Mutasynthesis approaches to the preparation of streptorubin B analogues

Prodiginines are a large family of red-pigmented tripyrrole-based antibiotics. Their biosynthesis in Streptomyces coelicolor A3(2), by enzymes encoded in the red gene cluster, involves the condensation of 4-methoxy-2,2′-bipyrrole-5- carboxaldehyde (MBC) and 2-undecylpyrrole, catalysed by the RedH enzyme to give undecylprodiginine. This is followed by the mechanistically interesting oxidative carbocyclisation, catalysed by RedG to give streptorubin B. In this thesis prodiginine analogues have been generated by a mutasynthetic approach, in which chemically synthesised analogues of intermediates MBC and 2-undecylpyrrole were fed to mutant strains of S. coelicolor in which one of the genes required to biosynthesise MBC or 2-undecylpyrrole (redM or redL respectively) have been deleted. RedH and RedG have been shown to display relatively broad substrate tolerances and several analogues of both undecylprodiginine and streptorubin B have been generated by this approach. A variety of factors which are potentially limiting to substrate tolerances have been probed, including the steric size, alkyl chain hydrophobicity and introduction of π-electrons. The absolute stereochemistry of streptorubin B has been investigated by a mutasynthetic approach in which 2-undecylpyrrole, stereospecifically labelled with deuterium, is fed to a mutant strain of S. coelicolor unable to biosynthesise 2-undecylpyrrole. During the course of the investigation streptorubin B was analysed on a homochiral stationary phase HPLC and evidence of both ent- and dia-streptorubin B was discovered in the natural product isolated from S. coelicolor. When the position of the deuterium label from the mutasynthesis experiment was investigated by 1H-NMR and 2H-NMR the partial epimerization of the synthetic material became apparent. This made the definitive determination of the absolute stereochemistry of streptorubin B impossible. However, a tentative assignment of the absolute stereochemistry was possible. This was supported by comparison with the related natural products metacycloprodigiosin, the stereochemistry of which was established here by CD spectroscopy and chiral HPLC analyses, and roseophilin

Elucidation of the prodiginine biosynthetic pathway in Streptomyces coelicolor A3(2)

The prodiginine antibiotics are produced by eubacteria, in particular members of the actinomycete family. Interest in this group of compounds has been stimulated by their antitumour, immunosuppressant and antimalarial activities at non-toxic levels. Streptomyces coelicolor A3(2) produces two prodiginines: undecylprodiginine and its carbocyclic derivative streptorubin B, which are both derived from the two intermediates 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC) and 2-undecylpyrrole (2-UP). The red gene cluster of S. coelicolor contains 23 genes responsible for prodiginine biosynthesis. PCR-targeting was used to generate rapid in-frame deletions or replacements of several genes in the S. coelicolor red cluster. Using this method redI, redJ, redK, the A domain encoding region of redL, redT and redV were disrupted. Prodiginine production by these mutants was analysed by LC-MS allowing roles for the genes investigated to be hypothesised. A major focus was investigating the function of RedH (proposed to catalyse the condensation of 2-UP and MBC) and RedG (proposed to be responsible for the oxidative carbocyclisation of undecylprodiginine to form streptorubin B) by genetic complementation of existing mutants and heterologous expression of the genes in S. venezuelae coupled with feeding of synthetic MBC and 2-UP. The results of these experiments clearly defined the roles of RedH in the condensation of MBC and 2-UP and RedG in the oxidative carbocyclisation of undecylprodiginine. Streptomyces longispororuber is known to produce undecylprodiginine (like S. coelicolor) and a carbocyclic undecylprodiginine derivative called metacycloprodigiosin (streptorubin A), which contains a 12-membered carbocycle instead of the 10-membered carbocycle of streptorubin B. A S. longispororuber fosmid library was constructed, from which a clone containing a previously identified redG orthologue was isolated and partially sequenced. Expression of the S. longispororuber redG orthologue in the S. coelicolor redG mutant resulted in production of metacycloprodigiosin instead of streptorubin B showing that RedG and its S. longispororuber orthologue catalyse carbocyclisation reactions during prodiginine biosynthesis. Another aim of the work was to investigate redU, a gene from the red cluster that encodes a phosphopantetheinyl transferase (PPTase). PPTases are responsible for post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). A pre-existing redU mutant and two newly constructed mutants lacking PPTases encoded elsewhere in the S. coelicolor genome were analysed to investigate the role of PPTases in S. coelicolor metabolite biosynthesis. Production of prodiginines, actinorhodins, methylenomycins, calcium dependent antibiotics, coelichelin and grey spore pigment was investigated as ACPs and PCPs are involved in biosynthesis of these compounds. Different specific PPtases were found to be required to modify the ACP/PCP domains/proteins in the biosynthesis of these metabolites

Elucidation of the prodiginine biosynthetic pathway in Streptomyces coelicolor A3(2)

The prodiginine antibiotics are produced by eubacteria, in particular members of the actinomycete family. Interest in this group of compounds has been stimulated by their antitumour, immunosuppressant and antimalarial activities at non-toxic levels. Streptomyces coelicolor A3(2) produces two prodiginines: undecylprodiginine and its carbocyclic derivative streptorubin B, which are both derived from the two intermediates 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC) and 2-undecylpyrrole (2-UP). The red gene cluster of S. coelicolor contains 23 genes responsible for prodiginine biosynthesis. PCR-targeting was used to generate rapid in-frame deletions or replacements of several genes in the S. coelicolor red cluster. Using this method redI, redJ, redK, the A domain encoding region of redL, redT and redV were disrupted. Prodiginine production by these mutants was analysed by LC-MS allowing roles for the genes investigated to be hypothesised. A major focus was investigating the function of RedH (proposed to catalyse the condensation of 2-UP and MBC) and RedG (proposed to be responsible for the oxidative carbocyclisation of undecylprodiginine to form streptorubin B) by genetic complementation of existing mutants and heterologous expression of the genes in S. venezuelae coupled with feeding of synthetic MBC and 2-UP. The results of these experiments clearly defined the roles of RedH in the condensation of MBC and 2-UP and RedG in the oxidative carbocyclisation of undecylprodiginine. Streptomyces longispororuber is known to produce undecylprodiginine (like S. coelicolor) and a carbocyclic undecylprodiginine derivative called metacycloprodigiosin (streptorubin A), which contains a 12-membered carbocycle instead of the 10-membered carbocycle of streptorubin B. A S. longispororuber fosmid library was constructed, from which a clone containing a previously identified redG orthologue was isolated and partially sequenced. Expression of the S. longispororuber redG orthologue in the S. coelicolor redG mutant resulted in production of metacycloprodigiosin instead of streptorubin B showing that RedG and its S. longispororuber orthologue catalyse carbocyclisation reactions during prodiginine biosynthesis. Another aim of the work was to investigate redU, a gene from the red cluster that encodes a phosphopantetheinyl transferase (PPTase). PPTases are responsible for post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). A pre-existing redU mutant and two newly constructed mutants lacking PPTases encoded elsewhere in the S. coelicolor genome were analysed to investigate the role of PPTases in S. coelicolor metabolite biosynthesis. Production of prodiginines, actinorhodins, methylenomycins, calcium dependent antibiotics, coelichelin and grey spore pigment was investigated as ACPs and PCPs are involved in biosynthesis of these compounds. Different specific PPtases were found to be required to modify the ACP/PCP domains/proteins in the biosynthesis of these metabolites.EThOS - Electronic Theses Online ServiceUniversity of WarwickGBUnited Kingdo

Development of contractive synthesis of cyclobutanes from pyrrolidines

Carbocycles are omnipresence in chemical pharmaceuticals, biologically active natural products and organic functional materials. Construction of structurally intriguing, highly functionalized small carbocycles with congested stereocenters remain to be an intricate task in organic chemistry. Consistent endeavor such as innovation of chemical methodology and development of synthetic tactics has been made to improve the synthetic efficiency to these complex structures. In particular, the synthesis of carbocycle through ring contraction, which complies with the concept of synthetic efficiency in modern organic chemistry, has been widely applied in organic synthesis of complex architectures. In this thesis, the unprecedented, stereospecific and contractive synthesis of multi-substituted cyclobutanes from corresponding pyrrolidines is discussed. The reaction mechanism is investigated and the reaction was applied to the synthesis of cytotoxic natural product piperarborenine B

The influence of structure on reactivity in alkene metathesis

Abstract Alkene metathesis has grown from a niche technique to a common component of the synthetic organic chemistry toolbox, driven in part by the development of more active catalyst systems, or those optimized for particular purposes. While the range of synthetic chemistry achieved has been exciting, the effects of structure on reactivity have not always been particularly clear, and rarely quantified. Understanding these relationships is important when designing new catalysts, reactions, and syntheses. Here, we examine what is known about the effect of structure on reactivity from two perspectives: the catalyst, and the substrate. The initiation of the precatalyst determines the rate at which active catalyst enters the catalytic cycle; the rate and selectivity of the alkene metathesis reaction is dependent on how the substrate and active catalyst interact. The tools deployed in modern studies of mechanism and structure/activity relationships in alkene metathesis are discussed

The synthesis and potential applications of asymmetric silacycles

Although the use of silicon-based reagents has undergone rapid development during the last twenty years, the application of organosilicon chemistry to asymmetric synthesis has been somewhat slower to develop. The many problems associated with the use of 'Si-centred' chiral organosilicon compounds has led to the application of 'C-centred' chiral organosilicon compounds. This work has been aimed at the synthesis and application of cyclic silicon species. Routes towards the synthesis of medium-sized rings have been investigated as a potential application of enantiomerically pure silacycles. This work has led to the discovery of an unusual tandem cycloaddition-bond fragmentation reaction of 3-(dienylacyloxy)cycloalk-2- en-l-ones, which affords a-tetralone as the principal product. Most work has been directed at the synthesis of asymmetric silacycles. Two routes have been explored. Firstly, the double asymmetric hydrosilylation of dienes, catalysts based on many transition metals were used but little evidence of hydrosilylation was observed. The second route is that of the double asymmetric hydroboration of divinylsilanes. Asymmetric stoichiometric hydroboration led to products of moderate to high enantiomeric excess, whilst rhodium-catalysed hydroboration led to high yields of the achiral syn isomer. The diastereoselectivity has been found to vary according to the length of the tether between two phosphine ligands, with maximum diastereoselectivity being observed for butanodiphosphines. NMR studies have investigated the possibility that this is related to the stability of a divinylsilane-diphosphine rhodium complex. Finally, the formation of a variety of silacycles has been attempted. Boron- redistribution of the product of hydroboration with (-)-diisopinocampheylborane has been shown to occur with retention of stereochemistry and subsequent carbonylation led to the formation of asymmetric silacyclohexanones. Oxidation of the hydroboration product led to the formation of a silyldiol species. Reactions of this silyldiol have provided the basis for encouraging preliminary attempts at the formation of other heterosilacycles

Novel ruthenium indenylidene catalysts : from homogeneous to heterogeneous

Nowadays a number of ruthenium metathesis catalysts have been developed owing to their accessibility, remarkable activity and selectivity, connected with good tolerance towards functional groups, air and moisture. Innovative development in the class of ruthenium metathesis catalysts coordinated with NHC has been experienced which mainly directed toward tuning their catalytic activity and selectivity through altering both steric and electronic properties. The unsymmetrical NHC ligands in particular, have been introduced to induce dissymmetry, a key for achieving higher level of selectivity in different reactions. A great number of the ruthenium complexes bearing unsymmetrical NHC ligands have been developed up to this moment. The bis-coordinated ruthenium indenylidene developed in this work showed moderate activity at higher temperature in RCM, ROMP and other kind of reactions such as isomerization of allylic alcohols and isomerization of alkenes. Failure of these catalysts to work at room temperature has been attributed to the lack of labile ligand.This call for further research which will focus on tuning of unsymmetrical NHC ligands to achieve more active and selective ruthenium complexes coordinated with non-labile NHC ligand with the labile one. The research should go in hand with design and synthesis of heterogeneous catalysts that can be recovered from the reaction mixture and be recycled. Although the support materials used in this study proved to be not suitable for metathesis, the obtained results can be considered as a challenge in the journey toward designing stable and active heterogeneous ruthenium indenylidene catalysts. Up to now a number of solid materials have been developed and successfully utilized in the immobilization of ruthenium benzylidene complexes. It is expected that the same materials can act as the suitable supports for ruthenium indenylidene and therefore, a study about development heterogeneous ruthenium indenylidene analogs would be of great interest

Total Synthesis of Clavilactones

Clavilactones A, B, and D are epidermal growth factor receptor tyrosine kinase inhibitors that were isolated from cultures of the fungus <i>Clitocybe clavipes</i>. Here, we report full details of the total synthesis of these clavilactones. A key feature of our synthetic approach is a ring-opening/ring-closing metathesis strategy that allows the concise transformation of a cyclobutenecarboxylate into a γ-butenolide. Coupled with enantioselective Ti/BINOL-catalyzed alkynylation of a multisubstituted benzaldehyde and ring-closing metathesis of a diene-bearing silylene acetal to construct the 10-membered carbocycle, this strategy enabled the total synthesis of the natural enantiomers (+)-clavilactone A and (−)-clavilactone B. In addition, the correct structure of clavilactone D was determined by the synthesis of two newly proposed structures. This research resulted in the asymmetric synthesis of the revised (+)-clavilactone D

Methodologies of Bicyclo[2.2.2]octane Compounds and Progress Toward the Total Synthesis of Parthenolide

In Chapter 1, rearrangements of 4-substituted bicyclo[2.2.2]oct-2-enyl-5-selenophenyl esters are explored. Reduction of the esters under radical-generating conditions initially generates a bicyclo[2.2.2]oct-5-en-2-yl radical which can rearrange to a bicyclo[3.2.1]oct-6-en-2-yl radical via a cyclopropylcarbinyl radical. Although the bicyclo[3.2.1]octene system exhibits greater strain than does the bicyclo[2.2.2]octene system, radical-stabilizing substituents can reverse this preference. The product ratios are influenced by the interplay of ring strain and radical stability. In addition, the rearranged products favored the equatorial over the axial isomers, which can be explained by torsional steering. In Chapter 2, different approaches to the synthesis of the optically active bicyclo[2.2.2]octane-2,5-diones are presented. A stereoselective Diels-Alder cycloaddition was explored to construct the bicyclo[2.2.2]octane skeleton in an efficient manner. This approach was hindered by unexpected polymerization and lack of stereoselectivity. A different method of synthesizing the optically active bicyclo[2.2.2]octane-2,5-dione featuring a diastereoselective 1,4-addition is also presented. In Chapter 3, progress toward the total synthesis of parthenolide is described. The synthetic route relies on ring-closing metathesis to close the 10-membered carbocycle of the natural product. The efforts leading up to and including the key ring-closing step are described. Specific attention is given to different methods for overcoming the difficulties associated with medium-sized ring formation