Solid phase peptide synthesis of modified Zf3 constructs

With the determination of two incorporation sites, modified Zf3 peptides were synthesized by means of solid phase peptide synthesis (SPPS), including the artificial amino acids. The SPPS approach dates back to the work of MERRIFIELD in 1963.^[77] It relies on the use of an insoluble solid support (resin) that is modified with an acid labile linker to which the C-terminus of the first amino acid, bearing a base labile N-terminal Fmoc protecting group, is attached. In contrast to protein biosynthesis, in which ribosomes elongate the sequence from the N-terminus to the C-terminus, the elongation in the SPPS approach is performed in opposite direction. The procedure starts with the removal of the resins N-terminal Fmoc protecting group by the addition of an organic base, such as piperidine or piperazine. The first amino acid is coupled to the resin by means of a suitable coupling cocktail containing reagents for the activation of the terminal carboxyl group by forming an active ester (Oxyma Pure^®/DIC, HATU/HOAt or HBTU/HOBt). The process of Fmoc deprotection and amino acid coupling is repeated until the desired sequence is generated on the solid support (Figure 2.7). The major advantage of SPPS is the facilitated washing procedure after each reaction step.^[78] The soluble side products can be easily separated, whereas the growing peptide attached to the resin remains in the filter tube. This also allows the use of all coupling reagents in excess that further increases the crude purity of the peptide and avoids the formation of deletion or truncated sequences. Finally, the peptide is cleaved from the resin by the addition of a strong acid, such as trifluoroacetic acid (TFA), which simultaneously removes all acid-labile side chain protecting groups.

Figure 2.7 Schematic representation of the solid-phase peptide synthesis (SPPS) cycle (SC = amino acid side chain).

The zinc finger constructs in the present study were synthesized by means of the Fmoc/tert-butyl strategy described above using a Fmoc-L-lysine(Boc)-OH pre-loaded Wang-resin. The peptides were either synthesized using a microwave-assisted peptide synthesizer (Liberty Blue or Liberty 12 from CEM), or microwave-assisted manual peptide synthesis when particular emphasis was put on the incorporation of the bulky building blocks was applied.

Standard amino acids were coupled using a coupling cocktail containing HOBt/HBTU or HOAt/HATU with DIPEA as activator base or applying the system Oxyma Pure^®/DIC as indicated in the experimental section. Special attention was payed on the incorporation of the unnatural amino acids. The increased steric demand of the building blocks made it necessary to use a slightly modified coupling protocol, which was applied by manual microwave peptide synthesis. In this sense, the coupling times for the building blocks as well as for the subsequent amino acid were extended from 4 min (90 °C, 25 W) to 2 x 20 min (65 °C, 12 W), respectively. In addition, the coupling mixture for the concerned amino acids was adapted from the literature, which recommended the use of a solution containing DIPEA as activator base and PyBOP/HOBt as coupling reagents.^[79] PyBOP is a very potent peptide coupling reagent that reduces racemization during elongated coupling times for critical amino acids. It is commonly used for challenging cyclization reactions and for the coupling of hindered amino acids, such as 2-aminoisobutyric acid (Aib) or bulky amino acids, as for instance phosphorylated threonine (Fmoc-Thr(PO3BzlH)-OH).^[80] Peptide synthesis was completed by manual coupling of the remaining amino acids under conventional condition with microwave support. The zinc finger mutants were cleaved from the resin by a mixture of TFA/EDT/H2O/TIS (94:2.5:2.5:1) and purified by RP-HPLC. The target peptides could be isolated in yields ranging between 15 – 38%. However, the HPLC chromatograms revealed the formation of several deletion and truncation sequences derived from the incorporation site of the building blocks as confirmed by mass spectrometry of the isolated compounds. It can therefore be assumed that the bulky building blocks negatively affect the subsequent peptide couplings. This was slightly improved by applying a double coupling procedure at decreased temperatures (50 °C) and extended reaction time (2 x 15 min) for the remaining amino acids.

Nonetheless, the dinuclear building blocks 14 and 16 were successfully incorporated into the native sequence of Zf3 at two different positions (Arg70 and Ser75, Figure 2.8).

Figure 2.8 Zinc finger mutants synthesized in this work. The BMIA modified building block (a) and the BPA modified building block (b) were incorporated at the arginine position 70 and the serine position 75.

It is to note that all Zf3 mutants were obtained in the oxidized form having a disulfide bond between the two N-terminal cysteine residues. This was disadvantageous for two reasons.

First, the subsequent native chemical ligation of Zf3 with the expressed Zf12 peptide requires a free N-terminal thiol moiety of the cysteine residue in Zf3, which attacks the C-terminal thioester of Zf12. Second, the peptides are only able to bind Zn(II) with both cysteine residues in their reduced state in order to receive their correct secondary structure. Hence, in all experiments presently described, the zinc fingers were reduced by the addition of the disulfide reducing agent tris(2-carboxyethyl)phosphine (TCEP) for 90 min prior to the experiment. TCEP belongs to the group of non-metal-coordinating reducing agents.^[81] In contrast, several studies revealed that other commonly used reducing agents, such as dithiothreitol (DTT) or 2-mercaptoethanol (2-ME), have a pronounced tendency to coordinate divalent metal ions due to their multidentate binding sites composed of sulfur and oxygen ligands at neutral pH.^[82] For this reason, the use of the latter substance had been deliberately abandoned in order to avoid interactions with the dinuclear metal complex of the building blocks. These interactions would be undesirable since the dinuclear metal complexes could occupy the allocated phosphate binding site with the reducing agent that may prevent proper substrate activation.