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Helical oligomers made up of the achiral amino acid Aib (2-aminoisobutyric acid) have great potential in relaying stereochemical information over nanometre distances by control of their macromolecular conformation. In a synthetic signalling pathway, the helical domain acts as a transducer, mediating the information flow between an input and an output. This thesis describes the advancement of these helical systems into functioning, dynamic transduction pathways capable of nanoscale information processing by a variety of different means. Through studying the effect of C-terminal chiral amino acids, alanine was found to give maximal control over the helical screw-sense. It was also discovered that the hydrogen bonding preference of the terminal group (ester, amide etc.) determined the screw-sense direction. By modifying the terminus, this directional preference was inverted using light or pH as secondary stimuli. Non-covalent interactions with solvent were found to be important in controlling the flow of information through the helical domain. In less polar solvents (THF, chloroform) a single screw sense can prevail for up to 200 monomers, leading to very low signal loss. A zinc binding site was developed to allow a chiral ligand to act as the input in the signalling process. Both amino and phosphoric acids led to remarkable levels of screw-sense induction. Using the reversible ligand binding interaction a pH switch was set up to moderate the binding of these two ligands, creating a ternary information switch. Significant progress was also made towards a light switch using a spiropyran as a competing ligand. Finally, a novel light-responsive switch capable of releasing a signalling molecule in solution was conceived and developed.

The reaction of tert-butyllithium with chloromethylphenylvinylsilane at low temperatures in hexane gave a 48% yield of a mixture of the five isomers of 1,3-dimethyl-1,3-diphenyl-2,4-dineopentyl-1,3-disilacyclobutane, formed by the head-to-tail dimerization of both E- and Z-1-methyl-1-phenyl-2-neopentylsilenes, along with an acyclic dimer. These were separated and their stereochemistry was established by ('1)H- and ('13)C-NMR spectroscopy. The E- and Z-silenes were also trapped as their {4 + 2} cycloadducts with cyclopentadiene, 2,3-dimethyl-1,3-butadiene and anthracene, which also were separated and stereochemically characterized. A consistent mole ratio of 70:30 for the E- and Z-silene adducts is interpreted as evidence for stereochemical induction in the silene generation reaction. It is also suggested that the dimerization of the silenes to give the 1,3-disilacyclobutanes occurs by a nonstereospecific stepwise pathway. When E- or Z-1-methyl-1-phenyl-2-neopentylsilene was generated by the retro-Diels-Alder flow vacuum thermolysis of its corresponding cyclopentadiene or anthracene adduct at temperatures between 400 and 600(DEGREES)C and then trapped with 2,3-dimethyl-1,3-butadiene, the stereochemical distribution of the products is independent of the stereochemistry of the silene precursor, indicating that the silene is not configurationally stable towards cis-trans isomerization at these temperatures. Evidence that the intermolecular ene reaction and the {4 + 2} cycloaddition which occur with 2,3-dimethyl-1,3-butadiene are concerted is presented. When either the E- or Z-silene, generated by the sealed tube thermolysis of its anthracene adduct by 300(DEGREES)C, was trapped with trimethylmethoxysilene, the diastereomer obtained depended on the stereochemistry of the silene precursor, showing that the silene is configurationally stable towards cis-trans isomerization up to 300(DEGREES)C. The temperature dependence of the ratio of the two diastereomers obtained when the silene formed from the pure E- or Z-anthracene adduct was trapped at higher temperatures permitted the determination of an activation energy for the silene isomerization. The activation energies for the E- and Z- and Z- to E-silene isomerization are 45 (+OR-) 6 and 20 (+OR-) 4 kcal mol('-1), respectively. The significance of these values is discussed.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21049号 / 薬科博第92号 / 新制||薬科||10(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 竹本 佳司, 教授 高須 清誠 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Microbial degradation of aromatic hydrocarbons is imperative for maintaining the global carbon cycle and removing potentially toxic aromatic xenobiotics. This thesis focuses on the characterization of a pyruvate-specific class II aldolase (BphI) and acetaldehyde dehydrogenase (BphJ), the final two enzymes of the bph meta-cleavage pathway in Burkholderia xenovorans LB400. This pathway is responsible for the degradation of the industrial pollutant polychlorinated biphenyls (PCB) and therefore mechanistic characterization of these enzymes can be applied to improve pollutant degradation. BphI catalyzes the aldol cleavage of 4-hydroxy-2-oxoacids to pyruvate and an aldehyde while BphJ transforms aldehydes to acyl-CoA, using NAD+ and CoASH as cofactors. Size-exclusion chromatography was used to determine that the oligomeric unit of the BphI-BphJ complex is a heterotetramer. The aldolase BphI was shown to exhibit a compulsory order mechanism and utilize 4-hydroxy-2-oxoacids with an S configuration at C4. The generation of BphI active site variants allowed for the proposal of a catalytic mechanism and a greater understanding as to how stereospecificity occurs. Using steady-state kinetic assays, Arg-16 was demonstrated to be essential for catalysis. Molecular modeling of the substrate and pH dependency (wild-type pKa of ~7, lost in H20A and H20S variants) were used to identify His-20 as the catalytic base. Tyr-290 was originally proposed to be the catalytic acid. However, this was refuted as a Tyr-290 (Y290F) variant did not affect the catalytic efficiency of the enzyme. Instead, the variant was observed to exhibit a loss of stereochemical control. From the crystal structure of an orthologous aldolase-dehydrogenase complex, solvent isotope effect studies, and a proton inventory, a water molecule was implicated as the catalytic acid. Based on their position within the crystal structure, Leu-87 and Leu-89 were implicated in substrate specificity. Replacement of Leu-89 with alanine effectively increased the length of the active site, allowing for the accommodation of longer aldehyde substrates. In contrast, Leu-87 was responsible for hydrophobic stabilization of the C4-methyl of the substrate. Double variants L87N;Y290F and L87W;Y290F were constructed to enable the binding of 4(R)-hydroxy-2-oxoacids. Polarimetric analysis confirmed that the double variants were able to synthesize 4-hydroxy-2-oxoacids of up to 8 carbons in lengths, which were of the opposite stereoisomer to those produced by the wild-type enzyme. Cys-131 was identified as the catalytic thiol that forms an acyl-enzyme intermediate in the dehydrogenase, BphJ. This enzyme was shown to exhibit similar specificity constants for acetaldehyde and propionaldehyde and utilize aliphatic aldehydes from two to five carbons in length as substrates. The enzyme was able to use either NAD+ or NADP+ as the cofactor. Finally, we demonstrated that aldehydes produced in the aldolase reaction are not released into the bulk solvent but are channeled directly to the dehydrogenase, providing the first biochemical determination of substrate channeling in any aldolase-dehydrogenase complex. / Chapter 3 - Reprinted (adapted) with permission from Baker, P., Carere, J., and Seah, S. Y. (2011) Probing the Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis in the Class II Pyruvate Aldolase, BphI, Biochemistry 50: 3559-3569. Copyright (2011) American Chemical Society. Chapter 4 - Reprinted (adapted) with permission from Baker, P., and Seah, S. Y. (2012) Rational design of stereoselectivity in the class II pyruvate aldolase BphI, J Am Chem Soc 134: 507-513. Copyright (2012) American Chemical Society. Chapter 6 - Reprinted (adapted) with permission from Baker, P., Hillis, C., Carere, J., and Seah, S. Y. (2012) Protein-protein interactions and substrate channeling in orthologous and chimeric aldolase-dehydrogenase complexes, Biochemistry 51: 1942-1952. Copyright (2012) American Chemical Society. / National Science and Engineering Research Council of Canada (NSERC), Ontario Graduate Scholarship in Science and Technology

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PART I RESOLUTION AND DESYMMETRISATION Chapter I. ‘A Novel Racemate Resolution’. This describes a novel resolution strategy as applied to racemic α-amino acids in the solid state. The strategy is based on the possibility that second order asymmetric transformations (SOAT) would be more likely in the case of achiral molecules that form chiral crystals (i.e. a non- centrosymmetric space group).1 In such cases, a fundamental requirement of SOAT – that the molecules racemise in solution prior to crystallization – is obviated. Furthermore, the resulting enantiomerically-enriched crystals may be employed to effect a solid-state kinetic resolution of a different racemate (composed of chiral molecules). This strategy was explored with crystalline succinic anhydride (1, Scheme 1), which not only exists in a non-centrosymmetric space group (P212121) but also possesses reactive functionality to effect the resolution step.2 Thus, a finely-ground mixture of 1 (0.5 eqiv.) and a racemic α-amino acid (2, 1.0 eqiv.) was heated at ~ 70 oC over ~ 5 h without solvent. The resulting N-succinoyl derivative (3) was separated from the unreacted 2, which was found to possess significant levels of optical purity (typically ~ 70%). The strategy was applied to several common α-amino acids, the results being summarized in Table 1. These results, apart from establishing ‘proof-of-concept’ and the viability of the resolution strategy, indicate that crystalline succinic anhydride (1) is enantiomerically enriched as originally hypothesized. Chapter II. ‘Enantiospecific Alkylation and Desymmetrisations’. This deals with two enolate-mediated strategies of asymmetric synthesis: one describes approaches towards the alkylation of the stereogenic centre in benzoin without loss of stereogenicity (Section A), and the other the desymmetrisation of a meso tartarate derivative with a chiral base catalyst (Section B). Section A. This describes exploratory studies aimed at achieving the enantiospecific α-alkylation of optically-active benzoin (4, Scheme 2) via its enolate anion 5. The strategy depends on the possibility that 5 would exist in atropisomeric forms, because of steric interactions between the vicinal phenyl groups. (This is indicated in the crystal structure of the analogous enediol carbonate derived from racemic 4.)3 In such a case, remarkably, 5 would be chiral, despite its planar enediolate core! Thus, possibly, the configurational chirality in 4 (by virtue of the C2 stereogenic centre) would be transformed to the helical chirality in 5 (by virtue of the atropisomerism). Furthermore, enantioface-selective alkylation of 5 with achiral alkylating agents would, in principle, be possible. Preliminary studies were then directed towards establishing that controlled deprotonation of optically-active 4, followed by the protonation of the resulting enediolate 5, leads back to the original 4. (+)-Benzoin (4) was prepared via resolution,4 and deprotonated with KH in THF.5 The resulting enediolate (5) was neutralized with acetic acid at -70 oC/THF to recover 4, but with insignificant levels of optical activity (e.e. ~ 12%). The results possibly indicate that ortho-substituted benzoin analogs may show greater retention of chirality upon deprotonation, as the racemisation of the enediolate atropisomers would be suppressed by steric hindrance between the aryl moities. Section B. This describes studies directed towards the catalytic desymmetrisation of meso dimethyl tartarate (6, Scheme 3). The strategy involves the formation of the acetonide derivative 7 and its regioselective α-deprotonation with a chiral base catalyst. The enantioface-selective protonation of the resulting enolate (8) would lead to the chiral analog 9. The overall sequence offers a possible alternative to catalytic deracemisation, which is normally unviable for thermodynamic reasons.6 The above strategy hinges on the meso derivative 7 being thermodynamically less stable than the enantiomeric 9, which would thus be favoured at equilibrium. In fact, this is likely as the eclipsing interactions between the syn ester moieties in 7 would be relieved in 9, in which the ester moieties are anti. However, deprotonation of 7 at the other α position would compete to varying extents, depending on the selectivity induced by the chiral base. At total equilibrium, the sequence would occur via deprotonation at both α sites at equal rates, and no net optical induction would be observed. (This is a thermodynamic requirement via the principle of microscopic reversibility.) Thus, the success of the above strategy depends on stalling the deprotonation-protonation sequence at a quasi-equilibrium stage involving only one of the enantiomers (9).6 The other operational requirement was the compatibility of the pKa’s of 7 and the chiral base employed: too low a pKa of the base would result in inefficient deprotonation and slow overall rate, but a high pKa would generate a large quantity of the enolate 8 at equilibrium. After due consideration, the lithiated chiral fluorene derivative 11 (pKa ~ 22) was chosen as the chiral base catalyst [11 was prepared from fluorene (10) as indicated]. Treating 7 with 0.2 equivalent of 10 in THF at -65 oC over 2 h, led to the formation of a mixture of 7 and 9 in a 45:55 ratio (isolated in 85% total yield). Chromatographic separation of the mixture led to the isolation of pure (+)-9, which was identified spectrally; it was found to possess [α]D24 = +21.84 (c 1.0, CHCl3), corresponding to e.e. = 64%. (This implies the indicated (4S, 5S) configuration for 1, 3-dioxolane 9, as previously reported.)7 These results, despite the moderate e.e. levels obtained, indicate the viability of the above catalytic desymmetrisation strategy, bearing in mind the mechanistic ambiguities mentioned above. PART II SYNTHESES OF ALDEHYDES AND AMINO ACIDS Chapter III. ‘An Asymmetric Synthesis of Aldehydes’. This describes an oxazoline approach to the synthesis of chiral aldehydes. The oxazoline methodology for the synthesis of homochiral α-alkylated carboxylic acids is well known,8 and it was of interest to adapt this to the synthesis of the corresponding aldehydes. Essentially, it was envisaged that the reaction sequence could be diverted towards aldehydes via reduction of the alkylated oxazoline intermediate (Scheme 4). Thus, 2-ethyl-4(S)-methoxymethyl-5(R)-phenyl-1,3-oxazoline (12) was deprotonated with lithium diisopropylamide in THF, and the resulting anion treated with various alkyl halides, in the reported manner.8 The resulting alkylated product (13) was N-methylated with MeI in refluxing MeNO2 over 6 h, to obtain the quaternary salt 14. This was reduced with NaBH4 in MeOH to obtain the expected N- methyl oxazolidine 15, which was hydrolyzed in refluxing aqueous oxalic acid to the free aldehydes 16. These were isolated in moderate yields and e.e. values as shown. Chapter IV. ‘A Darzens Route to α-Amino Acids’. This describes a novel route to α-amino acids, based on the classical Darzens glycidic ester synthesis.9 In this approach (Scheme 5), the glycidic ester (19) was prepared from benzaldehyde (17) and t-butyl bromoformate (18), with KOH in THF as base, and tetrabutylammonium bromide (TBAB) as phase transfer catalyst.9b The oxirane ring in 19 was cleaved via nucleophilic attack with an amine (20), to furnish the two regio-isomeric hydroxy- amino acids (21) and (22). Generally, the β-hydroxy-α-amino acid product (21) predominated over the α-hydroxy-β-amino acid product (22), the two being separated chromatographically. The hydroxyl group in 21 was reductively cleaved via its xanthate derivative (23), by refluxing it in toluene with AIBN (10 mol %) over 4 h. The resulting α-amino acid derivatives (24) were obtained in moderate yields (< 60 %) upon chromatographic purification. (The β-amino analog 22, would lead to the corresponding β-amino acid, but this was not pursued further.) This strategy lends itself to creating structural diversity at the β-centre in the α- amino acid, drawing upon the wide scope of the well-established Darzens condensation reaction. Also, the introduction of the amino moiety is facilitated by the enhanced reactivity at the α-centre of the oxirane ring in the glycidic ester (19), presumably for both electronic and steric reasons.

This thesis deals with the development of new reaction methodology as well as stereochemical investigations. The first part concerns the investigation of 1,2- and merged 1,2- and 1,3- asymmetric induction in Mukaiyama aldol additions to α-heteroatom and α,β- heteroatom substituted aldehydes respectively. In particular, the unexpected 1,2-syn selectivity obtained in the addition of sterically hindered nucleophiles to α-chloroaldehydes is examined, and an explanation for the observed stereochemical trends is proposed. The second part describes the development of a novel entry to α-amino-β- hydroxy esters by a 1,3-dipolar cycloaddition reaction of aldehydes and azomethine ylides, generated by thermolysis of aziridines. The third part deals with our efforts to develop a novel entry to vicinal all- carbon quaternary centers, based on an intramolecular domino Heck- carbonylation reaction using tetrasubstituted olefins. / QC 20120611