Solid-phase peptide synthesis (SPPS)

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Chapter 2 Methodology

This chapter provides background information about the individual experimental techniques reported in this thesis. Detailed experimental procedures utilising these techniques are mentioned in Chapter 3 to Chapter 5. The linear lipopeptides (GZ3.130, GZ3.27, GZ3.38, GZ3.26 and GZ3.37) described in Chapter 3 were synthesised using solid-phase peptide synthesis (SPPS) and the cyclic lipopeptides (GZ3.15, GZ3.21, GZ3.19, GZ3.40, GZ3.55) were synthesised using a combination of SPPS and solution-phase peptide synthesis. The synthesised peptides were characterised using mass spectrometry and purified using reverse-phase high performance liquid chromatography (RP-HPLC).
The antimicrobial activities of the synthetic peptides against bacterial pathogens were determined using a broth dilution minimum inhibitory concentration (MIC) assay. An in vitro colorimetric assay and scanning electron microscopy (SEM) was used to elucidate the mechanism of action of the antimicrobial peptides. Structure of the most active lipopeptide was thoroughly investigated using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies.
The most active lipopeptide was conjugated onto solid surfaces and these surface conjugations were characterised using water contact angle measurements, ellipsometry and X-Ray photoelectron spectroscopy (XPS).

Solution-phase peptide synthesis

In solution-phase peptide synthesis, two protected peptide fragments, dissolved in an appropriate solvent, are condensed in the presence of coupling reagents, to form the desired amide bond and peptide sequences (Scheme 2.1).
Scheme 2.1 General synthetic scheme for solution-phase peptide synthesis. AA denotes to amino acid.
Prior to the peptide bond formation, the reactive side-chains of the peptide and N- and C-terminal fragments are masked with protecting groups to prevent any undesired side reactions such as oligomerization. The newly formed peptide sequence can then be identified and purified by mass spectrometry and RP-HPLC respectively, before coupling it to another appropriately protected peptide fragment. Once the desired sequence has been synthesised, the remaining protecting groups are removed to produce the final deprotected peptide.
This technique has been successfully used, in the past, to synthesise complex peptides. For example, oxytocin is a naturally occurring cyclic hormonal peptide, used to induce labour in pregnant females. In 1953, Du Vigneaud et al. successfully synthesised the nine residue cyclic peptide oxytocin using solution-phase peptide synthesis, and in the subsequent year, was awarded the Nobel prize in Chemistry for the isolation, characterisation and total synthesis of this peptide.178 Furthermore Yajima et al. successfully synthesised the challenging 124 residue peptide, namely ribonuclease A, using solution-phase synthesis.179
Due to several limitations, solution phase synthesis is currently employed less frequently to generate peptides. These limitations include prolonged reaction conditions, solubility issues of the peptide fragments, low peptide yields due to several purification cycles and the increased risk of side reactions, such as peptide racemisation.180 Thus peptides are frequently synthesised by solid-phase peptide synthesis (SPPS) (see section 2.2). However, for the preparation of more complex peptides a combination of SPPS and solution-phase chemistry is frequently utilised.

Solid-phase peptide synthesis (SPPS)

In 1963, Bruce Merrifield reported the synthesis of a tetrapeptide on an insoluble solid support.181 This ground breaking invention by Merrifield simplified the synthesis of long (<50 amino acids) and complex peptides, and Merrifield was awarded the Nobel prize in Chemistry, in 1984, for this discovery.182
During SPPS, the peptide chain is assembled from the C-terminus (carboxy terminus) to the N-terminus (amino terminus), on an insoluble solid support (resin). A linker is incorporated between the growing peptide chain and the resin to act as a spacer and to provide the required functionality to the C-terminal end of the peptide. The amino terminus of the growing peptide chain is protected with temporary protecting groups (PGs), while the reactive side-chains of the amino acids are protected with PGs that are stable during the removal of the N-terminal PG. The peptide chain is elongated by removing the N-terminal protecting group and forming a selective peptide bond with the free carboxyl end of the incoming amino acid. Any unreacted amino acids are simply filtered off and this process of chain elongation continued on the resin. Once the required sequence is assembled, the peptide is cleaved from the resin, with simultaneous removal of the side-chain protecting groups (Scheme 2.2). The crude peptide is then analysed and purified.
Scheme 2.2 General scheme illustrating the synthesis of a peptide using SPPS. AA denotes to amino acid.
SPPS has numerous advantages over solution-phase synthesis. Synthesis of peptides via SPPS is faster due to the use of excess starting materials that drive the reaction to completion, which can also minimise racemisation. Crude peptides can be isolated from the solid support in high purity, as any unreacted starting materials are simply filtered off from the resin. Currently, two dominant SPPS strategies employed in the literature are Boc-SPPS, discovered by Merrifield and Fmoc-SPPS reported by Sheppard. 181,183
The Merrifield method, originally reported in 1963, involves the use of benzyl based side-chain protecting groups, with the N-terminus protected by the more acid-labile, tert- butoxycarbonyl (Boc) group.181 The temporary Boc group is removed under acidic conditions, e.g., using trifluoroacetic acid (TFA) whilst the side-chain benzyl groups and the chloromethyl resin (Merrifield resin) are resistant to TFA. The desired peptide is cleaved from the resin and the benzyl side-chain protecting groups removed under strong acidic conditions, such as using hydrogen fluoride (HF).181
The Sheppard method, or Fmoc SPPS, employs base-labile protecting groups, such as 9-fluorenylmethyloxycarbonyl (Fmoc), for the α -amine, while the side-chains are protected by acid-labile, tert-butyl (tBu)-type protecting groups. The peptide chain is elongated by removing the Fmoc group under basic conditions, and is followed by selective peptide bond formation with the C-terminus of the subsequent amino acid. The desired peptide sequence, along with the acid-labile protecting groups, is removed from the resin by treating it with TFA. The Merrifield methodology relies on extremely toxic HF to release the resin-bound peptide; therefore, the Sheppard methodology is more frequently used by peptide chemists and reported in the literature. The lipopeptides reported in this thesis were synthesised via the Fmoc/tBu method.

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General overview of Fmoc SPPS

The first step involves the conjugation of the C-terminal amino acid (Fmoc-AA1) onto the inert resin, via a linker (Scheme 2.3). The Fmoc group of the resin-bound AA1 is deprotected using 20% piperidine in N,N-dimethylformamide (DMF).183-184 The resin is thoroughly washed with DMF, to remove any excess piperidine. The N-protected subsequent amino acid (Fmoc-AA2), at a concentration four times in excess to the resin, is then coupled to the unprotected N-terminus of the resin-bound AA1. The peptide bond formation between AA1 and AA2 is facilitated by converting the C-terminal carboxylic acid moiety of AA2 into a more reactive ester, using excess coupling reagents (3.9 eq. to the resin), in the presence of the tertiary amine base N,N-diisopropylethylamine (DIPEA). The Fmoc deprotection and coupling for each subsequent amino acid is repeated until the desired sequence is assembled on the resin. The peptide is then released from the resin, while simultaneously cleaving off the orthogonal protecting groups, under acidic conditions such as TFA.

Chapter 1: Introduction
1.1 Antibiotics
1.2 Biofilms: formation, pathogenicity and health consequences
1.2.1 Biofilm resistance mechanisms
1.3 Antimicrobial Peptides (AMP)
1.4 Therapeutic limitations of AMPs
1.5 Improving AMP stability through peptide modification
1.6 Improving AMP stability using lipidation
1.7 Overall Aim (s) of the project.
Chapter 2: Methodology
2.1 Solution-phase peptide synthesis
2.2 Solid-phase peptide synthesis (SPPS)
2.3 Mass spectrometry
2.3.1 Electrospray ionisation (ESI)
2.3.2 Matrix-assisted laser desorption/ionization (MALDI)
2.4 Reversed-phase high performance liquid chromatography (RP-HPLC)
2.5 Broth dilution minimum inhibitory concentration (MIC) assay
2.6 Scanning electron microscopy (SEM)
2.7 Circular dichroism (CD)
2.7.1 Characteristics of CD spectra
2.8 Nuclear magnetic resonance spectroscopy (NMR)
2.9 Contact angle
2.10 Ellipsometry
2.11 X-Ray photoelectron spectroscopy (XPS)
Chapter 3: Synthesis of battacin and its analogues
3.1 Synthetic strategy of battacin 3.14
3.2 Synthesis of battacin analogues
3.3 Summary
3.4 Experimental
Chapter 4: Antibacterial activity of battacin and its analogues
4.1 Rationale behind the selection of the pathogens
4.2 In vitro antibacterial activity of battacin peptides
4.3 Time-kill assay of GZ3.27
4.4 Inhibition and eradication of biofilms using GZ3.27
4.5 Structure-activity relationship (SAR) studies
4.6 Haemolysis of mouse blood cells by selected battacin analogues
4.7 Mechanistic studies of GZ3.27
4.8 Solution conformation of GZ3.27
4.9 Mechanism of action of GZ3.27
4.10 Summary
4.11 Experimental
Chapter 5: Surface immobilisation studies of GZ3.27
5.1. Immobilisation of antimicrobial compounds to surfaces
5.2 Immobilisation of antimicrobial peptides onto surfaces
5.3 Immobilisation of battacin analogue, GZ3.27
5.4 Surface immobilisation of GZ3.163
5.5 Surface characterisation of immobilised peptide
5.6 Antibacterial activity of immobilised peptide
5.7 Summary
5.8 Experimental
Chapter 6: Overall summary and future work
6.1 Overall summary
6.2 Future Work
Appendices
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