Academic literature on the topic 'Biochemistry|Organic chemistry'

To better understand the relationship between the structure and electronic properties of the iron center and the functions of heme proteins, both naturally occurring heme proteins and synthetically prepared heme models have been studied. The reduction potential (E°') and the pH dependence of E°' of nitrophorin 1 (NP1) and myoglobin (Mb) were determined by spectroelectrochemical techniques. The difference in the electrostatic interaction of the Fe(III) center with buried charged groups in the heme pocket of Mb and NP1 is the major factor that causes the 300 mV difference in E°. The pH dependence of the E°' determined between pH 5.5 and 7.5 is small for both Mb and NP1 because they have the same axial ligands. Three meso-ortho-phenyl substituted porphyrins, (o-F), (o-CF₃) and (2,6-Cl₂)(p-OCH₃)₃TPP and one meso-para-phenyl substituted porphyrin, (p-OCH₃)₄TPP, were synthesized as models of cytochrome b₅ . Cyclic voltammetry was used to measure their reduction potentials. The overall formation constants, logβ₂III and logβ₂II, have been calculated based on the reduction potentials of the iron(III)/(II) couple as a function of N-methylimidazole concentration. The values of logβ₂III are in the order of o-F > o-CF₃ > p-OCH₃ ≈ 2,6-Cl₂, indicating that the electron-donating ability is in the order of o-F < o-CF₃ < p-OCH₃ ≈ 2,6-Cl₂. The overlap and direct transfer of electron density from the halogen to the iron in the product reduces the Lewis acidity of iron(III), resulting in decreased logβ₂III. The order of logβ₂II for N-methylimidazole complexed Fe(II) porphyrinates is similar to that of the Fe(III) complexes, indicating no major difference in the Lewis acidity of Fe(II) as compared to Fe(III). Basket handle porphyrinates with covalently bound methionine and aliphatic amine model ligands (RCH₂SCH₃, RCH₂SCH₃ and RNH₂, RNH₂) were chosen as precusor of cytochrome c and f. The Fe(III) complexes were to be prepared and investigated by electrochemical and spectroscopic techniques. The precursor porphyrin was synthesized. Several schemes were investigated for the synthesis of the handles having methylthioether and aliphatic amine without success, and it was decided not to continue this project. Therefore, no final basket handle porphyrin was available for further characterization.

Oligoribonucleotides comprising of 2',5' -linked internucleotide linkages are known to bind selectively to RNA over DNA. The ability to bind to RNA renders them suitable as probes for many biological applications, such as 'antisense technology'. Little is known about the effect of sugar structure (and conformation) on the binding properties of 2',5'-linked oligonucleotides. To get insight into the role of sugar conformation, 2 ',5'-linked oligonucleotides modified at the C3'-position of the furanose ring were synthesized via solid phase synthesis and their binding to complementary single stranded DNA and RNA was studied. Their application as antisense oligonucleotides was also evaluated.<br>The first analogue studied was the C3'-epimer of 2',5'-linked ribonucleic acids (2 ',5'-RNA), that is, an oligonucleotide in which the ribofuranose sugar is replaced by xylofuranose (2' ,5'-XNA). This was followed by the synthesis and analysis of the C3'-fluorinated xylofuranose analogue (2',5'-FXNA). The sugar conformation in these oligonucleotides are believed to have a very high population of the C3'-endo ('extended') conformation. Consistent with this notion, CD structural studies indicated that 2 ',5,'-linked XNA and FXNA show structural similarities to the 'extended' C2'- endo form of DNA. We found that both 2',5 '-XNA and 2',5'-FXNA bound weakly to complementary single stranded DNA and RNA. Neither of the xylooligomers resulted in RNaseH activated degradation of RNA.<br>The last modified oligonucleotide to be studied was the C3' -fluorinated-2',5'-linked ribonucleic acids (2',5'-FRNA), which has a compact C2'-endo sugar conformation. In contrast to 2',5'-FXNA, 2 ',5'-FRNA bound strongly to complementary oligonucleotides and showed structural similarities to RNA (CD spectroscopy). Our studies showed that it did not cause RNaseH based degradation of RNA.<br>These studies are consistent with the notion that the effect of sugar conformation in 2',5'-oligonucleotides is opposite to that of 3',5'-oligonucleotides. In other words, a C3'-endo sugar conformation in 2',5'-oligomers renders the oligonucleotide as 'extended' and portrays itself equivalent to the 'extended' DNA conformation (which has C2'-endo conformation), whereas a C2'-endo 2 ',5'-oligonucleotide adopts a 'compact' conformation that is equivalent to that seen in 3',5 '-oligonucleotides adopting the C3'- endo pucker (e.g. RNA).

Occasionally, researchers need to modify enzymes through amino acid substitutions to make them more efficient catalysts for organic synthesis. There is still debate over the best protein engineering strategy for improving enzyme enantioselectivity: rational design or directed evolution. Rational design experiments focus mutations close to the active site, while directed evolution experiments often find mutations far from the active site.<br>In this thesis, a combination of the two strategies improved Pseudomonas fluorescens esterase (PFE) for production of a useful synthetic building block for organic synthesis. Random mutagenesis within the active site increased enantioselectivity more effectively (up to 5-fold reaching E = 61) than random mutagenesis of the entire protein (only 1.5-fold reaching E = 19). A general survey of previously published enzyme improvements showed that closer mutations were more effective than distant mutations for improving enantioselectivity. On this basis, we proposed that random mutagenesis focused in the active site may dramatically increase the success rate in future directed evolution experiments. The X-ray crystal structures of three improved PFE mutants showed that mutations directly in the active site can increase enantioselectivity without significantly altering the shape of the binding pocket. For rationalizing the improved enantioselectivity, a crystal structure of a transition state analogue-complex provided the conformation of the fast reacting enantiomer and computer modeling determined the conformation of the slow reacting enantiomer.<br>When novel esterases are discovered from directed evolution experiments, they are screened with libraries of esters to identify their preferred substrates. A convenient method for the parallel synthesis of esters was developed by using solid-supported reagents to eliminate traditional purification.<br>Acetyl xylan esterase (AxeA) was examined as a potential catalyst for the production of chitosan, a biopolymer with many commercial applications. Screening for chitin deacetylase activity showed that AxeA preferentially deacetylates chitosan oligosaccharides over alkali-treated chitin and crystalline chitin.

The N3 position of thymidine was alkylated with different sized bromoalkyl phthalimide linkers in generally good yields, with no alkylation observed on the sugar ring. The free amino group, available after methylamine deprotection of the phthalimide linker, was protected with the levulinyl protecting group (e.g., 20). Branched 'Y'-shaped nucleic acids containing branching monomer 20 were synthesized using standard solid phase synthetic methodology and complexes of these branched nucleic acids with one and two mole-equivalents of linear complement dA$ sb{10}$ are investigated by thermal melting. At the branching point the levulinic amide exhibited poor lability to Letsinger's hydrazine solution and proved to be incompatible with the solid phase synthesis of branched nucleic acids. The primary amino group provided a handle which was used to extend the size of the linker incorporating a primary hydroxyl group at the site of levulinic protection (e.g., 37, 38). The levulinic ester showed much better lability to hydrazinolysis rendering it more compatible with the solid phase synthesis of branched nucleic acids. An extensive one and two dimensional NMR characterization of the functionalized nucleosides is reported.

Researchers use enzymes for enantio- and regioselective reactions because of their high selectivity and activity toward natural substrates. However, researchers sometimes need to modify the reaction system or the enzyme itself to get reliable selectivity and activity when they deal with unnatural substrates. To obtain researcher's need, one can change the solvent, modify the substrates, or alter the enzyme itself. These processes are called medium, substrate, and protein engineering, respectively.<br>This thesis deals with hydrolases, which are classified by EC 3. We applied the proper approach to improve their activity and selectivity depending on the reactions. For the first approach, highly polar ionic liquids were applied to lipase-catalyzed acylation. Ionic liquids worked reliably in enantio- and regioselective lipase-catalyzed reactions. In particular, ionic liquids dissolved polar substrates such as glucose and L-ascorbic acid, thereby facilitating their acylations. In the second approach to improving enantioselectivity of CAL-B (Candida antarctica lipase B) in beta-lactam ring opening reactions, we changed the nucleophile from water to a range of alcohols. Longer, secondary alcohols increased the reaction rate as well as the enantioselectivity. Molecular modeling revealed that the high enantioselectivity of CAL-B and the critical role of alcohols. For the last approach, structure-guided random mutagenesis was applied to increase the enantioselectivity of PFE ( Pseudomonas fluorescens esterase) toward MBMP (methyl 3-bromo-2-methylpropionate). The homology model was used to select amino acid residues for mutagenesis near the stereocenter of the docked tetrahedral intermediate of the substrate. Randomization of these residues yielded a Val122Ser mutant with E increased to 61 (from 12 of wild type enzyme), as well as a Val122Met mutant to 36.

In order to establish if binding affinity could be improved by conformationally restricting the amide backbone, exo-amide-linked and endo-amide-linked 2$ sp prime$,3$ sp prime$-cyclopropanated dinucleoside analogs (e.g. dimers 35, 54, and 55) have been studied. The dimers were synthesized by coupling the corresponding cyclopropyl acids (5$ sp prime$-end building unit) with aminothymidines (3$ sp prime$-end building unit) by standard peptide synthesis methodology. After proper functionalizations, the dimers were incorporated into DNA sequences, and the effects of their incorporation into DNA-strands on binding to complementary DNA and RNA were evaluated.<br>Different strategies were explored to prepare the carboxylic acid-functionalized 2$ sp prime,3 sp prime$-cyclopropanated nucleoside analogs as the 5$ sp prime$-end building block. It was found that reaction of the $ alpha, beta$-unsaturated selenonyl uridine with the anions of 2-substituted acetates could efficiently yield the ester-functionalized cyclopropanes (e.g. 32 and 49) in a stereoselective manner, through a Michael-type cyclopropanation mechanism. Proper transformations of the ester precursor successfully provided the desired acid derivatives (e.g. 33, 52 and 53).

The antisense principle bases its premise in the exquisite complementarity of a synthetic, chemically-modified oligonucleotide to tightly bind with a unique target RNA sequence. Rapid and selective genetic discrimination, as driven by the formation of multiple points of target contact, constitutes a central goal of oligonucleotide therapies. Most synthetic designs have, however, provided little structural insight on the role of the antisense oligonucleotide (AON) in triggering RNA cleavage of preformed hybrids, as catalyzed by a ubiquitous, intracellular enzyme known as ribonuclease H. The use of RNase H to assist AON inhibition of gene expression is crucial to mainstream antisense technologies, yet the precise mode by which this enzyme acts on AON/RNA duplexes remains unclear.<br>To address the role of substrate structure on enzyme activation, a dominant theme of this thesis highlights the design, synthesis and structural studies of novel AONs comprised of rigid 2'-deoxy-2'-fluoroarabino (2'F-ANA) or native (DNA) nucleotides, containing interspersed flexible (e.g. "2',3'-seconucleotides") or anucleosidic (e.g. butyl) residues. This unique AON class combines both pre-organization & flexibility within the hosting heteroduplex, which on their own usually prove detrimental towards enzyme trigger. Their combination, however, synergistically activates both E. coli and human RNases H, leading to potent destruction of duplexed RNA. These compounds thus represent the first examples of modified AONs lacking deoxyribose sugars that elicit RNase H activity comparably to the native (DNA) systems. DNA-derived AONs with acyclic residues also amplify enzyme-catalyzed target degradation, suggesting the added flexibility imparted to the substrate structure to be vital for ameliorating the protein/nucleic acid interaction. Melting and circular dichroic experiments have revealed that the enhanced dynamics associated with a particular acyclic modification remain globally undetectable, indicating the acyclic residues induce only local structural deformations to the helix architecture.<br>Intricate comparisons of the structural and biological properties of various acyclic residues (e.g. butyl, propyl and ethyl interresidue spacers) designed to locally compress or expand the AON helix backbone at a defined axial site has enabled a deeper understanding of the conformational factors that underlie the observed enhancements.

Urea, N-methylurea and carbamate linked thymidine dimers, 38, 37 and 39 respectively, were synthesized efficiently and incorporated into 12- and 19-mer DNA oligonucleotide strands, via solid-phase synthesis. Thermal denaturation studies utilizing complementary single stranded DNA, RNA and duplex DNA, indicated selectivity of binding of all three backbone modified oligomers to single stranded DNA. The oligomer containing dimer 37 exhibited the strongest binding to single stranded DNA. Carbamate and 5$ sp prime$-N-methylurea modified dimers suitable for the preparation of longer nucleoside homopolymers were efficiently prepared from either thymidine or 3$ sp prime$-azido-3$ sp prime$-deoxythymidine.<br>Three efficient syntheses of precursors (7, 33, 36) of carbamoylpolyoxamic acid starting from L-arabinose were developed. Utilizing uridine as the starting material, an expedient synthesis of a precursor (44) to the nucleoside moiety of polyoxin L was also achieved. The conversion of cyanides to carboxylic acids was extensively investigated.<br>Protected polyoxamic acid 17 was synthesized in 11 steps from L-arabinose.* ftn*Please refer to the dissertation for diagrams.

Enantioselective synthesis is one of the most important challenges of today's synthetic chemists. In particular, the hydroxylation of non-activated C-H bonds remains a significant challenge that few chemical catalysts have succeeded to overcome. The P450 enzymes, a family of heme-containing monooxygenases including more than 5000 known isoforms, are gaining considerable attention due to their ability to catalyze the very difficult regio- and stereo-selective oxidation of inactivated C-H bonds. The use of such enzyme is however limited by their functional complexity, low activity, need for cofactors, and poor stability. In this thesis, we elected to study the human P450 CYP3A4, because of its high substrate promiscuity. The first part of the project involved the replacement of the required cofactors (NADPH and cytochrome P450 reductase) by some cheap hydrogen peroxide donors or organic peroxides. Several surrogates, such as sodium percarbonate and cumene hydroperoxide, were found to be efficient at replacing the natural cofactor, without a significant loss of stability and activity. The second part of this thesis deals with optimization of the lyophilization conditions. Among the numerous additives tested, some sugars led to significant lyoprotection during the freeze-drying process. Finally, in the third part, the effect of the presence of organic solvents and ionic liquids on CYP3A4 activity was evaluated.