Alison M. Hill

Antimycobacterial calixarenes enhance innate defense mechanisms in murine macrophages and induce control of Mycobacterium tuberculosis infection in mice.

ABSTRACT Tuberculosis remains the leading cause of death among infectious diseases, accounting for more than two million deaths annually. The incidence of the disease is increasing globally, partially because of the resurgence of drug-resistant strains of Mycobacterium tuberculosis. Calixarenes are macrocyclic oligomers, some of which are able to modify the growth of M. tuberculosis in infected cells. Most experimental work has been carried out with Macrocyclon, also known as HOC 12.5EO. In this study, we demonstrate that Macrocyclon is effective in controlling M. tuberculosis infections, and we provide evidence that its effect is partially mediated by an l-arginine-dependent mechanism of macrophage activation that involves the activity of the inducible nitric oxide synthase. We also show that Macrocyclon is effective in athymic and major histocompatibility complex class II−/− mice and synthesized a number of structurally related calixarenes expressing significant antimycobacterial activity.

Primary and secondary metabolism regulates lipolysis in appressoria of Colletotrichum orbiculare.

The conidia of Colletotrichum orbiculare, the causal agent of cucumber anthracnose, develop appressoria that are pigmented with melanin for host plant infection. Premature appressoria contain abundant lipid droplets (LDs), but these disappear during appressorial maturation, indicating lipolysis inside the appressorial cells. The lipolysis and melanization in appressoria require the peroxin PEX6, suggesting the importance of peroxisomal metabolism in these processes. To investigate the relationships between appressorial lipolysis and fungal metabolic pathways, C. orbiculare knockout mutants of MFE1, which encodes a peroxisomal multifunctional enzyme, were generated in this study, and the phenotype of the mfe1 mutants was investigated. In contrast to the wild-type strain, which forms melanized appressoria, the mfe1 mutants formed colorless nonmelanized appressoria with abundant LDs, similar to those of pex6 mutants. This indicates that fatty acid β-oxidation in peroxisomes is critical for the appressorial melanization and lipolysis of C. orbiculare. Soraphen A, a specific inhibitor of acetyl-CoA carboxylase, inhibited appressorial lipolysis and melanization, producing phenocopies of the mfe1 mutants. This suggests that the conversion of acetyl-CoA, derived from fatty acid β-oxidation, to malonyl-CoA is required for the activation of lipolysis in appressoria. Surprisingly, we found that genetically blocking PKS1-dependent polyketide synthesis, an initial step in melanin biosynthesis, also impaired appressorial lipolysis. In contrast, genetically or pharmacologically blocking the steps in melanin synthesis downstream from PKS1 did not abolish appressorial lipolysis. These findings indicate that melanin biosynthesis, as well as fatty acid β-oxidation, is involved in the regulation of lipolysis inside fungal infection structures.

Investigation of the early stages in soraphen A biosynthesis

The unusual benzoate starter unit in soraphen A derives from phenylalanine via cinnamate in a beta-oxidative (plant-like) pathway; 3-phenyl-3-hydroxypropanoate incorporates directly into soraphen by loading onto module 2 of the PKS and indirectly from the beta-oxidative pathway to generate benzoyl CoA.

Investigation of glycerol incorporation into soraphen A

Glycerol has been incorporated mid-chain into the polyketide soraphen A 1 at C-3,4 and C-11,12; the pro-(S)-hydroxymethyl group of glycerol is lost and one of the hydrogens in the pro-(R)-hydroxymethyl group is retained at C-11 which excludes hydroxymalonate as the immediate precursor to the vicinal methoxy groups at C-11,12.

The selective functionalisation and difunctionalisation of p-substituted calix[6]arene and calix[8]arenes using hydrophilic moieties

Methodologies to access water soluble large ringed calixarenes in good yield using efficient synthetic procedures have been investigated. Symmetrical partial functionalisations at the lower rim are described using activated [n]ethylene glycol chains and the addition behaviour contrasted with that of bromoalkanenitriles which proceeds with no observed regioselectivity. Full functionalisations of the calixarenes bearing hydrophilic groups are then investigated and a two-step procedure established which appears to be generally applicable for the addition of different [n]ethylene glycol chains. Furthermore, difunctionalisation under different reaction conditions are described. Throughout, strategies for the characterisation of these high mass compounds are outlined.

A highly efficient synthesis of [1-13C, 18O]- and [1-13C, 2H2]-glycerol for the elucidation of biosynthetic pathways

Abstract Labeled glycerol is a widely used biochemical probe to investigate biosynthetic pathways. A highly efficient synthesis of [1- 13 C, 18 O]- and [1- 13 C, 2 H 2 ]-glycerol is described in which the 13 C label is introduced using cyanide. The 18 O label was introduced by a Pinner synthesis and reduction of the ester 5 allowed incorporation of the 2 H labels.

Type I Modular PKS

Polyketides (PKs) are secondary metabolites produced by bacteria, fungi, and plants. Many of these metabolites have important biological activity and have found clinical use as antibiotics, antifungals, anticancer drugs, and immunosuppressants. In this chapter, we discuss the modular type I polyketide synthases (PKSs) using the 6-deoxyerythronolide B synthase (DEBS) to illustrate reactions and enzymes used in PK synthesis, and the protein–protein interactions that are required for this process. A detailed discussion of the quaternary structure of PKSs is given, including evidence from both solution and structural studies, and from intact synthases and isolated domains. Other PKSs are discussed to show different strategies for chain initiation, extension, and termination. The importance of reaction kinetics for control of fidelity in product formation is discussed, and two alternative effects, residence-time control and occupancy-level control, are introduced. Some commercial applications of genetic manipulation of modular PKSs are also discussed.

The pentaketide hydroxyacyl intermediate in aspyrone biosynthesis in Aspergillus melieus is shown to be the (S)-enantiomer using deuterium-labelled precursors and 2H NMR

Following administration of both enantiomers of the pentaketide intermediate in the biosynthesis of aspyrone labelled with deuterium to Aspergillus melieus, it is shown by 2H NMR that only the (S)-isomer is incorporated intact into the natural product.

Investigation of the stereochemistry of the tri- and tetra-ketide hydroxyacyl intermediates in the biosynthesis of the polyketide aspyrone in Aspergillus malleus using deuterium labelling and deuterium NMR spectroscopy

Following administration of both enantiomers of the hydroxyacyl tri- and tetra-ketide intermediates in the biosynthesis of aspyrone labelled with deuterium to Aspergillus melleus, it is shown by 2H NMR that in each case only the (S)-isomer is incorporated intact into the natural product.