Chemoenzymatic approach toward the synthesis of 3-O-(α/β)-glucosylated 3-hydroxy-β-lactams

Glycosylation significantly alters the biological and physicochemical properties of small molecules. beta-Lactam alcohols comprise eligible substrates for such a transformation based on their distinct relevance in the chemical and medicinal community. In this framework, the unprecedented enzymatic glycosylation of the rigid and highly strained four-membered beta-lactam azaheterocycle was studied. For this purpose, cis-3-hydroxy-beta-lactams were efficiently prepared in three steps by means of a classical organic synthesis approach, while a biocatalytic step was implemented for the selective formation of the corresponding 3-O-alpha- and -beta-glucosides, hence overcoming the complexities typically encountered in synthetic glycochemistry and contributing to the increasing demand for sustainable processes in the framework of green chemistry. Two carbohydrate-active enzymes were selected based on their broad acceptor specificity and subsequently applied for the alpha- or beta-selective formation of beta-lactam-sugar adducts, using sucrose as a glucosyl donor

Sequence determinants of nucleotide binding in Sucrose Synthase : improving the affinity of a bacterial Sucrose Synthase for UDP by introducing plant residues

Sucrose Synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate (NDP) into NDP-glucose and fructose. Biochemical characterization of several plant and bacterial SuSys has revealed that the eukaryotic enzymes preferentially use UDP whereas prokaryotic SuSys prefer ADP as acceptor. In this study, SuSy from the bacterium Acidithiobacillus caldus, which has a higher affinity for ADP as reflected by the 25-fold lower K-m value compared to UDP, was used as a test case to scrutinize the effect of introducing plant residues at positions in a putative nucleotide binding motif surrounding the nucleobase ring of NDP. All eight single to sextuple mutants had similar activities as the wild-type enzyme but significantly reduced K-m values for UDP (up to 60 times). In addition, we recognized that substrate inhibition by UDP is introduced by a methionine at position 637. The affinity for ADP also increased for all but one variant, although the improvement was much smaller compared to UDP. Further characterization of a double mutant also revealed more than 2-fold reduction in K-m values for CDP and GDP. This demonstrates the general impact of the motif on nucleotide binding. Furthermore, this research also led to the establishment of a bacterial SuSy variant that is suitable for the recycling of UDP during glycosylation reactions. The latter was successfully demonstrated by combining this variant with a glycosyltransferase in a one-pot reaction for the production of the C-glucoside nothofagin, a health-promoting flavonoid naturally found in rooibos (tea)

Quantifying the average number of nucleic acid therapeutics per nanocarrier by single particle tracking microscopy

Nucleic acid biopharmaceuticals are being investigated as potential therapeutics. They need to be incorporated into a biocompatible carrier so as to overcome several biological barriers. Rational development of suitable nanocarriers requires high-quality characterization techniques. While size, concentration, and stability can be very well measured these days, even in complex biological fluids, a method to accurately quantify the number of nucleic acid therapeutics encapsulated in nanocarriers is still missing. Here we present a method, based on concentration measurements with single particle tracking microscopy, with which it is possible to directly measure the number of plasmid DNA molecules per nanoparticle, referred to as the plasmid/NP ratio. Using DOTAP/DOPE liposomes as a model carrier, we demonstrate the usefulness of the method by investigating the influence of various experimental factors on the plasmid/NP ratio. We find that the plasmid/NP ratio is inversely proportional with the size of the pDNA and that the plasmid/NP decreases when lipoplexes are prepared at lower concentrations of pDNA and nanocarrier, with values ranging from 6.5 to 3 plasmid/NP. Furthermore, the effect of pre- and post-PEGylation of lipoplexes was examined, finding that pre-PEGylation results in a decreased plasmid/NP ratio, while post-PEGylation did not alter the plasmid/NP ratio. These proof-of-concept experiments show that single particle tracking offers an extension of the nanoparticle characterization toolbox and is expected to aid in the efficient development of nanoformulations for nucleic acid-based therapies

Stability analysis of a plant glycosyltransferase

Background: Glycosylation can significantly improve physicochemical or biological properties of small molecules such as vitamins, fragrances and antibiotics. Glycosyltransferases (GTs) are carbohydrate-active enzymes that can efficiently catalyse such reactions. We recently discovered a promising biocatalyst (GT-1) for the glycosylation of a wide variety of compounds, but its thermostability will be crucial for industrial applications. Materials and Methods: GT-1 was recombinantly expressed and purified via Ni-NTA chromatography. Thermal half-life time (t50(50°C)) at 50 °C was assessed by sampling at fixed intervals until the residual activity had dropped to 50%, while the melting temperature (Tm) was determined via differential scanning fluorimetry. The bio-informatic tools YASARA and 3DM were used to select target positions for engineering. Results: GT-1 was succesfully expressed and yields up to 40 mg purified protein per liter culture were obtained. Further, t50(50°C) and Tm values of respectively 90 min and 56 °C were found. Discussion: The kinetic stability of GT-1 was rather low. Indeed, after 1 min of incubation at 50 °C, residual activity already dropped to 70%. Clearly, enzyme engineering strategies need to be developed. Therefore, a homology model was constructed and structural analysis revealed a strikingly large amount of open spaces. Filling these cavities with larger amino acids should increase hydrophobic packing, yielding a more stable enzyme. Conclusion: The kinetic and thermal stability of GT-1 was assessed. Engineering the enzyme’s stability by a combination of smart (sequence-based) and rational (structure-based) mutagenesis will be key to biocatalyst development

Developmental coordination disorder before the age of three : a longitudinal retrospective study in a Belgian Center for Developmental Disabilities

This study aimed to explore the association between developmental coordination disorder (DCD) diagnosed after the age of three and both a standardized motor test-the Alberta Infant Motor Scale (AIMS)-and non-standardized observation of movement quality carried out before the age of three. Children at risk or with developmental concerns were studied retrospectively. Children were excluded in case of a diagnosis, excluding DCD, e.g., cerebral palsy, or IQ < 70. Of the 503 included children, 246 were diagnosed with (at-risk) DCD. Multivariate binary logistic regression revealed a significant association between DCD diagnosis after the age of three and male gender and with different aspects of poor movement quality in different age groups before the age three. Univariate analyses revealed an association between DCD diagnosis and the number of poor movement-quality descriptions at 0-6 months, 6-12 months, and 18 months-3 years but not with the AIMS scores. The MABC-2 scores after the age of three were significantly correlated with the number of poor movement-quality descriptions in age groups 0-6 months and 18 months-3 years and with the AIMS scores in age groups 6-12 months and 12-18 months. The results suggest that DCD can be associated with poor movement quality before the age of three

Stability of motor problems in young children with or at risk of autism spectrum disorders, ADHD and or developmental coordination disorder

Aim: The aim of this study was to investigate the stability of motor problems in a clinically referred sample of children with, or at risk of, autism spectrum disorders (ASDs), attention-deficit-hyperactivity disorder (ADHD), and/or developmental coordination disorder (DCD). Method: Participants were 49 children (39 males, 10 females; mean age 5y 6mo, SD 10mo) with various developmental problems, a Movement Assessment Battery for Children (M-ABC) score on or below the 15th centile, and an IQ of 70 or more. Sixteen children were at risk of developing ADHD, 15 children had a diagnosis of, or were at risk of developing ASD, and 18 children had neither diagnosis. Children were reassessed 2 to 3 years later. Results: At follow-up (mean age 7y 11mo; SD 1y), the mean M-ABC score was significantly increased, and in 22 children was no longer below the 15th centile. A general linear model to compare the difference in M-ABC scores in the three groups of children demonstrated a significant difference between groups (p=0.013), with the age at the initial assessment as a significant covariate (p=0.052). The group of children with or at risk of ASD showed less improvement in motor performance. Interpretation: Motor problems among preschool age children are not always stable, but appear to be so in most children with ASDs

Enzymatic glycosylation of phenolic antioxidants: phosphorylase-mediated synthesis and characterization

Although numerous biologically active molecules exist as glycosides in nature, information on the activity, stability, and solubility of glycosylated antioxidants is rather limited to date. In this work, a wide variety of antioxidants were glycosylated using different phosphorylase enzymes. The resulting antioxidant library, containing alpha/beta-glucosides, different regioisomers, cellobiosides, and cellotriosides, was then characterized. Glycosylation was found to significantly increase the solubility and stability of all evaluated compounds. Despite decreased radical-scavenging abilities, most glycosides were identified to be potent antioxidants, outperforming the commonly used 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT). Moreover, the point of attachment, the anomeric configuration, and the glycosidic chain length were found to influence the properties of these phenolic glycosides