Aldehyde functional linear PEG conjugation

It is reported that amine groups, such as those occurring on lysine residues or at the N- terminal amine within a peptide, will react with carbonyl groups such as aldehydes in the presence of suitable reducing agents in a two-step reductive amination process to form stable secondary amines (scheme 2.2). The first step isviathe formation of a Schiff base / imine linked intermediate which is in equilibrium with the aldehyde and amine. Commonly the intermediate is then reduced by the addition of a reducing agent, which forms a stable secondary amine linkage. Sodium cyanoborohydride is a common reducing agent used in this manner and is preferable to sodium borohydride as the reducing agent.16,17 _{Due to}

milder reductions being performed, the Schiff base reductions occur efficiently but there is no reduction of any remaining aldehyde, which would potentially prevent higher yields in the reaction. Aldehyde modification of peptide or protein amino groups may also be preferable to NHS reactions, as potential charges can be maintained on the secondary amine product, which are not the case for the amide linkages associated with NHS conjugates.

Scheme 2.2. Schiff base formation of reaction between aldehyde and amine followed by reduction by NaCNBH3forming stable secondary amine linkage.

Multiple different chain length spacers between the aldehyde functionality and polymer chain have been used for the reductive amination of biomolecules by aldehyde conjugation, with varying levels of success (figure 2.4). The use of acetaldehyde reagents

have previously been used to couple to peptides, although these can be ineffective due to a limited stability, particularly under basic conditions, and a susceptibility to form undesirable by-products. Functional mPEG-propionaldehyde and mPEG-butyraldehyde contain longer linker chains between the aldehyde functionality and the PEG chain and have proven to have higher stabilities proving more useful under a variety of conditions for reductive amination.18–20

Figure 2.4.Commercially available aldehyde functional PEG reagents.

A further property that is often utilised within these reductive amination conjugation reactions is the tuning of pH for more selective targeting of amine groups on proteins or peptides. As there are two different commonly occurring types of amines that can be presented within peptides and proteins, those found on lysine residues throughout the structure or the singular amine located at theN-terminus, it can be beneficial to selectively target theN-terminus for a singular attachment.1,13,21_{The pK}

avalues for the lysine amines

(10 - 10.2) are much higher than for theN-Terminus (pKa ≈ 7) and under acidic or neutral

conditions, the terminal amine can usually be selectively modified.

For the discussion of the conjugation of these polymers onto oxytocin pH controls are not required as there are no lysine groups within the structure, which have associated problems with regards to multiple sites of attachment. In the case of the oxytocin, the singular available amine within the peptide that could potentially react in this manner is that located at theN-Terminus and will lead to a site specific attachment.

2.2.2.1. Oxytocin conjugation using aldehyde functional PEG

Scheme 2.3.Formation of stable linear PEGylated oxytocin following reaction with aldehyde PEG and subsequent reduction by NaCNBH3.

Two different molecular weights (Mw= 2 kDa & Mw= 5 kDa) of α-methoxy-ω-formyl

poly(ethylene glycol) (aldehyde PEG) were conjugated onto oxytocin with reduction by NaCNBH3(scheme 2.3). Oxytocin was dissolved in sodium phosphate buffer (pH 7.5, 100

mM) and reacted with 1 equivalent of the PEG reagent and a freshly prepared 25 mM solution of NaCNBH3(2.5 eq.) added before the reaction proceeded at 10 °C overnight.

After the conjugation an aliquot of the solution (equivalent to 0.2 mM oxytocin) was removed and diluted in water for RP-HPLC monitoring of the reaction. The HPLC UV

coupled (λ = 280 nm) chromatogram revealed the appearance of a newly formed broad

peak at a longer retention time (t = 14.0 minutes) to oxytocin (t = 7.4 minutes) alongside and approximate 60 % decrease in the native peptide remaining in the solution. RP-HPLC analysis of the unconjugated (aldehyde functional) polymer showed the appearance of one narrow peak at a retention time of t = 5.2 minutes. Post conjugation analysis of the reaction solution revealed that the presence of this peak is still observed alongside evidence of unreacted peptide for both Mwof PEGs conjugated (figure 2.5).

Figure 2.5. RP-HPLC monitoring of conjugation reaction of aldehyde PEG (2 kDa) onto oxytocin, after stirring for 24 hours at T = 10 °C.

In the same method as the succinimydyl conjugated oxytocin, a portion of this newly formed aldehyde conjugated oxytocin was removed and purified by semi-preparative RP- HPLC. The gradient of the semi-preparative system was optimised for efficient peak separation allowing efficient purification of the product. After lyophilisation, analysis of the purified polymer conjugate by analytical RP-HPLC revealed the appearance of a single broad peak (figure 2.6). There is some peak tailing leading to a shoulder appearing on the RP-HPLC chromatogram for the purified conjugate, potentially due to side reactions occuring, leading to the formation of additional products, which will be discussed later in this chapter.

MALDI-TOF MS analysis was performed on this oxytocin-polymer conjugate using DHB as the MALDI matrix and compared to the native aldehyde functional polymer. Similarly to the other linear (succinimidyl ester) conjugate, the peaks observed by MALDI-TOF MS were in good agreement with those expected for the stable (reduced with NaCNBH3) aldehyde

PEG – oxytocin conjugate. The peaks in the MALDI-TOF MS show very similar calculated (theoretical) and observed (experimental) values (figure 2.7 for n = 43 and n = 44 for the PEGnchain) and the ethylene glycol difference between the peaks is maintained (44.03 Da).

There is a clear shift in molecular weight from the unconjugated aldehyde polymer to the peptide-polymer conjugate corresponding to the molecular weight of the peptide (1007 Da).

Figure 2.7.MALDI-TOF MS analysis of oxytocin-polymer (aldehyde) 2 kDa conjugate.

These results assist in confirmation of the conjugate structure, ensuring that covalent attachment of the polymer onto oxytocin was in a site-specific manner with only one unit of polymer per peptide (mono-addition).