Effect of ethanol on the synthesis of large-ring cyclodextrins by cyclodextrin glucanotransferases

Cyclodextrin glucanotransferase (EC 2.4.1.19, CGTase) synthesizes cyclodextrins (CD) composed of 6 to more than hundred glucose units from amylose by an intramolecular transglycosylation reaction. The addition of ethanol to the reaction medium resulted in an increase of the yield of large-ring CD obtained with a CGTase from Bacillus sp. BT3-2 and Bacillus macerans. The presence of 15% ethanol in the reaction mixture with the CGTase from Bacillus sp. BT3-2 resulted in a 30% increase of the amounts of CD₁₀–CD₁₃ synthesized after 5 h of reaction. The addition of 20% ethanol increased the yield of CD₁₄–CD₂₁ up to 1000%. The hydrolysis of the large-ring CD by the CGTases was suppressed in the presence of ethanol. The ring-opening coupling cyclization reactions of the CGTase were effected differently by the organic solvent which may contribute to the observed increase of the yield and size of the CD obtained in the synthesis reactions.     

Effect of organic solvents on the yield and specificity of cyclodextrins by recombinant cyclodextrin glucanotransferase (CGTase) from Anaerobranca gottschalkii

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an enzyme that degrades starch and starch related glucans into cyclodextrins (CDs) by intramolecular transglycosylation reaction. The biochemical activity of recombinant CGTase from Anaerobranca gottschalkii for the yield and product specificity of cyclodextrins was investigated in the presence of organic solvents. Compared with the control of starch bioconversion, addition of various organic solvents generally increased the total CD and product specificity by affecting product inhibition and/or intermolecular transglycosylation reaction. The highest conversion (45 %) of starch to CDs was obtained in the presence of ethanol, while the simultaneous addition of two organic solvents, decanol-ethanol, comparatively showed a reduced total yield of 39 %. Despite this, the highest product ratio of 91 % α-CD, and 64 % β-CD was obtained in the presence of decanol and cyclohexane respectively. The effect of organic solvents on the yield and specificity of CD was attributed mainly to their effect on product inhibition and transglycosylation reaction. Although the use of two organic solvents showed almost a significant increase in total yield of CDs, it resulted in a comparatively lower specific product yield compared to their respective individual effect. Generally, normal enzyme activity was favoured at higher temperature of 65 °C, but the addition of organic solvents, in most cases, was found to decrease the bioconversion. Thus, the preferred optimal condition was reduced to 40 °C, where the maximal conversion of starch to CDs in general and α-CD in particular was achieved.     

Synthesis of Large-Ring Cyclodextrins by Cyclodextrin Glucanotransferases from Bacterial Isolates

The synthesis of large-ring cyclodextrins (LR-CD) with cyclodextrin glucanotransferase (CGTase) preparations from the bacterial isolates BT3, BT4, BT25 and BT57 was investigated. All CGTase preparations catalyzed the synthesis of LR-CD with 5% soluble starch or 2% synthetic amylose as substrate. The amount and size of LR-CD synthesized depended on the reaction time and on the particular CGTase preparation used. The yield of LR-CD obtained with the enzyme preparation from BT3 and synthetic amylose as substrate was about 18% of the total glucan amount after a reaction time of 23 h.     

Synthesis of novel silylated 1,2,4-triazin-5-ones

The reaction of various α-silyl-α-keto esters with thiosemicarbazide at 50 °C in ethyl acetate was found to give α-silyl-substituted thiosemicarbazone-acetic acid esters in good yield. These may then be converted to their corresponding silyl-substituted 1,2,4-triazin-5-ones by cyclization under basic conditions.     

Biochemical properties and cyclodextrin production profiles of isoforms of cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) catalyzes the conversion of starch to cyclodextrin (CD), an important host molecule for the study of host?Cguest interactions. CGTase from Paenibacillus sp. RB01 and its recombinant form showed the same isoform pattern. The three isoforms, two major (isoforms I and II) and one minor (isoform III), all had a different net charge but the same molecular mass. The aim of this work was to characterize the three isoforms, and especially to compare their CD production profiles. Isoforms I and II were separated on a FPLC Mono Q column and showed the same optimum pH (pH 5 for dextrinizing and pH 6?C7 for cyclization activity) and optimum temperature (65?C70 °C for both activities). However, the two isoforms differed in their catalytic efficiency of the coupling reaction with variable concentrations of the ??-CD donor in the presence of a fixed amount of cellobiose acceptor, with k_cat/K_m values of 3.46 × 10^?3 and 2.20 × 10^?3 mM^?1 min^?1, for isoforms I and II, respectively. Both isoforms I and II were found to have ??-CGTase activity and gave a similar CD6:CD7:CD8 product ratio of 0.2:1.0:0.6, with an increase in the ratio of the small-ring to the large-ring CDs from 1.0:0.5 to 1.0:0.3 from a 6 to 24 h reaction time. However, in terms of maximal CD yields, the two isoforms differed in their optimal reaction temperature and time required, the optimal conditions being at 40 °C for 6 h for isoform I and at 60 °C for 24 h for isoform II.     

Production and purification of CGTase of alkalophylicBacillus isolated from Brazilian soil

Alkalophylic bacilli that produce cyclodextringlycosyltransferase (CGTase) were isolated from Brazilian soil, with a scheme of two plating steps. In the first step, the bacterial isolate forms a halo in the cultivation medium that contains γ-cyclodextrin (CD) complexing dyes. The CGTase of an isolate was purified 157-fold by biospecific affinity chromatography, with β-CD showing a mol wt of 77,580 Daltons. It produces a γ- to β-CD ratio of 0.156 and a small amount of α-CD, using maltodextrin 10% as substrate, at 50°C, pH 8.0 and 22 h reaction time, reaching 21.4% conversion of the substrate to cyclodextrins. In the second screening step, the isolates chosen give larger halos with β-CD complexing dyes, and smaller halos with β-CD complexing dyes, leading to a 30% improvement in γ-CD selectivity, although at lower total yield for cyclodextrins (11.5%).     

New synthetic approach to substituted isoindolo[2,1-a]quinoline carboxylic acids via intramolecular Diels-Alder reaction of 4-(N-furyl-2)-4-arylaminobutenes-1 with maleic anhydride

Acylation of substituted 4-(furyl-2)-4-arylaminobut-1-enes with maleic anhydride provided 2-allyl-6-carboxy-4-oxo-3-aza-10-oxatricyclo[5.2.1.0^1,5]dec-8-enes in high yield under mild reaction conditions. The Diels-Alder adducts are formed via an initial amide formation followed by a stereoselective intramolecular [4+2] exo-cycloaddition reaction. Treatment of the tricyclic compounds with phosphoric acid at high temperatures (70-120 °C) promoted cyclic ether opening, intramolecular cyclization and aromatization to give the corresponding tetracyclic compounds, 5,6,6a,11-tetrahydro-10-carboxyisoindolo[2,1-a]quinolines, in moderate yields. The influence of the acid and the reaction temperature on the cyclization reactions are also discussed.     

Enhancement of selectivity for producing γ-cyclodextrin

The production of cyclodextrins (CDs) by cyclodextrin-glycosyl-transferase (CGTase) from Bacillus firmus was studied, with respect to the effect of the source of starch upon CD yield and on the selectivity for producing γ-CD. Cyclodextrin production tests were run for 24 h at 50°C, pH 8.0, and 1 mg/L of CGTase, and substrates were maltodextrin or the starches of rice, potato, cassava, and corn hydrolyzed up to D. E. 10. Cornstarch was the best substrate for producing γ-CD. Later, glycyrrhizin (2.5% [w/v]), which forms a stable complex with γ-CD, was added to the cornstarch reaction medium and increased the yield of γ-CD to about four times that produced with only maltodextrin, but the total yield of CDs remained practically unchanged. Therefore, the results showed that the studied CGTase is capable of giving relatively high yield of γ-CD in the presence of glycyrrhizin as complexant and cornstarch as substrate.     

乙醇对环糊精葡萄糖基转移酶催化作用的影响

采用紫外吸收光谱法测定了乙醇、环已烷与β-环糊精包合反应稳定常数及自由能变化, 确定乙醇与β-环糊精的包合反应稳定常数K_s=4.71 L/mol, ΔG=-3.84 kJ/mol; 环己烷与β-环糊精包合反应的K_s=19.56 L/mol, ΔG=-7.37 kJ/mol. 环己烷与β-环糊精的结合能力大大高于乙醇, 故用乙醇提高其产率的机理不同于传统工艺中所添加的环己烷. 通过测定乙醇对环糊精葡萄糖基转移酶催化作用的影响进一步探究其作用机理. 结果表明, 乙醇不仅能提高环糊精葡萄糖基转移酶环化作用的活力, 并在一定程度上阻止对转化环糊精有抑制作用的小分子糖的形成, 还可以减轻环糊精葡萄糖基转移酶对环糊精的水解及偶合作用, 从而提高环糊精的产率.     

Purification and Properties of a New Thermostable Cyclodextrin Glucanotransferase from Bacillus pseudalcaliphilus 8SB

A new cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB was studied in respect to its γ-cyclizing activity. An efficient conversion of a raw corn starch into only two types of cyclodextrins (β- and γ-CD) was achieved by the purified enzyme. Crude enzyme obtained by ultrafiltration was purified up to fivefold by starch adsorption with a recovery of 62% activity. The enzyme was a monomer with a molecular mass 71 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The CGTase exhibited two pH optima, at pH 6.0 and 8.0, and was at most active at 60 °C and pH 8.0. The enzyme retained more than 80% of its initial activity in a wide pH range, from 5.0 to 11.0. The CGTase was strongly inhibited by 15 mM Cu(2+), Fe(2+), Ag(+), and Zn(2+), while some metal ions, such as Ca(2+), Na(+), K(+), and Mo(7+), exerted a stimulating effect in concentration of 5 mM. The important feature of the studied CGTase was its high thermal stability: the enzyme retained almost 100% of its initial activity after 2 h of heating at 40-60 °C; its half-life was 2 h at 70 °C in the presence of 5 mM Ca(2+). The achieved 50.7% conversion of raw corn starch into 81.6% β- and 18.4% γ-CDs after 24 h enzyme reaction at 60 °C and pH 8.0 makes B. pseudalcaliphilus 8SB CGTase industrially important enzyme for cyclodextrin production.     

Efficient solid-phase synthesis of cyclic RGD peptides under controlled microwave heating

Cyclic RGD peptides are potent antagonists for the α_vβ₃ integrin receptor. In this Letter, microwave-assisted solid-phase synthesis of cyclic RGD peptides is described. In a coupling reaction between Fmoc-Arg(Pbf)-OH and high-loading H-Gly-Trt(2-Cl) resin, multiple coupling reactions were required for completion under the conventional HBTU activation. We found that the use of COMU, a new coupling reagent, under microwave heating to 50 °C accelerated the reaction even inside the resin. This method was applicable to the synthesis of linear pentapeptides, H-Asp(OtBu)-Xxx-Yyy-Arg(Pbf)-Gly-OH (Xxx = d-Phe(p-Br) or d-Tyr, Yyy = Lys(Boc) or MeVal). Cyclization of these peptides followed by deprotection gave the desired cyclic RGD peptides with high purity.     

Temperature dependent selective synthesis of linear 2-bromo and 2-alkoxyfuro[2,3-b]quinolines: reaction of 3-(2,2-dibromovinyl-)quinolin-2(1H)-ones with alcoholic KOH

Base promoted and temperature dependent reactions of 3-(2,2-dibromovinyl)quinolin-2(1H)-ones have been described. At 50 °C, the cyclization reactions afforded 2-bromofuro[2,3-b]quinolines in good yields. However, at elevated temperature (80 °C) the domino reaction proceeded affording 2-alkoxyfuro[2,3-b]quinolines via cyclization and nucleophilic substitution reactions.     

Direct glycosylation: synthesis of alpha-indoline ribonucleosides

A selective synthesis of α-anomers of indoline nucleosides is described. Ribonucleosides of indoline, dimethylindoline and 5-bromoindoline are readily prepared in good yield by reacting indoline bases directly with the protected sugar, 2,3-O-(1-methylethylidene) 5-O-(triphenylmethyl)-D-ribofuranose in dry ethanol or methylene chloride in presence of molecular sieves at 40-60 °C.     

A novel amylomaltase from Corynebacterium glutamicum and analysis of the large-ring cyclodextrin products

Amylomaltase catalyzes the formation of large-ring cyclodextrins (LR-CDs) from starch. This study aims to construct the recombinant amylomaltase from Corynebacterium glutamicum and to characterize the purified enzyme with the emphasis on the profile of LR-CDs production. A novel amylomaltase from Corynebacterium glutamicum ATCC 13032 was cloned and expressed in Escherichia coli BL21 (DE3) using the expression vector pET-19b. The open reading frame of amylomaltase gene of 2,121 bp (encoding the polypeptide of 706 amino acid residues) was obtained with the N-terminal His-tag fragment of 69 bp attached before the start codon of the amylomaltase gene. The deduced amino acid sequence showed a low sequence identity (20?C25%) to those thermostable amylomaltases from Thermus sp. The maximum enzyme activity was obtained when the recombinant cells were cultured at 37 °C for 2 h after induction with 0.4 mM isopropyl thio-??-D-galactoside (IPTG). The enzyme was 11-fold purified with a yield of 30% by a HiTrap affinity column. The purified amylomaltase showed a single band of 84 kDa on a 7.5% SDS-PAGE. When the enzyme acted on pea starch, it catalyzed an intramolecular transglucosylation (cyclization) reaction that produced LR-CDs or cycloamyloses (CA). The product profile was dependent on the incubation time and the enzyme concentration. Shorter incubation time gave larger LR-CDs as principal products. At 4 h incubation, the product was composed of a mixture of LR-CDs in the range of CD19?CCD50, with CD27?C28 as products with highest amount. It is noted that CD19 was the smallest product in all conditions tested. The enzyme also catalyzes intermolecular transglucosylation on various malto-oligosaccharides, with maltose as the smallest substrate.     

Tuning the Outcome of Enzyme-Mediated Dynamic Cyclodextrin Libraries to Enhance Template Effects

Enzyme-mediated dynamic combinatorial chemistry combines the concept of thermodynamically controlled covalent self-assembly with the inherent biological relevance of enzymatic transformations. A system of interconverting cyclodextrins has been explored, in which the glycosidic linkage is rendered dynamic by the action of cyclodextrin glucanotransferase (CGTase). External factors, such as pH, temperature, solvent, and salinity are reported to modulate the composition of the dynamic cyclodextrin library. Dynamic libraries of cyclodextrins (CDs) could be obtained in wide ranges of pH (5.0–9.0), temperature (5–37 °C), and salinity (up to 7.5 m NaNO₃), and with high organic solvent content (50 % by volume of ethanol), showing that enzyme-mediated dynamic systems can be robust and not limited to physiological conditions. Furthermore, it is demonstrated how strategic choice of reaction conditions can enhance template effects, in this case, to achieve highly selective production of α-CD, an otherwise challenging target due to competition from the structurally similar β-CD.     

Reaction of 2-Alkoxypropenals with 2-Mercaptoethanol

The reaction between 2-alkoxypropenals and 2-mercaptoethanol was studied at 20 and 60°C by means of ^1HNMR and GC-MS methods. Under kinetically controlled conditions (20°C, 7-30 days) with no catalyst the addition of 2-mercaptoethanol to 2-alkoxypropenals occurs along Markownikoff rule. The arising 2'-hydroxyethylthio-2-alkoxypropanal undergoes isomerization into the 2-hydroxy-3-alkoxy-3-methyl-1,4-oxathiane that at heating in the presence of catalytic amounts of acids is converted into 2-methyl-2-formyl-1,3-oxathiolane. The reaction of 2-alkoxypropenals with 2-mercaptoethanol at heating (60°C, 3 h) in the presence of acids affords 2-methyl-2,2'-bi(1,3-oxathiolane) even at 2-mercaptoethanol deficit. At the double excess of the latter the 2-methyl-2,2'-bi(1,3-oxathiolane) was obtained in quantitative yield. The presumable schemes of conversion of 2-hydroxy-3-alkoxy-3-methyl-1,4-oxathiane into 2-methyl-2-formyl-1,3-oxathiolane and 2-acetyl-1,3-oxathiolane are discussed.     

A simple and efficient one-step, regioselective, enzymatic glucosylation of arbutin by α-glucosidase

4-Hydroxyphenyl-β-isomaltoside has been synthesized by α-glucosidase assisted transglycosylation between arbutin as acceptor and sucrose as donor molecules, respectively. Optimum conditions for the transglucosylation reaction were 40 °C for 20 h with 10 mM arbutin and 1.5 M sucrose in a 100 mM sodium citrate/phosphate buffer at pH 5.0. The new glucoside was obtained in a 50% molar yield with respect to arbutin.     

Donor-acceptor complexes in copolymerization. L. Preparation and microstructure of chlorine-containing alternating conjugated diene–acceptor monomer copolymers

Neighboring monomer units cause significant shifts in the infrared absorption peaks attributed to cis- and trans-1,4 units in conjugated diene-acceptor monomer copolymers. Conjugated diene-maleic anhydride alternating copolymers apparently have a predominantly cis-1,4-structure, while alternating diene-SO₂ copolymers have a predominantly trans-1,4 structure. Alternating copolymers of butadiene, isoprene, and pentadiene-1,3 with α-chloroacrylonitrile and methyl α-chloroacrylate, prepared in the presence of Et_1.5AlCl_1.5(EASC), have trans-1,4 unsaturation. Alternating copolymers of chloroprene with acrylonitrile, methyl acrylate, methyl methacrylate, α-chloroacrylonitrile, and methyl α-chloroacrylate prepared in the presence of EASC-VOCl₃ have trans-1,4 configuration. The reaction between chloroprene and acrylonitrile in the presence of AlCl₃ yields the cyclic Diel-Alder adduct in the dark and the alternating copolymer under ultraviolet irradiation. The equimolar, presumably alternating, copolymers of chloroprene with methyl acrylate and methyl methacrylate undergo cyclization at 205°C to a far lesser extent than theoretically calculated, to yield five and seven-membered lactones. The polymerization of chloroprene in the presence of EASC and acetonitrile yields a radical homopolymer with trans-1,4 unsaturation.     

One-pot synthesis of iodine-substituted 1,4-oxazepines

A facile one-pot method for the synthesis of iodine-substituted 1,4-oxazepines is reported. When reacted with ZnCl₂ and I₂ in DCM at 40?°C, N-propargylic β-enaminones, prepared by the conjugate addition of propargylamine to α,β-alkynic ketones, underwent 7-exo-dig cyclization by zinc chloride and concomitant reaction with molecular iodine to afford 2-(iodomethylene)-2,3-dihydro-1,4-oxazepines in good to high yields. This cyclization was found to occur with broad scope of substrates and high tolerance of functional groups. The resulting iodine-containing 1,4-oxazepines can be further elaborated to more complex structures by subsequent cross-coupling reactions, which may provide a platform for biological studies.     

Wide bite angle diphosphine rhodium complexes: Synthesis, structure, and catalytic 1,4-addition of arylboronic acids to enones

A rhodium complex [ClRh(CO)(L1)] featuring a wide bite angle diphosphine ligand (L1 = 1,3-bis(2-diphenylphosphinomethylphenyl)benzene) has been synthesized and structurally characterized. L1 supports a bite angle (P-M-P angle, β) of 171.4° in the trans-square planar complex. L1 was tested in Rh-catalyzed 1,4-addition reactions of arylboronic acids (six examples) to α,β-unsaturated ketones (five examples). In mixed aqueous/cyclohexane solution at 60 °C, addition reactions proceed in up to quantitative yield with a 1:1 arylboronic acid/enone ratio. Yields as high as 77% can be acquired even when one of the coupling partners is sterically encumbered 2,4,6-trimethylphenylboronic acid.