Activated ester functional polymer conjugation

The conjugation of these synthesised polyPEG ‘comb’ α-succinimide functional polymers

could be carried out in a site-specific manner similar to the conjugation carried out for the linear polymers (scheme 2.6). This would again lead to an amide linked oxytocin conjugate, except that with polyPEGylation the PEG density (and ultimately the peptide-polymer architecture) is different to that of the linear example. This difference in structure would likely lead to different properties being observed such as in the potential stability of the peptide, and pharmacokinetic properties or retained biological activity.3,32

Scheme 2.6.Conjugation of α-succinimide functional poly(mPEGA480) polymer onto oxytocin.

To prevent the previously observed readily occurring hydrolysis of the polymer end group from occurring before conjugation onto the peptide, an ‘in-situ’ method was initially developed to minimise water interaction. An aliquot of the polymerisation solution (with

no purification of the polymer) was added to the peptide in anhydrous solvents, thereby not exposing the conjugatable polymer to an aqueous environment. A colour change was observed during the reaction as the solution changed from a faint green (as within the polymerisation solution) to a brown colour. Monitoring of the conjugation by RP-HPLC

analysis (λ = 280 nm) showed the formation of a number of side products, with minimal

concentration changes of the peak on the RP-HPLC chromatogram representing the native peptide (figure 2.11). There is the presence of some potential peptide-polymer conjugate (a broad peak at retention time t = 26.6 minutes), however, the number of impurities in the solution mean that purification and further conjugate confirmation would be difficult.

Figure 2.11.RP-HPLC trace of conjugation of NHS-poly(mPEGA)20from ‘in-situ’ conjugation of aliquot of polymerisation solution.

An alternative method was established for conducting this conjugation, which involved prior purification of a second batch of succinimide polymer synthesised targeting a molecular weight of 6.5 kDa. This succinimide functional polymer was synthesised using the same conditions for the polymerisation as for the kinetic experiment but with [M]:[I] ratio = 13:1. After polymerisation the removal of residual monomers and purification of the polymer was carried out by repeated precipitation into Et2O: hexane (1: 1) whilst

carefully minimising the chance of hydrolysis of the succinimide ester end group. The

retention of the α-end group succinimide ester functionality was confirmed by 1_{H NMR of} the purified polymer, where the peak at δ= 2.81 ppm represents the 4 protons in the

succinimide ring (figure 2.12). The experimental molecular weight could be calculated by

1_{H NMR (CDCl}

3) by a comparison between these protons and the protons from the first CH2 of the PEG repeat unit (δ = 4.1 ppm) yielding an average DPn of 13 (a molecular weight of

6500 g mol-1_{). Analysis of the purified polymer by SEC confirmed that the polymer}

molecular weight distribution remained narrow and mono-modal with final Mnof 6400 g

mol-1_{and dispersity = 1.08.}

Figure 2.12.1_{H NMR (CDCl3, 300.13 MHz) of succinimidyl ester functional poly(mPEGA480)13.}

Conjugation of purified succinimide – poly(mPEGA)

After confirmation that the α-end group functionality was retained the polymer was

subsequently conjugated onto oxytocin using the same conditions as for the linear succinimide ester conjugation (1.5 equivalents of polymer in anhydrous DMF containing 1 % TEA). However, when the reaction was sampled at t = 24 hours, or at longer reaction

formation of any desired conjugate product (figure 2.13). It was again believed that this was potentially due to hydrolysis of the succinimide ester end group meaning the functionality required for an efficient conjugation was no longer present.

As the conjugation can only occur at one position on the peptide structure, the addition of a large amount of polymer should have no adverse effect on the conjugation reaction,24

although would lead to a higher amount of the polymer as an impurity in the final product. Therefore, in an alternative procedure, a large excess of the succinimide functional polymer (10 equivalents) was added to oxytocin; it is noted that this is similar to industry used reaction conditions. The high amounts of equivalents were used in order to maintain some level of polymer end functionality and therefore allow conjugation to occur in the expected manner. Although, there is a large excess of polymer, some amount of this is not expected to be reactive with NHS end functionalisation due to hydrolysis.

Figure 2.13.RP-HPLC trace monitoring conjugation of NHS-poly(mPEGA)13with 1 or 10 equivalences.

Analysis of the reaction by RP-HPLC (UV λ= 280 nm) revealed the appearance of a

characteristic broad conjugate peak at a longer retention time (t = 23.8 minutes) than the native peptide (t = 12.2 minutes), attributed to the poly(mPEGA)-oxytocin conjugate (figure 2.13). RP-HPLC analysis of the unconjugated polymer did not reveal any peaks with high absorptions, therefore the appearance of a new broad conjugate peak can be attributed to

the desired conjugation occurring. The resulting conjugate was purified by dialysis against water (1 kDa MWCO, 3 days) to remove unreacted peptide and N-hydroxysuccinimide released during conjugation, after which RP-HPLC analysis revealed one single broad product (figure 2.14). It must be noted however, that there would still be a significant amount of the polyPEG reagent remaining in the purified conjugate.

Figure 2.14.RP-HPLC of oxytocin-poly(mPEGA) conjugate after purification.

It should be noted that in the original work regarding the synthesis and conjugation of NHS functional poly(mPEGMA) by Haddleton and co-workers, similar issues were raised with the conjugation of polymers wherein no conjugation was observed after 24 hours.24

However, on changing the initiator functionality from that of a methyl propionate group to a propionate, the reactivity was enhanced and the coupling could take place more efficiently.