Hybrid Lantibiotics: Combining Synthesis and Biosynthesis

The synthesis of two sets of different orthogonally protected lanthionine ready for incorporation into solid phase peptide synthesis to form cyclised peptides is described in this thesis, along with the cyclisation of individual rings D and E and the overlapping rings D and E. Previously developed orthogonally protected lanthionine containing Aloc, allyl, Fmoc and tBu protecting groups was synthesised using published synthetic route developed by Tabor’s group. A novel orthogonally protected lanthionine containing Teoc, TMSE, Fmoc and Tce group derivative has also been synthesised, after carrying several synthetic pathways. Both lanthionine residues contain protecting groups which are orthogonal to each other, which are also orthogonal to the transient Fmoc and permanent Boc/tBu protecting groups which are used in Fmoc based solid phase peptide synthesis. Incorporation of the previously developed lanthionine with Aloc/allyl protecting groups was carried out to form an analogue of ring E of nisin for the first time. Deprotection of the Aloc/allyl protecting groups were carried out with Ph(PPh3)4 using N’,N-dimethyl-barbituric acid (NDMBA). The second orthogonally protected lanthionine was also incorporated into solid phase peptide synthesis to synthesise an analogue of ring D of nisin. This was also to see whether this can be used to synthesise lanthionine-containing thio-ether bridged cyclic peptide by solid phase peptide synthesis. Teoc and TMSE deprotection was carried out in the presence of TBAF without effecting the other side chain and Fmoc protecting groups. Full characterisation of individual rings D and E were obtained. Quadruply orthogonal protecting group strategy was used to synthesise bicyclic peptide with two overlapping lanthionine bridges rings D and E. An effective methodology has been developed for the synthesis of the overlapping rings D and E of nisin by solid phase peptide synthesis

High throughput screening of monoamine oxidase (MAO-N-D5) substrate selectivity and rapid kinetic model generation

Full kinetic models provide insight into enzyme mechanism and kinetics and also support bioconversion process design and feasibility assessment. Previously we have established automated microwell methods for rapid data collection and hybrid kinetic modelling techniques for quantification of kinetic constants. In this work these methods are applied to explore the substrate selectivity and kinetics of monoamine oxidase, MAO-N-D5, from Aspergillus niger. In particular we examine the MAO-N-D5 variant Ile246Met/Asn336Ser/Met348Lys/Thr384Asn to allow the oxidation of secondary amines Initial screening showed that MAO-N-D5 enabled the selective oxidation of secondary amines in 8 and 9 carbon rings, as well as primary ethyl and propyl amines attached to secondary amines of indolines and pyrrolidines. Subsequently we developed a first kinetic model for the MAO-N-D5 enzyme based on the ping-pong bi-bi mechanism (similar to that for the human MAO-A enzyme). The full set of kinetic parameters were then established for three MAO-N-D5 substrates namely; 3-azabicyclo[3,3,0]octane, 1-(2 amino ethyl) pyrrolidine and 3-(2,3-dihydro-1H-indole-1-yl)propan-1-amine. The models for each amine substrate showed excellent agreement with experimentally determined progress curves over a range of operating conditions. They indicated that in each case amine inhibition was the main determinant of overall reaction rate rather than oxygen or imine (product) inhibition. From the perspective of larger scale bioconversion process design, the models indicated the need for fed-batch addition of the amine substrate and to increase the dissolved oxygen levels in order to maximize bioconversion process productivity

Cytotoxicity of some synthetic bis(arylidene) derivatives of cyclic ketones towards cisplatin-resistant human ovarian carcinoma cells

Symmetrical α,αʹ-bis(arylidene)ketones were prepared by acid-catalyzed aldol condensations between aliphatic ketones (e.g., cyclopentanone, 4-alkylcyclohexanones, tetrahydropyran-4-one, and tetrahydrothiopyran-4-one) and two equivalents of an aromatic hydroxyaldehyde (e.g., 4-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, vanillin, isovanillin, and 3-fluoro-4-hydroxybenzaldehyde). Most of the compounds were cytotoxic towards the cisplatin-resistant human ovarian cancer cell line A2780-CP70 as well as the non-resistant line A2780

Synthesis, antimicrobial activity, and membrane permeabilizing properties of C-terminally modified conjugates accessed by CuAAC

Functionalization of the lantibiotic nisin with fluorescent reporter molecules is highly important for the understanding of its mode of action as a potent antimicrobial peptide. In addition to this, multimerization of nisin to obtain multivalent peptide constructs and conjugation of nisin to bioactive molecules or grafting it on surfaces can be attractive methods for interference with bacterial growth. Here, we report a convenient method for the synthesis of such nisin conjugates and show that these nisin derivatives retain both their antimicrobial activity and their membrane permeabilizing properties. The synthesis is based on the Cu(I)-catalyzed alkyne−azide cycloaddition reaction (CuAAC) as a bioorthogonal ligation method for large and unprotected peptides in which nisin was C-terminally modified with propargylamine and subsequently efficiently conjugated to a series of functionalized azides. Two fluorescently labeled nisin conjugates together with a dimeric nisin construct were prepared while membrane insertion as well as antimicrobial activity were unaffected by these modifications. This study shows that C-terminal modification of nisin does not deteriorate biological activity in sharp contrast to N-terminal modification and therefore C-terminally modified nisin analogues are valuable tools to study the antibacterial mode of action of nisin. Furthermore, the ability to use stoichiometric amounts of the azide containing molecule opens up possibilities for surface tethering and more complex multivalent structures