Characterization of the aldehyde reactive probe reaction with AP-sites in DNA: influence of AP-lyase on adduct stability

Alkoxyamines react with the open-chain aldehyde form of AP-sites in DNA to produce open-chain aldehyde oximes. Here we characterize the effect of AP-site cleavage by yeast AP-endonuclease 1 (APN1) or T4 pyrimidine dimer DNA glycosylase/AP-lyase (T4 Pdg) on the efficiency and stability of the alkoxyamine aldehyde reactive probe (ARP) condensation reaction with AP-sites. The results indicate that (1) reaction of ARP with the open-chain aldehyde equilibrium form of the AP-site was less efficient than with the 3'-alpha,beta-unsaturated aldehyde produced by T4 Pdg; (2) the dRP moiety was least reactive with ARP; (3) both the AP-site and 3'-alpha,beta-unsaturated aldehyde were stable with regard to reaction with ARP over a 30-min incubation period at 37 degrees C; and (4) ARP adducted to the open-chain aldehyde form of the AP-site could be replaced by methoxyamine, but the 3'-alpha,beta-unsaturated aldehyde ARP oxime was stable against methoxyamine attack.     

Stereocontrolled Facile Synthesis and Biological Evaluation of (3′S) and (3′R)-3′-Amino (and Azido)-3′-Deoxy Pyranonucleosides

This article describes the synthesis of (3 ′S) and (3 ′R)-3 ′-amino-3 ′-deoxy pyranonucleosides and their precursors (3 ′S) and (3 ′R)-3 ′-azido-3 ′-deoxy pyranonucleosides. Azidation of 1,2:5,6-di-O-isopropylidene-3-O-toluenesulfonyl-α-D-allofuranose followed by hydrolysis and subsequent acetylation afforded 3-azido-3-deoxy-1,2,4,6-tetra-O-acetyl-D-glucopyranose, which upon coupling with the proper silylated bases, deacetylation, and catalytic hydrogenation, obtained the target 3 ′-amino-3 ′-deoxy-β-D-glucopyranonucleosides. The desired 1-(3 ′-amino-3 ′-deoxy-β-D-allopyranosyl)5-fluorouracil was readily prepared from the suitable imidazylate sugar after azidation followed by a protection/deprotection sequence and reduction of the unprotected azido precursor. No antiviral activity was observed for the novel nucleosides. Moderate cytostatic activity was recorded for the 5-fluorouracil derivatives.     

Efficiency of repair of an abasic site within DNA clustered damage sites by mammalian cell nuclear extracts

Ionizing radiation induces clustered DNA damage sites which have been shown to challenge the repair mechanism(s) of the cell. Evidence demonstrating that base excision repair is compromised during the repair of an abasic (AP) site present within a clustered damage site is presented. Simple bistranded clustered damage sites, comprised of either an AP-site and 8-oxoG or two AP-sites, one or five bases 3' or 5' to each other, were synthesized in oligonucleotides, and repair was carried out in xrs5 nuclear extracts. The rate of repair of an AP-site when present opposite 8-oxoG is reduced by up to 2-fold relative to that when an AP-site is present as an isolated lesion. The mechanism of repair of the AP-site shows asymmetry, depending on its position relative to 8-oxoG on the opposite strand. The AP-site is rejoined by short-patch base excision repair when the lesions are 5' to each other, whereas when the lesions are 3' to one another, rejoining of the AP-site occurs by both long-patch and short-patch repair processes. The major stalling of repair occurs at the DNA ligase step. 8-OxoG and an AP-site present within a cluster are processed sequentially, limiting the formation of double-strand breaks to <4%. In contrast, when two AP-sites are contained within the clustered DNA damage site, both AP-sites are incised simultaneously, giving rise to double-strand breaks. This study provides new insight into understanding the processes that lead to the biological consequences of radiation-induced DNA damage and ultimately tumorigenesis.     

Oxidation of methyl α-d-galactopyranoside by galactose oxidase: products formed and optimization of reaction conditions for production of aldehyde

The reaction conditions of galactose oxidase-catalyzed, targeted C-6 oxidation of galactose derivatives were optimized for aldehyde production and to minimize the formation of secondary products. Galactose oxidase, produced in transgenic Pichia pastoris carrying the galactose oxidase gene from Fusarium spp., was used as catalyst, methyl α-d-galactopyranoside as substrate, and reaction medium, temperature, concentration, and combinations of galactose oxidase, catalase, and horseradish peroxidase were used as variables. The reactions were followed by ¹H NMR spectroscopy and the main products isolated, characterized, and identified. An optimal combination of all the three enzymes gave aldehyde (methyl α-d-galacto-hexodialdo-1,5-pyranoside) in approximately 90% yield with a substrate concentration of 70 mM in water at 4 °C using air as oxygen source. Oxygen flushing of the reaction mixture was not necessary. The aldehyde existed as a hydrate in water. The main secondary products, a uronic acid (methyl α-d-galactopyranosiduronic acid) and an α,β-unsaturated aldehyde (methyl 4-deoxy-α-d-threo-hex-4-enodialdo-1,5-pyranoside), were observed for the first time to form in parallel. Formation of uronic acid seemed to be the result of impurities in the galactose oxidase preparation. ¹H and ¹³C NMR data of the products are reported for the α,β-unsaturated aldehyde for the first time, and chemical shifts in DMSO-d₆ for all the products for the first time. Oxidation of d-raffinose (α-d-galactopyranosyl-(1-6)-α-d-glucopyranosyl-(1-2)-β-d-fructofuranoside) in the same optimum conditions also proceeded well, resulting in approximately 90% yield of the corresponding aldehyde.     

NADPH-dependent reductases involved in the detoxification of reactive carbonyls in plants

Reactive carbonyls, especially α,β-unsaturated carbonyls produced through lipid peroxidation, damage biomolecules such as proteins and nucleotides; elimination of these carbonyls is therefore essential for maintaining cellular homeostasis. In this study, we focused on an NADPH-dependent detoxification of reactive carbonyls in plants and explored the enzyme system involved in this detoxification process. Using acrolein (CH(2) = CHCHO) as a model α,β-unsaturated carbonyl, we purified a predominant NADPH-dependent acrolein-reducing enzyme from cucumber leaves, and we identified the enzyme as an alkenal/one oxidoreductase (AOR) catalyzing reduction of an α,β-unsaturated bond. Cloning of cDNA encoding AORs revealed that cucumber contains two distinct AORs, chloroplastic AOR and cytosolic AOR. Homologs of cucumber AORs were found among various plant species, including Arabidopsis, and we confirmed that a homolog of Arabidopsis (At1g23740) also had AOR activity. Phylogenetic analysis showed that these AORs belong to a novel class of AORs. They preferentially reduced α,β-unsaturated ketones rather than α,β-unsaturated aldehydes. Furthermore, we selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as α,β-unsaturated aldehydes in chloroplasts. These results suggest that different classes of NADPH-dependent reductases cooperatively contribute to the detoxification of reactive carbonyls.     

Synthesis and Anti-HIV Activity of Novel 4′-Trifluoromethylated 5′-Deoxycarbocyclic Nucleoside Phosphonic Acids

Efficient synthetic route to novel 4′-trifluoromethylated 5′-deoxycarbocyclic nucleoside phosphonic acids was described from α-trifluoromethyl-α,β-unsaturated ester. Coupling of purine nucleosidic bases with cyclopentanol using a Mitsunobu reaction gave the nucleoside intermediates which were further phosphonated and hydrolyzed to reach desired nucleoside analogs. Synthesized nucleoside analogs were tested for anti-HIV activity as well as cytotoxicity. Adenine analog 22 shows significant anti-HIV activity (EC₅₀ = 8.3 μM) up to 100 μM.     

Depolymerization of heparin with diazomethane. Structure of N,O-methylated,even-numbered oligosaccharides produced by β-eliminative cleavage of the 2-amino-2-deoxyglycosylic linkage

The long-period reaction of heparin with excess diazomethane at 20° resulted in cleavage at the β-position of the uronic acid carboxyl group to give a mixture of methyl α- and β-glycosides of N,O-methylated di-, tetra-, and hexa-saccharides having a 4,5-unsaturated uronic acid, nonreducing end-group. The major disaccharides obtained were methyl O-(4-deoxy-3-O-methyl-α-l-threo-hex-4-enopyranosyluronic acid 2-sulfate)-(1→4)-2-deoxy-3-O-methyl-2-(N-methylsulfoamino)-α- and -β-d-glucopyranoside. The reaction of heparin at 4° yielded a mixture of methylated, higher-molecular-weight oligosaccharides, which retained some affinity for antithrombin III-Sepharose.     

DABCO-based chiral ionic liquids as recoverable and reusable organocatalyst for asymmetric Diels–Alder reaction

New DABCO-based chiral ionic liquids were synthesized and evaluated in asymmetric Diels–Alder reaction of cyclopentadiene with α,β-unsaturated aldehydes or 4-phenyl-3-buten-2-one. Chiral ionic liquid of modified MacMillan catalyst having a DABCO cation and hexafluorophosphate anion acts as organocatalyst (5 mol%) for the Diels–Alder reaction of crotonaldehyde and cyclopentadiene producing 98% of the product and 87% ee (endo) in CH₃CN/H₂O (95/5) at 25°C in 2 h. The scope and limitations of the catalysis were also studied by using cyclopentadiene and α,β-unsaturated aldehydes, and the Diels–Alder products were obtained in 18%–92% yields with 68%–93% ee. The catalyst was recycled and reused up to 6 cycles with a slight drop in ee and conversion of the product.     

Biocatalytic Knoevenagel reaction using alkaline protease from Bacillus licheniformis

Abstract

BLAP (alkaline protease from Bacillus licheniformis) was used as a biocatalyst in the Knoevenagel condensations of aromatic, hetero-aromatic and α;β-unsaturated aldehydes with less reactive acetylacetone or ethyl acetoacetate. The reactions were performed in organic solvent in the presence of water. The functionalized trisubstituted alkenes and α,β,γ,δ-unsaturated carbonyl compounds could be obtained in moderate to good yields with E/Z selectivities up to >99:1. This biocatalytic reaction provided an alternative pathway for Knoevenagel condensation, which also demonstrates a novel case of unnatural activity of existing enzymes.     

An example of enzymatic promiscuity: the Baylis–Hillman reaction catalyzed by a biotin esterase (BioH) from Escherichia coli

Ten lipases and esterases have been examined to catalyse the reaction between p-nitrobenzaldehyde and methyl vinyl ketone, the Baylis–Hillman reaction, to form 3-[hydroxyl-(4-nitrophenyl)-methyl]-but-3-en-2-one. Among these enzymes, Escherichia coli BioH esterase had the best activity. Optimal conditions for this reaction were: 0.1 mmol aldehyde, 0.1 mmol activated alkene, 30 mg E. coli BioH, 1 ml acetonitrile at 30 °C for 96 h. In addition to the named substrates, four other aldehydes and three activated alkenes were also investigated to determine the substrate range of the enzyme. The structures of nine products were confirmed by NMR and yields of the corresponding products ranged from 21 to 46 %.     

Phenylpropanoids from Juglans mandshurica exhibit cytotoxicities on liver cancer cell lines through apoptosis induction

Three new phenylpropanoids (1–3) together with six known congeners (4–9) were isolated from the bark of Juglans mandshurica Maxim using anti-hepatoma activity as a guide. Their structures were determined by comprehensive NMR and HRESIMS spectroscopic data analyses. All the isolated compounds were evaluated for their growth inhibitory activities against two kinds of liver cancer cell lines (HepG2 and Hep3B). Among them, compound 4 showed moderate cytotoxic activities against HepG2 and Hep3B cell lines with IC₅₀ values of 58.58 and 69.87 μM. Compound 5 exhibited 50% cell death rate in HepG2 and Hep3B cell lines at 63.70 and 46.45 μM, respectively. Further observation of morphological changes and Western blot demonstrated that compounds 4 and 5 exhibited their cytotoxic activities through the induction of apoptosis. A structure-activity relationship study suggested that an α, β-unsaturated aldehyde might be the most important functional group.     

Selective oxidation of steroidal allylic alcohols

Pyridinium chlorochromate in CH₂Cl₂ containing pyridine (2%) at 2—3°C has been found to effect the high yield selective oxidation of the hydroxyl function of a number of steroidal allylic alcohols. Under these conditions the oxidation of cholest-4-cn-3β-ol to the corresponding ketone was effected in 92% yield. Only the allylic hydroxyl function of 5α-cholest-8(14)-ene-3β,15α-diol, 5α-cholest-8(14)ene-3β,15β-diol and 5α-cholest-8(14)-ene-3β,7β-diol was oxidized under these conditions to give the corresponding α,β-unsaturated ketones in high yields. 5α-Cholest-8(14)-ene-3β,7α,15α-triol gave 5α-cholest-8(14)-ene-3β,7α-diol-15-one in 82% yield. Attempted oxidations of the 5α-cholestan-3β,15α-diol and 5α-cholest-7-ene-3β,15α-diol, both lacking an allylic hydroxyl function, under these conditions, were unsuccessful. Selective oxidation of the allylic alcohol function of 5α-cholest-8(14)-ene-3β,15β-diol using activated manganese dioxide gave 5α-cholest-8(14)-en-3β-ol-15-one in high yield while oxidation of the corresponding 15α-hydroxy epimer using manganese dioxide was unsuccessful.     

Fungal Reduction of C6 α,β-Unsaturated Carboxylic Acids

The metabolism of sorbic acid (trans-2,trans-4-hexadienoic acid) and its related compounds by Mucor sp. A-73 was investigated. Sorbic acid was reduced by this fungus to trans-4-hexenol (more than 90% yield). In a series of hexamonoenoic acids, carboxyl groups and α,β-double bond were reduced, but β,γand γ,δ double bonds were hardly reduced. The reduction of cis-2-hexenoic acid was slower than that of the corresponding trans isomer. Sorbic alcohol, one of α,β-unsaturated alcohols, was converted well to trans-4-hexenol by the fungus. These results showed that this fungus could carry out two independent reductions: (i) carboxyl group→alcohol, (ii) α,β-unsaturated alcohol→αβ-saturated one. Furthermore, α,β-unsaturated alcohols were temporarily detected in the course of fungal reductions of some α,β-unsaturated acids. The fact suggested that the reduction of α,β-unsaturated acids to α,β-saturated alcohols was initiated by the reaction (i) and followed by (ii). The biological hydrogenation of α,β-unsaturated alcohols is a new reaction.     

A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair

A number of endogenous and exogenous agents, and cellular processes create abasic (AP) sites in DNA. If unrepaired, AP sites cause mutations, strand breaks and cell death. Aldehyde-reactive agent methoxyamine reacts with AP sites and blocks their repair. Another alkoxyamine, ARP, tags AP sites with a biotin and is used to quantify these sites. We have combined both these abilities into one alkoxyamine, AA3, which reacts with AP sites with a better pH profile and reactivity than ARP. Additionally, AA3 contains an alkyne functionality for bioorthogonal click chemistry that can be used to link a wide variety of biochemical tags to AP sites. We used click chemistry to tag AP sites with biotin and a fluorescent molecule without the use of proteins or enzymes. AA3 has a better reactivity profile than ARP and gives much higher product yields at physiological pH than ARP. It is simpler to use than ARP and its use results in lower background and greater sensitivity for AP site detection. We also show that AA3 inhibits the first enzyme in the repair of abasic sites, APE-1, to about the same extent as methoxyamine. Furthermore, AA3 enhances the ability of an alkylating agent, methylmethane sulfonate, to kill human cells and is more effective in such combination chemotherapy than methoxyamine.     

An expeditious synthesis of blood-group antigens,ABO histo-blood group type II antigens and xenoantigen oligosaccharides with amino type spacer−arms

Blood group oligosaccharides are one of the most clinically important antigen families and they may also act as secondary ligands for bacterial toxins from Escherichia coli and Vibrio cholerae. Herein we report the synthesis of spacered (sp = CH₂CH₂CH₂NH₂) glycosides of A antigen {α-D-GalNAc-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-}, B antigen{α-D-Gal-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-}, LewisX{α-D-Gal-(l→4)-[α-L-Fuc-(l→3)]-β-D-GlcNAc-}, A type-II {α-D-GalNAc-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-(1→4)-β-D-GlcNAc-}, B type-II {α-D-Gal-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-(1→4)-β-D-GlcNAc-}, H type-II{α-L-Fuc-(l→2)-β-D-Gal-(1→4)-β-D-GlcNAc-}, xenoantigen {α-D-Gal-(l→3)-β-D-Gal-(1→4)-[α-L-Fuc-(l→2)]-β-D-GlcNAc-} and Linear B Type II {α-D-Gal-(l→3)-β-D-Gal-(1→4)-β-D-GlcNAc-} useful for a range of biochemical investigations. This linker was chosen so as to facilitate the future conjugation of the antigens to proteins or other molecules. We also measured the affinities of some synthesized oligosaccharides against El Tor CTB strain from V. cholera.     

Reaction of apurinic/apyrimidinic sites with [C]methoxyamine: A method for the quantitative assay of AP sites in DNA

A simple and rapid method is described for the determination of AP (apurinic/apyrimidinic) sites in DNA. The method involves the reaction of [¹⁴C]methoxyamine with the aldehyde group present in the deoxyribose moiety after a base loss. Studies with alkylated-depurinated DNA and with uracil-containing polydeoxyribonucleotides depyrimidinated by uracil-DNA glycosylase show that methoxyamine reacts with both apurinic and apyrimidinic sites in a rapid and exhaustive way. Under standard conditions (30-min incubation with 5 mM methoxyamine at 37°C, pH 7.2) untreated DNA is almost unreactive and the [¹⁴C]methoxyamine incorporation in DNA is proportional to the number of AP sites. Since the methoxyamine reaction is free from any degradative effect on DNA, AP sites may be estimated from a simple determination of the acid-insoluble radioactivity.     

Stereoselectivity of raney nickel catalyst in hydrogenolysis of a steroidal α,β-unsaturated epoxide. Chemical synthesis of 3β-benzoyloxy-5α-cholest-8(14)-en-15α-ol

Hydrogenation of 3β-benzoyloxy-14α, 15α-epoxy-5α-cholest-7-ene in benzene over a Raney nickel catalyst gave 3β-benzoyloxy-5α-cholest-8(14)-en-15α-ol and 3β-benzoyloxy-5α-cholest-8(14)-ene in 39% and 46% yields, respectively. Hydrogenation of the same α,β-unsaturated epoxy steryl ester under the same conditions except with the inclusion of triethylamine (4%) gave 3β-benzoyloxy-5α-cholest-8(14)-en-15α-ol in 89% yield.     

Synthesis and Properties of ApA Analogues with Shortened Phosphonate Internucleotide Linkage

A complete series of the 2 ′–5 ′ and 3 ′–5 ′ regioisomeric types of r(ApA) and 2 ′-d(ApA) analogues with the α-hydroxy-phosphonate C3 ′-O-P-CH(OH)-C4 ″ internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5 ′-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5 ′-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3 ′(2 ′)-O-P-CH₂-C4 ″ linkage lacking the 5 ′-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5 ′-carbon atom configuration. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed.     

Synthesis of Novel 4′α-Trifluoromethyl-2′β-C-Methyl-Carbodine Analogs as Anti-Hepatitis C Virus Agents

Novel 4?′α-trifluoromethyl-2?′β-methyl carbocyclic nucleoside analogs have been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell cultures. Construction of cyclopentene intermediate 10a was achieved via sequential Johnson–Claisen orthoester rearrangement and ring-closing metathesis starting from the α-trifluoromethyl-α,β-unsaturated ester 5. Stereoselective dihydroxylation and desilylation yielded the target carbodine analogs. The synthesized nucleoside analogs mentioned above (18 and 19) were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line (LucNeo#2). However, the synthesized nucleosides showed neither significant antiviral activity nor toxicity up to 50 μM.     

A simple and efficient synthesis of puromycin, 2,2′-anhydro-pyrimidine nucleosides,cytidines and 2′,3′-anhydroadenosine from 3′,5′-O-sulfinyl Xylo-nucleosides

Synthesis of antibiotics, puromycin and 3 ′-amino-3 ′-deoxy-N ⁶,N ⁶-dimethyladenosine 11 was achieved by utilizing the cyclic sulfite 6a of the xylo-3 ′,5 ′-dihydroxy group as a new protective group. The key synthetic step is the deprotection of the sulfite moiety through the intramolecular cyclization of 2-α-carbamate 7. In a similar manner, 2,2 ′-anhydro-pyrimidine nucleosides 15, ribo-cytidines 17 and 2 ′,3 ′-anhydroadenosine 14 were prepared in high yields from the corresponding sulfites 4, 5, and 6b, respectively.