Preparation,structure, and properties of periodate-oxidised ATP,a potential affinity-labelling reagent

(1) Periodate oxidation of ATP yields a single product which has been purified and characterised. Periodate-oxidised ATP (o-ATP) behaves as a single compound during TLC analysis, but NMR spectral studies show that it exists in aqueous solution as an equilibrium mixture of three dialdehyde monohydrates and a dihydrate. Little free aldehyde is present. The dialdehyde monohydrates are in the form of diastereomeric cyclic hemiacetals. (2) The dialdehyde grouping of o-ATP can be reduced with sodium borohydride, producing a dialcohol. (3) o-ATP has been frequently used in attempts to affinity label nucleotide-binding sites on proteins. The proposed structure of o-ATP is discussed in relation to this use for o-ATP.     

Periodate Oxidation of Some Carbohydrates as Examined by NMR Spectroscopy

Periodate oxidation of some sugar alcohols, methyl glycosides and a synthetic glucan in an amount of 5 ~ 20 mg was performed in ca. 0.2 ~ 0.4 ml of D₂O involving NaIO₄ (1.5 ~ 2.0 moles excess) in a NMR sample tube, and the reaction products were examined in the course of oxidation by NMR spectroscopy.

In addition to proton signals of formyl and formaldehyde (in acetal), proton signals at hemiacetal carbons were identified in the periodate oxidation. Splitting and change in O-methyl and N-acetate-methyl signals indicated presence of more than one structures for each of the reaction products in the periodate oxidations of methyl α-d-glucopyranoside and methyl N-acetyl-α-d-glucosaminide. A condensation product was detected in the periodate oxidation of glycolaldehyde, d,l-glyceraldehyde and d-galactitol. A synthetic glucan was found to have a structure of 1,6-linkage in a DP = 15?17.     

Morpholino spin-labeling for base-pair sequencing of a 3'-terminal RNA stem by proton homonuclear Overhauser enhancements: yeast ribosomal 5S RNA

Base-pair sequences for 5S and 5.8S RNAs are not readily extracted from proton homonuclear nuclear Overhauser enhancement (NOE) connectivity experiments alone, due to extensive peak overlap in the downfield (11-15 ppm) proton NMR spectrum. In this paper, we introduce a new method for base-pair proton peak assignment for ribosomal RNAs, based upon the distance-dependent broadening of the resonances of base-pair protons spatially proximal to a paramagnetic group. Introduction of a nitroxide spin-label covalently attached to the 3'-terminal ribose provides an unequivocal starting point for base-pair hydrogen-bond proton NMR assignment. Subsequent NOE connectivities then establish the base-pair sequence for the terminal stem of a 5S RNA. Periodate oxidation of yeast 5S RNA, followed by reaction with 4-amino-2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO-NH2) and sodium borohydride reduction, produces yeast 5S RNA specifically labeled with a paramagnetic nitroxide group at the 3'-terminal ribose. Comparison of the 500-MHz 1H NMR spectra of native and 3'-terminal spin-labeled yeast 5S RNA serves to identify the terminal base pair (G1 . C120) and its adjacent base pair (G2 . U119) on the basis of their proximity to the 3'-terminal spin-label. From that starting point, we have then identified (G . C, A . U, or G . U) and sequenced eight of the nine base pairs in the terminal helix via primary and secondary NOE's.     

Improved synthesis and purification of methylated amanitins using diazomethane

We have examined the conditions of methylating alpha-amanitin with diazomethane with the intent of producing 6'-O-methyl-alpha-amanitin (meAMA). Under the appropriate conditions meAMA was afforded as the sole product in nearly quantitative yield. By exceeding the stoichiometries designed for optimal meAMA synthesis, a dimethylated amanitin, 1'-N-, 6'-O-dimethyl-alpha-amanitin (dimeAMA), was also produced. Both products were characterized, following HPLC, by ultraviolet and n.m.r. spectroscopy. Based upon their inhibitory potential against wheat germ RNA polymerase II, apparent dissociation constants of 4.3 nM and 5.4 nM were estimated for meAMA and dimeAMA, respectively.     

Reversal of murine alveolar macrophage-mediated suppression of plaque-forming cell response by sodium periodate

Murine alveolar macrophages (AM) have been shown to suppress the in vitro plaque-forming cell (PFC) response of spleen cells previously primed with sheep erythrocytes (SRBC) in a dose-dependent manner. Mild oxidation of cell membranes on viable AM with sodium periodate resulted in total abrogation of AM-mediated suppression of the PFC response, while periodate treatment of spleen cells resulted only in partial reduction of the suppression. Pretreatment of AM with sodium periodate followed by addition of the aldehyde blocking agent, hydroxylamine, resulted in restoration of the PFC-suppressing activity of AM. Periodate treatment of AM also resulted in significantly increased macrophage-T-cell binding and cluster formation. These observations suggest that the generation of aldehyde moieties on AM membrane sialoglycoconjugates promotes positive macrophage-lymphocyte interactions, resulting in abrogation of AM-mediated suppression of the PFC response.     

A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins.

A new approach for removing O-glycosidically linked carbohydrate side chains from glycoproteins is described. Periodate oxidation of the C3 and C4 carbons in peptide-linked N-acetylgalactosamine (GalNAc) residues generates a dialdehyde product which, under mild alkaline conditions, undergoes a beta-elimination which releases carbohydrate and leaves an intact peptide core. The pH and time dependence, and intermediates of the elimination, have been extensively followed by carbon-13 NMR spectroscopy and amino acid analysis using ovine submaxillary mucin (OSM) as the substrate. The deglycosylation of OSM is complete and provides apomucin in high yield with an amino acid composition identical to the starting material. Carboxymethylated OSM when deglycosylated by this method gives an apomucin with an apparent molecular weight of ca. 700 x 10(3). The molecular weight is the same as that calculated for the peptide core of the starting mucin, demonstrating the absence of peptide core cleavage. This contrasts with the use of trifluoromethanesulfonic acid (TFMSA), which generates apomucin products of lower molecular weights. Oligosaccharide side chains substituted at C3 of the peptide-linked GalNAc residue are resistant to the oxidation and elimination. Glycoproteins containing these more complex side chains can be deglycosylated by pretreatment with TFMSA under mild (0 degree C) conditions, which removes peripheral sugars (while leaving the peptide-linked GalNAc residue intact), followed by oxidation and beta-elimination. Studies on the deglycosylation of porcine submaxillary mucin and human tracheobronchial mucin indicate that this approach provides more efficient removal of carbohydrate and less peptide core degradation than a more vigorous (25 degrees C) treatment with TFMSA alone. 13C NMR spectroscopic studies and carbohydrate analysis of the deglycosylation intermediates of the human mucin indicate that certain sialic acid containing and N-acetylglucosamine-containing oligosaccharides have elevated resistance to TFMSA treatment at 0 degrees C. By the use of neuraminidase, repeated mild TFMSA treatments, and multiple oxidations and beta-eliminations, the human mucin can be nearly completely deglycosylated. It is expected that all mucins and most glycoproteins containing O-glycosidic linkages can be readily and nearly completely deglycosylated using this combined approach.     

A one-step method of 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid synthesis by soybean lipoxygenase

A product of lipoxygenase (LOX) oxidation of docosahexaenoic acid (DHA), 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diH(P)DHA] was obtained through various reaction pathways that involved DHA, 17(S)-hydro(pero)xydocosahexa-4Z,7Z,11Z,13Z,15E,19Z-enoic acid [17(S)-H(P)DHA], soybean lipoxygenase (sLOX), and potato tuber lipoxygenase (ptLOX) in various combinations. The structure of the product was confirmed by HPLC, ultraviolet (UV) light spectrometry, GC-MS, tandem MS, and NMR spectroscopy. It has been found that 10,17(S)-diH(P)DHA formed by sLOX through direct oxidation of either DHA or 17(S)-H(P)DHA was apparently identical to the product of ptLOX oxidation of the latter. The sLOX- and ptLOX-derived samples of 10,17-diHDHAs coeluted under the conditions of normal, reverse, and chiral phase HPLC analyses, displayed identical UV absorption spectra with maxima at 260, 270, and 280 nm, and had similar one-dimensional and two-dimensional proton NMR spectra. Analysis of their NMR spectra led to the conclusion that 10,17-diHDHA formed by sLOX had solely 11E,13Z,15E configuration of the conjugated triene fragment, which was identical to the previously published structure of its ptLOX-derived counterpart. Based on the cis,trans geometry of the reaction products, the conclusion is made that in the tested conditions sLOX catalyzed direct double dioxygenation of DHA. Compared with the previously described two-enzyme method that involved sLOX and ptLOX, the current simplified one-enzyme procedure uses only sLOX as the catalyst of both dioxygenation steps.     

Synthesis of betulonic aldehyde derivatives

Selective functionalization of betulonic aldehyde (the oxidation product of betulin) was studied with the aim of obtaining new physiologically active substances. We developed a method for the synthesis of azomethine derivatives at the C-28 aldehyde group and benzylidene derivatives at the 2-methylene group of the A ring. The structure of the synthesized products was proved by ¹H NMR.     

Oxidation of pyrimidine bases and their derivatives by sodium peroxodisulfate

Treatment of 1,3-dimethyluracil (1) with sodium peroxodisulfate in water at 80 degrees C under nitrogen gave 5-hydroxy-1,3-dimethyluracil (6) as a main product. Under similar conditions, oxidation of 5-fluoro-1,3-dimethyluracil (2) gave a 6,6'-dimeric compound (10) together with other products. Similar oxidation of 5-bromo-1,3-dimethyluracil (3) and 1,3,6-trimethyluracil (4) was also investigated. Furthermore, reaction of 1,3-dimethylthymine (5) and adenine in the presence of sodium peroxodisulfate gave coupling products (12) and (13).     

TEMPO-mediated oxidation of native cellulose. The effect of oxidation conditions on chemical and crystal structures of the water-insoluble fractions

Cellulose cotton linter was oxidized with sodium hypochlorite with catalytic amounts of sodium bromide and 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) under various conditions. After this TEMPO-mediated oxidation, water-insoluble fractions were collected and characterized in terms of carboxylate and aldehyde contents, crystallinities and crystal sizes, degrees of polymerization, morphology, and water retention values. Carboxylate and aldehyde groups were introduced into the water-insoluble fractions up to about 0.7 and 0.3 mmol/g, respectively, by the oxidation, where recovery of the water-insoluble fractions were generally higher than 80%. Crystallinities and crystal sizes of cellulose I were nearly unchanged during the oxidation, and thus, carboxylate and aldehyde groups were introduced selectively on crystal surfaces and in disordered regions of the water-insoluble fractions. Water retention values of cotton linter can be increased from 60% to about 280% through the introduction of hydrophilic carboxylate groups and morphological changes from fibrous forms to short fragments by the TEMPO-mediated oxidation.     

The identity of a cyanogen bromide fragment of bovine dentin collagen containing the site of an intermolecular cross-link.

A peptide fraction isolated from a cyanogen bromide digest of bovine dentin collagen had a molecular weight of 46000. Its size and amino acid composition indicated that it could not consist of peptides derived from the cleavage of a single alpha chain. On reduction with tritiated sodium borohydride, radioactivity was incorporated primarily into 5, 5'-dihydroxylysinonorleucine without degradation at the peptide backbone. Periodate cleavage of the reduced or nonreduced peptide fraction generated one fragment of molecular weight 28000 and one of 18000 completely accounting for the size of the parent peptide. On amino acid analysis the constituent single-chain peptides were determined to be alpha2CB4 and alpha1CB6. Both peptides isolated after periodate oxidation of the tritiated borohydride reduced cross-link peptide were found to contain (3H)hydroxynorvaline. These data show that some hydroxylysine of alpha2CB4, a helical region peptide, was present in aldehyde form and could act as the aldehyde donor icross-link, Schiff's base formation. The only cross-linkage of this alpha2CB4 acting as an aldehyde donor peptide to alpha1CB6 would be a helical region to helical region bond, perhaps accounting for the unusual stability and low solubility of dentin collagen.     

The specificity of lipoxygenase-catalyzed lipid peroxidation and the effects of radical-scavenging antioxidants

The oxidation of low density lipoprotein (LDL) by lipoxygenase has been implicated in the pathogenesis of atherosclerosis. It has been known that lipoxygenase-mediated lipid peroxidation proceeds in general via regio-, stereo- and enantio-specific mechanisms, but that it is sometimes accompanied by a share of random hydroperoxides as side reaction products. In this study we investigated the oxidation of various substrates (linoleic acid, methyl linoleate, phosphatidylcholine, isolated LDL, and human plasma) by the arachidonate 15-lipoxygenases from rabbit reticulocytes and soybeans aiming at elucidating the effects of substrate, lipoxygenase and reaction milieu on the contribution and mechanism of random oxidation and also the effect of antioxidant. The specific character of the rabbit 15-lipoxygenase reaction was confirmed under all conditions employed here. However, the specificity by soybean lipoxygenase was markedly dependent on the conditions. When phosphatidylcholine liposomes and LDL were oxygenated by soybean lipoxygenase, the product pattern was found to be exclusively regio-, stereo-, and enantio-random. When free linoleic acid was incorporated into PC liposomes and oxidized by soybean lipoxygenase, the free acid was specifically oxygenated, whereas esterified linoleate gave random oxidation products exclusively. Radical-scavenging antioxidants such as alpha-tocopherol, ascorbic acid and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol selectively inhibited the random oxidation but did not influence specific product formation. It is assumed that the random reaction products originate from free radical intermediates, which have escaped the active site of the enzyme and thus may be accessible to radical scavengers. These data indicate that the specificity of lipoxygenase-catalyzed lipid oxidation and the inhibitory effects of antioxidants depend on the physico-chemical state of the substrate and type of lipoxygenase and that they may change completely depending on the conditions.     

次氯酸与不饱和脂肪酸氧化机制和转化产物的理论研究

摘要 目的：揭示次氯酸与不饱和脂肪酸的氧化反应机制及转化产物。方法：运用Gaussian 16软件包,采用密度泛函方法M06-2X(D3),结合6-31+G(d)基组,在SMD液相水模型水平下进行计算。结果：次氯酸与单不饱和脂肪酸油酸的氧化反应是先形成氯鎓离子中间体,氯鎓离子再与水分子反应生成氯醇,第一步氯鎓离子的形成是控速步骤,其反应活化自由能~8 kcal/mol。环氧化合物和短链的醛是两种转化产物,前者由氯醇脱氯化氢而来,而后者由环氧化合物和氯醇通过系列与次氯酸根的反应而得到,生成它们的控速步骤的反应活化自由能分别为23 和24 kcal/mol。选取两个乙基为取代基的乙烯为油酸模型,其与次氯酸反应的活化自由能仅比油酸高1 kcal/mol。计算得到次氯酸与亚油酸、顺-9,反-11 亚油酸、梓树酸和花生四烯酸模型氧化反应生成氯醇的活化自由能分别是~10、13、16和14 kcal/mol。结论：氯鎓离子中间体机制是次氯酸与不饱和脂肪酸氧化反应的主要机制,反应的活化自由能通常低于15 kcal/mol,意味着此氧化反应动力学上容易发生。氧化产物氯醇能转化为环氧化合物和短链的醛,但活化自由能较高,约23和24 kcal/mol。选取距离双键3个碳以内的结构为不饱和脂肪酸模型,它能够很好地反映不饱和脂肪酸的反应活性。     

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.     

18-Substituted steroids. Part 15. 6 beta-Hydroxylation of aldosterone by liver

6 beta-Hydroxyaldosterone and 6 beta-hydroxy-17-isoaldosterone, characterized by high-field NMR studies, are among the major polar metabolites formed from aldosterone by incubation with rat liver slices or microsomal fraction. It is uncertain at present whether the 17-iso product results from an enzymatic or a chemical inversion of configuration. Periodate degradation of the 6 beta-hydroxyaldosterone gave 6 beta-hydroxyaldosterone gamma-lactone, identical with a synthetic sample.     

Enzymatic Improvement of Food Flavor IV. Oxidation of Aldehydes in Soybean Extracts by Aldehyde Oxidase

Aldehyde oxidase (aldehyde: oxygen oxidoreductase, EC 1.2.3.1) was partially purified from bovine liver. The enzyme irreversibly oxidized various aldehydes to the corresponding acids by using dissolved oxygen as an electron acceptor. Although the Km value for n-hexanal was low (6 µm), that for acetaldehyde was high (20 mm).

Medium-chain aldehydes such as hexanal and pentanal appear to be mainly responsible for green beany odor of soybean products. A great reduction in the beany odor was observed after the soybean extract was incubated with aldehyde oxidase under aerobic conditions. Dissolved oxygen was utilized as the electron acceptor throughout the enzyme-catalyzed oxidation of aldehydes and none of other cofactors were found to be required.

It has been shown that bovine liver mitochondrial aldehyde dehydrogenase oxidizes the soybean protein-bound aldehyde with a rate comparable to that for free n-hexanal (Agric. Biol. Chem., 43, in press). Comparative studies of aldehyde oxidase and aldehyde dehydrogenase with respect to oxidation-rates of free aldehydes and the soybean protein-bound aldehydes indicated that aldehyde oxidase acted on the bound aldehyde with a much slower rate.     

Galactosyl residue in exopolysaccharide from Rhizobium meliloti JJ-1 exposed to manganese is furanoid

Exopolysaccharide (EPS) elaborated by Rhizobium meliloti JJ-1 in a manganese supplemented medium was isolated. Periodate oxidation, reduction with sodium borohydride, followed by hydrolysis and subsequent capillary gas liquid chromatography of the derived alditol acetates revealed that D-galactose in this complex biopolymer is in furanoid form. This observation was further confirmed by ¹³carbon nuclear magnetic resonance (¹³C NMR).     

Oxidatively generated DNA damage after Cu(II) catalysis of dopamine and related catecholamine neurotransmitters and neurotoxins: Role of reactive oxygen species

There is increasing evidence supporting a causal role for oxidatively damaged DNA in neurodegeneration during the natural aging process and in neurodegenerative diseases such as Parkinson and Alzheimer. The presence of redox-active catecholamine neurotransmitters coupled with the localization of catalytic copper to DNA suggests a plausible role for these agents in the induction of oxidatively generated DNA damage. In this study we have investigated the role of Cu(II)-catalyzed oxidation of several catecholamine neurotransmitters and related neurotoxins in inducing oxidatively generated DNA damage. Autoxidation of all catechol neurotransmitters and related congeners tested resulted in the formation of nearly a dozen oxidation DNA products resulting in a decomposition pattern that was essentially identical for all agents tested. The presence of Cu(II), and to a lesser extent Fe(III), had no effect on the decomposition pattern but substantially enhanced the DNA product levels by up to 75-fold, with dopamine producing the highest levels of unidentified oxidation DNA products (383±46 adducts/10(6) nucleotides), nearly 3-fold greater than 8-oxo-7,8-dihydro-2'-deoxyguanosine (122±19 adducts/10(6) nucleotides) under the same conditions. The addition of sodium azide, 2,2,6,6-tetramethyl-4-piperidone, tiron, catalase, bathocuproine, or methional to the dopamine/Cu(II) reaction mixture resulted in a substantial decrease (>90%) in oxidation DNA product levels, indicating a role for singlet oxygen, superoxide, H(2)O(2), Cu(I), and Cu(I)OOH in their formation. Whereas the addition of N-tert-butyl-α-phenylnitrone significantly decreased (67%) dopamine-mediated oxidatively damaged DNA, three other hydroxyl radical scavengers, ascorbic acid, sodium benzoate, and mannitol, had little to no effect on these oxidation DNA product levels, suggesting that free hydroxyl radicals may have limited involvement in this dopamine/Cu(II)-mediated oxidatively generated DNA damage. These studies suggest a possible contributory role of oxidatively generated DNA damage by dopamine and related catechol neurotransmitters/neurotoxins in neurodegeneration and cell death. We also found that a naturally occurring broad-spectrum antioxidant, ellagic acid, was substantially effective (nearly 50% inhibition) at low doses (1μM) at preventing this dopamine/Cu(II)-mediated oxidatively generated DNA damage. Because dietary ellagic acid has been found to reduce oxidative stress in rat brains, a neuroprotective role of this polyphenol is plausible.     

Glycolipids and glycoproteins formed from UDP-galactose by pea membranes

Pea membranes were incubated with UDP-[¹⁴C]galactose and sequentially extracted with lipid solvents and 2% sodium dodecyl sulfate (SDS). At least three-quarters of the products were SDS-soluble. All fractions contained some [¹⁴C]glucose, indicating the presence of an active epimerase which, however, could be inhibited by ADP-ribose. The chloroform-methanol extract contained mainly neutral galactosyl lipids and a small amount of dolicyl monophosphoryl glucose. The chloroform-methanol-water extract contained trace amounts of lipid-linked galactosyl oligosaccharide with properties comparable to polyisoprenyl pyrophosphoryl derivatives. Polyacrylamide gel electrophoresis of SDS-soluble products indicated the formation of both immobile and mobile components with similar size distribution (Sepharose CL-6B). The mobile component only was susceptible to hydrolysis by protease. Periodate oxidation analysis of SDS-soluble and -insoluble products indicated that they were composed mainly of 1 → 6 galactosyl residues, i.e. as in many arabinogalactan proteins and arabinogalactans.