Gordon Lowe

The Cysteine proteinases

Abstract The cysteine proteinases form a group of enzymes which depend for their activity on the thiol group of a cysteine residue. They occur in mammals plants and bacteria and fulfil a wide variety of functions. This report focuses attention on the plant proteinases, papain, ficin and stem-bromelain, and streptococcal proteinase from haemolytic streptococci. Papain is the most extensively studied member of this family and its tertiary structure is known. The review highlights therefore the present state of knowledge of the structure, specificity and mechanism of action of this enzyme.

4-Picoline-2,2′:6′,2″-terpyridine-platinum(II) — A potent intercalator of DNA

4‐Picoline‐2,2′:6′,2″‐terpyridine‐platinum(II) is shown in a ligation assay to unwind and so intercalate into DNA. Circular dichroism is used to determine an equilibrium binding constant of approximately 2 × 107 M−1 for the most stable binding mode of 4‐picoline‐2,2′:6′,2″‐terpyridine‐platinum(II) to poly[d(A‐T)2] with a site size of about 4 base pairs, and about 1 × 106 M−1 for a second binding mode with a site size of about 2 base pairs. Fluorescence spectroscopy provides further evidence for the strong equilibrium binding constant of 4‐picoline‐2,2′:6′,2″‐terpyridine‐platinum(II) in that it displaces ethidium bromide bound to DNA. The double positive charge on 4‐picoline‐2,2′:6′,2″‐terpyridine‐platinum(II), together with the intercalative binding mode is probably responsible for the large binding constant.

Aryl hydrazides as linkers for solid phase synthesis which are cleavable under mild oxidative conditions

Abstract We have developed and acid/base stable aryl hydrazide linker which is readily coupled to solid phase resins. Cleavage is specific and facile, requiring a copper (II) catalyst, base and a nucleophile to proceed. The conditions are compatible with all 20 proteinogenic amino acids and quantitative cleavage is achieved within 2 hours at 20°C to give peptides with C-terminal acid, amide or ester functionalities. Aryl hydrazides also offer scope as simple “traceless” linkers.

Evaluation of resins for on-bead screening: a study of papain and chymotrypsin specificity using PEGA-bound combinatorial peptide libraries.

TentaGel, ArgoGel and PEGA resins were evaluated for on-bead biological screening, using a fluorescently-labelled peptide attached to each and assayed for papain activity. Peptide attached to PEGA was cleaved in near quantitative yield at the expected sites, whilst an identical sequence on TentaGel and ArgoGel beads was hydrolysed in very low yields and nonspecifically on ArgoGel. The compatibility of PEGA with enzymes was further demonstrated by the determination of subsite specificities of papain and chymotrypsin using PEGA-bound peptide libraries, which proved to be similar to those observed in free solution.

Structure and properties of TentaGel resin beads: implications for combinatorial library chemistry.

SummaryIn view of the widespread use of TentaGel resin beads for the synthesis of combinatorial libraries, the properties of TentaGel resin have been examined using a combination of confocal laser microscopy and NMR spectroscopy. Evidence is presented that trypsin, a 23.5-kDa enzyme, can penetrate to the core of 90-μm TentaGel beads, and that the matrix of such beads permits molecular motion at a similar rate to that in solution. The beads act as a separate gel phase rather than as a porous solid. These conclusions have important implications for the bioassay of on-bead combinatorial chemical libraries.

A study of D52S hen lysozyme‐G1cNAc oligosaccharide complexes by NMR spectroscopy and electrospray mass spectrometry

The production of a mutant hen lysozyme is described in which Asp‐52, one of the catalytically important residues, is replaced by Ser. The mutant enzyme has very low catalytic activity but NMR studies show that its structure is closely similar to that of the wild‐type protein. NMR experiments also show that well defined complexes are formed with GlcNAc4 and GlcNAc6 bound in the active Site of the mutant enzyme. Then complexes have been examined using electrospray mass spectrometry (ESMS). The most intense peaks arise from the uncomplexed protein indicating that dissociation takes place in the mass spectrometer under the conditions used here. Peaks from minor species corresponding to complexes between the protein and the oligosaccharides are, however, also observed. The possibility that the latter arise from novel covalent enzyme‐saccharide complexes is discussed.

An investigation of the binding of protein proteinase inhibitors to trypsin by electrospray ionization mass spectrometry

The binding of BPTI and SBTI with trypsin has been investigated by ESI MS, using the mutant K15V‐BPTI and the chemically modified RcamBPTI as controls. Although high cone voltages (+80 V) produce sharp spectra of BPTI, RcamBPTI, SBTI and trypsin alone, the complexes of BPTI, RcamBPTI and SBTI with trypsin undergo partial dissociation due to collisional activation. At lower cone voltages (+40 V) these non‐covalent complexes are stable. The charge distribution on the trypsin and the inhibitors produced by gas phase dissociation of the complexes are markedly different from those of the components alone, indicating that ESI MS provides a novel probe for exploring the ionic interactions at the contact surface of proteins. Moreover, by determining the cone voltage at which the gas phase dissociation of complexes occurs it may be possible to use ESI MS to compare the binding energies of closely related complexes.

Synthesis and properties of novel pyrrolidinyl PNA carrying β-amino acid spacers

Abstract Novel pyrrolidinyl peptide nucleic acids comprising alternate sequences of nucleobase-modified d -proline and β-amino acid spacers selected from l -aminopyrrolidine-2-carboxylic acid, d -aminopyrrolidine-2-carboxylic acid, (1 R ,2 S )-2-aminocyclopentane carboxylic acid and β-alanine were synthesized using solid phase methodology. Gel-binding shift assay revealed that only the homothymine PNA decamer bearing d -aminopyrrolidine-2-carboxylic acid spacer binds with (dA) 10 .

Synthesis and properties of chiral peptide nucleic acids with a N-Aminoethyl-d-proline backbone

A synthon of D-proline substituted at the 4-position by thymine and at N by a flexible aminoethyl linker, has been used to prepare a novel chiral peptide nucleic acid (cPNA) with (2R,4R) stereochemistry using solid phase methodology. The homothymine decamer cPNA binds to complementary polyadenylic acid to form a 2:1 hybrid with high affinity and specificity according to UV and CD studies, whereas no binding to the corresponding polydeoxyadenylic acid was observed.

Synthesis and configurational assignment of some novel bicyclic sulphamidites and sulphamidates

Abstract ( S )-Prolinol and 2( RS )-piperidine-methanol each react with thionyl chloride to give a pair of diastereoisomeric bicyclic sulphamidites which differ only in their configuration at sulphur. By contrast 2( RS )-pyrrolidine-ethanol and 2( RS )-piperidine-ethanol each react with thionyl chloride to give single diastereoisomeric products. The configurations of these bicyclic sulphamidites, and the sulphamidates derived from them, have been determined.