Sporopollenin, the least known yet toughest natural biopolymer

© 2015 Mackenzie, Boa, Diego-Taboada, Atkin and Sathyapalan. Sporopollenin is highly cross-linked polymer composed of carbon, hydrogen, and oxygen that is extraordinarily stable and has been found chemically intact in sedimentary rocks some 500 million years old. It makes up the outer shell (exine) of plant spores and pollen and when extracted it is in the form of an empty exine or microcapsule. The exines resemble the spores and pollen from which they are extracted, in size and morphology. Also, from any one plant such characteristics are incredible uniform. The exines can be used as microcapsules or simply as micron-sized particles due to the variety of functional groups on their surfaces. The loading of a material into the chamber of the exine microcapsule is via multi-directional nano-diameter sized channels. The exines can be filled with a variety of polar and non-polar materials. Enzymes can be encapsulated within the shells and still remain active. In vivo studies in humans have shown that an encapsulated active substance can have a substantially increased bioavailability than if it is taken alone. The sporopollenin exine surface possesses phenolic, alkane, alkene, ketone, lactone, and carboxylic acid groups. Therefore, it can be derivatized in a number of ways, which has given rise to applications in areas, such as solid supported for peptide synthesis, catalysis, and ion-exchange chromatography. Also, the presence of the phenolic groups on sporopollenin endows it with antioxidant activity

Sulfonated sporopollenin as an efficient and recyclable heterogeneous catalyst for dehydration of D-xylose and xylan into furfural

The natural acidity of sporopollenin, the biopolymer coating the outer walls of pollen grains, was enhanced by the sulfonation of its surface. Modified sporopollenin displaying sulfonic acid groups has been prepared, characterized by elemental analysis, SEM, EDX, FTIR and XPS and tested as a heterogeneous catalyst in the dehydration of D-xylose and xylan to produce furfural. The optimal reaction conditions involve 10 wt % of sulfonated sporopollenin in the presence of 1.5 mmol of NaCl in a biphasic water-CPME system. When heated at 190 °C, the reaction affords furfural in a yield of 69% after 40 min under microwave irradiation. The time dependence of the dehydration and influence of temperature, pentose loading and positive effect of chloride ions on the reaction rate are reported. It was found that the catalytic system, recharged with the pentose and solvent, could be recycled ten times without loss of performance. The transformation of xylan into furfural at 190 °C for 50 min gave furfural in a yield of 37%

How does iron interact with sporopollenin exine capsules? An X-ray absorption study including microfocus XANES and XRF imaging

Sporopollenin exine capsules (SECs) derived from plant spores and pollen grains have been proposed as adsorption, remediation and drug delivery agents. Despite many studies there is scant structural data available. This X-ray absorption investigation represents the first direct structural data on the interaction of metals with SECs and allows elucidation of their structure–property relationships. Fe K-edge XANES and EXAFS data have shown that the iron local environment in SECs (derived from Lycopodium clavatum) reacted with aqueous ferric chloride solutions is similar to that of ferrihydrite (FeOOH) and by implication ferritin. Fe Kα XRF micro-focus experiments show that there is a poor correlation between the iron distribution and the underlying SEC structure indicating that the SEC is coated in the FeOOH material. In contrast, the Fe Kα XRF micro-focus experiments on SECs reacted with aqueous ferrous chloride solutions show that there is a very high correlation between the iron distribution and the SEC structure, indicating a much more specific form of interaction of the iron with the SEC surface functional groups. Fe K-edge XANES and EXAFS data show that the FeII can be easily oxidised to give a structure similar to, but not identical to that in the FeIII case, and that even if anaerobic conditions are used there is still partial oxidation to FeIII

Glycogen Content Regulates Peroxisome Proliferator Activated Receptor-∂ (PPAR-∂) Activity in Rat Skeletal Muscle

Performing exercise in a glycogen depleted state increases skeletal muscle lipid utilization and the transcription of genes regulating mitochondrial β-oxidation. Potential candidates for glycogen-mediated metabolic adaptation are the peroxisome proliferator activated receptor (PPAR) coactivator-1α (PGC-1α) and the transcription factor/nuclear receptor PPAR-∂. It was therefore the aim of the present study to examine whether acute exercise with or without glycogen manipulation affects PGC-1α and PPAR-∂ function in rodent skeletal muscle. Twenty female Wistar rats were randomly assigned to 5 experimental groups (n = 4): control [CON]; normal glycogen control [NG-C]; normal glycogen exercise [NG-E]; low glycogen control [LG-C]; and low glycogen exercise [LG-E]). Gastrocnemius (GTN) muscles were collected immediately following exercise and analyzed for glycogen content, PPAR-∂ activity via chromatin immunoprecipitation (ChIP) assays, AMPK α1/α2 kinase activity, and the localization of AMPK and PGC-1α. Exercise reduced muscle glycogen by 47 and 75% relative to CON in the NG-E and LG-E groups, respectively. Exercise that started with low glycogen (LG-E) finished with higher AMPK-α2 activity (147%, p<0.05), nuclear AMPK-α2 and PGC-1α, but no difference in AMPK-α1 activity compared to CON. In addition, PPAR-∂ binding to the CPT1 promoter was significantly increased only in the LG-E group. Finally, cell reporter studies in contracting C2C12 myotubes indicated that PPAR-∂ activity following contraction is sensitive to glucose availability, providing mechanistic insight into the association between PPAR-∂ and glycogen content/substrate availability. The present study is the first to examine PPAR-∂ activity in skeletal muscle in response to an acute bout of endurance exercise. Our data would suggest that a factor associated with muscle contraction and/or glycogen depletion activates PPAR-∂ and initiates AMPK translocation in skeletal muscle in response to exercise

Protein free microcapsules obtained from plant spores as a model for drug delivery: Ibuprofen encapsulation, release and taste masking

Sporopollenin exine capsules (SEC) extracted from Lycopodium clavatum spores were shown to encapsulate ibuprofen as a drug model, with 97 ± 1% efficiency as measured by recovery of the loaded drug and absence of the drug on the SEC surface by scanning electron microscopy (SEM). The encapsulated ibuprofen was shown to be unchanged from its bulk crystalline form by solid state NMR, FTIR and XRD. Essential for drug delivery applications, SEC were shown to be non-toxic to human endothelial cells and free of allergenic protein epitopes by MALDI-TOF-MS and ESI-QqToF-MS. Potential application for targeted release into the intestinal region of the gastrointestinal tract (GIT) was demonstrated by 88 ± 1% of the drug being retained in simulated gastric fluid (SGF) after 45 minutes and 85 ± 2% being released after 5 min in buffer (PBS; pH 7.4). The SEC were shown to provide significant taste masking of encapsulated ibuprofen in a double blind trial with 10 human volunteers. © The Royal Society of Chemistry 2013

Sporopollenin exine capsules (SpECs) derived from Lycopodium clavatum provide practical antioxidant properties by retarding rancidification of an ω-3 oil

In recent years the use of natural antioxidants in foodstuffs and personal care products has become increasingly important for consumers and therefore manufacturers. In this work, sporopollenin exine capsules (SpECs), extracted from spores of the common club moss Lycopodium clavatum L, have been shown to protect an ω-3 oil from oxidation caused by natural light or accelerated oxidation with UV irradiation. The mechanism of action has been shown to be principally by free radical quenching as opposed to light shielding, supported by evidence of similarity in levels of protection when the ratio of SpECs to oil was 0.2 % w/v compared with 50 % w/w. The antioxidant effect is not materially altered by the extraction process from the raw material and is clearly an inherent property of the sporopollenin contained in the spores of L. clavatum due to the accessible phenolic groups on the surface on the SpECs. These results provide promising evidence that SpECs could be useful as a bio-sourced antioxidant for protecting ω-3 oils and related oxidation-prone molecules

Microencapsulation of Baker’s Yeast in Gellan Gum Beads Used in Repeated Cycles of Glucose Fermentation

The purpose of this work is to prepare ionically cross-linked (with CaCl2) gellan particles with immobilized yeast cells for their use in repeated fermentation cycles of glucose. The study investigates the influence of ionic cross-linker concentration on the stability and physical properties of the particles obtained before extrusion and during time in the coagulation bath (the cross-linker solution with different CaCl2 concentrations). It was found that by increasing the amount of the cross-linker the degree of cross-linking in the spherical gellan matrix increases, having a direct influence on the particle morphology and swelling degree in water. These characteristics were found to be very important for diffusion of substrate, that is, the glucose, into the yeast immobilized cells and for the biocatalytic activity of the yeast immobilized cells in gellan particles. These results highlight the potential of these bioreactors to be used in repeated fermentation cycles (minimum 10) without reducing their biocatalytic activity and maintaining their productivity at similar parameters to those obtained in the free yeast fermentation. Encapsulation of Saccharomyces cerevisiae into the gellan gum beads plays a role in the effective application of immobilized yeast for the fermentation process

Sporopollenin as an efficient green support for covalent immobilization of a lipase

Sporopollenin exine capsules (SECs), derived from the spores of Lycopodium clavatum, have been functionalised with 1,n-diamines and the resulting aminoalkyl microcapsules used to immobilize Candida antarctica lipase B (Cal B) via a glutaradehyde-based diimine covalent linker. The supported enzyme efficiently catalyzes the esterification of oleic acid with ethanol. Initial rates using the SEC-CalBs were comparable to the commercial enzyme Novozym 435, but displayed up to 20-fold higher specific activity. The supported enzymes could also be recycled and after four cycles displayed only a modest decrease in conversions. In a kinetic resolution the SEC-CalBs efficiently acetylated rac-1-phenylethanol, with conversions up to 37% after 5 hours and product enantiomeric excesses of >99%. Related to this, the dynamic resolution of rac-1-phenylethylamine, in the presence of Pd-BaSO₄ and ammonium formate, led to the acetylated amine with a 94% conversion and >99% ee

Disorders of sex development : insights from targeted gene sequencing of a large international patient cohort

Background: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously. Results: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46, XY DSD and 48 with 46, XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46, XY DSD. In patients with 46, XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management. Conclusions: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes

Analogues of bredinin : synthesis of 5-hydroxyimidazoles from acyclic precursors

Compared to 1-substituted-5-aminoimidazoles relatively few 1-substituted-5-hydroxyimidazoles have been reported; however, an interesting 1-substituted-5-hydroxyimidazole is the immunosuppressive agent bredinin, which shows antiviral, antimalarial, antitumour and antiarthritic activities. Bredinin is obtained naturally by extraction from a fermentation process, and current synthetic strategies for the synthesis are ambiguous and don’t easily allow for the synthesis of analogues. Surprisingly, relatively few analogues of bredinin are known hence a convenient route to such types of compounds would be useful in providing a library of compounds for structural activity studies to be made.The aim of the present study was to develop a synthetic strategy which employed inexpensive and readily available acyclic precursors to synthesise a library of analogues of bredinin. An advantage of using acyclic precursors to synthesise imidazoles is that the introduction of substituents, in particular, those in the 1-position is unambiguous. Therefore, our approach has been to explore an efficient and general route to 1-substitued-5-hydroxyimidazoles employing inexpensive and readily available startingmaterials.Our initial target was to synthesise ethyl 1-substituted-5-hydroxyimidazole-4-carboxylates since such ester intermediates have the potential to be converted to a wide variety of related 4-substituted imidazoles. A variety of ethyl 1-substituted-5-hydroxyimidazole-4-carboxylates were synthesised. Those synthesised using lipophilic amines at position 1 of the imidazole ring (structures 39-43), were obtained in high yields and easily purified. However, those synthesised using polar amines at position 1 (structures 43-45), were more difficult to isolate and purify due to their increased hydrophilic nature.In addition, the adopted strategy offers the opportunity to introduce a variety of substituents in the 2-position of the imidazole ring (structures 48-49), however this increase in chain length at position 2 had a direct effect on the yield of the imidazole obtained. The longer the chain length, the lower the yields, due to the steric hindrance experienced in the cyclisation step of the synthetic sequence.In summary a novel convergent synthetic strategy was devised and both ethyl 1,2-substituted-5-hydroxyimidazole-4-carboxylates, with both aryl and alkyl groups atposition 1, and benzyl 1-benzyl-5-hydroxyimidazole-4-carboxylate were successfully synthesised. Both lipophilicity and steric bulk were found to play a crucial role in the successful cyclisation and subsequent isolation and purification of the imidazoles, and a compromise between the two must be achieved to give the optimal reaction conditions.In an attempt to gain a greater understanding of these ethyl 1-substituted-5-hydroxyimidazole-4-carboxylates systems, their reactivity under different reactions conditions was assessed. Ethyl 1-benzyl-5-hydroxyimidazole-4-carboxylate was used as the model system and was found to be stable to all targeted manipulation at position 2 and 4 of the imidazole ring. Additionally, all attempts to protect the 5-hydroxyl function, to allow the subsequent manipulation of the ethyl ester, were also unsuccessful. The unexpected benzylation of the nitrogen at position 3 of the imidazole ring, to form the quaternary ammonium salt, confirms that this is the most nucleophilic atom within the imidazole ring system.A novel linear synthetic strategy using acyclic precursors was developed which enabled the addition of substituents in positions 1, 2, 4 and 5 of the imidazole ring unambiguously. A particular advantage of this route is that it would be possible to synthesise 4-substituted-5-hydroxyimidazoles.An interesting difference between the two synthetic routes is that nucleosides of the 5-hydroxyimidazoles were successfully synthesised using the linear synthetic strategy, however they could not be synthesised using the convergent strategy. This indicates the important role nucleophilicity could play in the intramolecular cyclisation step of the convergent synthetic strategy.The most important implications of the present study are the development of two separate, unambiguous synthetic routes to the 5-hydroxyimidazoles, employinginexpensive acyclic precursors. The routes could provide access to a variety of substituted 5-hydroxyimidazoles in order to build a library of these compounds.EThOS - Electronic Theses Online ServiceEPSRC (Sponsor)The University of Hull (Sponsor)Department of Chemistry, The University of Hull (Sponsor)GBUnited Kingdo