Biomedical subjects
Publications and source records attributed to Rein V Ulijn.
PEGA supports functionalised with permanent charges show superior swelling properties in aqueous media when compared to neutral PEGA; a novel positively charged PEGA resin significantly improves penicillin G amidase (PGA) catalysed biotransformation on solid support, by favouring accessibility of the negatively charged enzyme.
A protease (thermolysin) was used to directly synthesise a number of dipeptides from soluble Fmoc-amino acids onto a solid support (PEGA1900) in bulk aqueous media, often in very good yields. This shift in equilibrium toward synthesis is remarkable because for soluble dipeptides in aqueous solution hydrolysis rather than synthesis is observed. Three possible reasons for the equilibrium shift were considered: (i) using a solid support makes it easy to use an excess of reagents, so mass action contributes towards synthesis; (ii) reduction in the unfavourable hydrophobic hydration of the Fmoc group within the solid support compared with the free amino acid in solution and (iii) suppression of the ionization of amino groups linked to the solid phase due to mutual electrostatic repulsion. It was found that under the conditions studied the second effect was most important.
By taking advantage of the reversibility of the thermolysin-catalysed amide synthesis-hydrolysis reaction on a solid support, both L,L and L,D diastereoisomers of dipeptides and L-amino acids are accessible in good yields starting from enantiomeric mixtures of amino acids.
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Thermolysin catalyzed solid-to-solid synthesis of the model peptide Z-L-Phe-L-Leu-NH(2) is practically feasible in water and a range of organic solvents with different physicochemical properties. Excellent overall conversions were obtained in acetonitrile, ethyl acetate, n-hexane, methanol, 2-propanol, tert-amyl alcohol, tetrahydrofuran, toluene and water, while no product precipitation was observed in dichloromethane resulting in a much lower yield. In precipitation driven synthesis the product accumulates both in solution and in the solid phase. It was shown that the highest overall yields (yield in the liquid plus yield in the solid) can be expected in solvents where the substrate solubilities are minimized. The best yields of solid product can be expected in solvents where both product and substrate solubilities are lowest. This was in agreement with experimental observations and should be generally valid.
Thermolysin is catalytically inactive in mixtures of 10-15 % acetonitrile in aqueous buffer. Unexpectedly, dilution of the inactive enzyme with acetonitrile leads to complete recovery of the catalytic activity in a similar way to dilution with aqueous buffer. Circular dichroism and fluorescence studies of thermolysin in the same solvent mixtures reveal discontinuous changes in the overall secondary and tertiary protein structure that correlate well with the reversible differences in catalytic activity. The spectra on either side of the minimum activity point are different from each other, a fact indicating that the enzyme may be able to access two active conformations which are thermodynamically stable in different solvent environments.
The direct enzymatic synthesis of peptides from amino acids is widely used as a useful alternative to chemical synthesis. However, good yields of such enzyme-catalyzed reactions require altered reaction conditions to overcome the bias for hydrolysis in aqueous medium. We argue that the synthesis/hydrolysis equilibrium can be shifted toward synthesis in aqueous medium by immobilizing the amine on solid support. In this report, we show the first examples of solid-phase peptide synthesis catalyzed by a protease in bulk aqueous buffer.
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