Controlled Pore Glass Research Articles

Optimal immobilization of trypsin from the spleen of albacore tuna (Thunnus alalunga) and its characterization

Trypsin purified from the spleen of albacore tuna was immobilized onto Octyl Sepharose CL-4B, glutaraldehyde activated silica and 5′-4,4′-dimethyltryptamine-thymidine-succinyl controlled pore glass. Trypsin was highly and efficiently immobilized onto Octyl Sepharose CL-4B, with the highest activity (6.26 U/g support) and specific activity (1.45 U/mg bound protein). The optimum conditions for trypsin immobilization onto Octyl Sepharose CL-4B were 40 mg/mL trypsin solution, pH 7 at 4 °C for 6 h of incubation time. The optimal temperature and pH for the hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) by the immobilized trypsin were 55 °C and 8.5, both of which were higher than that of the free form. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and organic solvents. The immobilized enzyme was less sensitive to inhibition by the soybean trypsin inhibitor compared with the free soluble form of the enzyme. According to the results, the immobilized trypsin and free enzyme retained 83% and 47% of their activity, respectively, when they were incubated with 1 μM of the soybean trypsin inhibitor. For the reusability study, the immobilized trypsin maintained 60% of its activity after 4 periods of activity, indicating that the immobilized trypsin had appropriate stability and could be reused.

Acceleration of decomposition of CL-20 explosive under nanoconfinement

The thermal properties of CL-20 explosive in the bulk and confined in controlled pore glass matrices to nanoscale dimensions were studied using dynamic differential scanning calorimetry. The decomposition reaction of the CL-20 was found to be accelerated in 12-nm-diameter pores compared to the bulk CL-20 with the onset of the decomposition occurring 16–24 °C lower and a fourfold to sevenfold larger reaction rate constant. The total heat of decomposition was found to be independent of pore size and heating rate, and the average activation energy for all samples was found to be 160 ± 7 kJ mol−1.

Novel Bisimidazole-Containing Peptidomimetic Molecules for Мetal-Independent RNA Cleavage: Synthesis and Solid-Phase Screening Method

Novel bisimidazole-containing peptidomimetic molecules, which can efficiently cleave RNA in the absence of divalent metal ions have been synthesized. The thymidine 5'-monophosphate derivatives modified with these peptidomimetics have been obtained on CPG (controlled pore glass) using a solid phase azide-alkyne cycloaddition. The ability of all synthesized derivatives to cleave RNA has been demonstrated by a solid-phase screening method. The resulting conjugates have provided a 20–100% cleavage of the RNA target in 18 hours. The results allow us to consider the described strategy as a useful approach for the rapid selection of potentially promising peptidomimetic molecules in order to synthesize reactive constructs on their basis.

Construction of Pyrimidine Bases Bearing Carboxylic Acid Equivalents at the C5 Position by Postsynthetic Modification of Oligonucleotides.

This unit describes postsynthetic modification of oligonucleotides (ONs) containing 2'-deoxy-5-trifluoromethyluridine and 2'-deoxy-5-trifluoromethylcytidine. In ONs, the trifluoromethyl group at the C5 position of pyrimidine bases is converted into a variety of carboxylic acid equivalents using alkaline and amine solutions. In addition, treating fully protected and controlled pore glass (CPG)-attached ONs with methylamine and sodium hydroxide aqueous solution results in deprotection of all protecting groups (except the 4,4'-dimethoxytrityl group), cleavage from CPG, and simultaneous conversion of the trifluoromethyl group to afford the corresponding ONs containing 5-substituted pyrimidine bases. © 2019 by John Wiley & Sons, Inc.

Nanoconfinement Effects on the Glass Transition and Crystallization Behaviors of Nifedipine.

The impact of nanoconfinement on the crystallization and glass transition behaviors of nifedipine (NIF) has been investigated using differential scanning calorimetry. Nanoconfinement was provided by imbibing the NIF into a porous medium (controlled pore glass, CPG), and results were compared with the unconfined bulk material. Consistent with previous results from the literature, both glass transition temperature Tg and melting temperature Tm decrease with decreasing pore size. The melting temperature was found to decrease with the reciprocal of pore diameter and could be analyzed with the Gibbs-Thomson equation. In addition, for confinement sizes of 7.5 and 12 nm, it was found that no cold-crystallization occurs upon heating from the glassy state to above the expected melting transition. Finally, at intermediate confinements we find evidence of a possible new, confinement-induced polymorph of NIF.

Development of nanofiber based immunosorbent surface for the removal of fluoxetine from breast milk

In this study, novel bioactive material has been identified for the removal of fluoxetine from breast milk. For this purpose, the poly (vinyl alcohol)/poly (acrylic acid) based nanofiber with an antibody immobilized has been developed. The efficiency of this nanofiber was compared with the control pore glass (CPG) either in milk. The bioactive nanofiber removed fluoxetine significantly higher than the CPG. The efficiency of the fluoxetine removal with the nanofiber was 98% in breast milk. In conclusion, the nanofiber based immunosorbent surface that is developed in this study was found to be efficient for chemical sensing of fluoxetine.

Post-synthetic modification of oligonucleotides containing 5-trifluoromethylpyrimidine bases

A practical and operationally simple post-synthetic modification of oligonucleotides containing 5-trifluoromethylpyrimidine bases is described. Trifluoromethyl group was used as a post-synthetic precursor and 5-trifluoromethylpyrimidine bases within oligonucleotides were converted into the corresponding 5-carboxy-, 5-cyano-, 5-amidinyl-, and 5-carbamoyl derivatives by treatment with an alkaline solution and amines. Moreover, post-synthetic treatment of fully protected and controlled pore glass (CPG)-attached oligonucleotides proceeded successfully with the simultaneous removal of all protecting groups, cleavage from CPG, and conversion of the trifluoromethyl group to afford the corresponding modified oligonucleotides.

Probe material choice for nuclear magnetic resonance cryoporometry (NMRC) measurements of the nano-scale pore size distribution of unconventional reservoirs

Nuclear magnetic resonance cryoporometry is a newly developed technique that can characterize the pore size distribution of nano-scale porous materials. To date, this technique has scarcely been used for the testing of unconventional oil and gas reservoirs; thus, their micro- and nano-scale pore structures must still be investigated. The selection of the probe material for this technique has a key impact on the quality of the measurement results during the testing of geological samples. In this paper, we present details on the nuclear magnetic resonance cryoporometric procedure. Several types of probe materials were compared during the nuclear testing of standard nano-scale porous materials and unconventional reservoir geological samples from Sichuan Basin, Southwest China. Gas sorption experiments were also carried out on the same samples simultaneously. The KGT values of the probe materials octamethylcyclotetrasiloxane and calcium chloride hexahydrate were calibrated using standard nano-scale porous materials to reveal respective values of 149.3 Knm and 184 Knm. Water did not successfully wet the pore surfaces of the standard controlled pore glass samples; moreover, water damaged the pore structures of the geological samples, which was confirmed during two freeze-melting tests. The complex phase transition during the melting of cyclohexane introduced a nuclear magnetic resonance signal in addition to that from liquid in the pores, which led to an imprecise characterization of the pore size distribution. Octamethylcyclotetrasiloxane and calcium chloride hexahydrate have been rarely employed as nuclear magnetic resonance cryoporometric probe materials for the testing of an unconventional reservoir. Both of these materials were able to characterize pore sizes up to 1 μm, and they were more applicable than either water or cyclohexane.

Controlled Pore Glass-based oligonucleotide affinity support: towards High Throughput Screening methods for the identification of conformation-selective G-quadruplex ligands

Target selectivity is one of the main challenges in the search for small molecules able to act as effective and non-toxic anticancer and/or antiviral drugs. To achieve this goal, handy, rapid and reliable High Throughput Screening methodologies are needed. We here describe a novel functionalization for the solid phase synthesis of oligonucleotides on Controlled Pore Glass, including a flexible hexaethylene glycol spacer linking the first nucleoside through the nucleobase via a covalent bond stable to the final deprotection step. This allowed us preparing fully deprotected oligonucleotides still covalently attached to their supports. In detail, on this support we performed both the on-line synthesis of different secondary structure-forming oligonucleotides and the affinity chromatography-based screenings of conformation-selective G-quadruplex ligands. By using a fluorescent core-extended naphthalene diimide with different emitting response upon binding to sequences folding into G-quadruplexes of different topologies, we have been able to discriminate not only G-quadruplex vs. duplex DNA structures, but also different G-quadruplex conformations on the glass beads by confocal microscopy.

Effect of nanopore confinement on the thermal and structural properties of heneicosan

The effect of nano-confinement on the phase transitions of heneicosane (n-C21H44, C21) were investigated using differential scanning calorimetry (DSC) and variable temperature powder X-ray diffraction (XRD). When C21 is confined within the pores of controlled pore glasses (CPGs), SBA-15 or other mesoporous materials, the solid-solid (Ts) and solid-liquid (Tm) transition temperatures are depressed. The depression of the phase transition temperatures (ΔTs, ΔTm) for C21 in CPGs and SBA-15, the degree of supercooling and enthalpy change (ΔHs, ΔHm) for C21 in CPGs, exhibited a linear dependence on the inverse pore size. The ΔTm values were insensitive to the polarity pore wall but varied with the pore geometry. XRD analysis revealed that the lamellar ordering characteristic for the solid C21 was disturbed in the nanopores, with the extent increasing as the pore size decreased. A new rotator phase RII is found for C21 confined in CPG (12 nm) and SBA-15 (14.4 nm).

Copper Nanoparticles on Controlled Pore Glass and TEMPO for the Aerobic Oxidation of Alcohols

AbstractHerein, we report on the facile synthesis of a heterogeneous copper nanocatalyst and its combination with 2,2,6,6‐tetramethyl‐1‐piperidinyloxy (TEMPO) for the aerobic oxidation of alcohols to their corresponding carbonyl compounds. This low cost copper nanocatalyst was found to exhibit excellent recyclability, making it a highly attractive catalytic system from an economical and environmental point of view. Extensive characterization of the catalyst by a number of techniques revealed that it was comprised of well‐dispersed Cu(I/II) nanoparticles with an average size of around 6 nm.

Ultrafast Laplace NMR with hyperpolarized xenon gas

Laplace NMR, consisting of diffusion and relaxation experiments, provides detailed information about dynamics of fluids in porous materials. Recently, we showed that two-dimensional Laplace NMR experiments can be carried out with a single scan based on spatial encoding. The method shortens the experiment time by one to three orders of magnitude, and therefore it is called ultrafast Laplace NMR. Furthermore, the single-scan approach facilitates significantly the use of nuclear spin hyperpolarization for boosting the sensitivity of the experiment, because a laborious hyperpolarization procedure does not need to be repeated. Here, we push the limits of the ultrafast Laplace NMR method by applying it, for the first time, in the investigation of a gas phase substance, namely hyperpolarized xenon gas. We show that, regardless of the fast diffusion of gas, layer-like spatial encoding is feasible, and an ultrafast diffusion – T2 relaxation correlation experiment reveals significantly different signals of free gas and gas adsorbed in a mesoporous controlled pore glass (CPG). The observed diffusion coefficients are many orders of magnitude larger than those detected earlier from liquid phase substances, emphasizing the extended application range of the method. The challenges in the methodology, caused by the fast diffusion, are also discussed.

Phosphatidylcholine determination in dietary supplement by coupled enzymes immobilized in a single bioreactor

Abstract A rapid and selective method for the determination of phosphatidylcholine (PC) is described. It is based on the use of a single packed bed reactor in which phospholipase C, alkaline phosphatase and choline oxidase are co-immobilized on long chain-alkylamino controlled pore glass. The detection of hydrogen peroxide, that is the final product of subsequent enzymatic reactions occurring within the bioreactor, is used for the determination of PC in a dietary supplement. The results of triplicate analysis show a coefficient of variation of 5%. The calibration curve is linear over a concentration range of 10–60 mg/L (R2 = 0.942) and the detection limit is 4.25 mg/L. The bioreactor resulted to be stable for at least 1 month.

A ‘catch and release’ strategy towards HPLC-free purification of synthetic oligonucleotides by a combination of the strain-promoted alkyne-azide cycloaddition and the photocleavage

A convenient strategy to purify oligonucleotides (ONs) synthesized by solid phase synthesis on an automatic DNA/RNA synthesizer was described. By attaching a photocleavable azide linker as the last phosphoramidite unit in the ON synthesis, only the desired full-length sequence was ‘caught’ on a controlled pore glass (CPG) resin possessing an aza-dimethoxycyclooctyne (DIBAC) derivative. Washing the resulting CPG resin to remove all unbounded species, the subsequent photoirradiation allowed the pure ONs to be ‘released’ without leaving any chemical modifications on native ON structure or chemical reagents from the solid phase ON synthesis.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-<em>O</em>-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5'-[CUA CGG AAU CAU]-3' containing zero, one, or two modifications located at the C2'-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2'-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism.

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5'-[CUA CGG AAU CAU]-3' containing zero, one, or two modifications located at the C2'-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2'-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Diffusive Transport in Pores. Tortuosity and Molecular Interaction with the Pore Wall

The self-diffusion of neat water, dimethyl sulfoxide (DMSO), octanol, and the molecular components in a water–DMSO solution was measured by 1H and 2H NMR diffusion experiments for those fluids imbibed into controlled pore glasses (CPG). Their highly interconnected structure is scaled by pore size and shows invariant pore topology independent of the size. The nominal pore diameter of the explored CPGs varied from 7.5 to 72.9 nm. Hence, the about micrometer mean-square diffusional displacement during the explored diffusion times was much larger than the individual pore size, and the experiment yielded the average diffusion coefficient. Great care was taken to establish the actual pore volumes of the CPGs. Transverse relaxation experiments processed by inverse Laplace transformation were performed to verify that the liquids explored filled exactly the available pore volume. Relative to the respective diffusion coefficients obtained in bulk phases, we observe a reduction in the diffusion coefficient that is independent of pore size for the larger pores and becomes stronger toward the smaller pores. Geometric tortuosity governs the behavior at larger pore sizes, while the interaction with pore walls becomes the dominant factor at our smallest pore diameter. Deviation from the trends predicted by the Renkin equation indicates that the interaction with the pore wall is not a just simple steric one but is in part dependent on the specific features of the molecules explored here.

A High-Throughput Process for the Solid-Phase Purification of Synthetic DNA Sequences.

An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5'-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5'-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. © 2017 by John Wiley & Sons, Inc.

Quantifying the adsorption of flowing gas mixtures in porous materials by remote detection NMR

Traditional adsorption measurements are carried out at static conditions for a single gas component, and multi-component adsorption measurements are challenging and time-consuming. Here we introduce an efficient remote detection NMR method for in situ analysis of adsorption of flowing gas in mesoporous materials. We investigated adsorption of continuously flowing propane and propene gases as well as their mixture in packed beds of controlled pore glass and silica gel materials. Remote detection provided from 300 to 700 -fold sensitivity enhancement as compared to a direct experiment carried out by a large coil around the packed bed. The unique time-of-flight information obtained using this method was utilized in flow velocity determination, and the velocities were converted into amount of adsorbed gas. As the detection was performed outside the packed bed region, the spectra were not influenced by the porous materials. Because resonances of each gas component were well-resolved in the spectra, the amount of adsorption of each gas component could be determined from the same data, measured in a few minutes.

A Simple Post-Polymerization Modification Method for Controlling Side-Chain Information in Digital Polymers.

A three-step post-polymerization modification method was developed for the design of digitally encoded poly(phosphodiester)s with controllable side groups. Sequence-defined precursors were synthesized, either manually on polystyrene resins or automatically on controlled pore glass supports, using two phosphoramidite monomers containing either terminal alkynes or triisopropylsilyl (TIPS) protected alkyne side groups. Afterwards, these polymers were modified by stepwise copper-catalyzed azide-alkyne cycloaddition (CuAAC). The terminal alkynes were first reacted with a model azide compound, and after removal of the TIPS groups, the remaining alkynes were reacted with another organic azide. This simple method allows for quantitative side-chain modification, thus opening up interesting avenues for the preparation of a wide variety of digital polymers.