Resin screening

Although it is true that all linkers cleaved by acidic conditions will result in a resin bound cation, the propensity for this species to participate in the back alkylation of sensitive nucleophilic residues is not necessarily equal. The Wang linker is particularly prone to this side reaction, most likely due to the exposed nature of the cation. An alternative, often recommended for the synthesis of peptides containing tryptophan, is the 2-chlorotrityl linker.^{172, 173} The reduced occurrence of back alkylation to the indole side chain of tryptophan, has been attributed to a steric shielding effect provided by the triphenylmethyl structure that encapsulates the cleavage site.¹⁴⁵ This shielding effect has also been shown to effectively supress diketopiperazine formation, another common side reaction which can lead to near quantitative losses in the synthesis of sensitive peptide sequences.¹⁷⁴ It was therefore decided to resynthesize the lysine analogue 39, on a number of different resins, and compare the recovery. In order to reduce the influence of handling errors, this would be achieved by conducting the synthesis for each resin, on the same scale, using identical reaction conditions, and analysing the crudes by analytical reverse phase HPLC, after cleavage. Although there would be no internal standard, and therefore estimation of yield would not be possible, by preparing the HPLC samples in an identical manner, the area of the product peak could serve as a qualitative comparison of recovery between each resin.

133 In addition to the original Wang resin, and the suspected superior candidate 2-chlorotrityl resin, the screen would also include Rink amide MBHA resin,¹⁷⁵ and NovaSyn^® TGA resin.¹⁷⁶ The structures of the lysine substituted resins are shown in figure 3.5.

Figure 3.5 Structures of the different lysine substituted resins.

134 NovaSyn® TGA resin also contains the Wang linker, however instead of being attached directly to the polystyrene polymer by a benzyl ether, it is spaced by a polyethylene glycol chain. Such polyethylene glycol functionalised resins, are said to provide a more favourable matrix environment, for solid phase synthesis.¹⁷⁷ The polyethylene spacers help to improve the solvation of the growing peptide chains, and protect against aggregation which can reduce the coupling efficiencies of susceptible sequences.

Association of the hydrophobic duocarmycin structure with polystyrene resins might be a foreseeable issue when considering the synthesis of more complex analogues in the future. It was also speculated that the improved mass transfer kinetics of the matrix environment of polyethylene glycol functionalised resins, may facilitate the escape of products from the matrix before they could react with the cleavage cation.

Rink amide MHBA resin, contains the Rink amide linker, attached via a norleucine spacer to a polystyrene based 4-methylbenzhydrylamine resin. This spacer protects against alternative detachment of the Rink amide linker at the ether during cleavage. This can occur when the Rink amide linker is attached directly to polystyrene, in an analogous fashion to release of p-quinone methide observed with Wang resin. Unlike the other linkers, cleavage provides the product as a terminal amide, as opposed to a carboxylic acid. This was deemed to have potential useful applications in the future. Furthermore, the two phenyl groups surrounding the cleavage site may provide some protection against back alkylation. In fact, it could be said to offer a level of steric protection which is intermediary between the well shielded cleavage site of the 2-chlorotrityl linker, and the very exposed cleavage site of the Wang linker.

For the screening, the synthesis was conducted on a 0.038 mmol scale, using the reaction conditions already described in section 3.3. The unnatural enantiomer of 11 was used as this was deemed less valuable. After cleavage, the crudes were evaporated to dryness and dissolved in 1 mL of MeOH. 100 µL of the resulting solutions were diluted with 900 µL of MeOH and analysed by reverse phase HPLC. A comparison of product peak area for each resin is shown as a bar chart in figure 3.6.

It can be seen that recovery was greatest with 2-chlorotrityl resin. The two resins containing the Wang linker produced the lowest recovery, and were essentially identical.

Rink amide MHBA fell somewhere in between. This is consistent with the degree of steric shielding for each resin. Furthermore, it would seem that the improved mass transfer kinetics of the NovaSyn® TGA resin provided no detectable protection against back alkylation with the Wang linker. In fact, the polyethylene glycol spacer may be detrimental

135 as it increases the distance of the carbocation from the polystyrene core, which itself can act to scavenge this reactive species.

Although it is clear that 2-chlorotrityl resin provides the greatest recovery of product, we cannot be sure from these results, how large the difference is, as the relationship between concentration and absorbance at 254 nm may not be linear. Furthermore, the product from Rink amide cleavage is different to the other resins, being the amide rather than the carboxylic acid. It is not known what effect this minor change has on the absorbance character of the compound. However, as it is the shared aromatic structure that will be responsible for the majority of absorbance at 254 nm, this is unlikely to have a significant impact.

These results at least confirmed the suspicion that 2-chlorotrityl resin is a more appropriate choice than Wang resin for syntheses incorporating 11. The increased recovery is most likely due to the reduced occurrence of back alkylation of the resin with the indole scaffold on cleavage. 2-Chlorotrityl resin holds further advantages. Firstly, it is cleaved in only one place. This means it does not produce a soluble linker derived carbocation, which can alkylate the indole scaffold to produce soluble side products. It is possible this also reduces the overall burden on the scavengers. In addition, the triphenylmethyl structure produces a highly stabilised carbocation, and as such 2-chlorotrityl resin can be cleaved under far milder conditions. For example cleavage when

Figure 3.6 Bar chart comparing the HPLC product peak area between different resins (see text).

HPLC conditions: 10 µL injection. Agilent Eclipse XDB-C18 column, 4.8 x 150 mm, 5 µm. Solvent A: [Water and 0.05 % TFA], Solvent B: [MeOH and 0.05 % TFA]. Gradient: 0% [B] to 95 % [B], from

136 simultaneous t-butyl side chain protection is not required, can be affected with a TFA concentration as low as 1%.

Due to the above advantages, and the results of the screening, 2-chlorotrityl resin would be chosen for future work, when a carboxylic acid end group was desired.