ENMD-2076 can be used to carry out sequential reactions on immobilized substrates in multiple solvents

A major advantage of solid phase chemistry and biocatalysis over solution phase reactions is the ability to perform sequential reactions without purification of intermediate products from ENMD-2076 the reaction mixture. To demonstrate that solid phase biocatalysis can be used to carry out sequential reactions on immobilized substrates in multiple solvents, the halogenated bergenin derivative was subsequently acylated by EXT SC in diisopropyl ether with 500 mM excess vinyl butyrate. EXT SC was selected over its DS counterpart based on its higher solution phase activity in IPE, the solvent in which the highest overall EXT SC activity was observed. The resulting product was cleaved from the CPG via concentrated LipB dissolved in aqueous buffer in both 0 and 10% acetonitrile co solvent, resulting in a 10% post cleavage recovery yield of 7 bromo 4 butyrylbergenin under optimal cleavage conditions.
To our knowledge, this represents the first example of a two step aqueous:organic biocatalytic reaction sequence for modifying a solidsupported natural product. This approach can likely be expanded to produce libraries of solidsupported natural products in both aqueous and non aqueous media, which could serve to be Y-27632 a valuable tool for compound generation and lead optimization in drug discovery. Extraction Native protein was dissolved in aqueous buffer at 1 mg/mL and then added to an equivalent volume of 2 mM sodium bis sulfosuccinate dissolved in isooctane. The vial was vortexed for 10 s and placed in an orbital shaker at 30 and 250 rpm for 45 min. The biphasic mixture was then immediately centrifuged at 2700 ? g for 5 min to separate the two phases.
Following phase separation, the organic phase was removed and evaporated to dryness by a flowing air stream and the enzyme/surfactant complex organic phase were determined spectrophotometrically by scanning from 220 320 nm, recording the peak absorbance at 280 nm, and subtracting a baseline absorbance reading at 310 nm. The extinction coefficient for protein solubilized in organic solvent was experimentally determined to be the same as for protein dissolved in H2O. Direct Solubilization Directly solubilized proteins were prepared according to the procedure described by Akbar et al.30 In particular, a small amount of nano filtered water was added to a flame dried vial, followed by addition of 2 mM AOT dissolved in isooctane. Native protein was added to the isooctane/water mixture to yield a final concentration of 1 mg/mL suspended enzyme and the contents were magnetically stirred for ca.
45 min. After stirring, the vial was centrifuged at 2700 ? g for 5 min and the supernatant was transferred to a separate vial and evaporated to dryness before being dissolved in the final reaction solvent. Bergenin Immobilization and Cleavage on CPG In a flame dried vial, 30 mM bergenin was dissolved in a 1:1 DMF:DCM solution, followed by addition of 1 eq. 1 hydroxybenzotriazole, 0.2 eq. 4 dimethyl aminopyridine, and 0.25 eq. carboxy CPG. After allowing the resin to settle to the bottom of the vial, the liquid above the resin was sampled for LC analysis of initial bergenin concentration and 1 eq. 1,3 diisopropyl carbodiimide was added to initiate the immobilization reaction. Reaction vials were incubated for 50 h at 25, 100 rpm.

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