23 research outputs found

    Biomimetic Radical Chemistry and Applications 2021

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    The high importance of free radical chemistry for a variety of biological events, including ageing and inflammation, has attracted considerable interest in understanding the related mechanistic steps at the molecular level. Modelling the free radical chemical reactivity of biological systems is an important research area. When studying free-radical-based chemical mechanisms, biomimetic chemistry and the design of established biomimetic models come into play to perform experiments in a controlled environment, suitably designed to be a similar as possible to cellular conditions. This Special Issue provides readers with a wide overview of biomimetic radical chemistry, where molecular mechanisms have been defined and molecular libraries of products are developed to be used as traces for the discoveries of some relevant biological processes. Several subjects are presented, with five articles and five reviews written by specialists in the fields of DNA, proteins, lipids, biotechnological applications and bioinspired synthesis, with “free radicals” as the common denominator

    Novel Diagnostic and Therapeutic Approaches for Mitochondrial Disorders

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    Mitochondrial disorders are among the most common inherited genetic disorders, with a combined prevalence of 1:5,000. These are genetically, biochemically, and clinically heterogeneous disorders affecting any organ or tissue in the body. A poor understanding of gene-to-phenotype relationships and pathophysiological mechanisms has resulted in sometimes years-long diagnostic odysseys and a lack of curative therapies. Consequently, outcomes are often poor with most patients dying in early childhood. The aim of this project is to improve patient outlooks by using novel tools to address both the diagnostic and therapeutic challenges associated with mitochondrial disease. The diagnostic aspect of the study involved the creation of four interactive diagnostic resources which can complement next generation sequencing (NGS) technologies to achieve more rapid diagnoses for patients. MitoEpilepsy Map, MitoCardio Map, MitoLiver Map, and MitoMedicine Map were created to aid in the diagnosis of mitochondrial epilepsy, cardiomyopathy, liver disease, and the entirety of mitochondrial disease, respectively. These, maps were accurate in identifying candidate genes from clinical vignettes of genetically confirmed cases of mitochondrial disease in 69-100% of cases. These maps will be valuable resources for interpreting NGS results, hopefully facilitating quicker and more accurate genetic diagnoses for affected patients. The therapeutic aspect of the project aimed to develop a new treatment strategy for mitochondrial disease caused by nonsense mutations. Translational read-through therapy involves pharmacological incorporation of a near-cognate amino acid in place of a premature stop codon during translation. A systematic in vitro proof-of-principle study was performed in patient fibroblasts harbouring bi-allelic nonsense mutations in ten different mitochondrial disease genes. In five patient cell cultures, translational read-through therapy was able to restore transcript, protein, and mitochondrial function, thus demonstrating in vitro efficacy and paving the way for future clinical development. Together, these approaches help improve outcomes for patients suffering from mitochondrial disease

    Organophosphorus Chemistry 2018

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    Organophosphorus chemistry is an important discipline within organic chemistry. Phosphorus compounds, such as phosphines, trialkyl phosphites, phosphine oxides (chalcogenides), phosphonates, phosphinates and >P(O)H species, etc., may be important starting materials or intermediates in syntheses. Let us mention the Wittig reaction and the related transformations, the Arbuzov- and the Pudovik reactions, the Kabachnik–Fields condensation, the Hirao reaction, the Mitsunobu reaction, etc. Other reactions, e.g., homogeneous catalytic transformations or C-C coupling reactions involve P-ligands in transition metal (Pt, Pd, etc.) complex catalysts. The synthesis of chiral organophosphorus compounds means a continuous challenge. Methods have been elaborated for the resolution of tertiary phosphine oxides and for stereoselective organophosphorus transformations. P-heterocyclic compounds, including aromatic and bridged derivatives, P-functionalized macrocycles, dendrimers and low coordinated P-fragments, are also of interest. An important segment of organophosphorus chemistry is the pool of biologically-active compounds that are searched and used as drugs, or as plant-protecting agents. The natural analogue of P-compounds may also be mentioned. Many new phosphine oxides, phosphinates, phosphonates and phosphoric esters have been described, which may find application on a broad scale. Phase transfer catalysis, ionic liquids and detergents also have connections to phosphorus chemistry. Green chemical aspects of organophosphorus chemistry (e.g., microwave-assisted syntheses, solvent-free accomplishments, optimizations, and atom-efficient syntheses) represent a dynamically developing field. Last, but not least, theoretical approaches and computational chemistry are also a strong sub-discipline within organophosphorus chemistry

    Nucleoside Analogues for Positron Emission Tomography Imaging and to Study Radiation Mediated Generation of Radicals from Azides

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    Gemcitabine is a potent anticancer cytidine analogue used to treat solid tumors. Its efficacy is diminished by rapid deamination to a toxic uridine derivative by cytidine deaminase. To overcome this limitation and add radioactive nuclei (18F or 68Ga) for PET imaging, I synthesized two 4-N­-alkylgemcitabine analogues i) bearing β-keto tosylate moiety for subsequent 18F-fluorination and ii) having SCN-Bn-NOTA chelator to complex 68Ga. The first was synthesized by replacement of tosylamide in 4-N­-tosylgemcitabine with 1-amino-10-undecene, followed by elaboration of terminal alkene through dihydroxylation, regioselective tosylation and oxidation. Subsequent fluorination using KF in presence of 18-Crown-6 at 75°C for 1 hr gave 4-N­-alkylgemcitabine fluoromethyl ketone. The second was synthesized by analogous replacement of tosylamide with N-Boc-1,3-propanediamine, followed by deprotection with TFA. The reactive terminal amine was condensed with SCN-Bn-NOTA, giving 4-N­-alkylgemcitabine-SCN-Bn-NOTA ligand, which efficiently complexed Ga or 68Ga for in vivo PET studies in rats. Clofarabine is a highly effective chemotherapeutic adenosine analogue used for treatment of acute lymphoblastic leukemia. Clofarabine undergoes rate limiting phosphorylation from its 5\u27-monophosphate to 5\u27-diphosphate by purine monophosphate kinase, and possible dephosphorylation of its respective 5\u27-monophosphate by 5\u27-nucleotidases. Synthesis of clofarabine diphosphate prodrugs, and potentially their 18F-radiolabeled analogues, were undertaken to overcome these limitations. Successful synthesis of model adenosine diphosphate prodrug, by coupling adenosine monophosphate with bis(benzoyloxybenzyl) phosphoramidite in presence of 5-phenyl-1-H­-tetrazole activator was achieved. The aminyl radical generated from azide moiety in 3\u27-azido-3\u27-deoxythymidine (3\u27-AZT) or 5-azidomethyl-2\u27-deoxyuridine (AmdU), upon addition of radiation-produced electrons, is thought to be the source of their radiosensitizing effects. Herein, I report synthesis of azido-modified purine and pyrimidine analogues for EPR study of formation of reactive aminyl radical in guanine, adenine and cytidine bases. The EPR studies of electron addition to 2-azidoguanosine (i.e. 2-azidoinsoine), protected 4-azidocytidine and 4-tetrazolocytidine analogues clearly establish that the position of the azide in base moiety dictates reactivity. The azide directly attached to nucleobases at ortho/para position to ring nitrogens produce stable RN3•- that does not rapidly convert to aminyl radical, except in the excited state. Hence, these did not display much radiosensitizing effects in in vivo biological studies in MDA-MB-231, MCF7 and U87 cell lines

    Structure-function Studies of Kinetoplastid Proteins

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    The class kinetoplastida include parasites responsible for devastating diseases like African sleeping sickness, Chagas’s disease and Leishmaniases, mainly effecting people in the developing world. Current treatments are highly toxic and inefficient, leading to an urgent need of novel anti-parasitic compounds. This thesis focuses on the structural characterisation of potential drug targets against these parasites, namely adenosine kinase from Trypanosoma brucei (TbAK), thymidine kinases from T. brucei (TbTK) and Leishmania major (LmTK) and the leucyl aminopeptidases from T. brucei (TbLAP-A), T. cruzi (TcLAP-A) and L. major (LmLAP-A). Structures of TbAK were solved in two conformations, open (apo) and closed (in complex with adenosine and ADP), both to 2.6 Å. Comprised of a big α/β-domain and a small lid domain, the structures confirm the large conformational change of the lid domain upon substrate binding. The structures of C-terminally truncated versions of LmTK and TbTK were determined as ligand-bound complexes with resolutions up to 2.4 Å and 2.2 Å, respectively. They show high similarity to structures of homologues in the PDB. The structures solved in this thesis give valuable information about ligand binding and aid rational drug design. Leucyl aminopeptidase (LAP-A) was evaluated as a potential drug target in T. brucei parasites. It is not essential for T. brucei parasites grown in vitro, shown by generation and analysis of LAP-A-depleted parasites. Although this does not support LAP-A as a drug target in T. brucei, no conclusions can be drawn about the potential in T. cruzi and L. major. Several structures of the LAP-As were solved, the highest resolution ones to 2.3 Å, 2.3 Å and 2.5 Å for TbLAP-A, TcLAP-A and LmLAP-A, respectively. These enzymes are hexameric and show the typical two-domain architecture of M17 LAPs. Although the physiological function remains elusive, the work in this thesis provides a firm basis for future studies

    Structural studies of allosteric regulation in the class Ia Ribonucleotide reductase from Escherichia coli

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    Thesis (Ph. D. in Biological Chemistry)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013.Cataloged from PDF version of thesis. Vita.Includes bibliographical references.Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, the building blocks for DNA replication and repair. The E. coli class Ia enzyme requires two subunits to catalyze the radical-based reduction reaction. [beta]2 houses a diferric-tyrosyl radical cofactor and [alpha]2 contains the active site and two allosteric effector binding sites. Allosteric control of RNR fine-tunes both the relative ratios (via substrate specificity regulation) and the total amount (via activity regulation) of deoxyribonucleotides (dNTPs) in the cell. The molecular basis of this regulation has been enigmatic, largely due to a lack of structural information about how the [alpha]2 and [beta]2 subunits interact. Here, we present the structure of a complex between the [alpha]2 and [beta]2 subunits in the presence of negative activity effector dATP, revealing an [alpha]4[beta]4 ring-like structure. Using electron microscopy (EM), small-angle X-ray scattering (SAXS), and analytical ultracentrifugation (AUC) we show how activity regulation is achieved by modulating the distributions of active [alpha]2[beta]2 and inhibited [alpha]4[beta]4, an interconversion that requires dramatic subunit rearrangements. The X-ray crystal structure of the dATP-inhibited RNR and a second structure obtained using a mechanism based inhibitor reveal that [alpha]4[beta]4 rings can interlock to form an ([alpha]4[beta]4)2 megacomplex. We use SAXS to understand the solution conditions that contribute to the observed concatenation and present a mechanism for the formation of these unusual structures. We also present the first X-ray crystal structures of [alpha]2 with ATP or dATP bound at both allosteric sites, and discuss how observed differences in their binding influence the modulation between [alpha]2[beta]2 and [alpha]4[beta]4. Finally, we present structures that comprise a full set of cognate substrate/specificity effector pairs bound to the E. coli class Ia RNR. These structures allow us to describe how binding of dNTP effectors at the specificity site promotes binding of a preferred substrate. With these structural data, we describe in molecular detail, how the binding of allosteric effectors influences RNR activity and substrate specificity.by Christina Marie Zimanyi.Ph.D.in Biological Chemistr

    Enzyme Kinetics of the Mitochondrial Deoxyribonucleoside Salvage Pathway Are Not Sufficient to Support Rapid mtDNA Replication

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    Using a computational model, we simulated mitochondrial deoxynucleotide metabolism and mitochondrial DNA replication. Our results indicate that the output from the mitochondrial salvage enzymes alone is inadequate to support a mitochondrial DNA replication duration of as long as 10 hours. We find that an external source of deoxyribonucleoside diphosphates or triphosphates (dNTPs), in addition to those supplied by mitochondrial salvage, is essential for the replication of mitochondrial DNA to complete in the experimentally observed duration of approximately 1 to 2 hours. For meeting a relatively fast replication target of 2 hours, almost two-thirds of the dNTP requirements had to be externally supplied as either deoxyribonucleoside di- or triphosphates, at about equal rates for all four dNTPs. Added monophosphates did not suffice. However, for a replication target of 10 hours, mitochondrial salvage was able to provide for most, but not all, of the total substrate requirements. Still, additional dGTPs and dATPs had to be supplied. Our analysis of the enzyme kinetics also revealed that the majority of enzymes of this pathway prefer substrates that are not precursors (canonical deoxyribonucleosides and deoxyribonucleotides) for mitochondrial DNA replication, such as phosphorylated ribonucleotides, instead of the corresponding deoxyribonucleotides. The kinetic constants for reactions between mitochondrial salvage enzymes and deoxyribonucleotide substrates are physiologically unreasonable for achieving efficient catalysis with the expected in situ concentrations of deoxyribonucleotides

    Ribonucleotide reduction and redox regulation in Archaea : surprising twists on a common theme

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    Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.Vita.Includes bibliographical references.To study the nucleotide reduction system in Archaea, we have expressed the Archaeoglobus fulgidus gene encoding the adenosylcobalamin (AdoCbl) dependent ribonucleotide reductase (afuRNR) protein. Initial characterization of the recombinant afuRNR indicates that it reduces CDP to dCDP in an AdoCbl dependent manner. One astonishing finding is the capacity of afuRNR to reduce both diphosphate and triphosphate nucleosides to their respective deoxynucleosides. This is the first instance of a RNR displaying dual substrate selectivity. Our investigation of redox regulation focused on thioredoxin reductase (TrR) and thioredoxin (Tr). In addition to providing the reducing equivalents for many RNRs, TrRs are involved in maintaining the intracellular redox environment. This process typically involves the transfer of electrons from NADPH to Trs. Here the characterization of a putative TrR (Ta0984) and Tr (Ta0866) from Thermoplasma acidophilum are presented. To explore the apparent inability of taTrR to use NADPH or NADH as a reductant, we carried out a complete electrochemical characterization, which ruled out redox potential as the source of this non-reactivity. A 2.35 A resolution structure of taTrR, also presented here, shows that despite the overall structural similarity to the wellcharacterized TrR from E. coli, the "NADPH binding pocket" is not conserved. E. coli TrR residues implicated in NADPH binding, H175, R176, R177, and R181 have been substituted with E185, M186, Y187, and M191 in the ta protein. Thus, we have identified a Tr and TrR protein system from T. acidophilum for which the TrR shares overall structural and redox properties with other TrRs, but lacks the appropriate binding motif to use the standard NADPH reductant. Our discovery of a TrR that does not use NADPH provides a new twist in redox regulation.Further, four A. fulgidus genes encoding four Trs (afuTr 1, afuTr 2, afuTr 3, and afuTr 4) proteins have been identified and their proteins expressed. Electrochemical characterization via protein-film voltammetry shows that the afuTrs reduction potentials are -32 mV for afuTr 1, -301 mV for afuTr 2, -287 mV for afuTr 3, and -309 mV for afuTr 4. This set of reduction potentials found in this Archaeon represents the greatest range in a specific organism.by Hector Hugo Hernandez.Ph.D

    Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate

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    Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2007.Vita.Includes bibliographical references.Efforts towards the Synthesis of Fully N-Differentiated Heparin-like Glycosaminoglycans. Heparin-like glycosaminoglycans (HLGAGs) are complex information-carrying biopolymers and are an important component of the coagulation cascade. They have also been implicated in interactions with growth factors, cytokines, virus entry, and other functions. Currently, no general synthesis of arbitrary HLGAG sequences has been demonstrated. The modular synthesis of glycosaminoglycans requires straightforward methods for the production of large quantities of protected uronic acid building blocks. An efficient route to methyl 3-0- benzyl-1,2-O-isopropylidene-a-L-idopyranosiduronate from diacetone glucose in nine steps and 36% overall yield is described. Additionally, a general method for the conversion of glycals to the corresponding 1,2-cis-isopropylidene-a-glycosides is reported. Epoxidation of glycals with dimethyldioxirane followed by ZnC12-catalyzed addition of acetone converted a variety of protected glycals into 1,2-cis-isopropylidene-a-glycosides in good yield. The reaction is compatible with a range of protecting groups, as well as free hydroxyl groups. This method has been applied to develop a synthesis of 3-O-benzyl-1,2-O-isopropylidene-P-D-glucopyranosiduronate in seven steps and 32% overall yield.(cont.) These compounds are useful as glycosyl acceptors and as intermediates that may be further elaborated into uronic acid trichloroacetimidate glycosyl donors for the assembly of glycosaminoglycan structures. The glucosamine residues in HLGAGs have been found to exist as amines, acetamides, and N-sulfonates. In order to develop a completely general, modular synthesis of heparin, three degrees of orthogonal nitrogen protection are required. Reported is a strategy for the synthesis of fully N-differentiated heparin oligosaccharides in the context of target octasaccharide 3-1, which contains an N-acetate, N-sulfonates, and a free amine. The protecting group scheme used in the synthesis blocked the N-acetate as a N-diacetate, the N-sulfonates as azido groups, and the amine as a N-CBz; free hydroxyls were masked as benzyl ethers and O-sulfonates as acetate esters. Disaccharide and tetrasaccharide modules were synthesized using this strategy; however, the union of tetrasaccharide trichloroacetimidate 3-4 with disaccharide acceptor 3-5 unexpectedly formed the undesired P-linked glycoside in addition to the a-linkage anticipated for iduronic acid nucleophiles, resulting in an inseparable 6:1 a: p mixture of products. Detailed studies into the basis for this unexpected result were conducted and are also reported.(cont.) Investigations into the Mechanism of Inactivation of RTPR by Gemcitabine Triphosphate. Ribonucleoside triphosphate reductase (RTPR) is an adenosylcobalamin (AdoCbl) dependant enzyme that catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates via controlled radical chemistry. The antitumor agent 2',2'-difluoro-2'- deoxycytidine (gemcitabine, F2C) has been shown to owe some of its in vivo activity to inhibition of human RNR by the 5'-diphosphate (F2CDP). Previous studies have shown that RTPR is rapidly inactivated by one equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP). This inactivation is associated with the release of two equivalents of fluoride and modification of RTPR by a Co-S bond between C419 and the cobalamin cofactor. In order to further characterize this inactivation, isotopically labeled derivatives of F2CTP were synthesized: radiolabeled 1'-[3H]-F2C and mass labeled 1'-[2H]-F2C and 3'-[2H]-F2C. These compounds were converted to F2CTP through a set of enzymatic phosphorylation steps which overcome difficulties found using traditional, chemical methods. Biochemical investigations were performed using these labeled derivatives to track the fate of the base and sugar during RTPR inactivation by F2CTP.(cont.) The release of cytosine base, previously overlooked in this system, was detected utilizing 5-[3H]-F2CTP: 0.7 equiv. of cytosine were released, with 0.15-0.2 equiv. of unreacted F2CTP remaining. Size exclusion chromatography (SEC) was used to quantify covalent labeling of RTPR by F2CTP: 0.15 equiv. were detected using 5-[3H]-F2CTP, 0.45 equiv. were detected using 1'-[3H]-F2CTP. A small molecule nucleotide product was identified in inactivation mixtures quenched with NaBH4 and identified as an isomer of cytidine, indicating the loss of both fluorides and the addition of an oxygen at the 2' carbon. RTPR inactivated with 1'-[3H]-F2CTP was digested with trypsin and peptides containing radioactivity purified. Identical peptides were prepared using partially deuterated F2CTP, allowing identification by MALDI-MS. Post source decay (PSD) MS/MS methods were used to further characterize these peptides, identifying the site of label as the C-terminal tryptic peptide of RTPR at C731 and C736. The cysteines were labeled through conjugate addition with a furanone-like precursor that had lost cytosine, triphosphate, and both fluorines. The results of these studies have allowed for the first time the proposal of a mechanistic hypothesis for RTPR inactivation by F2CTP.by Gregory J.S. Lohman.Ph.D
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