David Gani

The cleavage activities of aphthovirus and cardiovirus 2A proteins.

The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A proteins cleaving C-terminally. Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa. We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/beta-glucuronidase [CAT-2A-GUS]) polyprotein system devoid of any other FMDV sequences with high (approximately 85%), although not complete, cleavage. In this paper we show that insertion of upstream FMDV capsid protein 1 D sequences increases the activity. In addition, we have demonstrated that the cardiovirus Theilers murine encephalomyelitis virus(TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high efficiency--if not completely. The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into the CAT/GUS artificial polyprotein system (in a single ORF). This recombinant [CAT-deltaTME2A-GUS] polyprotein was able to mediate cleavage with high (approximately 85%) efficiency--directly comparable to the activity observed when FMDV 2A was inserted. A similar insertion into [CAT-GUS] of the C-terminal 19 aa of the cardiovirus encephalomyocarditis virus (EMC) 2A, together with the N-terminal proline residue of protein 2B, produced a [CAT-delta EMC2A-GUS] polyprotein which also mediated cleavage at approximately 85%. Analysis of the products of expression of these artificial polyproteins in a prokaryotic translation system did not, apparently, reveal any GUS cleavage product.

Resin-immobilised benzyl and aryl vinyl sulfones: New versatile traceless linkers for solid-phase organic synthesis

Abstract New polystyrene-based resins containing benzyl and aryl vinyl sulfone groups are described. The vinyl sulfone group reacts efficiently with 2° amines, via conjugate addition, and the resin-bound 3° amine products can be quatermised through alkylation. Subsequent deamination to give 3° amines and the regenerated vinyl sulfone occurs in moderate to good yield. Both systems can be recycled and show moderate stability to acids and high stability to nucleophiles including Grignard reagents.

Enantiospecific synthesis of 3-substituted aspartic acids via enzymic amination of substituted fumaric acids

Abstract The use of the enzyme 3-methylaspartase in the synthesis of L-aspartic acids containing 3-halogeno- or 3-alkyl- substituents, in the (S)-configuration, and also some of the corresponding C-3 deuteriated isotopomers is described.

Synthesis of stereochemically defined phosphonamidate-containing peptides: Inhibitors for the HIV-1 proteinase

Abstract Phosphonamidate-containing peptidic substrate analogues of the HIV-1 gag-pol proteinase-reverse transcriptase junction {-Phe-ψ[PO2-N]-(S)-Pro- and -Phe-ψ[P(OMe)O-N]-(S)-Pro-}, mimicks for the transition states for proteolysis, have been synthesised. The absolute stereochemistry at C-1 of the phosphonophenylalanine residue was determined by X-ray crystallography. Boc-(S)-Asn-Phe-ψ[PO2-N]-(S)-Ile-NH-i-Bu and Boc-(S)-Asn-(R)-Phe-ψ[P(OMe)O-N]-(S)-Pro-(S)-Ile-NH-i-Bu inhibit the HIV-1 proteinase.

Overexpression, purification, crystallization and data collection of 3-methylaspartase from Clostridium tetanomorphum

3-Methylaspartase (E.C. 4.3.1.2) catalyses the reversible anti elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic acid as well as a slower syn elimination from the (2S,3R)-epimer, L-erythro-3-methylaspartic acid. The anti-elimination reaction occurs in the second step of the catabolic pathway for glutamic acid in Clostridium tetanomorphum. The reverse reaction is of particular interest because the addition of ammonia to substituted fumaric acids is highly stereoselective and gives highly functionalized amino acids. The mechanism of the transformation is unusual and of considerable interest. 3-Methylaspartase from C. tetanomorphum has been overexpressed and purified from Escherichia coli. Crystals of the enzyme have been obtained by sitting-drop vapour diffusion. Two native data sets have been collected, one in-house on a rotating-anode generator to 3.2 A and one at the European Synchrotron Radiation Facility to 2.0 A. A 2.1 A data set has been collected on a crystal of selenomethionine protein. Combining the data sets identify the space group as P2(1)2(1)2, with unit-cell parameters a = 110.3, b = 109.9, c = 67.2 A, alpha = beta = gamma = 90 degrees. The asymmetric unit contains two monomers with 42% solvent. A self-rotation function indicates the presence of a twofold axis, consistent with a biological dimer.

Synthesis of d- and l-myo-inositol 1-phosphorothioate, substrates for inositol monophosphatase

Abstract The D- and L- enantiomers of myo-inositol 1-phosphorothioate have been synthesized from 2,3,4,5,6-pentakis-O-benzyl myo-inositol in 6 steps, both compounds are substrates for inositol monophosphatase. D-glucopyranose 6-phosphorothioate did not serve as a substrate for the enzyme inositol synthase in an alternative synthesis of L-myo-inositol 1-phosphorothioate.

Butyrate metabolism in streptomycetes. Characterization of an intramolecular vicinal interchange rearrangement linking isobutyrate and butyrate in Streptomyces cinnamonensis

The incorporations of various carbon-13 and deuterium labelled forms of isobutyrate into the polyether antibiotic monensin-A have provided evidence for the existence of a novel rearrangement in whole cells of Streptomyces cinnamonensis, which leads to the conversion of isobutyrate into butyrate. This rearrangement is shown to proceed in an intramolecular fashion by migration of the carboxy carbon of isobutyrate to the 2-pro-S methyl, with a concomitant back migration of a hydrogen atom from this methyl group predominantly into the 3-pro-R position in butyrate. Formally, therefore, the carboxy carbon is replaced with overall retention of configuration, in a vicinal interchange rearrangement. The significance of these observations with regard to the coenzyme requirements of the rearrangement, and its relationship to polyether and macrolide antibiotic production in Streptomycetes is discussed.

Stereospecific synthesis of α-deuteriated α-amino acids: regiospecific deuteriation of chiral 3-isopropyl-2,5-dimethoxy-3,6-dihydropyrazines

Base-catalysed deuteriation of (3R)- or (3S)-3-isopropyl-2,5-dimethoxy-3,6-dihydropyrazines in refluxing CH3O2H–2H2O gives the [6-2H2]-isotopomer in excellent yields without disturbing the stereogenic centre at C-3. These compounds provide convenient and efficient access to a range of (R)- and (S)-α-deuteriated α-amino acids, including serine, aspartic acid, allylglycine and phenylalanine, via alkylation of the butyllithium generated C-6 anion.

A method for the quantification of resin loading using 19F gel phase NMR spectroscopy and a new method for benzyl ether linker cleavage in solid phase chemistry

A simple and efficient method for monitoring and quantifying the extent of loading onto polymer resin supports for solid-phase synthesis using 19F gel-phase NMR spectroscopy is described. This assay was utilised in the synthesis of an inositol monophosphatase inhibitor on Merrifield resin. A series of Merrifield resin derived benzylic ethers were prepared and were cleaved from the resin when treated with SnCl4 at room temperature to give the expected alcohol, phenol or olefin. This new cleavage method was used to remove the inositol monophosphatase inhibitor from the resin. A method for quantifying polymer support loading, using 19F gel phase NMR spectroscopy and a facile method for the cleavage of Merrifield resin derivatives is described in the context of a solid phase synthesis of phosphatase inhibitor 6.

Syntheses of (2S,3R)- and (2S,3R)[3-2h]- 3-methylaspartic acid: slow substrates for a syn-elimination reaction catalysed by methylaspartase.

Abstract Methylaspartase catalyses the slow syn -elimination of ammonia from the (2S,3R)-[L- erythro ]-diastereomer of the natural substrate, (2S,3S)-3-methylaspartic acid, to give mesaconic acid. To provide material of sufficient stereochemical purity to probe the mechanism of the reaction, two synthetic routes to (2S,3R)- and (2S,3R)[3- 2 H]- 3-methylaspartic acid were devised. The use of these (2S,3R)-3-methylaspartic adds revealed that the enzymic reaction does not involve C-3 epimerisation followed by normal anti -elimination, ruling-out the possibility of a carbanion intermediate. Conversely, the substrate displayed very large primary deuterium isotope effects indicating rate-limiting CH bond cleavage.