Construcciones de teorías

The peptoids investigated in this thesis were synthesised by members of the Cobb Group at Durham University. From their extensive peptoid library a small subset was selected to examine in detail the relationships between sequence, structure and activity. The selected peptoids were chosen for either their interesting structural rearrangements or notable biological activity.

3.2.1

Side Chain Groups

In this section we introduce a library of peptoid sequences containing a small selection of different aromatic and charged side chain groups. While a significant proportion of aromatic side chains containing a chiral center are required to induce the formation of a helical secondary structure, inclusion of charged side chains can also promote water solubil- ity. The main aromatic residue considered in this work is N spe, which has been discussed widely in the peptoid literature and is known to induce helical secondary structure. We also briefly consider the substitution of N spe for the opposite handed equivalent, N rpe and a similar aromatic but achiral side chain, N phe. Two types of charged side chains are considered, N ae and N Lys. The N Lys residue is the peptoid equivalent of the nat- urally occurring amino acid lysine. N ae differs from N Lys only in that the side chain contains two fewer carbon atoms and is therefore referred to as ”shorter” in discussion. The structures and full chemical names of each of these side chains are shown in Table 3.1.

3.2.2

Primary Sequences

In this work we exclusively consider linear peptoid sequences, all of which are 12 residues in length and thus relatively short in comparison to many antimicrobial peptides. We

3. Experimental Secondary Structural Characterisation

Side Chain Structure Full Name

N ae N -(2-aminoethyl)glycine

N Lys N -(4-aminobutyl)glycine

N spe N -(S-phenylethyl)glycine

N rpe N -(R-phenylethyl)glycine

N phe N -(benzyl)glycine

Tab. 3.1: Chemical structure, full chemical name and shorthand name of each peptoid monomer discussed in the experimental and computational results within this thesis.

divide our complete library of peptoids into two sub libraries: the repeat motif sequences and the scrambled sequences. The repeat motif peptoids were selected to investigate due to their biological activity against a range of bacteria and parasites. The details of this activity are discussed in Chapter 5. The scrambled sequence library contains a series of rearrangements of the primary sequence of two of the repeat motif peptoids. Every sequence in the library has an unmodified N-terminal group and an amidated C-terminal group.

Repeat Motif Sequences

The repeat motif peptoids contain mixtures of the aforementioned charged and aromatic side chains in varying proportions, arranged in three different repeat motif sequences which are shown in Figure 3.9, where blue circles represent a charged residue (N ae or N Lys) and yellow circles represent an aromatic residue (N spe, N rpe or N phe). Throughout the thesis we use this colour scheme to represent charged and aromatic residues non-specifically. In

3. Experimental Secondary Structural Characterisation

some cases we also use pink circles to specifically represent N ae residues and teal circles to specifically represent N Lys residues. The different repeat motif peptoid sequences

Fig. 3.9: Arrangements of charged (blue) and aromatic (yellow) residues in repeat motif peptoid sequences.

have different net charges in aqueous solution due to varying proportions of charged and aromatic residues between motifs. Motif 1 has a total charge of +6 and aromatic content of 50%, motif 2 a charge of +4 and aromatic content of 67% and motif 3 a charge of +3 and aromatic content of 75%. In this chapter we examine how the relative ratios and positioning of charges affect peptoid secondary structure in different solvent conditions. Later, in Chapter 5 we investigate how these variations affect the antimicrobial activity of the peptoids.

Scrambled Sequences

The scrambled sequences are peptoids with primary structures that are a series of re- arrangements of the motif 2 peptoids. The motif 2 peptoids were initially selected as interesting molecules to study due to their significant antimicrobial properties. The inves- tigation of the scrambled sequences allows insight into whether the distinct patterning of charged and aromatic residues is necessary for the peptoids to maintain the biophysical properties to which their biological activity is at least partially attributed. The scram-

Fig. 3.10: Arrangements of charged (blue) and aromatic (yellow) residues in scrambled peptoid sequences. Numbers 1-7 refer to sequences where the charged side chain is N ae and 8-14 refer to sequences where the charged side chain is N Lys.

bled sequences have the same proportions of charged and aromatic residues as the motif 2 peptoids (a charge of +4 and 67% aromatic content). The key difference between the peptoids in this library is therefore the distribution of charged and aromatic residues along the peptoid backbone. The charges are distributed fairly evenly along the backbone and

3. Experimental Secondary Structural Characterisation

none of the sequences contain more than 2 neighbouring charged residues. Some of the sequences do contain regions with up to 4 neighbouring aromatic residues.

Summary of Complete Peptoid Library and Naming Convention

Table 3.2 lists the full library of primary sequences considered in this chapter and through- out the thesis. The following naming convention is adopted for all future references to individual peptoids: each peptoid is allocated a number prefaced with letters referring to the sub-library to which it belongs. The first of the repeat motif peptoid sequences is therefore referred to as RM1 and the first of the scrambled sequences as S1. Note

Peptoid Reference Name Primary Sequence

RM1 (NaeNspe)6 RM2 (NLysNspe)6 RM3 (NaeNspeNspe)4 RM4 (NLysNspeNspe)4 RM5 (NaeNspeNspeNspe)3 RM6 (NLysNspeNspeNspe)3 RM7 (NaeNrpeNrpe)4 RM8 (NLysNpheNphe)4 S1 (NaeNspeNspe)4

S2 (NspeNae)2(Nspe)4(NaeNspe)2 S3 Nae(Nspe)4(Nae)2(Nspe)4Nae S4 Nae(Nspe)4(NaeNspe)3Nspe S5 Nae(Nspe)4Nae(Nspe)2(NaeNspe)2 S6 (NspeNspeNae)3Nae(Nspe)2 S7 (NspeNspeNae)2(NaeNspeNspe)2

S8 (NLysNspeNspe)4

S9 (NspeNLys)2(Nspe)4(NLysNspe)2 S10 NLys(Nspe)4(NLys)2(Nspe)4NLys S11 NLys(Nspe)4(NLysNspe)3Nspe S12 NLys(Nspe)4NLys(Nspe)2(NLysNspe)2 S13 (NspeNspeNLys)3NLys(Nspe)2 S14 (NspeNspeNLys)2(NLysNspeNspe)2

Tab. 3.2: Primary Sequences of each peptoid investigated in this thesis. For clarity, N ae residues are shown in pink, N Lys residues in teal, N spe residues in black, N rpe residues in gold and N phe residues in grey.

that under this naming system, two peptoids actually appear twice: (NaeNspeNspe)₄ and (NLysNspeNspe)₄ appear as RM3 and S1 and RM4 and S8 respectively (due to their belonging to both the repeat motif and scrambled libraries).

3. Experimental Secondary Structural Characterisation

3.2.3

Experimental Aims

By investigating this library of peptoids we aim to address two key questions about the relationship between sequence and structure that have not yet been fully explored in the literature. Firstly, the repeat motif sequences allow us to investigate how the relative proportion of different charged and aromatic residues affects the peptoid structure and whether the nature of the cationic side chain is influential. Secondly, we use the scram- bled sequences to examine how the secondary structure varies in sequences with identical proportions of charged and aromatic residues but arranged in different orders, hence giving insight into how the charge distribution along the chain affects the secondary structure of the peptoid.