Efficient enzymatic systems for synthesis of novel α-mangostin glycosides exhibiting antibacterial activity against Gram-positive bacteria

Two enzymatic systems were developed for the efficient synthesis of glycoside products of α-mangostin, a natural xanthonoid exhibiting anti-oxidant, antibacterial, anti-inflammatory, and anticancer activities. In these systems, one-pot reactions for the synthesis of UDP-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with a glycosyltransferase (GT) from Bacillus licheniformis DSM-13 to afford C-3 and C-6 position modified glucose and 2-deoxyglucose conjugated novel α-mangostin derivatives. α-Mangostin 3-O-β-D-glucopyranoside, α-mangostin 6-O-β-D-glucopyranoside, α-mangostin 3,6-di-O-β-D-glucopyranoside, α-mangostin 3-O-β-D-2-deoxyglucopyranoside, α-mangostin 6-O-β-D-2-deoxyglucopyranoside, and α-mangostin 3,6-di-O-β-D-2-deoxyglucopyranoside were successfully produced in practical quantities and characterized by high-resolution quadruple time-of-flight electrospray ionization-mass spectrometry (HR-QTOF ESI/MS), ¹H and ¹³C NMR analyses. In excess of the substrate, the maximum productions of three α-mangostin glucopyranosides (4.8 mg/mL, 86.5 % overall conversion of α-mangostin) and three α-mangostin 2-deoxyglucopyronosides (4.0 mg/mL, 79 % overall conversion of α-mangostin) were achieved at 4-h incubation period. All the α-mangostin glycosides exhibited improved water solubility, and their antibacterial activity against three Gram-positive bacteria Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus was drastically enhanced by the glucosylation at C-3 position. In this study, diverse glycosylated α-mangostin were produced in significant quantities by using inexpensive starting materials and recycling co-factors within a reaction vessel without use of expensive NDP-sugars in the glycosylation reactions.     

Syntheses of α-tocopheryl glycosides by glucosidases

Enzymatic syntheses of water-soluble alpha-tocopheryl glycosides were carried out in di-isopropyl ether using amyloglucosidase from Rhizopus mold or beta-glucosidase isolated from sweet almond. Optimum conditions for the amyloglucosidase were: alpha-tocopherol 0.5 mmol, D-glucose 0.5 mmol, 400 activity unit (AU) amyloglucosidase, 0.2 mM pH 7 phosphate buffer and 72 h; and for the beta-glucosidase: alpha-tocopherol 0.5 mmol, D: -glucose 0.5 mmol, 110 AU beta-glucosidase, 0.1 mM pH 6 phosphate buffer and 72 h. Out of 11 carbohydrates employed, amyloglucosidase reacted only with D-glucose to give 50% of 6-O-(alpha-D-glucopyranosyl)alpha-tocopherol. However, the beta-glucosidase gave 6-O-(beta-D-glucopyranosyl)alpha-tocopherol, 6-O-(alpha-D-galactopyranosyl)alpha-tocopherol, 6-O-(beta-D-galactopyranosyl)alpha-tocopherol, 6-O-(alpha-D-mannopyranosyl)alpha-tocopherol and 6-O-(beta-D-mannopyranosyl)alpha-tocopherol in yields ranging from 10-25%. Water solubility of 6-O-(alpha-D-glucopyranosyl)alpha-tocopherol was 26 g/l at 25 degrees C. alpha-Tocopheryl glycosides showed antioxidant activities with IC(50) values from 0.5 to 1 mM and angiotensin-converting enzyme (ACE) inhibitory activity with IC(50) values from 1.3 to 2.6 mM.     

Use of an activated carbon column for the synthesis of disaccharides by use of a reversed hydrolysis activity of β-galactosidase

Summary An activated carbon column was utilized for the synthesis of disaccharides by use of a reversed hydrolysis activity of an immobilized -galactosidase column in order to shift the equilibrium to the direction of condensation. The yields of lactulose and allo-lactulose from galactose and fructose, and N-acetyl lactosamine and N-acetyl allolactosamine from galactose and N-acetyl glucosamine, were 11.3% and 10.0%, respectively.     

Efficient synthesis of α(2–8)-linkedN-acetyl andN-glycolylneuraminic acid disaccharides from colominic acid

Controlled acid hydrolysis of poly-(2,8)-linked homopolymers ofN-acetylneuraminic acid (colominic acid) and its homologous poly-N-glycolylneuraminic acid afforded high yields of the corresponding disaccharides useful in block synthesis of disialylated gangliosides. The poly-N-glycolylanalog was derived from de-N-acetylated colominic acid by two different reaction sequences. The first one involved reaction with acetoxyacetyl chloride followed by de-O-acetylation. The second and most interesting one requiredN-acryloylation and reductive ozonolysis.     

Structures of the novel α-glucosyl linked diterpene glycosides from Stevia rebaudiana

From the commercial extract of the leaves of Stevia rebaudiana, two new minor diterpene glycosides having α-glucosyl linkage were isolated besides the known steviol glycosides including stevioside, steviolbioside, rebaudiosides A–F, rubusoside and dulcoside A. The structures of the two compounds were identified as 13-[(2-O-(3-α-O-d-glucopyranosyl)-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (1), and 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (2), on the basis of extensive NMR and MS spectral data as well as chemical studies.     

Availability of tyrosine amide for α-chymotrypsin-catalyzed synthesis of oligo-tyrosine peptides

Oligo-tyrosine peptides such as Tyr-Tyr having angiotensin I-converting enzyme (ACE) inhibitory activity could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media. However, peptide yield in the reaction was below 10%. Since l-tyrosine amide showed highly nucleophilic activity for the deacylation of enzyme through which a new peptide bond was made, its application to the enzymatic peptide synthesis was evaluated in this study. Addition of tyrosine amide into the reaction produced Tyr-Tyr-NH₂, of which yield exceeded 130% on the basis of tyrosine ethyl ester. Although purified Tyr-Tyr-NH₂ did not inhibit ACE activity, α-chymotrypsin could act on the dipeptide amide and convert about 40% of it to Tyr-Tyr. The use of both ester and amide forms of tyrosine is expected to be a potent procedure for α-chymotrypsin-catalyzed synthesis of antihypertensive peptides.     

Localization and synthesis of α-fetoprotein in the chicken

Summary The localization and sites of synthesis of -fetoprotein in chick embryos throughout development have been investigated using the combined techniques of immunofluorescence microscopy and tissue culture in the presence of radiolabelled amino acids, followed by immunoautoradiographic analysis.Alpha-fetoprotein is present in a range of embryonic tissues and especially concentrated in the yolk sac, liver and connective tissue. Analysis of culture fluids revealed that the yolk sac is the major site of -fetoprotein synthesis with smaller, but significant quantities being produced by the liver.These results are discussed in relation to mammalian -fetoprotein, and the merits of the chick embryo for studies on the biological function of AFP are considered.Supported by an award from the Science Research Council, to whom grateful acknowledgement is made     

Localisation and synthesis of α-fetoprotein in the rabbit

Summary The cellular location and sites of synthesis of -fetoprotein (AFP) in the foetal, neonatal and maternal rabbit, were studied by the fluorescent antibody technique and by culturing tissuesin vitro with labelled amino acids. AFP was found to be localised intracellularly within liver hepatocytes and yolk sac endoderm of the foetus, and within the maternal uterine epithelium. Analysis of extracts of the cultured tissues for incorporation of radioactivity into serum proteins separated by polyacrylamide gel electrophoresis or analysed by autoradiography of immuno-precipitation lines, confirmed that the foetal liver and yolk sac splanchnopleur were the principal sites of primary synthesis of AFP. Localisation of AFP in the uterine epithelium and other foetal organs was consistent with a secondary derivation from the uterine fluid or from the blood circulation. These findings are discussed in relationship to findings in man and other mammals.Supported by an award from the Medical Research Council to whom grateful acknowledgement is made.     

A cell-free system for the study of α-amylase synthesis in barley aleurone layers

1. A cell-free system capable of alpha-amylase synthesis has been obtained from the aleurone layers of germinating barley. 2. This system requires potassium chloride, sucrose and an amino acid mixture in order to function. The crude preparation does not require calcium chloride. Chloramphenicol inhibits alpha-amylase synthesis as indicated both by increase in measurable enzyme activity and incorporation of l-[U-(14)C]glutamic acid.     

An Application of Serially Balanced Designs for the Study of Known Taste Samples with the α-ASTREE Electronic Tongue

The α-ASTREE e-Tongue instrument uses seven sensors to characterize taste signals associated with a liquid sample. The instrument was used to study eight test preparations (comprised of a blank, four preparations corresponding to four known tastes and Sodium Topiramate in three concentrations known to have a bitter taste) and eight washes. Serially balanced residual effects designs were used to order the samples to estimate residual and main effects. The design provided for eight repeated measurements per test preparation. The experimental results suggested the following: (1) The seven sensors can be separated into three groups according to the ability to discriminate test preparations, and three of the sensors contributed little or no information. Further investigation suggested the lack of differentiability might be due to the age of the sensors. (2) The sensors discriminated known tastes from blank. The residual effect due to test preparations might appear after repeated usage. (3) Exploratory principal component analysis of the data indicated that nearly 90% of the total variability across the seven sensors could be explained by a single principal component. (4) The four standard taste preparations did not correspond to orthogonal dimensions in the principal component axes. (5) The three Sodium Topiramate test preparations could neither be associated with the corresponding known bitter taste sample nor could the three doses be shown to follow a quantitative dose-response relationship on the e-Tongue measurement scale. The practical interpretation of the results of the statistical analysis indicates only poor discriminative ability of the e-Tongue to distinguish clearly between increasing concentrations of a known bitter compound such as Sodium Topiramate. No apparent linear relationship could be discerned over increasing concentrations that would allow the quantification of bitterness.KEY WORDS: cluster analysis, electronic tongue, principal component analysis, residual effects designs, serially balanced design     

A novel convergent method for the synthesis of α-acyl-γ-hydroxylactams and its application in the total synthesis of PI-090 and 091

A novel convergent method for the synthesis of α-acyl-γ-hydroxylactams utilizing the aldol reaction of N-Boc-protected γ-methoxylactams was developed. As the first application of this method for the synthesis of biologically active natural products, the total synthesis of platelet aggregation inhibitors PI-090 and PI-091 were also investigated and successfully achieved.     

Application of solid-phase Ellman''s reagent for preparation of disulfide-paired isomers of α-conotoxin SI

Disulfide-paired regioisomers of -conotoxin SIcan be accessed by orthogonal schemes using thecombination of S-9H-xanthen-9-yl (S-Xan) and S-acetamidomethyl (S-Acm)groups for cysteine protection. Following solid-phaseassemblies of the linear precursors, the peptides werecleaved from the solid support concurrent with removalof S-Xan protecting groups. The first disulfidebridges were formed in solution, using either thetraditional DMSO method or a recently introducedapproach featuring a solid-phase Ellman's reagent. The second disulfide bridges were oxidized by threedifferent methods: reactions mediated by thalliumtrifluoroacetate, iodine, or a sulfoxide/silylmixture. In general, yields depended primarily onwhich regioisomer was the target, rather than thespecific chemistry used for either disulfide-formingstep. However, the selectivities towards the desiredregio-isomers were reproducibly better using thesolid-phase Ellman's reagent, by comparison to theDMSO method. In the most favorable cases, completeselectivity was achieved, while even in cases wherethe net results using DMSO gave considerablescrambling, the corresponding experiments with thesolid-phase Ellman's reagent were more selective. Possible reasons why choice of oxidation method forthe first step affects the selectivity at the secondstep are discussed.     

Application of solid-phase Ellman's reagent for preparation of disulfide-paired isomers of α-conotoxin SI

Summary Disulfide-paired regioisomers of α-conotoxin SI can be accessed by orthogonal schemes using the combination ofS-9H-xanthen-9-yl (S-Xan) andS-acetamidomethyl (S-Acm) groups for cysteine protection. Following solidphase assemblies of the linear precursors, the peptides were cleaved from the solid support concurrent with removal ofS-Xan protecting groups. The first disulfide bridges were formed in solution, using either the traditional DMSO method or a recently introduced approach featuring a solid-phase Ellman's reagent. The second disulfide bridges were oxidized by three different methods: reactions mediated by thallium trifluoroacetate, iodine, or a sulfoxide/silyl mixture. In general, yields depended primarily on which regioisomer was the target, rather than the specific chemistry used for either disulfide-forming step. However, the selectivities towards the desired regioisomers were reproducibly better using the solid-phase Ellman's reagent, by comparison to the DMSO method. In the most favorable cases, complete selectivity was achieved, while even in cases where the net results using DMSO gave considerable scrambling, the corresponding experiments with the solid-phase Ellman's reagent were more selective. Possible reasons why choice of oxidation method for the first step affects the selectivity at the second step are discussed.     

Improvement of the transfucosylation activity of α-l-fucosidase from Thermotoga maritima for the synthesis of fucosylated oligosaccharides in the presence of calcium and sodium

The influence of CaCl₂ and NaCl in the hydrolytic activity and the influence of CaCl₂ in the synthesis of fucosylated oligosaccharides using α-l-fucosidase from Thermotoga maritima were evaluated. The hydrolytic activity of α-l-fucosidase from Thermotoga maritima displayed a maximum increase of 67% in the presence of 0.8 M NaCl with water activity (a_w) of 0.9672 and of 138% in the presence of 1.1 M CaCl₂ (a_w 0.9581). In addition, the hydrolytic activity was higher when using CaCl₂ compared to NaCl at a_w of 0.8956, 0.9581 and 0.9672. On the other hand, the effect of CaCl₂ in the synthesis of fucosylated oligosaccharides using 4-nitrophenyl-fucose as donor substrate and lactose as acceptor was studied. In these reactions, the presence of 1.1 M CaCl₂ favored the rate of transfucosylation, and improved the yield of synthesis duplicating and triplicating it with lactose concentrations of 58 and 146 mM, respectively. CaCl₂ did not significatively affect hydrolysis rate in these reactions. The combination of the activating effect of CaCl₂, the decrement in a_w and lactose concentration had a synergistic effect favoring the synthesis of fucosylated oligosaccharides.

     

Polymerization of mannosyl tricyclic orthoesters for the synthesis of α(1–6) mannopyranan—the backbone of lipomannan

Tuberculosis (TB) remains a major health problem worldwide. Understanding the interactions between the surface components of Mycobacterium tuberculosis (Mtb), the main causative agent of TB, with host immune response will be critical for developments of effective treatments and prevention of TB. Chemically defined mimics of the bacterial envelope components serve as important tools for biological studies of the bacterial interactions with mammalian hosts. We report here a rapid synthetic approach utilizing mannosyl tricyclic orthoesters as monomers for regio- and stereo-controlled polymerizations to generate α(1–6) mannopyranan—the backbone of lipomannan. The polymerizations generated multiple glycosidic bonds in a single chemical transformation in regio- and stereo-selective manners. TMSOTf is the optimum catalyst to promote the selective and high yielding polymerization when compared with other Lewis acids. In addition, the monomers 3,4-O-benzyl-β-d-mannopyranose 1,2,6-orthobenzoate (1) and 3,4-O-benzyl-β-d-mannopyranose 1,2,6-orthopivalate (2) can be synthesized in multiple-gram scale and in a rapid fashion. Characterizations by GPC and NMR indicate the identity of α(1–6) mannopyranan with DPn (degree of polymerization) = 20.     

Ammonia lyases and aminomutases as biocatalysts for the synthesis of α-amino and β-amino acids

Ammonia lyases catalyse the reversible addition of ammonia to cinnamic acid (1: R=H) and p-hydroxycinnamic (1: R=OH) to generate L-phenylalanine (2: R=H) and L-tyrosine (2: R=OH) respectively (Figure 1a). Both phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) are widely distributed in plants, fungi and prokaryotes. Recently there has been interest in the use of these enzymes for the synthesis of a broader range of L-arylalanines. Aminomutases catalyse a related reaction, namely the interconversion of α-amino acids to β-amino acids (Figure 1b). In the case of L-phenylalanine, this reaction is catalysed by phenylalanine aminomutase (PAM) and proceeds stereospecifically via the intermediate cinnamic acid to generate β-Phe 3. Ammonia lyases and aminomutases are related in sequence and structure and share the same active site cofactor 4-methylideneimidazole-5-one (MIO). There is currently interest in the possibility of using these biocatalysts to prepare a wide range of enantiomerically pure l-configured α-amino and β-amino acids. Recent reviews have focused on the mechanism of these MIO containing enzymes. The aim of this review is to review recent progress in the application of ammonia lyase and aminomutase enzymes to prepare enantiomerically pure α-amino and β-amino acids.     

Engineering Escherichia coli for the synthesis of short- and medium-chain α,β-unsaturated carboxylic acids

Concerns over sustained availability of fossil resources along with environmental impact of their use have stimulated the development of alternative methods for fuel and chemical production from renewable resources. In this work, we present a new approach to produce α,β-unsaturated carboxylic acids (α,β-UCAs) using an engineered reversal of the β-oxidation (r-BOX) cycle. To increase the availability of both acyl-CoAs and enoyl-CoAs for α,β-UCA production, we use an engineered Escherichia coli strain devoid of mixed-acid fermentation pathways and known thioesterases. Core genes for r-BOX such as thiolase, hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase were chromosomally overexpressed under the control of a cumate inducible phage promoter. Native E. coli thioesterase YdiI was used as the cycle-terminating enzyme, as it was found to have not only the ability to convert trans-enoyl-CoAs to the corresponding α,β-UCAs, but also a very low catalytic efficiency on acetyl-CoA, the primer and extender unit for the r-BOX pathway. Coupling of r-BOX with YdiI led to crotonic acid production at titers reaching 1.5 g/L in flask cultures and 3.2 g/L in a controlled bioreactor. The engineered r-BOX pathway was also used to achieve for the first time the production of 2-hexenoic acid, 2-octenoic acid, and 2-decenoic acid at a final titer of 0.2 g/L. The superior nature of the engineered pathway was further validated through the use of in silico metabolic flux analysis, which showed the ability of r-BOX to support growth-coupled production of α,β-UCAs with a higher ATP efficiency than the widely used fatty acid biosynthesis pathway. Taken together, our findings suggest that r-BOX could be an ideal platform to implement the biological production of α,β-UCAs.