Biomedical subjects
Publications and source records attributed to Sabine L Flitsch.
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.
Dolichols are polyisoprenoid lipid components of mammalian membranes consisting of an average of 20 head-to-tail linked isoprene units of which the first isoprene is fully saturated. The unusual size of these lipids is intriguing and poses questions about the role of dolichol structure in biological processes. In order to probe structure and function we have synthesised potential dolichyl analogues that retain only the first two isoprene units and carry a second functional group within the terminal lipid chain. Such analogues were evaluated as substrates for a key enzyme in the dolichyl-dependent pathway of glycan biosynthesis, dolichyl phosphate mannose (Dol-P-Man) synthase. It was shown that some functional groups, including labels such as biotin, could be tolerated. When the synthetic analogues were attached to a solid support they were still substrates for the Dol-P-Man system and thus allowed the enzymatic solid-phase synthesis of glycolipids.
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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Synthetic bifunctional analogues 4a, b and 14 of dolichol phosphate 1 were attached to solid support and were shown to be substrates for Dol-P-Man synthase.
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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[reaction: see text]. Glycoproteins are particularly suited to protein semisynthesis since homogeneous samples for biological analyses are not readily available using traditional recombinant techniques. Here we apply glycosyl iodoacetamides, normally used for the modification of bacterially derived proteins, to solid-phase glycopeptide synthesis. This provides access to glycopeptide alpha-thioesters, which may lend themselves to the semisynthesis of homogeneous N-linked glycoprotein mimics and novel glycopeptide libraries.
A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from Rhodococcus sp. strain NCIMB 9784 which is of unique primary structural organization. Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins. The reductase partner comprises flavin mononucleotide- and NADH-binding motifs and a [2Fe2S] ferredoxin-like center. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a soluble form. A recombinant strain of E. coli was able to mediate the O dealkylation of 7-ethoxycoumarin in good yield, despite the absence of any recombinant redox proteins. This unprecedented finding leads us to propose that P450RhF represents the first example of a new class of cytochromes P450 in which the reducing equivalents are supplied by a novel reductase in a fused arrangement.
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