Synthetically derived BiAux modulates auxin co-receptor activity to stimulate lateral root formation.

The root system plays an essential role in plant growth and adaptation to the surrounding environment. The root clock periodically specifies lateral root prebranch sites (PBS), where a group of pericycle founder cells (FC) is primed to become lateral root founder cells and eventually give rise to lateral root primordia or lateral roots (LRs). This clock-driven organ formation process is tightly controlled by modulation of auxin content and signaling. Auxin perception entails the physical interaction of TRANSPORT INHIBITOR RESPONSE 1 (TIR1) or AUXIN SIGNALING F-BOX (AFBs) proteins with AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to form a co-receptor system. Despite the apparent simplicity, the understanding of how specific auxin co-receptors are assembled remains unclear. We identified the compound bis-methyl auxin conjugated with N-glucoside, or BiAux, in Arabidopsis (Arabidopsis thaliana) that specifically induces the formation of PBS and the emergence of LR, with a slight effect on root elongation. Docking analyses indicated that BiAux binds to F-box proteins, and we showed that BiAux function depends on TIR1 and AFB2 F-box proteins and AUXIN RESPONSE FACTOR 7 activity, which is involved in FC specification and LR formation. Finally, using a yeast (Saccharomyces cerevisiae) heterologous expression system, we showed that BiAux favors the assemblage of specific co-receptors subunits involved in LR formation and enhances AUXIN/INDOLE-3-ACETIC ACID 28 protein degradation. These results indicate that BiAux acts as an allosteric modulator of specific auxin co-receptors. Therefore, BiAux exerts a fine-tune regulation of auxin signaling aimed to the specific formation of LR among the many development processes regulated by auxin.

A Novel Lipase from Streptomyces exfoliatus DSMZ 41693 for Biotechnological Applications.

Genome mining of Streptomyces exfoliatus DSMZ 41693 has allowed us to identify four different lipase-encoding sequences, and one of them (SeLipC) has been successfully cloned and extracellularly expressed using Rhodococcus sp. T104 as a host. SeLipC was purified by one-step hydrophobic interaction chromatography. The enzyme is a monomeric protein of 27.6 kDa, which belongs to subfamily I.7 of lipolytic enzymes according to its phylogenetic analysis and biochemical characterization. The purified enzyme shows the highest activity at 60 °C and an optimum pH of 8.5, whereas thermal stability is significantly improved when protein concentration is increased, as confirmed by thermal deactivation kinetics, circular dichroism, and differential scanning calorimetry. Enzyme hydrolytic activity using p-nitrophenyl palmitate (pNPP) as substrate can be modulated by different water-miscible organic cosolvents, detergents, and metal ions. Likewise, kinetic parameters for pNPP are: KM = 49.6 µM, kcat = 57 s-1, and kcat/KM = 1.15 × 106 s-1·M-1. SeLipC is also able to hydrolyze olive oil and degrade several polyester-type polymers such as poly(butylene succinate) (PBS), poly(butylene succinate)-co-(butylene adipate) (PBSA), and poly(ε-caprolactone) (PCL). Moreover, SeLipC can catalyze the synthesis of different sugar fatty acid esters by transesterification using vinyl laurate as an acyl donor, demonstrating its interest in different biotechnological applications.

Biocatalyzed Synthesis of Glycostructures with Anti-infective Activity.

Molecules containing carbohydrate moieties play essential roles in fighting a variety of bacterial and viral infections. Consequently, the design of new carbohydrate-containing drugs or vaccines has attracted great attention in recent years as means to target several infectious diseases.Conventional methods to produce these compounds face numerous challenges because their current production technology is based on chemical synthesis, which often requires several steps and uses environmentally unfriendly reactants, contaminant solvents, and inefficient protocols. The search for sustainable processes such as the use of biocatalysts and eco-friendly solvents is of vital importance. Therefore, their use in a variety of reactions leading to the production of pharmaceuticals has increased exponentially in the last years, fueled by recent advances in protein engineering, enzyme directed evolution, combinatorial biosynthesis, immobilization techniques, and flow biocatalysis. In glycochemistry and glycobiology, enzymes belonging to the families of glycosidases, glycosyltransferases (Gtfs), lipases, and, in the case of nucleoside and nucleotide analogues, also nucleoside phosphorylases (NPs) are the preferred choices as catalysts.In this Account, on the basis of our expertise, we will discuss the recent biocatalytic and sustainable approaches that have been employed to synthesize carbohydrate-based drugs, ranging from antiviral nucleosides and nucleotides to antibiotics with antibacterial activity and glycoconjugates such as neoglycoproteins (glycovaccines, GCVs) and glycodendrimers that are considered as very promising tools against viral and bacterial infections.In the first section, we will report the use of NPs and N-deoxyribosyltransferases for the development of transglycosylation processes aimed at the synthesis of nucleoside analogues with antiviral activity. The use of deoxyribonucleoside kinases and hydrolases for the modification of the sugar moiety of nucleosides has been widely investigated.Next, we will describe the results obtained using enzymes for the chemoenzymatic synthesis of glycoconjugates such as GCVs and glycodendrimers with antibacterial and antiviral activity. In this context, the search for efficient enzymatic syntheses represents an excellent strategy to produce structure-defined antigenic or immunogenic oligosaccharide analogues with high purity. Lipases, glycosidases, and Gtfs have been used for their preparation.Interestingly, many authors have proposed the use Gtfs originating from the biosynthesis of natural glycosylated antibiotics such as glycopeptides, macrolides, and aminoglycosides. These have been used in the chemoenzymatic semisynthesis of novel antibiotic derivatives by modification of the sugar moiety linked to their complex scaffold. These contributions will be described in the last section of this review because of their relevance in the fight against the spreading phenomenon of antibiotic resistance. In this context, the pioneering in vivo synthesis of novel derivatives obtained by genetic manipulation of producer strains (combinatorial biosynthesis) will be shortly described as well.All of these strategies provide a useful and environmentally friendly synthetic toolbox. Likewise, the field represents an illustrative example of how biocatalysis can contribute to the sustainable development of complex glycan-based therapies and how problems derived from the integration of natural tools in synthetic pathways can be efficiently tackled to afford high yields and selectivity. The use of enzymatic synthesis is becoming a reality in the pharmaceutical industry and in drug discovery to rapidly afford collections of new antibacterial or antiviral molecules with improved specificity and better metabolic stability.

Enzymatic Synthesis and Molecular Modelling Studies of Rhamnose Esters Using Lipase from Pseudomonas stutzeri.

Rhamnolipids are becoming an important class of glycolipid biosurfactants. Herein, we describe for the first time the enzymatic synthesis of rhamnose fatty acid esters by the transesterification of rhamnose with fatty acid vinyl esters, using lipase from Pseudomonas stutzeri as a biocatalyst. The use of this lipase allows excellent catalytic activity in the synthesis of 4-O-acylrhamnose (99% conversion and full regioselectivity) after 3 h of reaction using tetrahydrofuran (THF) as the reaction media and an excess of vinyl laurate as the acyl donor. The role of reaction conditions, such as temperature, the substrates molar ratio, organic reaction medium and acyl donor chain-length, was studied. Optimum conditions were found using 35 °C, a molar ratio of 1:3 (rhamnose:acyldonor), solvents with a low logP value, and fatty acids with chain lengths from C4 to C18 as acyl donors. In hydrophilic solvents such as THF and acetone, conversions of up to 99-92% were achieved after 3 h of reaction. In a more sustainable solvent such as 2-methyl-THF (2-MeTHF), high conversions were also obtained (86%). Short and medium chain acyl donors (C4-C10) allowed maximum conversions after 3 h, and long chain acyl donors (C12-C18) required longer reactions (5 h) to get 99% conversions. Furthermore, scaled up reactions are feasible without losing catalytic action and regioselectivity. In order to explain enzyme regioselectivity and its ability to accommodate ester chains of different lengths, homology modelling, docking studies and molecular dynamic simulations were performed to explain the behaviour observed.

Synthesis of Glycodendrimers with Antiviral and Antibacterial Activity.

Glycodendrimers are an important class of synthetic macromolecules that can be used to mimic many structural and functional features of cell-surface glycoconjugates. Their carbohydrate moieties perform key important functions in bacterial and viral infections, often regulated by carbohydrate-protein interactions. Several studies have shown that the molecular structure, valency and spatial organisation of carbohydrate epitopes in glycoconjugates are key factors in the specificity and avidity of carbohydrate-protein interactions. Choosing the right glycodendrimers almost always helps to interfere with such interactions and blocks bacterial or viral adhesion and entry into host cells as an effective strategy to inhibit bacterial or viral infections. Herein, the state of the art in the design and synthesis of glycodendrimers employed for the development of anti-adhesion therapy against bacterial and viral infections is described.

Structural insights into the desymmetrization of bulky 1,2-dicarbonyls through enzymatic monoreduction.

Benzil reductases are dehydrogenases preferentially active on aromatic 1,2-diketones, but the reasons for this peculiar substrate recognition have not yet been clarified. The benzil reductase (KRED1-Pglu) from the non-conventional yeast Pichia glucozyma showed excellent activity and stereoselectivity in the monoreduction of space-demanding aromatic 1,2-dicarbonyls, making this enzyme attractive as biocatalyst in organic chemistry. Structural insights into the stereoselective monoreduction of 1,2-diketones catalyzed by KRED1-Pglu were investigated starting from its 1.77 Å resolution crystal structure, followed by QM and classical calculations; this study allowed for the identification and characterization of the KRED1-Pglu reactive site. Once identified the recognition elements involved in the stereoselective desymmetrization of bulky 1,2-dicarbonyls mediated by KRED1-Pglu, a mechanism was proposed together with an in silico prediction of substrates reactivity.

Efficient Synthesis of Muramic and Glucuronic Acid Glycodendrimers as Dengue Virus Antagonists.

Carbohydrates are involved in many important pathological processes, such as bacterial and viral infections, by means of carbohydrate-protein interactions. Glycoconjugates with multiple carbohydrates are involved in multivalent interactions, thus increasing their binding strengths to proteins. In this work, we report the efficient synthesis of novel muramic and glucuronic acid glycodendrimers as potential Dengue virus antagonists. Aromatic scaffolds functionalized with a terminal ethynyl groups were coupled to muramic and glucuronic acid azides by click chemistry through optimized synthetic strategies to afford the desired glycodendrimers with high yields. Surface Plasmon Resonance studies have demonstrated that the compounds reported bind efficiently to the Dengue virus envelope protein. Molecular modelling studies were carried out to simulate and explain the binding observed. These studies confirm that efficient chemical synthesis of glycodendrimers can be brought about easily offering a versatile strategy to find new active compounds against Dengue virus.

Acceptor Specificity of ß-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation.

Fungal ß-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The ß-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model ß-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure-activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-ß-d-glucosaminide or 4-nitrophenyl N-acetyl-ß-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the ß(1-4) position, the galacto-acceptor afforded a ß(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on ß-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.

Chemoenzymatic synthesis of carbohydrates as antidiabetic and anticancer drugs.

Different chemoenzymatic strategies for the preparation of carbohydrates and analogues possessing antidiabetic or anticancer activity are summarized. In this sense, some examples illustrating the use of enzymes such as aldolases, lipases or glycosidases (in some cases improved by genetic engineering techniques) are presented, showing the advantages of the implementation of chemoenzymatic protocols, which combine the flexibility of chemical synthesis with the efficiency, selectivity and sustainability of biotransformations to obtain diverse complex carbohydrates, glycoconjugates and glycomimetics.

Chemoselective CaO-mediated acylation of alcohols and amines in 2-methyltetrahydrofuran.

Calcium oxide is proposed as an innocuous acid scavenger for the chemoselective synthesis of amide- and ester-type compounds. Although these molecules have wide spread applications in organic and pharmaceutical chemistry, and a large number of routes have been designed for their synthesis, the development of more efficient and environmentally friendly acylation strategies remains an ongoing challenge. The use of CaO allows for the stoichiometric acylation of primary alcohols in the presence of phenols or tertiary alcohols; amines can also be subjected to acylation reactions in the presence of hydroxyl groups. Chirality is obtained through acylation if the starting material is an optically pure alcohol or if a chiral acylating agent is used. Furthermore, the use of 2-methyltetrahydrofuran (2-MeTHF), a more ecofriendly solvent, leads to maximized yields. This protocol is successfully applied to the synthesis of an interesting N-aryloxazolidin-2-one intermediate for the preparation of linezolid-type compounds.

2-Methyltetrahydrofuran (2-MeTHF): a biomass-derived solvent with broad application in organic chemistry.

2-Methyl-tetrahydrofuran (2-MeTHF) can be derived from renewable resources (e.g., furfural or levulinic acid) and is a promising alternative solvent in the search for environmentally benign synthesis strategies. Its physical and chemical properties, such as its low miscibility with water, boiling point, remarkable stability compared to other cyclic-based solvents such as THF, and others make it appealing for applications in syntheses involving organometallics, organocatalysis, and biotransformations or for processing lignocellulosic materials. Interestingly, a significant number of industries have also started to assess 2-MeTHF in several synthetic procedures, often with excellent results and prospects. Likewise, preliminary toxicology assessments suggest that the use of 2-MeTHF might even be extended to more processes in pharmaceutical chemistry. This Minireview describes the properties of 2-MeTHF, the state-of-the-art of its use in synthesis, and covers several outstanding examples of its application from both industry and academia.

Biocatalytic strategies for the asymmetric synthesis of alpha-hydroxy ketones.

The development of efficient syntheses for enantiomerically enriched alpha-hydroxy ketones is an important research focus in the pharmaceutical industry. For example, alpha-hydroxy ketones are found in antidepressants, in selective inhibitors of amyloid-beta protein production (used in the treatment of Alzheimer's), in farnesyl transferase inhibitors (Kurasoin A and B), and in antitumor antibiotics (Olivomycin A and Chromomycin A3). Moreover, alpha-hydroxy ketones are of particular value as fine chemicals because of their utility as building blocks for the production of larger molecules. They can also be used in preparing many other important structures, such as amino alcohols, diols, and so forth. Several purely chemical synthetic approaches have been proposed to afford these compounds, together with some organocatalytic strategies (thiazolium-based carboligations, proline alpha-hydroxylations, and so forth). However, many of these chemical approaches are not straightforward, lack selectivity, or are economically unattractive because of the large number of chemical steps required (usually combined with low enantioselectivities). In this Account, we describe three different biocatalytic approaches that have been developed to efficiently produce alpha-hydroxy ketones: (i) The use of thiamine diphosphate-dependent lyases (ThDP-lyases) to catalyze the umpolung carboligation of aldehydes. Enantiopure alpha-hydroxy ketones are formed from inexpensive aldehydes with this method. Some lyases with a broad substrate spectrum have been successfully characterized. Furthermore, the use of biphasic media with recombinant whole cells overexpressing lyases leads to productivities of approximately 80-100 g/L with high enantiomeric excesses (up to >99%). (ii) The use of hydrolases to produce alpha-hydroxy ketones by means of (in situ) dynamic kinetic resolutions (DKRs). Lipases are able to successfully resolve racemates, and many outstanding examples have been reported. However, this approach leads to a maximum theoretical yield of 50%. As a means of overcoming this problem, these traditional lipase-catalyzed kinetic resolutions are combined with racemization of remnant substrate, which can be done in situ or in separate compartments. Examples showing high conversions (>90%) and enantiomeric excesses (>99%) are described. (iii) Whole-cell redox processes, catalyzed by several microorganisms, either by means of free enzymes (applying a cofactor regeneration system) or by whole cells. Through the use of redox machineries, different strategies can lead to high yields and enantiomeric excesses. Some enantiopure alpha-hydroxy ketones can be formed by reductions of diketones and by selective oxidations of vicinal diols. Likewise, some redox processes involving sugar chemistry (involving alpha-hydroxy ketones) have been developed on the industrial scale. Finally, the redox whole-cell concept allows racemizations (and deracemizations) as well. These three strategies provide a useful and environmentally friendly synthetic toolbox. Likewise, the field represents an illustrative example of how biocatalysis can assist practical synthetic processes, and how problems derived from the integration of natural tools in synthetic pathways can be efficiently tackled to afford high yields and enantioselectivities.

Relación entre hipertensión arterial sistémica y síndrome de apnea-hipopnea obstructiva del sueño y sus factores de resgo asociados, en población hipertensa de un centro médico: Cali (Colombia)2008 / Relation between systemic hypertension and apnea-hypopnea syndrome and its associated risk factors in hypertensive population from a medical center: Cali (Colombia) 2008

Introducción: La relación causa efecto entre la hipertensión arterial sistémica y un mayor riesgo para desarrollar síndrome de apnea-hipopnea obstructiva del sueño (SAHOS) es aún incierta. El desconocimiento de la influencia del SAHOS sobre la hipertensión arterial ha llevado al excesivo diagnóstico de hipertensión arterial idiopática, desmotivando la búsqueda de la real causa subyacente, que en la mayoría de los casos puede deberse a trastornos del sueño. Materiales y métodos: En el presente estudio se incluyen pacientes hipertensos quienes consultaron en el centro médico ONIRIS, especializado en sueño, y se evaluó en ellos variables como índice de masa corporal, índice cintura cadera, circunferencia del cuello, tabaquismo y alcohol, para determinar cuáles de ellos desarrollaron SAHOS. Resultados: De 309 pacientes estudiados, el 67,4 por ciento fue positivo para SAHOS, confirmando algún grado de asociación entre ésta y la hipertensión arterial. Además, variables incluidas como factores de riesgo arrojaron resultados muy similares a los obtenidos en otros estudios que han demostrado dicha asociación. Conclusiones: Tiene predominio por el sexo masculino, se presenta generalmente en mayores de 50 años y obesos; dichos factores ponen en un riesgo elevado al paciente de desarrollar eventos vasculares que disminuyen la calidad de vida e incluso llevarlo a la muerte.

Dynamic kinetic resolution of benzoins by lipase-metal combo catalysis.

The synthesis of some noncommercial racemic 1,2-diaryl-2-hydroxyethanones (benzoins) is described, optimizing the previously reported methodologies. In a further step, the kinetic resolution of these substrates is reported, obtaining conversions of around 50% and ee(p) higher than 99% in very short reaction times. As enzymatic catalyst, after screening of several enzymes, the lipase TL (from Pseudomonas stutzeri) was the most efficient, working in an organic solvent with a very low log P value, such as THF. Finally, the dynamic-kinetic resolution of different benzoins using a lipase-ruthenium-catalyzed transesterification in organic solvents is described for the first time, obtaining conversions up to 90% maintaining the excellent enantioselectivity in all cases.

Análisis de los factores de riesgo de la hipertensión arterial en Colima, México / Analysis of risk factors for hypertension in Colima, Mexico

OBJETIVO: Evaluar la posible asociación de la edad, el sexo, el sobrepeso, los antecedentes familiares de hipertensión arterial (HTA), el alcoholismo y el sedentarismo con la HTA en la población adulta de la ciudad de Colima, México. MÉTODOS: Estudio transversal analítico de base poblacional. Se aplicó una encuesta estructurada a 280 adultos mayores de 30 años que residían en la ciudad mexicana de Colima en 2001 y 2002. Las variables estudiadas fueron el sexo, la edad, el peso, la talla, los antecedentes familiares de HTA, la práctica de ejercicio físico, el tabaquismo y el consumo de alcohol. La presión arterial (PA) se midió por el método auscultatorio. Las mediciones limítrofes o dudosas se repitieron cuatro o cinco días después. Se consideró que había HTA cuando la PA sistólica era >140 mm Hg y la presión arterial diastólica era > 90 mm Hg, o la persona estaba bajo tratamiento antihipertensivo. Se calcularon las razones de posibilidades (odds ratios, RP) de las variables estudiadas y sus intervalos de confianza de 95 por ciento (IC95 por ciento). La asociación entre las variables y la HTA se estimó mediante regresión logística y la interacción mediante el coeficiente de productos de interacción. RESULTADOS: La prevalencia bruta de HTA fue de 28,6 por ciento. La prevalencia fue mayor en hombres que en mujeres (42,1 por ciento frente a 19,2 por ciento, respectivamente; RP = 3,04; IC95 por ciento: 1,8 a 5,2) y en personas mayores de 49 años que en personas de 30 a 49 años (36,8 por ciento frente a 21,9 por ciento, respectivamente; RP = 2,07; IC95 por ciento: 1,22 a 3,50). Los antecedentes familiares de HTA y el sobrepeso mostraron asociación con la HTA, mientras que la práctica de ejercicio físico tuvo un efecto protector (RP = 0,45; 0,23 a 0,86). Se encontró interacción entre la HTA y la edad (> 50 años), los antecedentes familiares de HTA, el sobrepeso y la práctica de ejercicio físico, particularmente en mujeres. CONCLUSIONES: La prevalencia de HTA en Colima es muy semejante a la encontrada a nivel nacional en México. Su fuerte asociación con el sexo masculino, independientemente de las otras variables, resalta la necesidad de promover campañas preventivas más enfocadas en los hombres.

[Analysis of risk factors for hypertension in Colima, Mexico]. / Análisis de los factores de riesgo de la hipertensión arterial en Colima, México.

OBJECTIVE: To evaluate the possible association that age, sex, excess weight, family history of hypertension, alcoholism, and sedentary lifestyle have with hypertension in the adult population of the city of Colima, Mexico. METHODS: This was a population-based analytic cross-sectional study. A structured survey was used with 280 adults older than 30 years of age who were living in the city of Colima in 2001 and 2002. The variables studied were sex, age, weight, height, family history of hypertension, engaging in physical exercise, smoking, and consuming alcohol. Blood pressure (BP) was measured with the auscultatory method. Borderline or doubtful measurements were checked again four or five days later. Hypertension was defined as systolic BP > or = 140 mm Hg and diastolic blood pressure > or = 90 mm Hg, or as the person being under antihypertensive treatment. The odds ratios (ORs) of the variables studied were calculated, along with their 95% confidence intervals (95% CIs). The association between the variables and hypertension was estimated through logistic regression, and their interaction through the coefficient of the interaction products. RESULTS: The overall prevalence of hypertension was 28.6%. The prevalence was higher in men than in women (42.1% vs. 19.2%; OR = 3.04, 95% CI: 1.8 to 5.2) and in people older than 49 years than in people 30 to 49 years old (36.8% vs. 21.9%; OR = 2.07, 95% CI: 1.22 to 3.50). A family history of hypertension and excess weight were associated with hypertension, while physical exercise had a protective effect (OR = 0.45; 95% CI: 0.23 to 0.86). There was interaction between hypertension and age > or = 50 years, a family history of hypertension, overweight, and physical exercise, especially among women. CONCLUSIONS: The prevalence of hypertension in Colima is very similar to that for Mexico as a whole. The strong association that hypertension had with male gender, regardless of the other variables, emphasizes the need for promoting prevention campaigns that focus more on men.