Synthesis of alternate 10/12-helical β-peptides

4.6.1 Heavy-fluorous-tagged solution phase synthesis

Unlike the 14- or 12-helical peptides, there have been very less reports on the effective synthesis of alternate 10/12-helical β-peptides of comparable sequence length. Many trials with different coupling conditions and different resins in microwave-assisted SPPS were completely unsuccessful. So, an unconventional method, based on heavy-fluorous-tagged solution phase strategy, for synthesizing the unique 10/12 helical peptides was adopted (Figure 4.9). ^[159]

Figure 4.9: Schematic representation of the heavy-fluorous-tag-assisted solution phase peptide synthesis for alternate 10/12-helcal peptides ^[159]

This method was fundamentally developed for the glass micro-reactor system and was comparatively more efficient in that system. However, alternate 10/12-helical β-peptides were tried no longer than 4-amino acid long sequences by this method. Since the typical glass-micro-reactor, system was not available for this project, so the method was tried with some minimal optimization of the conditions in solution phase using regular glass apparatus. It was a tedious method, as after each coupling step, the desired peptide fractions had to be purified by fluorous solid phase extraction (FSPE) technique. This also reduced the yield to considerable extent. But as there was no other reported method available, so it was the only option at that moment.

The method started with the attachment of the first N(Boc)-β³-D-amino acid to the heavy fluorous tag (a benzyl alcohol derivative). At the next step, the N(Boc)-was deprotected under acidic condition using TFA/DCM. All the β³ and β²-amino acids were converted into their acid fluoride counterparts by using cyanuric fluoride at -30⁰C for 3 hours. The acid fluorides were more reactive than that of the acids, and since the coupling between β³- β²-amino acids were energetically not favourable, so the coupling between the two amino acids were not effective and one of them had to converted to the acid fluoride. Therefore, the coupling between the fluorous-tagged β³-amino acid and the incoming β³-amino acid fluoride was carried out at 90⁰C for 10 minutes followed by standard work up and purification of the fluorous tagged-desired di-peptide fragment by FSPE. Next, the N(Boc)-deprotection was carried out using the same acidic condition followed by the coupling of an incoming β²-amino acid fluoride. The coupling between the fluorous-tagged β³-dipeptide and incoming β²-amino acid fluoride was performed at 120⁰C for 15 minutes followed by work-up and purification of the desired tri-peptide fragment by FSPE.

So, the cycle of coupling between β³ and β²-amino acids were continued until a tetrapetide was synthesized. At this stage, formation of the tetra-peptide was confirmed by RP-HPLC and ESI-MS after a small portion was cleaved from the fluorous-tag via hydrogenation with hydrogen and Pd/C overnight under 3 Bar pressure. But unfortunately, no trace of a penta-peptide product was obtained from RP-HPLC and ESI-MS. Therefore, only an alternate 10/12-helical tetra-peptide composed of alternating β³/β²-amino acids could successfully be accessed via this heavy-fluorous-tag-assisted solution phase synthesis strategy.

4.6.2 Non-microwave-assisted manual SPPS

After getting unsuccessful in synthesizing the target 10/12-helical transmembrane β-peptides with microwave-assisted SPPS as well as heavy-fluorous-tag-assisted solution phase peptide synthesis strategies, a new route was tried (Figure 4.10), as reported by Kolesinska et. al. ^[8]

Figure 4.10: Schematic representation of non-microwave-assisted manual SPPS synthetic strategy to synthesize alternate 10/12-helical transmembrane β-peptides composed of Val/Ala/Leu triads.

[8]

The synthesis by non-microwave-assisted SPPS was initially tried by block co-polymer synthesis strategy via coupling a resin-cleaved alternate 10/12-helical penta-peptide with another solid-supported 10/12-helical hexa-peptide under similar conditions as well as under an increased temperature to 50⁰C for 30 minutes. Since, the coupling was getting more and more inefficient with the increase of chain length starting from hexa-peptide, so the synthesis by block-copolymer strategy was taken into account. But none of the trials resulted in a successful synthesis of the desired alternate 10/12-helical transmembrane sequence. The higher temperature reduces the probability of random peptide aggregation during SPPS, so an elevated temperature was used. However, that did not help.

Hence, the synthesis was continued in linear chain coupling fashion, the temperature was kept at room temperature, and the coupling time was increased to 2 H along with a capping time for 1-1.5 H at room temperature. Therefore, the method begins with loading the first amino acid, β³ -D-Val/Ala/Leu or β²-D-Val/Ala/Leu, to 2-chlorotrityl chloride resin (loading capacity = 1.63 mmol/g) in presence of DIPEA at room temperature. The loaded resin was deprotected twice for 30 minutes with 20% piperidine in DMF. Now the loaded resin was ready to undergo further coupling cycles. The synthesis of 10/12-helical hexa-peptide composed of pure hydrophobic sequences of alternate β²/β³ (Val/Ala/Leu/ triad (P6 and P7) was carried out successfully with very promising yields. The loaded resin was coupled with the target β-amino acid residue (β² or β³-Val/Ala/Leu in alternate fashion) in presence of a newly developed coupling reagent, DMT/NMM/TsO^-, that had to synthesized at laboratory due to commercial unavailability^[160] and DIPEA at room temperature for 2 H. The coupling was followed by capping of the unreacted free amines by the capping cocktail, acetic anhydride/DIPEA/DMAP/DMF, at room temperature under pure N2-bubbling for 1-1.5 H. Next, the double deprotection was carried out; first with 20% piperidine in DMF for 20 min at room temperature and second with a cocktail of 20%

piperidine in DMF and 2& DBU in DMF for 10 min. at room temperature. This cycle of coupling-capping-deprotection was continued until the target sequence was obtained. The peptide was cleaved form the resin under acidic conditions using HFIP/DCM (3/7) to yield the desired peptide as an acid at the C-terminal. Therefore, the target alternate 10/12-helical peptides composed of only hydrophobic alternate β²/β³-Val/Ala/Leu triads was not a problem.

Nevertheless, the target peptides, P2 and P3, were 9-10 amino acid long. Synthesis of these

longer sequences proved to be extremely challenging. The initial challenge was to elongate the chain length from 6 to 10 amino acid. After that was accomplished, the next challenge was to insert a β³-D-Trp residue towards the C- or N-termini. The presence of Trp was necessary, as the plan was to use Trp-fluorescence to check the position of the peptide whether in aqueous region or inside lipid vesicle (hydrophobic region). However, after several trials with various coupling conditions, the coupling of β³-D-Trp to the 2-chlorotrityl resin or to other solid supported β³ -D-Val/Ala/Leu or β²-D-Val/Ala/Leu amino acid residues was not successful in non-microwave-assisted manual SPPS. Therefore, after loading the first β² or β³-Val/Ala/Leu hydrophobic amino acid residue to the resin by non-microwave-assisted manual SPPS, the strategy was changed to microwave-assisted manual SPPS and the desired Trp-residue was coupled to the solid supported β² or β³-amino acids by the method adapted to synthesize β³-D-Val-peptides (P4 and P10). The coupling reagent was changed to DIPEA/HATU/HOAT and the coupling was C-terminal of the growing alternate 10/12-helical peptide chain, the coupling strategy was again shifted to the non-microwave-assisted manual SPPS, just as the P6 and P7, and the coupling-capping-deprotection cycles were continued until the desired peptide sequences (P2 and P3) were obtained (Figure 4.10). The yield for the synthesis of P2 and P3 were comparatively much lower than that of P6 and P7, but the desired peptide could be purified by RP-HPLC and was confirmed by ESI-MS and HR-MS spectroscopy.