Floral Scent Production in Clarkia breweri1 : III. Enzymatic Synthesis and Emission of Benzenoid Esters

The fragrance of Clarkia breweri (Onagraceae), a California annual plant, includes three benzenoid esters: benzylacetate, benzylbenzoate, and methylsalicylate. Here we report that petal tissue was responsible for the benzylacetate and methylsalicylate emission, whereas the pistil was the main source of benzylbenzoate. The activities of two novel enzymes, acetyl-coenzyme A:benzylalcohol acetyltransferase (BEAT), which catalyzes the acetyl esterification of benzylalcohol, and S-adenosyl-l-methionine:salicylic acid carboxyl methyltransferase, which catalyzes the methyl esterification of salicylic acid, were also highest in petal tissue and absent in leaves. In addition, the activity of both enzymes in the various floral organs was developmentally and differentially regulated. S-Adenosyl-l-methionine:salicylic acid carboxyl methyltransferase activity in petals peaked in mature buds and declined during the next few days after anthesis, and it showed a strong, positive correlation with the emission of methylsalicylate. The levels of BEAT activity and benzylacetate emission in petals also increased in parallel as the buds matured and the flowers opened, but as emission began to decline on the 2nd d after anthesis, BEAT activity continued to increase and remained high until the end of the lifespan of the flower.

Enantioselective direct aldol reaction of α-keto esters catalyzed by (Sa)-binam-D-prolinamide under quasi solvent-free conditions

(Sa)-Binam-D-prolinamide (20 mol%), instead of (Sa)-binam-L-prolinamide, in combination with chloroacetic acid (100 mol%) is an efficient organocatalyst for the direct aldol reaction between α-keto esters as electrophiles and alkyl and α-functionalised ketones, under quasi solvent-free conditions, providing access to highly functionalised chiral quaternary γ-keto α-hydroxyesters with up to 92% ee.

Domino 1,3-Dipolar Cycloadditions of N-Alkyl-α-Amino Esters with Paraformaldehyde: A Direct Access to α-Hydroxymethyl α-Amino Acids

N-Alkyl-α-amino esters undergo a domino reaction, based on the iminium cation generation, with paraformaldehyde, followed by a 1,3-dipolar cycloaddition of the stabilized azometh­ine ylide with another equivalent of formaldehyde. The resulting products are oxazolidines, which can be transformed after hydrolysis into α-hydroxymethyl α-amino acid or its derivatives. The diastereoselective 1,3-dipolar cycloaddition was performed using sarcosine (–)-menthyl or (–)-8-phenylmenthyl esters affording the cyclic product with moderate enantiomeric ratio.

Facile synthesis of rhodamine esters using acetyl chloride in alcohol solution

The rhodamines are a highly fluorescent class of compound used in many different fields of research, from the lasing medium in dye lasers to biological stains and markers for cellular drug resistance. In this study, esters (2-7) of rhodamine 110 (1) were conveniently prepared via the addition of acetyl chloride to a solution of the free acid (1) in the appropriate alcohol. This method conferred several advantages over previous preparations, namely that for low boiling alcohols, simple evaporation of the solution afforded the ester in quantitative yield with no need for purification. For higher boiling point alcohols, a method has been developed which allows the separation of longer chain esters from the alcohol solvent.

Macrophage-specific expression of human lysosomal acid lipase corrects inflammation and pathogenic phenotypes in lal(-/-) mice

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in the cell. The downstream metabolites of these compounds serve as hormonal ligands for nuclear receptors and transcription factors. Genetic ablation of the lal gene in the mouse caused malformation of macrophages and inflammation-triggered multiple pathogenic phenotypes in multiple organs. To assess the relationship between macro phages and lal(-/-) pathogenic phenotypes, a macrophage-specific doxycycline-inducible transgenic system was generated to induce human LAL (hLAL) expression in the lal(-/-) genetic background under control of the 7.2-kb c-fins promoter/intron2 regulatory sequence. Doxycycline-induced hLAL expression in macrophages significantly ameliorated aberrant gene expression, inflammatory cell (neutrophil) influx, and pathogenesis in multiple organs. These studies strongly support that neutral lipid metabolism in macrophages contributes to organ inflammation and pathogenesis.

A blank slate? Layer-by-layer deposition of hyaluronic acid and chitosan onto various surfaces

Although poly(alpha-hydroxy esters), especially the PLGA family of lactic acid/glycolic acid copolymers, have many properties which make them promising materials for tissue engineering, the inherent chemistry of surfaces made from these particular polymers is problematic. In vivo, they promote a strong foreign-body response as a result of nonspecific adsorption and denaturation of serum proteins, which generally results in the formation of a nonfunctional fibrous capsule. Surface modification post-production of the scaffolds is an often-utilized approach to solving this problem, conceptually allowing the formation of a scaffold with mechanical properties defined by the bulk material and molecular-level interactions defined by the modified surface properties. A promising concept is the so-called blank slate: essentially a surface that is rendered resistant to nonspecific protein adsorption but can be readily activated to covalently bind bio-functional molecules such as extracellular matrix proteins, growth factors or polysaccharides. This study focuses on the use of the quartz crystal microbalance (QCM) to follow the layer-by-layer (LbL) electrostatic deposition of high molecular weight hyaluronic acid and chitosan onto PLGA surfaces rendered positively charged by aminolysis, to form a robust, protein-resistant coating. We further show that this surface may be further functionalized via the covalent attachment of collagen IV, which may then be used as a template for the self-assembly of basement membrane components from dilute Matrigel. The response of NIH-3T3 fibroblasts to these surfaces was also followed and shown to closely parallel the results observed in the QCM.

Design and characterisation of novel blends of poly(lactic acid)

This thesis is concerned with the effect of polymer structure on miscibility of the three component blends based on poly(lactic acid) (PLA) with using blending techniques. The examination of novel PLA homologues (pre-synthesised poly(a-esters)), including a range of aliphatic and aromatic poly(a-esters) is an important aspect of the work. Because of their structural simplicity and similarity to PLA, they provide an ideal system to study the effect of polyester structures on the miscibility of PLA polymer blends. The miscibility behaviour of the PLA homologues is compared with other aliphatic polyesters (e.g. poly(e-caprolactone) (PCL), poly(hydroxybutyrate hydroxyvalerate) (P(HB-HV)), together with a series of cellulose-based polymers (e.g. cellulose acetate butyrate (CAB)). The work started with the exploration the technique used for preliminary observation of the miscibility of blends referred to as “a rapid screening method” and then the miscibility of binary blends was observed and characterised by percent transmittance together with the Coleman and Painter miscibility approach. However, it was observed that symmetrical structures (e.g. a1(dimethyl), a2(diethyl)) promote the well-packing which restrict their chains from intermingling into poly(L-lactide) (PLLA) chains and leads the blends to be immiscible, whereas, asymmetrical structures (e.g. a4(cyclohexyl)) behave to the contrary. a6(chloromethyl-methyl) should interact well with PLLA because of the polar group of chloride to form interactions, but it does not. It is difficult to disrupt the helical structure of PLLA. PLA were immiscible with PCL, P(HB-HV), or compatibiliser (e.g. G40, LLA-co-PCL), but miscible with CAB which is a hydrogen-bonded polymer. However, these binary blends provided a useful indication for the exploration the novel three component blends. In summary, the miscibility of the three-component blends are miscible even if only two polymers are miscible. This is the benefit for doing the three components blend in this thesis, which is not an attempt to produce a theoretical explanation for the miscibility of three components blend system.

The polymerization of lactic acid anhydrosulphite by anionic initiators

This thesis is primarily concerned with the synthesis and polymerization of 5-methyl-1;3, 2-dioxathiolan-4-one-2-oxide (lactic acid anhydrosulphite (LAAS)) using anionic initiators under various conditions. Poly(lactic acid) is a biodegradable polymer which finds many uses in biomedical applications such as drug-delivery and wound-support systems. For such applications it is desirable to produce polymers having predictable molecular weight distributions and crystallinity, The use of anionic initiators offers a potential route to the creation of living polymers. The synthesis of LAAS was achieved by means of an established route though the procedure was modified to some extent and a new method of purification of the monomer using copper oxides was introduced, Chromatographic purification methods were also examined but found to be ineffective. An unusual impurity was discovered in some syntheses and this was identified by means of 1H and 13C NMR, elemental analysis and GC-MS. Since poly-α-esters having hydroxyl-bearing substituents might be expected to have high equilibrium water contents and hence low surface tension characteristics which might aid bio-compatibility, synthesis of gluconic acid anhydrosulphite was also attempted and the product characterised by 1H and 13C NMR. The kinetics of the decomposition of lactic acid anhydrosulphite by lithium tert-butoxide in nitrobenzene has been examined by means of gas evolution measurements. The kinetics of the reaction with potassium tert-butoxide (and also sec-butyl lithium) in tetrahydrofuran has been studied using calorimetric techniques. LAAS was block co-polymerized with styrene and also with 1,3-butadiene in tetrahydrofuran (in the latter case a statistical co-polymer was also produced).

Hydrothermal saline promoted grafting of periodic mesoporous organic sulfonic acid silicas for sustainable FAME production

Hydrothermal saline promoted grafting of sulfonic acid groups onto SBA-15 and periodic mesoporous organic silica analogues affords solid acid catalysts with high acid site loadings (>2.5 mmol g-1 H+), ordered mesoporosity and tunable hydrophobicity. The resulting catalysts show excellent activity for fatty acid esterification and tripalmitin transesterification to methyl palmitate, with framework phenyl groups promoting fatty acid methyl esters production. (Chemical Equation Presented)

Syntheses of aza analogues of kainic acid

The kainate receptors are one of the three major groups of ionotropic glutamate receptors in the mammalian central nervous system. They are so named after their most potent agonist, kainic acid (KA), a natural product isolated from the seaweed Diginea simplex. This compound shows both neuroexcitatory and excitotoxic activities, and is an important pharmacological tool for neurophysiological studies. We predict that the more synthetically accessible aza analogues of kainic acid, could act as functional mimics of KA. These could be produced by the 1,3-dipolar cycloaddition of diazoalkanes with trans glutaconate esters. ^ 1,3-Dipolar cycloadditions have been shown to produce 1-pyrazolines that isomerize into 2-pyrazolines. The 1- and 2-pyrazolines can be precursors to aza analogs of kainoids. The regioselectivity, relative stereochemistry and isomerization of the 1-pyrazolines into 2-pyrazolines have been evaluated. Reductions of the 1- and 2-pyrazolines produced aza analogs of kainoids. TMS diazomethane was used as the dipole in 1,3-dipolar cycloaddition reactions leading to aza KA analogs via 2-pyrazolines. A systematic study of cycloaddition-isomerization processes involving TMS-diazomethane and various α, β-unsaturated dipolarophiles has been undertaken. 1H-NMR monitoring of the reaction mixture compositions during the cycloaddition reaction revealed evidence of retro-dipolar cycloaddition processes. Faster formation of 4,5- trans-1-pyrazoline at the beginning of the reaction and subsequent isomerization of this product into 4,5-cis-1-pyrazoline via a retro-dipolar cycloaddition has been observed. Increased reaction time and/or reaction temperature preferentially caused the irreversible isomerization of 4,5-cis-1-pyrazoline into 4,5-cis-2-pyrazoline, which led to high yields of 4,5-cis-2-pyrazolines in the overall process. ^ Two syntheses of the 5-unsubstituted aza-kainic acid have been performed; first, via the reduction of the TMS-eliminated 2-pyrazoline from TMS diazomethane; second by the direct reduction of 1-pyrazoline with Hg/Al-amalgam. 5-Phenyl aza-kainic acid has been produced by direct reduction of 1-pyrazoline, obtained in the reaction of phenyldiazomethane and dibenzyl glutaconate, with Hg/Al-amalgam. ^ Current responses to aza kainate analogs in Aplysia whole cell buccal ganglia indicate potent neuroexcitatory activity. The repetitive exposure of neuronal cells to the 5-unsubstituted aza-kainic acid led to non-desensitizing current responses, showing both binding affinity and neuronal ion-channel activation by the synthesized agonist compound. ^

Remote Substituent Effects on the Stereoselectivity and Organocatalytic Activity of Densely Substituted Unnatural Proline Esters in Aldol Reactions

The organocatalytic activities of highly substituted proline esters obtained through asymmetric [3+2] cycloadditions of azomethine ylides derived from glycine iminoesters have been analyzed by 19F NMR and through kinetic isotope effects. Kinetic rate constants have been determined for unnatural proline esters incorporating different substituents. It has been found that exo-L and endo-L unnatural proline methyl esters yield opposite enantiomers in aldol reactions between cyclic ketones and aromatic aldehydes. The combined results reported in this study show subtle and remote effects that determine the organocatalytic behavior of these synthetic but readily available amino acid derivatives. These data can be used as design criteria for the development of new pyrrolidine-based organocatalysts.

Synthetic studies towards the total synthesis of hexacyclinic acid

In the first chapter of this thesis, published works found in the literature about hexacyclinic acid and FR182877 are reported and commented. A quick summary of the previous work done in the Prunet group is also described. In the second and third chapter, a more detailed account of the work undertaken during this PhD was given. Firstly, syntheses of two ABC tricycles incorporating tert-butyl and (trimethylsilyl)ethyl esters were undertaken. These syntheses include two key steps previously developed in the group, a diastereoselective Michael addition and a Snider cyclisation. Multiple conditions for the hydrolysis of the esters were attempted but none of them gave the desired product. The main part of this work is focused on the synthesis of a CDEF model and in particular about the development of the key step, the formation of a nine-membered ring. Several DEF fragments were synthesised in short synthetic sequences and as single isomers. Six different synthetic pathways were developed in total and a novel method, a Michael/elimination reaction, was found to be a very efficient way to close the desired medium-size ring. From the nine-membered ring, regioselective reduction and palladiumcatalysed allylic substitution led to the formation of the CDF tricycle. Final steps of the synthesis were fruitless and led only to decomposition. A synthesis of a chiral C-ring was also developed during this PhD. II Finally, another project was undertaken, not related to hexacyclinic acid. Methodology developed in the group for the diastereoselective formation of trisubstituted alkenes employing a temporary silicon-tethered ring-closing metathesis was extended to homoallylic alcohols. The first steps of the method were similar to the previous methodology but the end-game had to be modified in favour of an oxidation/reduction sequence to successfully obtain the desired products with the correct geometry. In the fourth chapter, procedures and analytical data for the synthesised compounds previously described are reported.

Catalytic upgrading of carboxylic acids and esters to bio fuels and bio chemicals

The research activity was focused on the transformation of methyl propionate (MP) into methyl methacrylate (MMA), avoiding the use of formaldehyde (FAL) thanks to a one-pot strategy involving in situ methanol (MeOH) dehydrogenation over the same catalytic bed were the hydroxy-methylation/dehydration of MP with FAL occurs. The relevance of such research line is related to the availability of cheap renewable bio-glycerol from biodiesel production, from which MP can be obtained via a series of simple catalytic reactions. Moreover, the conventional MMA synthesis (Lucite process) suffers from safety issues related to the direct use of carcinogenic FAL and depends on non-renewable MP. During preliminary studies, ketonization of carboxylic acids and esters has been recognized as a detrimental reaction which hinders the selective synthesis of MMA at low temperature, together with H-transfer hydrogenation with FAL or MeOH as the H-donor at higher temperatures. Therefore, ketonization of propionic acid (PA) and MP was investigated over several catalysts (metal oxides and metal phosphates), to obtain a better understanding of the structure-activity relationship governing the reaction and to design a catalyst for MMA synthesis capable to promote the desired reaction while minimizing ketonization and H-transfer. However, ketonization possesses scientific and industrial value itself and represents a strategy for the upgrade of bio oils from fast pyrolysis of lignocellulosic materials, a robust and versatile technology capable to transform the most abundant biomass into liquid biofuels. The catalysts screening showed that ZrO2 and La2O3 are the best catalysts, while MgO possesses low ketonization activity, but still, H-transfer parasitic hydrogenation of MMA reduces its yield over all catalysts. Such study resulted in the design of Mg/Ga mixed oxides that showed enhanced dehydrogenating activity towards MeOH at low temperatures. It was found that the introduction of Ga not only minimize ketonization, but also modulates catalyst basicity reducing H-transfer hydrogenations.

Upgrading Bio-Platform Molecules in the Gas-Phase: From Levulinic Acid to Bio-Chemicals

Levulinic acid (LA) is a polyfunctional molecule obtained from biomass. Because of its structure, the United States Department of energy classified LA as one of the top 12 building block chemicals. Typically, it is valorized through chemical reduction to γ-valerolactone (GVL). It is usually done with H2 in batch systems with high H2 pressures and noble metal catalysts, making it expensive and less applicable. Therefore, alternative approaches such as catalytic transfer hydrogenation (CTH) through the Meerwein–Ponndorf–Verley (MPV) reaction over heterogeneous catalysts have been studied. This uses organic molecules (alcohols) which act as a hydride transfer agent (H-donor), to reduce molecules containing carbonyl groups. Given the stability of the intermediate, reports have shown the batch liquid-phase CTH of levulinate esters with secondary alcohols, and remarkable results (GVL yield) have been obtained over ZrO2, given the need of a Lewis acid (LASites) and base pair for CTH. However, there were no reports of the continuous gas-phase CTH of levulinate esters. Therefore, high surface area ZrO2 was tested for gas-phase CTH of methyl levulinate (ML) using ethanol, methanol and isopropanol as H-donors. Under optimized conditions with ethanol (250 ℃), the reaction is selective towards GVL (yield 70%). However, heavy carbonaceous materials over the catalyst surface progressively blocked LASites changing the chemoselectivity. The in situ regeneration of the catalyst permitted a partial recovery of the LASites and an almost total recovery of the initial catalytic behavior, proving the deactivation reversible. Tests with methanol were not promising (ML conversion 35%, GVL yield 4%). As expected, using isopropanol provided complete conversion and a GVL yield of 80%. The reaction was also tested using bioethanol derived from agricultural waste. In addition, a preliminary study was performed for the hydrogenolysis of polyols to produce bioethanol, were Pd-Fe catalyst promoted the ethanol selective (37%) hydrogenolysis of glycerol.