WZ8040 was detected for either pore size CPG

The loading of Phe on both C3 and C10 CPG was determined using elemental analysis by measuring the difference in carbon and nitrogen levels before and after reaction with Phe. In the case of the C10 linker, no Phe attachment was detected for either pore size CPG, possibly due to crowding by the comparatively long linker within the smaller pores, and the smaller overall surface area for the larger pore CPG. To preclude WZ8040 both possible problems, subsequent reactions employed the 400 ? CPG functionalized by the C3 silane. Attachment of Phe to the modified CPG resulted in an amino acid loading of 63.9 mol/g according to elemental analysis. Bergenin coupling onto the Phe CPG was carried out using conditions analogous to those used for the solution phase coupling reaction between bergenin and Boc L Phe. The HATU/DIPEA combination yielded the highest solution coupling efficiency for the esterification reaction and therefore was used to attach bergenin to Phe CPG.
The maximum loading of bergenin to the Phe CPG was 34.2 mol/g, as determined by monitoring the reaction progress with LC/MS. Removal of attached bergenin was accomplished using VX-680 H2SO4, SC, and CT. Cleavage with CT gave a 9.3% cleavage yield, which was two times higher than SC. Although this result shows the feasibility of using a Phe linker for selective cleavage of bergenin using CT, this yield was lower than that obtained using solubilized lipase B or SC to remove bergenin directly from carboxy CPG in organic solvents. Thus, Phe CPG was not employed in subsequent solid phase biocatalytic reactions. Solid supported bergenin was halogenated by chloroperoxidase as the first step of a two step enzymatic modification.
When performed for 20 h, halogenation conversions of nearly 70% were obtained with solution phase bergenin. Although it was not expected that conversions for solid phase bergenin halogenation would be as high, successful aqueous phase modifications of immobilized natural products could prove to be particularly useful for substrates that are poorly soluble in water, as is typically the case with many potential pharmaceutical lead compounds. In the case of solid supported bergenin, halogenation was carried out under similar conditions to the solution phase reaction, with the following modifications: the CPO/bergenin and KBr/ bergenin ratios were increased by approximately 70 and 25 fold, respectively. Under these conditions, a solid phase halogenation conversion of 5.
9% was obtained, thereby demonstrating the ability of the CPO to access at least a portion of the solid supported bergenin. Efforts to improve this conversion included the following: 1 increasing the H2O2/CPO ratio, 2 running consecutive halogenation reactions with complete removal and replenishment of enzyme solution between reactions, and 3 enzyme preincubation in the absence of H2O2. It was thought that allowing the enzyme to diffuse through the porous CPG network prior to H2O2 addition may enable better access of CPO to the solid supported bergenin before enzyme inactivation by H2O2. Of these measures, only the enzyme pre incubation afforded any significant increase in solid phase halogenation conversion nearly a 3 fold increase from 5.9 to 17.3% indicating that slow diffusion of the enzyme into the CPG pores is limiting.