Scheme 7.10: Immobilisation of 156 and 158 onto the sepharose bead.
The aqueous washes, obtained by flushing the affinity column with three column volumes of distilled water, were extracted with DCM. This was to determine the amounts of unbound 156 and 158 and thus the amount of 156 and 158 immobilised on
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the bead. The organic DCM layer was dried, concentrated and analysed by NMR spectroscopy for the presence of 156 or 158. The affinity column was also washed with MeOH to ensure the removal of any excess unbound 156 and 158. The MeOH wash was also concentrated and analysed by NMR spectroscopy. The resultant residues, from the extracted aqueous washes and the MeOH washes, contained both 156 and 158
Table 7.1: Percentage of compounds 156 and 158 immobilized on Sepharose bead.
Analysis of the various washes indicated that immobilization had occurred. Although a number of publications do not report a method for verifying immobilization, other reports alluded to the use of certain tests for verification.180,181 In any case, we wanted to further test the immobilization process.
Sepharose beads are commonly used for the immobilization of peptides.182 The process of attaching peptides to the bead is similar to the one described above. However, to verify that the peptides have been immobilized, amino acid analysis is performed.183 This involves cleaving the bound molecule by means of hydrolysis and analysing the remaining residue. As this would be destructive to the ligand a different strategy was chosen.
McMahon et al. reported that the immobilization of a substance onto the sepharose bead was verified by means of an observed colour change.184 As 156 and 158 were both colourless oils, no such colour change was expected and the beads remained white.
To counteract this problem, a coloured derivative was synthesised to test the efficiency of the immobilization process. The glucose uptake assay uncovered a derivative, 42, which was active, but less active than 4. Compound 42, which contains a para nitro
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group instead of the para CF3 found in 4, was an orange solid and it was hypothesised that its attachment to the sepharose bead would result in a colour change.
Immobilization of 42 would leave the beads orange allowing verification of the immobilization protocol.184
7.1.4 Synthesis of a coloured derivative of 158.
The previous synthetic approaches used to attach 154 to the active derivatives, 4 and 79, could not be used for the functionalization of 42. As nitro groups are readily reduced to the corresponding amines, formylation of 42 followed by a reductive amination could result in the reduction of the nitro group. Therefore, an alternative route was needed.
The attempted synthesis of the 42-bead conjugate is shown in Scheme 7.11. This involved the formylation of 74 using the conditions previously described. The PEG linker was then attached to the formylated ester derivative (159) via a reductive amination to give compound 160. This was then deprotected using base catalysed ester hydrolysis revealing the carboxylic acid (161), which was verified by 1H NMR spectroscopy due to the absence of the methyl ester peak and by IR spectroscopy where the peak at 2111 cm-1 is indicative of the presence of an azide. Due to the presence of the secondary amine however, the acidic work up resulted in the formation of a salt, which was evident as the product was not found in the various organic washes but was recovered from the aqueous layer. The product was carried forward to the next reaction and an attempted coupling to 33 using HOBt and TBTU was performed. A complex mixture was obtained however, and 162 was not isolated. This approach was therefore abandoned.
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Scheme 7.11: Synthesis of 42-PEG3 linker 162 (i) Tin(IV)chloride, DCM, N2, rt, 1 hrs, 56% (ii) NaBH4, MeOH, rt, 3 hrs, 58% (iii) KOH, EtOH, reflux, 2 hrs, quant. (iv)
HOBt, TBTU, NEt3, DMF, N2, rt, overnight, product not isolated.
As it was hoped to verify the immobilization of the compounds to the bead using a coloured compound with a similar structure to 4 or 79, the nitro derivative of 79 was synthesised. It was hoped that the nitro derivative of 79 would allow easy coupling to the PEG linker. The synthetic route for this approach is outlined in Scheme 7.12. 1-(4-Nitrophenyl)piperazine was coupled to mono methylazelate using HOBt and TBTU to give 163. The ester was then hydrolysed to the acid using base catalysed ester hydrolysis. This gave 164 in a 79% yield. The Staudinger product, 164, was then
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attached via an amide bond to give 165. The amide product was then transformed to the azide as before using the Staudinger reduction. This afforded the orange amino compound 166 (Scheme 7.12). This method did not require the use of a reducing agent and as a result the para nitro group remained intact. The synthesis of 166 was confirmed by IR spectroscopy firstly by the absence of the azide peak but also due to the presence of the N-H stretch at 3389 cm-1 and the N-H bend at 1631 cm-1. The presence of the para nitro group was also evident form the IR spectrum due to the bands at 1597 cm-1 and 1321 cm-1. 1H NMR spectroscopy confirmed the presence of the phenyl group with the two doublets at 8.14 and 7.02 ppm, which are indicative of the protons ortho and meta to the piperazine ring.
Scheme 7.12: Synthesis of 166 (i) HOBt, TBTU, NEt3, DMF, rt, overnight, 81% (ii) KOH, EtOH, reflux, 4 hrs, 79% (iii) 154, HOBt, TBTU, NEt3, DMF, rt, overnight, 94%
(iv) 1 M HCl, Et2O, EtOAc, rt, overnight, 13%.
With a coloured PEG linked derivative now obtained, the next step was to attach the 166-PEG3 construct to the sepharose bead as before. As with the previous examples, the