Column Chromatography and Kinetics of Nucleosides and Nucleic Acid Bases on Immobilized Nickel- and Cobalt-CDAE-Sporopollenin

ABSTRACT

Sporopollenin, a natural polymer, has been modified for application as a ligand-exchange material. The ligand-exchange Chromatography with the functionalized Lycopodium clavatum is a useful method for the rapid separation of nucleosides and nucleic acid bases. The synthesis of tris(carboxymethyl) ethylene diamine Lycopodium clavatum has been described. The resin contained functional diaminoethane and carboxyl groups. Nickel(II) and cobalt(II) metal ions can easily be immobilized on this carboxylated-diamino-ethyl (CDAE)-sporopollenin. Physico-chemical and chelating properties of Lycopodium clavatum have been studied extensively. Ligand-exchange chromatography of CDAE-sporopollenin has also been compared to the conventional synthetic chelex-100 resin. The kinetics of cytidine in CDAE-sporopollenin resin has been investigated. The rate measurements have been made by a potentiometric technique. The relative rates at which nucleosides bind to the resin are determined by the actual chemical-exchange reaction between ligands and resin.     

Prodrugs of Nucleoside Triphosphates as a Sound and Challenging Approach: A Pioneering Work That Opens a New Era in the Direct Intracellular Delivery of Nucleoside Triphosphates

Synthetic nucleosides, designed to mimic naturally occurring nucleosides, are important antiviral and anticancer chemotherapeutic agents. However, nucleosides are not active as such and need to be metabolized, step by step, to their corresponding active nucleoside triphosphates (NTPs). This is mediated by phosphorylating enzymes, mainly host cellular kinases with strong specificity for their substrates; in many cases, this specificity prevents efficient conversion into the NTPs. To circumvent this metabolic handicap, successful nucleo(s/t)ide prodrugs have been developed as a valuable concept in the design of effective drugs. The unique concept of the TriPPPro approach, developed by Chris Meier and colleagues, is a powerful tool for the intracellular delivery of active NTPs, bypassing all the phosphorylation steps required by nucleosides to yield the active NTP metabolites. This concept is illustrated herein with general examples.     

Aryloxy Phosphoramidate Triesters: a Technology for Delivering Monophosphorylated Nucleosides and Sugars into Cells

Prodrug technologies aimed at delivering nucleoside monophosphates into cells (protides) have proved to be effective in improving the therapeutic potential of antiviral and anticancer nucleosides. In these cases, the nucleoside monophosphates are delivered into the cell, where they may then be further converted (phosphorylated) to their active species. Herein, we describe one of these technologies developed in our laboratories, known as the phosphoramidate protide method. In this approach, the charges of the phosphate group are fully masked to provide efficient passive cell‐membrane penetration. Upon entering the cell, the masking groups are enzymatically cleaved to release the phosphorylated biomolecule. The application of this technology to various therapeutic nucleosides has resulted in improved antiviral and anticancer activities, and in some cases it has transformed inactive nucleosides to active ones. Additionally, the phosphoramidate technology has also been applied to numerous antiviral nucleoside phosphonates, and has resulted in at least three phosphoramidate‐based nucleotides progressing to clinical investigations. Furthermore, the phosphoramidate technology has been recently applied to sugars (mainly glucosamine) in order to improve their therapeutic potential. The development of the phosphoramidate technology, mechanism of action and the application of the technology to various monophosphorylated nucleosides and sugars will be reviewed.     

Probing Ambiguous Base‐Pairs by Genetic Transformation with XNA Templates

The templating potential of anhydrohexitol oligonucleotides bearing ambiguous bases was studied in vivo, by using a selection screen for mosaic heteroduplex plasmids in Escherichia coli. 1,5‐Anhydro‐2,3‐dideoxy‐2‐(5‐nitroindazol‐1‐yl)‐D ‐arabino‐hexitol showed the greatest ambiguity among the three nucleosides tested. At most two successive ambiguous bases could be tolerated on hexitol templates read in bacterial cells. Hexitol nucleosides bearing simplified heterocycles thus stand as promising monomers for generating random DNA sequences in vivo from defined synthetic oligonucleotides.     

Production,Characterization and Synthetic Application of a Purine Nucleoside Phosphorylase from Aeromonas hydrophila

Purine nucleoside phosphorylase (PNP) from Aeromonas hydrophila encoded by the deoD gene has been over‐expressed in Escherichia coli, purified, characterized about its substrate specificity and used for the preparative synthesis of some 6‐substituted purine‐9‐ribosides. Substrate specificity towards natural nucleosides showed that this PNP catalyzes the phosphorolysis of both 6‐oxo‐ and 6‐aminopurine (deoxy)ribonucleosides. A library of nucleoside analogues was synthesized and then submitted to enzymatic phosphorolysis as well. This assay revealed that 1‐, 2‐, 6‐ and 7‐modified nucleosides are accepted as substrates, whereas 8‐substituted nucleosides are not. A few transglycosylation reactions were carried out using 7‐methylguanosine iodide ( 4 ) as a D ‐ribose donor and 6‐substituted purines as acceptor. In particular, following this approach, 2‐amino‐6‐chloropurine‐9‐riboside ( 2c ), 6‐methoxypurine‐9‐riboside ( 2d ) and 2‐amino‐6‐(methylthio)purine‐9‐riboside ( 2g ) were synthesized in very high yield and purity.     

Breakdown of polyethylene therepthalate microplastics under saltwater conditions using engineered Vibrio natriegens

Poly(ethylene terephthalate) (PET) is a highly recyclable plastic that has been extensively used and manufactured. Like other plastics, PET resists natural degradation, thus accumulating in the environment. Several recycling strategies have been applied to PET, but these tend to result in downcycled products that eventually end up in landfills. This accumulation of landfilled PET waste contributes to the formation of microplastics, which pose a serious threat to marine life and ecosystems, and potentially to human health. To address this issue, our project leveraged synthetic biology to develop a whole-cell biocatalyst capable of depolymerizing PET in seawater environments by using the fast-growing, nonpathogenic, moderate halophile Vibrio natriegens. By leveraging a two-enzyme system—comprising a chimera of IsPETase and IsMHETase from Ideonella sakaiensis—displayed on V. natriegens, we constructed whole-cell catalysts that depolymerize PET and convert it into its monomers in salt-containing media and at a temperature of 30°C.     

Synthesis and Antiviral Evaluation of TriPPPro‐AbacavirTP,TriPPPro‐CarbovirTP,and Their 1′,2′‐cis‐Disubstituted Analogues

Herein we describe the synthesis of lipophilic triphosphate prodrugs of abacavir, carbovir, and their 1′,2′‐cis‐substituted carbocyclic analogues. The 1′,2′‐cis‐carbocyclic nucleosides were prepared by starting from enantiomerically pure (1R,2S)‐2‐((benzyloxy)methyl)cyclopent‐3‐en‐1‐ol by a microwave‐assisted Mitsunobu‐type reaction with 2‐amino‐6‐chloropurine. All four nucleoside analogues were prepared from their 2‐amino‐6‐chloropurine precursors. The nucleosides were converted into their corresponding nucleoside triphosphate prodrugs (TriPPPro approach) by application of the H‐phosphonate route. The TriPPPro compounds were hydrolyzed in different media, in which the formation of nucleoside triphosphates was proven. While the TriPPPro compounds of abacavir and carbovir showed increased antiviral activity over their parent nucleoside, the TriPPPro compounds of the 1′,2′‐cis‐substituted analogues as well as their parent nucleosides proved to be inactive against HIV.     

Palladium‐Catalyzed Aryl Amination Reactions of 6‐Bromo‐ and 6‐Chloropurine Nucleosides

Palladium‐catalyzed C N bond forming reactions of 6‐bromo‐ as well as 6‐chloropurine ribonucleosides and the 2′‐deoxy analogues with arylamines are described. Efficient conversions were observed with palladium(II) acetate/Xantphos/cesium carbonate, in toluene at 100 °C. Reactions of the bromonucleoside derivatives could be conducted at a lowered catalytic loading [5 mol% Pd(OAc)₂/7.5 mol% Xantphos], whereas good product yields were obtained with a higher catalyst load [10 mol% Pd(OAc)₂/15 mol% Xantphos] when the chloro analogue was employed. Among the examples evaluated, silyl protection for the hydroxy groups appears better as compared to acetyl. The methodology has been evaluated via reactions with a variety of arylamines and by synthesis of biologically relevant deoxyadenosine and adenosine dimers. This is the first detailed analysis of aryl amination reactions of 6‐chloropurine nucleosides, and comparison of the two halogenated nucleoside substrates.     

Improvement of SNAr Reaction Rate by an Electron‐Withdrawing Group in the Crosslinking of DNA Cytosine‐5 Methyltransferase by a Covalent Oligodeoxyribonucleotide Inhibitor

DNA cytosine 5‐methyltransferase (DNMT) catalyzes methylation at the C5 position of the cytosine residues in the CpG sequence. Aberrant DNA methylation patterns are found in cancer cells. Therefore, inhibition of human DNMT is an effective strategy for treating various cancers. The inhibitors of DNMT have an electron‐deficient nucleobase because this group facilitates attack by the catalytic Cys residue in DNMTs. Recently, we reported the synthesis and properties of mechanism‐based modified nucleosides, 2‐amino‐4‐halopyridine‐C‐nucleosides (d^XP), as inhibitors of DNMT. To develop a more efficient inhibitor of DNMT for oligonucleotide therapeutics, oligodeoxyribonucleotides (ODNs) containing other nucleoside analogues, which react more quickly with DNMT, are needed. Herein, we describe the design, synthesis, and evaluation of the properties of 2‐amino‐3‐cyano‐4‐halopyridine‐C‐nucleosides (d^XP^CN) and ODNs containing d^XP^CN, as more reactive inhibitors of DNMTs. Nucleophilic aromatic substitution (S_NAr) of the designed nucleosides, d^XP^CN, was faster than that of d^XP, and the ODN containing d^XP^CN effectively formed a complex with DNMTs. This study suggests that the incorporation of an electron‐withdrawing group would be an effective method to increase reactivity toward the nucleophile of the DNMTs, while maintaining high specificity.     

A Conformational Mimetic Approach for the Synthesis of Carbocyclic Nucleosides as Anti‐HCV Leads

Computer‐aided approaches coupled with medicinal chemistry were used to explore novel carbocyclic nucleosides as potential anti‐hepatitis C virus (HCV) agents. Conformational analyses were carried out on 6‐amino‐1H‐pyrazolo[3,4‐d]pyrimidine (6‐APP)‐based carbocyclic nucleoside analogues, which were considered as nucleoside mimetics to act as HCV RNA‐dependent RNA polymerase (RdRp) inhibitors. Structural insight gained from the modeling studies revealed the molecular basis behind these nucleoside mimetics. The rationally chosen 6‐APP analogues were prepared and evaluated for anti‐HCV activity. RdRp SiteMap analysis revealed the presence of a hydrophobic cavity near C7 of the nucleosides; introduction of bulkier substituents at this position enhanced their activity. Herein we report the identification of an iodinated compound with an EC₅₀ value of 6.6 μM as a preliminary anti‐HCV lead.     

Synthesis and Biological Evaluation of Purine 2′‐Fluoro‐2′‐deoxyriboside ProTides as Anti‐influenza Virus Agents

2′‐Fluoro‐2′‐deoxyguanosine has been reported to have potent anti‐influenza virus activity in vitro and in vivo. Herein we describe the synthesis and biological evaluation of 6‐modified 2′‐fluoro‐2′‐deoxyguanosine analogues and their corresponding phosphoramidate ProTides as potential anti‐influenza virus agents. Whereas the parent nucleosides were devoid of antiviral activity in two different cellular assays, the 5′‐O‐naphthyl(methoxy‐L ‐alaninyl) ProTide derivatives of 6‐O‐methyl‐2′‐fluoro‐2′‐deoxyguanosine, 6‐O‐ethyl‐2′‐fluoro‐2′‐deoxyguanosine, and 2′‐deoxy‐2′‐fluoro‐6‐chloroguanosine, and the 5′‐O‐naphthyl(ethoxy‐L ‐alaninyl) ProTide of 6‐O‐ethyl‐2′‐fluoro‐2′‐deoxyguanosine displayed antiviral EC₉₉ values of ～12 μM . The antiviral results are supported by metabolism studies. Rapid conversion into the L ‐alaninyl metabolite and then 6‐modified 2′‐fluoro‐2′‐deoxyguanosine 5′‐monophosphate was observed in enzymatic assays with yeast carboxypeptidase Y or crude cell lysate. Evidence for efficient removal of the 6‐substituent on the guanine part was provided by enzymatic studies with adenosine deaminase, and by molecular modeling of the nucleoside 5′‐monophosphates in the catalytic site of a model of ADAL1, thus indicating the utility of the double prodrug concept.     

Biochemical Characterization of the Lysine Acetylation of Tyrosyl‐tRNA Synthetase in Escherichia coli

Aminoacyl‐tRNA synthetases (aaRSs) play essential roles in protein synthesis. As a member of the aaRS family, the tyrosyl‐tRNA synthetase (TyrRS) in Escherichia coli has been shown in proteomic studies to be acetylated at multiple lysine residues. However, these putative acetylation targets have not yet been biochemically characterized. In this study, we applied a genetic‐code‐expansion strategy to site‐specifically incorporate N^?‐acetyl‐l ‐lysine into selected positions of TyrRS for in vitro characterization. Enzyme assays demonstrated that acetylation at K85, K235, and K238 could impair the enzyme activity. In vitro deacetylation experiments showed that most acetylated lysine residues in TyrRS were sensitive to the E. coli deacetylase CobB but not YcgC. In vitro acetylation assays indicated that 25 members of the Gcn5‐related N‐acetyltransferase family in E. coli, including YfiQ, could not acetylate TyrRS efficiently, whereas TyrRS could be acetylated chemically by acetyl‐CoA or acetyl‐phosphate (AcP) only. Our in vitro characterization experiments indicated that lysine acetylation could be a possible mechanism for modulating aaRS enzyme activities, thus affecting translation.     

An Isocytidine Derivative with a 2‐Amino‐6‐methylpyridine Unit for Selective Recognition of the CG Interrupting Site in an Antiparallel Triplex DNA

Sequence‐specific recognition of duplex DNA mediated by triple helix formation offers a potential basis for oligonucleotide therapy and biotechnology. However, triplex formation is limited mostly to homopurine strands, due to poor stabilization at CG or TA base pairs in the target duplex DNA sequences. Several non‐natural nucleosides have been designed for the recognition of CG or TA base pairs within an antiparallel triplex DNA. Nevertheless, problems including low selectivity and high dependence on the neighboring bases remain unsolved. We thus synthesized N²‐arylmethyl isodC derivatives and incorporated them into triplex‐forming oligonucleotides (TFOs) for the selective recognition of the CG base pair within antiparallel triplex DNA. It was shown that an isodC derivative bearing a 2‐amino‐6‐methylpyridine moiety (AP‐isodC) recognizes the CG base pair with high selectivity in antiparallel triplex DNA irrespective of the flanking base pairs.     

Click‐Chemistry‐Mediated Rapid Microbubble Capture for Acute Thrombus Ultrasound Molecular Imaging

Bioorthogonal coupling chemistry has been studied as a potentially advantageous approach for molecular imaging because it offers rapid, efficient, and strong binding, which might also benefit stability, production, and chemical conjugation. The inverse‐electron‐demand Diels–Alder reaction between a 1,2,4,5‐tetrazine and trans‐cyclooctene (TCO) is an example of a highly selective and rapid bioorthogonal coupling reaction that has been used successfully to prepare targeted molecular imaging probes. Here we report a fast, reliable, and highly sensitive approach, based on a two‐step pretargeting bioorthogonal approach, to achieving activated‐platelet‐specific CD62p‐targeted thrombus ultrasound molecular imaging. Tetrazine‐modified microbubbles (tetra‐MBs) could be uniquely and rapidly captured by subsequent click chemistry of thrombus tagged with a trans‐cyclooctene‐pretreated CD62p antibody. Moreover, such tetra‐MBs showed great long‐term stability under physiological conditions, thus offering the ability to monitor thrombus changes in real time. We demonstrated for the first time that a bioorthogonal targeting molecular ultrasound imaging strategy based on tetra‐MBs could be a simple but powerful tool for rapid diagnosis of acute thrombosis.     

Frequency distributions of breaking load,tenacity, and ratio of cell wall thickness to ribbon width for single cotton fibers

Analysis of the frequency distributions of breaking load, tenacity, and ratio of cell wall thickness to ribbon width (C/R) for single cotton fibers in this study has indicated that they can all be represented by β-distributions. This suggests that a parallel model of element configurations exists in which there is a uniform distribution of elemental strengths in single cotton fibers before as well as after slack mercerization. In the case of breaking load and tenacity, the distributions are positively skewed and the skewness decreases on slack mercerization, suggesting that quite a few potentially weak places have been either completely removed or at least strengthened. The changes in C/R ratio on slack mercerization for cottons having a range of maturity have been examined and discussed. The dependence of properties such as breaking load, tenacity, and linear density on cell wall to ribbon width ratio (C/R) have also been studied.     

Difference in hydroxamic acid content in roots and root exudates of wheat (Triticum aestivum L.) and rye (Secale cereale L.): Possible role in allelopathy

Hydroxamic acids (Hx) produced by some cereal crops have been associated with allelopathy. However, the release of Hx to the soil by the producing plant-an essential condition for a compound to be involved in allelopathy-has not been shown. GC and HPLC analysis of roots and root exudates of wheat (Triticum aestivum L.) and rye (Secale cereale L.) cultivars, with high Hx levels in their leaves, demonstrated the presence of these compounds in the roots of all cultivars analyzed and in root exudates of rye. Moreover, bioassays employing root exudates collected from wheat and rye seedlings demonstrated that only rye exudates inhibited root growth of wild oats,Avena fatua L., a weed whose root growth is inhibited by Hx. These results suggest that rye could potentially interfere with the growth ofAvena fatua in nature and that this interference could be due to the release of Hx to the soil by way of roots.     

The Use of Copper Immobilized on Silica for Compound Class Fractionation of Polycyclic Aromatic Compounds in Environmental Samples

Abstract

Copper immobilized on silica has been investigated as an adsorbent for the fractionation of polycyclic aromatic compounds (PACs) into different compound classes. an open column chromatographic packing based on copper (II) bound to a silica substrate was used as a means of subdividing the PACs into less complex groups of similar compound types. the process has been applied to the fractionation of PACs extracted from a marine harbour sediment, for which the complex mixture of PACs could be separated into three subgroups, the contents of which were: (1) polycyclic aromatic hydrocarbons (PAHs), alkylated PAHs, and thiophenes; (2) compounds containing pyrrole-type (sp ³-hybridized) nitrogen (carbazoles), and also quinones and other oxygen containing species; and (3) compounds containing pyridine-type(sp ²-hybridized) nitrogen (acridine and its homologues). the fractions thus obtained have been characterized by gas chromatography-mass spectrometry (GC-MS). Extracted mass spectra, obtained from the crests of GC-MS peaks observed in reconstructed single ion chromatograms of selected m/z values, enabled unambiguous identification of many of the minor components of the mixture.     

Synthesis,isolation, and GC analysis of all the 6,8-to 13,15-<Emphasis Type="Italic">cis/trans</Emphasis> conjugated linoleic acid isomers

Octadecadienoic acids with conjugated double bonds are often referred to as conjugated linoleic acid, or CLA. CLA is of considerable interest because of potentially beneficial effects reported from animal studies. Analysis of CLA is usually carried out by GC elution of FAME. If the presence of low-level isomers is of interest, a complementary technique such as silverion HPLC is also used. These analyses have been hindered by a lack of well-characterized commercially available reference materials. Described here are the synthesis and isolation of selected 6,8-through 13,15-positional CLA isomers, followed by isomerization of these CLA isomers with iodine to produce all the possible cis,cis,cis,trans,trans,cis, and trans,trans combinations. Also present are the GC retention times of the CLA FAME relative to γ-linolenic acid (6c,9c,12c-octadecatrienoic acid) FAME using a 100-m CP Sil-88 capillary column (Varian Inc., Lake Forest, CA). These data include all the CLA isomers that have been identified thus far in foods and dietary supplements and should greatly aid in the future analysis of CLA in these products.     

The Peculiar Case of the Hyper-thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus**

The poor solubility of many nucleosides and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes that can withstand these conditions. Herein, we report that the pyrimidine nucleoside phosphorylase from Thermus thermophilus is active over an exceptionally broad pH (4–10), temperature (up to 100 °C) and cosolvent space (up to 80 % (v/v) nonaqueous medium), and displays tremendous stability under harsh reaction conditions with predicted total turnover numbers of more than 10⁶ for various pyrimidine nucleosides. However, its use as a biocatalyst for preparative applications is critically limited due to its inhibition by nucleobases at low concentrations, which is unprecedented among nonspecific pyrimidine nucleoside phosphorylases.