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The focus of the presented work is the engineering of polyketide-derived natural products in different stages of their biosynthesis. Within this context, one project centres on the engineering of a polyketide synthase (PKS) to enable the production of unnatural products, while another project focusses on unravelling intricacies of biosynthetic pathways and the understanding of tailoring enzymes. The iterative PKS TenS is known to produce the pentaketide-tyrosine hybrid tenellin. Products of iterative PKS can differ in reduction level, methylation pattern and chain length. Thus, programming must take place. In TenS, the programming of the chain length is known to be determined by the KR domain. Structure prediction of the KR domain enabled the identification of a relevant substrate binding helix, which was the focus of in vivo swap experiments. Swap experiments were conducted with analogous substrate binding helices from related PKS, which produce similar hexa- and heptaketides. The hybrid PKS products were analysed and indicated an influence of the helix swap on the programming. Further, the simultaneous swap of only four rationally selected amino acids showed a small effect on the chain length, as well as mutation of single isolated amino acids during a following alanine scan of the helix. The fungus Hypoxylon lienhwacheense is a producer of the tropolone alkyl citrate conjugates lienhwalides A-C and cordyanhydride B, consisting of three alkyl citrate units linked linearly. Genome analysis revealed two distinct gene clusters proposed to be responsible for the biosynthesis of tropolones and alkyl citrates, respectively. Both clusters were analysed bioinformatically, followed by the elucidation of both pathways by a combination of heterologous expression in the host Aspergillus oryzae NSAR1 and protein isolation with following in vitro assays. For alkyl citrates, the biosynthesis was revealed to be analogous to other known fungal alkyl citrate biosynthetic pathways.

Boron complexes based on tropolone, a seven-membered non-benzenoid aromatic compound, were synthesized. We revealed that the aromaticity of tropolone was enhanced by boron complexation. Importantly, all boron complexes exhibited photoluminescence, and room-temperature phosphorescence (RTP) was observed from the halogenated derivatives, BrTpB and ITpB in the crystalline states. Furthermore, a non-substituted complex, TpB, showed phosphorescence without any heavy atoms in the solution state at 77 K. DFT calculation data suggest that TpB should have large enough spin-orbit coupling (SOC) constants to accept the intersystem crossing. The RTP of halogenated complexes were achieved by heavy atom effects. Significantly, this is the first report of RTP from boron tropolonate complexes.

This review summarises the known structures, biological activities, and biosynthetic pathways of the tropolone sesquiterpenoid family of fungal secondary metabolites. Synthesis of this knowledge allows likely structural and stereochemical misassignments to be revised and shows how the compounds can be divided into three main biosynthetic classes based on the stereochemistry of key biosynthetic steps.

The sesquiterpene-tropolones belong to a distinctive structural class of meroterpene natural products with impressive biological activities, including anticancer, antifungal, antimalarial, and antibacterial. In this article, we describe a concise, modular, and cycloaddition-based approach to a series of sesquiterpene mono- and bistropolones, including (-)-epolone B, (+)-isoepolone B, (±)-dehydroxypycnidione, and (-)-10-epi-pycnidione. Alongside the development of a general strategy to access this unique family of metabolites were computational modeling studies that justified the diastereoselectivity observed during key cycloadditions. Ultimately, these studies prompted stereochemical reassignments of the pycnidione subclass and shed additional light on the biosynthesis of these remarkable natural products.

Tropolone sesquiterpenoids (TS) are an intriguing family of biologically active fungal meroterpenoids that arise through a unique intermolecular hetero Diels–Alder (hDA) reaction between humulene and tropolones. Here, we report on the combinatorial biosynthesis of a series of unprecedented analogs of the TS pycnidione 1 and xenovulene A 2. In a systematic synthetic biology driven approach, we recombined genes from three TS biosynthetic gene clusters (pycnidione 1, xenovulene A 2 and eupenifeldin 3) in the fungal host Aspergillus oryzae NSAR1. Rational design of the reconstituted pathways granted control over the number of hDA reactions taking place, the chemical nature of the fused polyketide moiety (tropolono- vs. monobenzo-pyranyl) and the degree of hydroxylation. Formation of unexpected monobenzopyranyl sesquiterpenoids was investigated using isotope-feeding studies to reveal a new and highly unusual oxidative ring contraction rearrangement. © 2020 Wiley-VCH GmbH

The inhibition and binding of three metal-binding pharmacophores (MBPs), 2-hydroxycyclohepta-2,4,6-trien-1-one (tropolone), 2-mercaptopyridine-N-oxide (1,2-HOPTO), and 2-hydroxycyclohepta-2,4,6-triene-1-thione (thiotropolone) to human carbonic anhydrase II (hCAII) and a mutant protein hCAII L198G were investigated. These MBPs displayed bidentate coordination to the active site Zn(II) metal ion, but the MBPs respond to the mutation of L198G differently, as characterized by inhibition activity assays and X-ray crystallography. The L198G mutation increases the active site volume thereby decreasing the steric pressure exerted on MBPs upon binding, allowing changes in MBP coordination to be observed. When comparing the binding mode of tropolone to thiotropolone or 1,2-HOPTO (O,O versus O,S donor sets), structural modifications of the hCAII active site were shown to have a stronger effect on MBPs with an O,O versus O,S donor set. These findings were corroborated with density functional theory (DFT) calculations of model coordination complexes. These results suggest that the MBP binding geometry is a malleable interaction, particularly for certain ligands, and that the identity of the donor atoms influences the response of the ligand to changes in the protein active site environment. Understanding underlying interactions between a MBP and a metalloenzyme active site may aid in the design and development of potent metalloenzyme inhibitors.

The inhibition and binding of three metal-binding pharmacophores (MBPs), 2-hydroxycyclohepta-2,4,6-trien-1-one (tropolone), 2-mercaptopyridine-N-oxide (1,2-HOPTO), and 2-hydroxycyclohepta-2,4,6-triene-1-thione (thiotropolone) to human carbonic anhydrase II (hCAII) and a mutant protein hCAII L198G were investigated. These MBPs displayed bidentate coordination to the active site Zn(II) metal ion, but the MBPs respond to the mutation of L198G differently, as characterized by inhibition activity assays and X-ray crystallography. The L198G mutation increases the active site volume thereby decreasing the steric pressure exerted on MBPs upon binding, allowing changes in MBP coordination to be observed. When comparing the binding mode of tropolone to thiotropolone or 1,2-HOPTO (O,O versus O,S donor sets), structural modifications of the hCAII active site were shown to have a stronger effect on MBPs with an O,O versus O,S donor set. These findings were corroborated with density functional theory (DFT) calculations of model coordination complexes. These results suggest that the MBP binding geometry is a malleable interaction, particularly for certain ligands, and that the identity of the donor atoms influences the response of the ligand to changes in the protein active site environment. Understanding underlying interactions between a MBP and a metalloenzyme active site may aid in the design and development of potent metalloenzyme inhibitors.

English: The aim of this study was to gain a further insight into the bonding modes of 1,5-cyclooctadiene to various middle to late transition metal centres. The intension was to design a theoretical model of the influences of the variations of the metal centres and ligands on the “Venus fly-trap” system. Two principle geometric features were classified in the 1,5- cyclo-octadine when co-ordinated, i.e. the (bite) angle of the olefinic moieties to the metal centre, and the (jaw) angle, i.e. the dihedral angle between the two planes formed between the alkane ethylene groups. The latter mimicking the “jaws” of the Venus flytrap. A range of 1,5-cyclo-octadiene metal complexes were successfully synthesized and characterized via NMR and IR spectroscopy, the metal centers chosen were that of rhodium(I), platinum(II) and palladium(II). The ligands selected were divided into three categories namely · β-diketonato · β-enaminonato · tropolonato From each of these categories ligands were selected to vary the electron withdrawing or donating ability. Both symmetrical and non-symmetrical ligands were made use of, to see the influence of the nitrogen trans effect on the 1,5-cyclo-octadiene moiety. Finally, the tropolonato category added the influence of the smaller five membered metal chelate ring. A single crystal X-ray crystallographic study of the complexes was undertaken. The reported X-ray crystallographic structure determinations include the following complexes: [Pd(cod)(acac)]PF6 (1, Monoclinic P21/n, R = 3.59 %), [Pd(cod)(acac)]BF4 (2, Orthorhombic Pca21, R = 2.24 %), [Pd(cod)(thtfac)]PF6 (3, Monoclinic P21/n, R = 2.43 %), [Pd(cod)(thtfac)]BF4 (4, Monoclinic P21/n, R = 2.83 %), [Pd(cod)(tfacac)]PF6 (5, Triclinic P1 , R = 13.41 %), [Pd(cod)(hfacac)]PF6 (6, Monoclinic P21/c, R = 2.60 %), [Pt(cod)(acac)] (7, Orthorhombic Pca21, R = 1.75 %), [Pt(cod)(acac)]PF6 (8, Monoclinic P21/n, R = 3.02 %), [Pt(cod)(dbm)]BF4 (9, Triclinic P1), [Pt(cod)(thtfac)]BF4 (10, Monoclinic P21/n, R, Afrikaans: Die doelwit van hierdie studie was om ‘n meer breedvoerige insig te verkry oor die verbindings van 1,5-siklo-oktadieen met verskeie middel- tot laat oorgangs metal kerne, met die doel om ‘n teoretiese model te skep. Hierdie model sal die invloed van die wisseling van die metal kerne en die ligande op die “Venus fly-trap” sisteem illustreer. Twee hoof geometriese karakter eienskappe was geklassifiseer wanneer die siklooktadieen gekoordineer is, (a) die (bite) hoek van die olefeniese gedeeltes na die metal kern, en (b) die (jaw) hoek wat die dihidriese hoek tussen die twee oppervlakkes is wat gevorm het tussen die alkaan eteen groepe. Laasgenoemde naboots die “jaws” van die “Venus fly-trap”. ‘n Reeks van 1,5-siklo-oktadieen metal komplekse was suksesvol gesintetiseer en geklassifiseer met behulp van KMR en IR spektroskopie. The metal kerne wat gekies was, is rhodium(I), platinum(II) en palladium(II). Die ligande wat gekies was kan gegroepeer word in drie afdelings, naamlik: · β-diketonato · β-enaminonato · tropolonato Ligande uit hierdie kategorieë is gekies om elektron ontrekking of skenking vermoeë te varieer. Beide simmetries en nie-simmetriese ligande is gebruik om die invloed van die stikstof trans effek op die 1,5-siklo-oktadieen gedeelte. Laastens het die tropolonato kategorie die invloed van die kleiner vyf-lid metal chelaat ring bygedra. ‘n Enkel kristal x-straal kristallografiese studie van die komplekse was ook onderneem. Die x-straal kristallografiese struktuur bepalings wat verkryg was het die volgende komplekse ingesluit: [Pd(cod)(acac)]PF6 (1, Monoklien P21/n, R = 3.59 %), [Pd(cod)(acac)]BF4 (2, Orthorombies Pca21, R = 2.24 %), [Pd(cod)(thtfac)]PF6 (3, Monoklien P21/n, R = 2.43 %), [Pd(cod)(thtfac)]BF4 (4, Monoklien P21/n, R = 2.83 %), [Pd(cod)(tfacac)]PF6 (5, Triklien P1 , R = 13.41 %), [Pd(cod)(hfacac)]PF6 (6, Monoklien P21/c, R = 2.60 %), [Pt(cod)(acac)] (7, Orthorombies Pca21, R = 1.75 %), [Pt(cod)(acac)]PF6 (8, Monoklien P21/n, R = 3.02, National Research Foundation (NRF)