DCL formation. This study has also further emphasised the poor stability of thioester lipids possessing an alcohol functionality in the presence of base, as evidence of the intramolecular acyl-migrated product was observed at the start of the 1H NMR spectroscopic progression studies.

Finally, two thioester lipids capable of self-assembling into vesicle structures were formed via trans-thioesterification reactions. The trans-thioesterification reactions were one-pot reactions. Successful synthesis required a reduction source (TCEP.HCl), basic conditions (pH 7.5), a thioester lipid and a thiol source. These conditions could have been met at the on at the site of pre-biotic hydrothermal vents. Given the relative pH of sea-water (~8.0), the reduction environment of hydrothermal vents on generation of H2 at high temperatures, and the proposed abundance of pre-biotic amphiphiles thioesters and thiols at the site of hydrothermal vents. It is plausible that derivatives of these thioester containing lipids were synthesised on pre-biotic Earth.

5.2 Future work

Future work on this project will address the multiple studies that proved unsuccessful over the course of the year. Initially, full characterisation of both self-assembling thioester lipids, 3a and 3b, needs to be completed. Given that 3a affords low intensity 1H NMR spectrums in D2O at an elevated temperature of 52°C, and broad spectrums in chloroform. 1H NMR spectroscopic analysis will need to be carried out at higher elevated temperatures

(80-90°C) in D2O. This would reduce the effect of slow molecular tumbling, by disrupting

114 the formation of self-assembled vesicles. Furthermore, full characterisation on all successfully synthesised compounds will need to be obtained.

An initial starting point for future work could be the synthesis of the oleoyle derivative 1c. This would allow the effect of unsaturated acyl-chains, on the resulting self-assembled structures formed by thioester lipids. Successful synthesis could be achieved by following the procedure of Lopez et al.26 in which palmitoyl chloride would be replaced with oleoyl chloride. Subsequent trans-thioesterification of the resulting thioester lipid 1c with 1e, would afford the di-substituted oleoyl equivalent 3c (See Figure 5.1)

Figure 5.1: Reaction scheme for the generation of 2c, by the trans-thioesterification reaction of 1c with 1e.

Given both 3a and 3b have been observed to self-assemble into vesicles, similar self- assembling behaviour would be expected for 3c.

Further research will also be required in developing a synthetic strategy to synthesise the disulfide lipid 3f via thiol-disulfide interchange reactions. As shown in this project, thioester lipids of sulfonic acid functionality were observed to self-assemble into vesicle structures. It would therefore be expected that disulfide lipids possessing sulfonic acid functionality would self-assemble into vesicles (See Figure 5.2).

115

Figure 5.2: Chemical structure of the disulfide lipid 3f.

Future experiments could also focus on the introduction of both sulfonic ester and phosphoric acid functionalities into the thioester lipids. As synthetic studies have shown in this project, the primary alcohol group of thioester lipids 2a and 2b is prone to undergo acyl-migration at the thioester functionalities. A possible route for intramolecular acyl migration-blocking would be to phosphorylate and or sulfonate and effectively ‘tie-up’ the hydroxyl group of 1d prior to thio-acylation. For this to proceed successfully, it would require prior cyclic dimerization protection of the thiols substituent, followed by subsequent disulfide reduction by TCEP.HCl (See Figure 5.3). If successful, the resulting self-assembled structures they form will be studied.

Figure 5.3: Reaction scheme for the introduction of a disulfide protection group into 1d.

Where a) is either POCl3, pyridine and H2O, or pyridine-suflur trioxide complex, b) is TCEP.HCl, and c) is the subsequent trans-thioesterification reaction with 1a and DIPEA.

116

Further DCC experiments will focus on the generation of DCLs from thioester lipids 3a and 3b. If successful, thioester lipids possessing both stearoyl and palmitoyl acyl-chains would be generated (See Figure 5.4) As lipids 3a and 3b have been observed to self- assemble into vesicle structures, this will allow the effect of the bilayer on the interchange of acyl-chains to be studied.

Figure 5.4: Chemical structure of the resulting mixed-acyl chained thioester lipids on DCL formation from thioester lipids 3a and 3b.

Further vesicle studies of 3a and 3b, should also be performed to further validate their proposed vesicle structures in aqueous media. For example, transmission electron microscopy could be employed to both image and size their resulting self-assembled structures. Moreover, differential scanning calorimetry should be used to obtain the resulting phase-transition temperatures (Tm) associated with both thioester lipids 3a and

3b. The resulting Tm would give an indication of the relative degree of packing acting between the acyl-chains of vesicles constituting 3a and 3b.

Given the technical issues associated with the UV-spectrometer used in this project, further work should repeat the trans-thioesterification kinetic investigation. This would allow further validation of the auto-catalytic behaviour observed on the trans-thioesterification reaction of 1a with 1e, along with corresponding rate-constants, rate-laws and extents of trans-thioesterification to be obtained.

117

Chapter 6

Annexes

118

1H NMR spectra 2-nitro-4-(palmitoylthio)benzoic acid

119

13C NMR spectra 2-nitro-4-(palmitoylthio)benzoic acid

120

1H NMR spectra 2-nitro-5-(stearoylthio)benzoic acid

121

13C NMR 2-nitro-5-(stearoylthio)benzoic acid

122

1H NMR (S,S’)-(3-hydroxypropane-1,2-diyl)dihexadecanethiolate

123

1H NMR spectra (S.S’)-(3-hydroxypropane-1,2-diyl)dioctadecanethiolate

124

1H NMR spectra S-(1-(palmitoyloxy)-3-(palmitoylthio)propan-2-yl)phosphorothiolate

125

13C NMR spectra S-(1-(palmitoyloxy)-3-(palmitoylthio)propan-2yl)phosphorothiolate

126

1H NMR spectra S-(1-(palmitoyloxy)-3-(palmitoylthio)propan-2-yl)sulfurothioate

127

1H NMR spectra (2,3)-bis(stearoylthio)propane-1-sulfonate

128

1H NMR spectra (2,3)-bis(palmitoylthio)propane-1-sulfonate

129

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