Primary Amine Nucleophilic Addition to Nitrilium Closo-Dodecaborate [B12H11NCCH3]–: A Simple and Effective Route to the New BNCT Drug Design

In the present work, a convenient and straightforward approach to the preparation of borylated amidines based on the closo-dodecaborate anion [B12H11NCCH3NHR]–, R=H, Alk, Ar was developed. This method has two stages. A nitrile derivative of the general form [B12H11NCCH3]− was obtained, using a modified technique, in the first stage. On the second stage the resulting molecular system interacted with primary amines to form the target amidine products. This approach is characterised by a simple chemical apparatus, mild conditions and high yields of the final products. The mechanism of the addition of amine to the nitrile derivative of the closo-dodecaborate anion was studied, using quantum-chemical methods. The interaction between NH3 and [B12H11NCCH3]− ammonia was chosen as an example. It was found that the structure of the transition state determines the stereo-selectivity of the process. A study of the biological properties of borylated amidine sodium salts indicated that the substances had low toxicity and could accumulate in cancer cells in significant amounts.

Characterisation, Manipulation and Directed Evolution of Non-Ribosomal Peptide Synthetase Enzymes

<p><b>Non-ribosomal peptide synthetases (NRPS) are large, modular enzymes that synthesisebiologically active secondary metabolites from amino acid precursors without the need for anucleic acid template. NRPS play an integral role in microbial physiology and also havepotential applications in the synthesis of novel peptide molecules. Both of these aspects areexamined in this thesis.</b></p> <p>Under conditions of iron starvation Pseudomonas syringae synthesises siderophores for activeuptake of iron. The primary siderophore of P. syringae is pyoverdine, a fluorescent moleculethat is assembled from amino acid (aa) precursors by NRPS. Five putative pyoverdine NRPSgenes in P. syringae pv. phaseolicola 1448a (Ps1448a) were identified and characterised insilico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Creation ofpyoverdine null Ps1448a enabled identification of a previously uncharacterised temperatureregulatedsecondary siderophore, achromobactin, which is NRPS independent and has loweraffinity for iron. Pyoverdine and achromobactin null mutants were characterised in regard toiron uptake, virulence and growth in iron-limited conditions. Determination of the substratespecificity for the seven adenylation (A) domains of the Ps1448a pyoverdine sidechain NRPSwas also attempted. Although ultimately unsuccessful, these attempts provided a rigorousassessment of methods for the expression, purification and biochemical characterisation of Adomains.</p> <p>The Ps1448a NRPS were subsequently employed in domain swapping experiments to testcondensation (C) domain specificity for aa substrates during peptide formation in vivo.</p> <p>Experiments in which the terminal C- and/or A-domain of the Pseudomonas aeruginosa(PAO1) pyoverdine NRPS system were replaced with alternative domains from Ps1448a andPAO1 were consistent with previous in vitro observations that C-domains exhibit strongsidechain and stereo-selectivity at the downstream aa position, but only stereo-selectivity atthe upstream aa position.</p> <p>These results prompted investigation into the role of inter-domain communication in NRPSfunction, to test the hypothesis that the thiolation (T) domain enters into specific interactionswith other domains, which might provide an alternative explanation for the diminished activityof recombinant NRPS enzymes. A recently characterised single-module NRPS, bpsA, waschosen as a reporter gene for these experiments based on its ability to generate blue pigment inEscherichia coli. Substitution of the native bpsA T-domain consistently impaired function,consistent with the hypothesis. It was shown that directed evolution could be applied to restorefunction in substituted T-domains. Mutations that restored function were mapped in silico, anda structural model for interaction between the thioester (TE) and T-domain of BpsA wasderived.</p> <p>The utility of bpsA for discovery and characterisation of phosphopantetheinyl transferase(PPTase) enzymes was also investigated. In vivo and in vitro assays for determination ofPPTase activity were developed and a high-throughput screen for discovery of new PPTases inenvironmental DNA libraries was successfully implemented.</p>

Characterisation, Manipulation and Directed Evolution of Non-Ribosomal Peptide Synthetase Enzymes

<p><b>Non-ribosomal peptide synthetases (NRPS) are large, modular enzymes that synthesisebiologically active secondary metabolites from amino acid precursors without the need for anucleic acid template. NRPS play an integral role in microbial physiology and also havepotential applications in the synthesis of novel peptide molecules. Both of these aspects areexamined in this thesis.</b></p> <p>Under conditions of iron starvation Pseudomonas syringae synthesises siderophores for activeuptake of iron. The primary siderophore of P. syringae is pyoverdine, a fluorescent moleculethat is assembled from amino acid (aa) precursors by NRPS. Five putative pyoverdine NRPSgenes in P. syringae pv. phaseolicola 1448a (Ps1448a) were identified and characterised insilico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Creation ofpyoverdine null Ps1448a enabled identification of a previously uncharacterised temperatureregulatedsecondary siderophore, achromobactin, which is NRPS independent and has loweraffinity for iron. Pyoverdine and achromobactin null mutants were characterised in regard toiron uptake, virulence and growth in iron-limited conditions. Determination of the substratespecificity for the seven adenylation (A) domains of the Ps1448a pyoverdine sidechain NRPSwas also attempted. Although ultimately unsuccessful, these attempts provided a rigorousassessment of methods for the expression, purification and biochemical characterisation of Adomains.</p> <p>The Ps1448a NRPS were subsequently employed in domain swapping experiments to testcondensation (C) domain specificity for aa substrates during peptide formation in vivo.</p> <p>Experiments in which the terminal C- and/or A-domain of the Pseudomonas aeruginosa(PAO1) pyoverdine NRPS system were replaced with alternative domains from Ps1448a andPAO1 were consistent with previous in vitro observations that C-domains exhibit strongsidechain and stereo-selectivity at the downstream aa position, but only stereo-selectivity atthe upstream aa position.</p> <p>These results prompted investigation into the role of inter-domain communication in NRPSfunction, to test the hypothesis that the thiolation (T) domain enters into specific interactionswith other domains, which might provide an alternative explanation for the diminished activityof recombinant NRPS enzymes. A recently characterised single-module NRPS, bpsA, waschosen as a reporter gene for these experiments based on its ability to generate blue pigment inEscherichia coli. Substitution of the native bpsA T-domain consistently impaired function,consistent with the hypothesis. It was shown that directed evolution could be applied to restorefunction in substituted T-domains. Mutations that restored function were mapped in silico, anda structural model for interaction between the thioester (TE) and T-domain of BpsA wasderived.</p> <p>The utility of bpsA for discovery and characterisation of phosphopantetheinyl transferase(PPTase) enzymes was also investigated. In vivo and in vitro assays for determination ofPPTase activity were developed and a high-throughput screen for discovery of new PPTases inenvironmental DNA libraries was successfully implemented.</p>

Synthesis of 1-Trifluorometylindanes and Close Structures: A Mini Review

This review describes methods for the synthesis of 1-trifluomethylindanes and close structures, which are still quite rare and scarcely available compounds. There are two main approaches to obtain 1-CF3-indanes. The first one is the construction of an indane system from CF3 precursors; the main methods are acid-mediated Friedel–Crafts cyclization, transition metal-catalyzed [3+2] annulation, and free-radical transformations. The second approach is the trifluoromethylation of a ready-made indane core by various CF3 sources, such as Ruppert–Prakash or Togni reagents. Many of these synthetic procedures possess high regio- and stereo-selectivity, allowing the preparation of unique 1-CF3-indane structures. In recent years, great attention has been paid to the synthesis of 1-CF3-indanes, due to the discovery of important biologically active properties for these compounds.

Synthesis of polysubstituted spiropyrrolidines using 2-acetylfuran, 2-acetylthiophene, and 2-acetylpyrrole

A series of novel spirooxindoles have been synthesized through three-component 1,3-dipolar cycloaddition of azomethine ylides generated in situ by the decarboxylative condensation of isatin and sarcosine with the dipolarophile 3-phenyl-1-(heteroaryl -2-yl)prop-2-en-1- one, synthesized by the Knoevenagel reaction using 2-acetylfuran, 2-acetylpyrrole, 2-acetylthiophene and substituted benzaldehydes. These compounds are used for the first time as dipolarophiles. This method has the advantages of mild reaction conditions, high atom economy, excellent yields, and high regio- and stereo-selectivity. The reaction was carried out by mixing equimolar amounts of enone and isatin, as well as a slight excess of sarcosine in isopropyl alcohol when heated to 60–70°C. Among the enones with various heterocyclic substituents, it is most convenient to use compounds containing a pyrrole fragment as dipolarophiles, since the products are obtained in a short amount of time in good yields. The use of enones obtained from 2-acetylthiophene leads to an increase in the reaction time, and from 2-acetylfuran - to a significant resinification of the reaction mixture. The structures of the compounds obtained were proved using a combination of 1H, 13C NMR spectroscopy data, as well as two-dimensional NMR experiments of heteronuclear correlation, HSQC and HMBC. Based on the data obtained, a mechanism for the formation of products has been proposed.

Highly Diastereoselective Cascade [5+1] Double Michael Reaction, a Route for The Synthesis of Spiro Thio-Oxindoles

The first diastereoselective synthesis of spiro-thiooxindole is reported via Michael reaction between thiooxindoles and divinyl ketones. The reaction was conducted without any catalyst or additive under green conditions, i.e., ethanol as the solvent and room temperature. This study showed the art of governing regioselectivity in which novel spiro frameworks were generated in high yields. In addition to simple and column chromatography-free purification, the high diastereoselectivity makes this protocol very robust. The regio- and stereo- selectivity of the reaction has been discussed in light of the theoretical calculations, and molecular docking has been performed on the reaction products. Our method presents the first synthesis of cis-spiro thiooxindoles, which can be used to generate cis-spiro oxindoles. According to our docking studies, the latter demonstrates higher binding affinities towards the LdUMPS than corresponding trans-spiro oxindoles and may be utilized to develop new hit compounds for leishmaniasis treatment.

Enzymatic Crosslinked Hydrogels for Biomedical Application

Self-assembled structures mostly arises through enzyme-regulated phenomena in nature under persistent conditions. Enzymatic reactions are one of main biological processes in fabrication and construction of supramolecular hydrogel networks required for biomedical applications. The enzymatic processes provide a unique opportunity to integrate hydrogel formation. In most of cases, structure and substrates of hydrogels are adjusted by enzyme catalysis due to the chemo-, regio- and stereo-selectivity of enzymes. Hydrogels processed by using various enzyme schemes showed remarkable characteristics as dynamic frames for cells, bioactive molecules and drugs in biomedical applications. A novel class of enzyme-mediated crosslinking hydrogels mimics the extracellular matrices by displaying unique physicochemical properties and functionalities like water-retention capacity, drug loading ability, biodegradability, biocompatibility, biostability, bioactivity, optoelectronic properties, self-healing ability, shape memory ability. In recent years, many enzymatic systems investigated hydrogel cross-linking. Results of biocompatible hydrogel products show that these mechanisms of crosslinking can fulfill requirements for variety of biomedical applications including tissue engineering, wound healing and drug delivery.

Genome-Wide Characterization of Dirigent Proteins in Populus: Gene Expression Variation and Expression Pattern in Response to Marssonina brunnea and Phytohormones

Marssonina brunnea causes a major disease that limits poplar growth. Lignin and lignan play essential roles in protecting plants from various biological stresses. Dirigent (DIR) proteins are thought to control the stereoselective coupling of coniferyl alcohol in the formation of lignan and lignin. DIR family members have been well studied in several plant species, but no previous detailed genome-wide analysis has been carried out in forest trees, such as poplar. We identified 40 PtDIR genes in Populus trichocarpa and classified them into three subgroups (DIR-a, DIR-b/d, and DIR-e) based on phylogenetic analyses. These genes are distributed on 11 poplar chromosomes, and 80% of PtDIRs (32/40) are intronless. The cis-element analysis inferred that PtDIRs possess many types of biological and abiotic stress-response cis-elements. We also analyzed intra- and inter-specific collinearity, which provided deep insights into the evolutionary characteristics of the poplar DIR genes. Analyses of the protein tertiary structure and critical amino acid residues showed that PtDIR7–10 and PtDIR13–16, which belong to the DIR-a subfamily, might be involved in the regio- and stereo-selectivity of bimolecular phenoxy radical coupling in poplars. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis revealed different expression patterns for the PtDIR genes of P. trichocarpa and the PeDIR genes of ‘Nanlin 895’ in various tissues. Additionally, we analyzed responses of PeDIRs to M. brunnea and different phytohormone treatments (abscisic acid, salicylic acid, methyl jasmonate, and ethylene) in ‘Nanlin 895’. The results showed that at least 18 genes responded strongly to M. brunnea, and these PeDIRs also showed significant responses to phytohormones. These results suggest that DIR genes are involved in the poplar defense response against M. brunnea, and this study will provide fundamental insights for future research on poplar DIR genes.

Current Advances in the Bacterial Toolbox for the Biotechnological Production of Monoterpene-Based Aroma Compounds

Monoterpenes are plant secondary metabolites, widely used in industrial processes as precursors of important aroma compounds, such as vanillin and (−)-menthol. However, the physicochemical properties of monoterpenes make difficult their conventional conversion into value-added aromas. Biocatalysis, either by using whole cells or enzymes, may overcome such drawbacks in terms of purity of the final product, ecological and economic constraints of the current catalysis processes or extraction from plant material. In particular, the ability of oxidative enzymes (e.g., oxygenases) to modify the monoterpene backbone, with high regio- and stereo-selectivity, is attractive for the production of “natural” aromas for the flavor and fragrances industries. We review the research efforts carried out in the molecular analysis of bacterial monoterpene catabolic pathways and biochemical characterization of the respective key oxidative enzymes, with particular focus on the most relevant precursors, β-pinene, limonene and β-myrcene. The presented overview of the current state of art demonstrates that the specialized enzymatic repertoires of monoterpene-catabolizing bacteria are expanding the toolbox towards the tailored and sustainable biotechnological production of values-added aroma compounds (e.g., isonovalal, α-terpineol, and carvone isomers) whose implementation must be supported by the current advances in systems biology and metabolic engineering approaches.

Molecular basis of regio- and stereo-specificity in biosynthesis of bacterial heterodimeric diketopiperazines

Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s (NasbB, NasS1868 and NasF5053) and reveal four critical residues (Qln65, Ala86, Ser284 and Val288) that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70–1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDKP-forming P450s, laying a solid foundation for rapid access to the molecular diversity of HTDKP frameworks through rational engineering of the P450s.