Understanding protease catalysed solid phase peptide synthesis

Abstract

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.

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

“…Amidation would be of particular interest because many natural amide synthetases,s uch as NRPS [14] and ATP-grasp enzymes [15,16] ,s uffer from very narrow substrate specificity,w hich limits their applications in biocatalysis.A lternatively,h ydrolases (such as lipases [17] and proteases [18] ), which are commonly used for amide formation, [2] require systems in which very little water is present, such as organic solvents,todrive the reaction towards amide bond formation. [19][20][21][22] Four CARc andidates (CARmm from Mycobacterium marinum, [3] CARni from Nocardia iowensis, [4] CARtp from Tsukamurella paurometabola, [10] and CARse from Segniliparus rotundus, [23] )were produced recombinantly in Escherichia coli,w ith the coexpressed gene for the Bacillus subtilis phosphopantetheinyl transferase (PPTase;S fp), [24] which is required for post-translational addition of the PPant group. [4] These CARs were purified (Supporting Information, Figure S1) and the amidation of carboxylic acid substrates 1-6 was tested.…”

Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides

“…It is known that related cyclic peptidic trypsin inhibitors such as BPTI 49,50 and SFTI-I 51 bind to the enzyme 30 active site in an inter-convertible mixture of non-covalent enzyme-peptide complex and covalently-bound peptidylenzyme, a non-productive analogue of the acyl-enzyme intermediate in proteolytic cleavage. Building on previous work that shows that proteases can be induced to synthesise a 35 peptide bond by careful control of the reaction conditions, [52][53][54][55] we reasoned that a linear cyclotide backbone acid bearing the P 1 residue at the C-terminus might be a viable substrate for protease-mediated ligation, thus enabling synthesis of the cyclic backbone without the need for a C-terminal thioester. 40 An initial proof-of-principle experiment was carried out to test whether this concept could be applied for total synthesis of the naturally-occurring MCoTI-II.…”

SPOT Synthesis of Peptide Arrays on Self‐Assembled Monolayers and their Evaluation as Enzyme Substrates

“…[20] In previous studies, we have shown that proteases can also be used for the synthesis of peptide bonds on polymer surfaces, such as PEGA (poly(ethyleneglycol) grafted onto polyacrylamide). [23,24] Detailed analysis of such reactions was troublesome because of the need for cleavable linkers, which were in some cases also labile in the presence of proteases. [25] The SAM platform proved to be much more successful (Figure 1 D).…”