Rationale

The treatment of the most important pathogen in CF, P. aeruginosa, with a pro-HDP targeted at the most common enzyme, NE, forms the basis of the prodrug model. The large quantities of the enzyme found in the CF lung would predict that the active sequence must be

synthesised from D-amino acids to ensure that it is not degraded.

Maintaining an L-amino acid linker will ensure that it is cleaved upon

application of the pro-HDP to the endobronchial space with the release of the active peptide. Based on our own review of the literature,

oligoglutamic acid is the most promising pro-moiety (135).

3.1.2 Peptide synthesis

Synthesis of the peptides in this project was carried by standard Solid Phase Peptide Synthesis (SPPS), using the Fmoc/t-Bu protection scheme. It involves incrementally synthesising the peptide from the C- to the N-terminus, in a manner that minimises racemisation and is easily automated, allowing large peptides to be synthesised quite rapidly. All amino acids used in the synthesis are temporarily protected

with Fmoc, a base-sensitive Nα-amino protecting group. The C-

terminal carboxylic acid is left free, while any side-chain functional groups of the amino acid are also semi-permanently protected, but with an acid-sensitive group instead of Fmoc. An example of the

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Figure 3.1: Fmoc-D-Arg(Pbf)-OH. The amine is protected by the base- labile Fmoc, while the side-chain is protected by the acid-labile Pbf group. The C-terminal carboxylic acid is left free.

The process of coupling multiple amino acids together to form a peptide begins with the attachment of the first amino acid onto an insoluble resin, a technique pioneered by Bruce Merrifield (169). The resin, which is retained by a filter in the reaction chamber, allows the application of multiple phases of excess reagents relative to the immobilised peptide. The excesses of reagents at each stage of the synthesis allow a very high level of conversion to be maintained. This is crucial when one considers that even a level of 99% coupling efficiency will lead to an overall synthetic yield of 82% for a 20 amino acid peptide. The coupling agent used, HATU, is one of the most efficient available and allows fast coupling with little loss of chiral integrity. This is important for the pro-HDPs for the maintenance of

distinct D- and L-amino acid motifs. The reaction mechanism is

illustrated in Figure 3.2 where the first amino acid has already been coupled to the resin and a second is being added. First, HATU reacts with the -COOH group of the second Fmoc-protected amino acid (AA2) (which is deprotonated by the base DIEA) [1]. This releases a

side product, -OAt, which reacts with the concomitantly-formed

isouronium ester to form another active ester [2], eliminating a substituted urea at the same time [3]. Both intermediate esters can next form an amide bond with resin-bound amino acid 1 (AA1) whose amine is not protected [4]. The coupling efficiency of HATU is superior

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hydrogen bond with the reacting amide [5] in a phenomenon known as

the “neighbouring effect” (170). As the amide bond is formed, -OAt is

eliminated, leaving a dipeptide coupled to the resin, but with the amino group protected by Fmoc [6].

Figure 3.2: Reaction scheme for the addition of amino acid 2 (AA2) to resin-bound amino acid 1 (AA1), using HATU/DIEA coupling

chemistry.

As stated above, Fmoc is base-sensitive (171), and can be removed with the application of 20% v/v piperidine. This allows intermittent steps of deprotection and coupling to be carried out (Figure 3.3A). To cleave the finished peptide, of n amino acids in length, and remove side-chain protecting groups, acidic conditions are used (Figure 3.3B). The conditions required to remove both sets of protecting groups

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of the side-chain functions) are mutually exclusive (described as orthogonal), which ensures that side-chain branches cannot form and that only the amine of the N-terminal amino acid is free to react.

Figure 3.3: Summary of SPPS. The peptide synthesis cycle involves incremental steps of coupling with HATU/DIEA onto an insoluble resin support followed by deprotection with piperidine. R = amino acid side- chain, PG = protecting group, and n = number of amino acids (A). The complete resin-bound peptide is cleaved from the resin in acidic conditions which also remove side-chain protecting groups (B). Electrophilic and reactive entities are released from the protecting groups during the acidolytic deprotection. To prevent them from recombining with the nucleophilic liberated side-chain functional

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mixture is required. Different protecting groups are required for each amino acid, and in the same way, different scavenging agents are required for each protecting group (Figure 3.4).

Figure 3.4: Side-chain protecting groups for each amino acid (A) with their corresponding scavenging agents (B). In addition Pbf is the protecting group for arginine. 1, 2 ethane-dithiol is required for cleavage reactions containing tryptophan (to protect its indole ring), and methionine and cysteine (to protect their sulphur-containing side- chains).

Synthesis of the entire D-amino acid active sequence is generally

possible using automated peptide synthesis. However, further

elongation with L-amino acid linker group and oligoglutamic acid pro-

moiety requires careful monitoring of coupling and deprotection by manual synthesis due to the potential aggregation of relatively long and hydrophobic (protected) peptide chains with the polymer matrix of the synthetic resin. Monitoring in this case is carried out by the Kaiser

test, which measures the presence of a free Nα-amino group. A

positive result indicates deprotection, while a negative indicates coupling. With this monitoring it is possible to determine if repeated

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coupling steps are required to complete the addition of an amino acid i.e. a double- or triple-coupling step. As the oligoglutamic acid

sequence is elongated, multiple coupling steps are often required for each additional residue, as aggregation of the peptide chain can be promoted by the tri-alanine motif.