On the other hand, Hapten 3 that was linked to DT through the 2-position, distal to that used for Hapten 4, had a very different Ab and affinity response

On the other hand, Hapten 3 that was linked to DT through the 2-position, distal to that used for Hapten 4, had a very different Ab and affinity response. converted through to ester 24 via Wittig reaction and hydrogenation over rhodium on alumina (Figure 5) (Figure S29, Figure S30). Ester 24 was reacted with the enolate of lactam 25 to give ketoamide 26 (Figure S31). Acidic hydrolysis with concomitant Y-33075 decarboxylation and condensation gave imine 27 (Figure S32) which was metallated with butyllithium leading to ring-closure to give racemic amine 28 (Figure S33). Methylation under reductive amination conditions gave 29 (Figure S34) while subsequent Heck coupling, hydrogenation and basic hydrolysis generated Hapten 9 as a racemic mixture (Figure S35, Figure S36, Figure S37). Open Klf2 Y-33075 in a separate window Figure 5 Synthesis of Hapten 9. Hapten 10 was synthesised via the commercially available bromide 32 utilising similar methodology to the previous compounds (Figure 6) (Figure S20, Figure S21, Figure S22, Figure S23). Open in a separate window Figure 6 Synthesis of Hapten 10. The degree of hapten coupling to DT was measured for all haptens using a reverse-phase HPLC (RP-HPLC) method utilizing a Waters C-18 X-Bridge column with a gradient of 0.1% triethylamine (TEA): 0.1% TEA in methanol (Table 1). In this process, haptens were uncoupled from DT by acid hydrolysis and analysis of pre-hydrolysis and post-hydrolysis levels used to determine the degree of conjugation per unit loading Y-33075 of DT. Table Y-33075 1 Hapten Loading of Different Hapten-DT Conjugates. to leave orange oils. These were dissolved in methanol (MeOH) and run through a 5 g SCX cartridge eluting with MeOH, then 4:1 MeOH: Ammonia (7M in MeOH). The appropriate fractions were evaporated and chromatographed on 4 g ISCO cartridges eluting with mixtures of dichloromethane (DCM), MeOH and ammonia (NH3) and evaporated to yield the protected haptens that were used in the next step without further purification or characterization. The protected version of Hapten 7.L1 was prepared from diethyl squarate, which was dissolved in ethanol (EtOH) and treated with BOC-ethylenediamine, Hnigs base and Y-33075 Hapten 7 and the reaction stirred at RT for 16 hours. The mixture was evaporated to leave an orange oil that was dissolved in MeOH and run through a 5 g SCX cartridge eluting with MeOH, then 4:1 MeOH: Ammonia (7M in MeOH). The appropriate fractions were evaporated and chromatographed as above. BOC-protected amines were deprotected to yield Haptens 7.L1 to 7.L12 by dissolving in DCM and cooling to 0 C. To this cooled solution, trifluoroacetic acid (TFA) was added and the reaction was allowed to warm to RT and stirred for 16 hours. The reaction mixtures were evaporated to leave orange gums that were dissolved in MeOH and loaded onto 5 g SCX cartridges and eluted with MeOH, then 4:1 MeOH: Ammonia (7M in MeOH). The solutions were evaporated under reduced pressure to yield the haptens depicted in Figure 7. The degree of hapten coupling to DT was measured for all haptens as outlined above (Table 2). Table 2 Hapten Loading of Different Hapten 7-DT Conjugates. function of the anti-nicotine antibodies in mice, as assessed by IV administration of radiolabeled nicotine, also varied with different hapten conjugates (p < 0.0001 and p = 0.0002, for plasma and brain, respectively). Hapten conjugates containing Haptens 2, 3, 6, 7 and 11 significantly increased levels of nicotine in the blood compared to non-immunized animals (p<0.05) (Figure 9B), and hapten conjugates containing Haptens 2, 5, 6, 7 and 8 significantly decreased levels of nicotine in the brain (p<0.05) (Figure 9C). Overall best responses were obtained with conjugates containing Haptens 2, 6 or 7 that had ~130, 81 and 156% greater retention of nicotine in the plasma, and 36, 27 and 40% decrease of nicotine in.