Adenosine 5'-diphosphate dialdehyde: an affinity labeling reagent for phenol-sulfotransferase

Adenosine 5′-diphosphate (5′-ADP) was oxidized with periodic acid to 2′-O-[(R)-formyl(adenin-9-yl)methyl]-3′-diphosphate-3′-deoxy-(S)-glyceraldehyde (ADP-dialdehyde). ADP-dialdehyde, but not 2′, 3′-acyclic ADP, inhibited phenol-sulfotransferase (PST). The inhibition of PST by ADP dialdehyde was irreversible. A kinetic analysis of the enzyme inactivation suggests the formation of a dissociable enzyme-inhibitor complex prior to the inactivation step. PST could be completely protected from inactivation by the inclusion of 3′-phosphoadenosine-5′-phosphosulfate in the preincubation mixture. These results are consistent with ADP-dialdehyde being an affinity labeling reagent for PST.     

Roles of β-d-ribofuranose ring and the functional groups of the d-ribose moiety of adenosylcobalamin in the diol dehydratase reaction

Four analogs of adenosylcobalamin (AdoCbl) modified in the d-ribose moiety of the Coβ ligand were synthesized, and their coenzyme properties were studied with diol dehydratase of Klebsiella pneumoniae ATCC 8724. 2′-Deoxyadenosylcobalamin (2′-dAdoCbl) and 3′-deoxyadenosylcobalamin (3′-dAdoCbl) were active as coenzyme. 2′,3′-Secoadenosylcobalamin (2′,3′-secoAdoCbl), an analog bearing the same functional groups as AdoCbl but nicked between the 2′ and 3′ in the ribose moiety, and its 2′,3′-dialdehyde derivative (2′,3′-secoAdoCbl dialdehyde) were totally inactive analogs of the coenzyme. It is therefore evident that the β-d-ribofuranose ring itself, possibly its rigid structure, is essential and much more important than the functional groups of the ribose moiety for coenzyme function (relative importance; β-d-ribofuranose ring ⪢ 3′-OH ⪢ 2′-OH ⪢ ether group). With 2′-dAloCbl and 3′-dAdoCbl as enzymes. an absorption peak at 478 nm appeared during enzymatic reaction, suggesting homolysis of the CCo bound to form cob(II)alamin as intermediate. In the absence of substrate, the complexes of the enzyme with these active analogs underwent rapid inactivation by oxygen. This suggests that their CCo bond is activated even in the absence of substrate by binding to the apoprotein. No significant spectral changes were observed with 2′,3′-secoAdoCbl upon binding to the apoenzyme. In contrast, spectroscopic observation indicates that 2′,3′-secoAdoCbl dialdehyde, another inactive analog, underwent gradual and irreversible cleavage of the CCo bond by interaction with the apodiol dehydratase, forming the enzyme-bound cob(II)alamin without intermediates.     

Neolignans from mezilaurus itauba

The wood bark of Mezilaurus itauba afforded in addition to seven known neolignans, three new compounds rel-(7R,8R,1′S,3′S)-Δ^5′,8′-5′-methoxy-3,4-methylenedioxy-1′,2′,3′,4′-tetrahydro-2′,4′-dioxo-7.3′,8.1′-neolignan, rel-(7S,8S,1′S, 2′S, 3′R, 4′S)-Δ^8′-2′,4′-dihydroxy-3,4-methylenedioxy-1′,2′,3′,4′,5′,6′-hexahydro-5′-oxo-7.3′,8.1′-neolignan and rel-(7S,8S)-Δ^8′-6′-hydroxy 5′-methoxy-3,4-methylenedioxy-7·O·2′,8.3′-neolignan. The latter compound has been detected previously in Aniba terminalis. The structures were elucidated by spectroscopic methods and comparison with related compounds.     

Eusiderins and other neolignans from an Aniba species

A previous report disclosed the presence of benzodioxan and bicyclo[3.2.1]octanoid neolignans in the benzene extract of the trunk wood of an Amazonian Aniba (Lauraceae) species. The chloroform extract of the same material contains additionally two new benzodioxan neolignans [rel-(7S,8R)-Δ^8′-7-hydroxy-3,4,5,5′-tetramethoxy-7.0.3′,8.0.4′-neolignan; rel-(7R,8R)-Δ^7′-3,4,5,5′-tetramethoxy-9′-oxo-7.0.3′,8.0.4′-neolignan], two new bicyclo[3.2.1]-octanoid neolignans [(7R,8S,1′S,2′S,3′S,4′R)-Δ^8′-2′,4′-dihydroxy-3,3′-dimethoxy-4,5-methylenedioxy-1′,2′,3′,4′,5′,6′-hexahydro-5′-oxo-7.3′,8.1′-neolignan; (7R,8S,1′R,2′S,3′S)-Δ^8′-2′-hydroxy-3,3′,5′-trimethoxy-4,5-methylenedioxy-1′,2′,3′,4′-tetrahydro-4′-oxo-7.3′,8.1′-neolignan] and a hydrobenzofuranoid neolignan [(7S,8R,1′S,5′S)-Δ^8′-3,3′,5′-tri-methoxy-4,5-methylenedioxy-1′,4′,5′,6′-tetrahydro-4′-oxo-7.0.2′,8.1-neolignan].     

Nucleosides. 124. 3-Amino-3-deoxyhexopyranosyl Nucleosides. 7. 1-(3-Deoxy-3-Nitro-β-D-glucopyranosyl) Uracil and Its Galactopyranosyl Isomer1

Abstract

Crystalline 1-(3-deoxy-3-nitro-β-D-glucopyranosyl) uracil (3), originally prepared by nitromethane condensation of “uridine dialdehyde,” was found to contain the galactosyl isomer (4). Each isomer was obtained in pure form by 4′,6′-O-benzylidenation of the mixture of 3 and 4, followed by chromatographic separation and subsequent O-debenzylidenation. The structure of each isomer was established by chemical conversion of the isomer into the corresponding known 3′-acetamido-2′,4′,6′-tri-O-acetyl derivative.     

Benzofuranoid Neolignans from Piper kadsura

In a continuing research for neolignans from Piper kadsura (Choisy) Ohwi, six benzofuranoid neolignans were isolated from the aerial part of the plant. Their structure determination were based on the spectroscopic analysis (UV, IR, MS, NMR and CD) and derivative synthesis. Three of the isolated compounds were identified as new structures: 7R, 8R, 1′S-△8′-3, 4-methylenedioxy-5′-methoxy-l′, 4′-dihydro-4′-oxo-7, 0, 2′, 8. l′-neolignan ( Ⅰ ), 7 R, 8 R, 1 ′ R- △8′ - 3,4- methylenedioxy- 1 ′- methoxy - 1′,6′- dihydro- 6′- oxo- 7.0.4′,8. 3′-neolignan (Ⅳ) and 7R, 8R, 1′S-△8′-3, 4-methylenedioxy-l′-methoxy-1′,6′-dihydro-6′-oxo-7.0.4′,8.3′-neolignan (Ⅴ). Known compounds among them are 7R, 8S,1′S-△8′-3, 4-methylenedioxy-5′-methoxy-1′, 4′-dihydro-4′-oxo-7. 0. 2′, 8. 1′-neolignan(Ⅱ), 7S, 8S, 1′R-△8′-3, 4, 5′-trimethoxy-1′, 4′-dihydro-4′-oxo-7.0. 2′, 8. 1′-neolignan (Ⅲ) and 75, 85, 1′S-△8′-3, 4, l′-trimethoxy-l′, 6′-dihydro-6′-oxo-7. 0. 4′, 8. 3′-neolignans (Ⅵ). All of them were isolated from the plant for the first time.     

An active site-directed, irreversible inactivation of yeast hexokinase by (R,S)2,3-epoxypropyl beta-D-glucopyranoside

(1) Only (R,S)2′,3′-epoxypropyl β-d-glucopyranoside of the complete series of mono (R,S)2′.3′-epoxypropyl ethers and glycosides of d-glucopyranose significantly inactivated yeast hexokinase.(2) (R,S)2′,3′-Epoxypropyl β-d-glucopyranoside inactivates yeast hexokinase in the absence of MgATP^2?, The rate of inactivation is unaffected by MgATP^2?.(3) The rate of inactivation of hexokinase with (R,S)2′,3′-epoxypropyl β-d-ilucopyranoside was much greater when hexokinase was present in a monomeric form than when it was present in a dimeric form.(4) (R,S)2′,3′-Epoxypropyl β-d-glucopyranoside has a high K_t (0.38 M) and at a saturating concentrarion, the first order rate constant for the inactivation of monomeric hexokinase is 8.3 · 10^?4 sec.(5) d-Glucose protects against this inactivation and this was used to derive a dissocistion constant of 0.21 mM for d-glucose in the absence of MgATP^2?.(6) The alkylation of yeast hexokinase by (R,S)2′,3′-epoxypropyl β-d-gluco-pyranoside was not specific to the active site. When the concentration of (R,S)2′,3′-epoxypropyl β-d-glucopyranoside was 50 mM two thiol groups outside the active site were also alkylated.(7) The reaction between 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and yeast hexokinase was examined in detail. Two thiol groups per monomer (mol. wt. 50000) reacted with a second order rate constant of 27 1 mole^?1 sec^?1. A third thiol group reacted more slowly with a second-order rate constant of 1.6 1 mole^?1 sec^?1 and a fourth thiol group reacted very slowly with inactivation of the enzyme. Tue second-order rate constant in this case was 0.1 1 mole^?1 sec^?1.     

Inactivation of phosphofructokinase by dialdehyde-ATP.

Rabbit muscle phosphofructokinase (PFK) is rapidly inactivated by a 2′,3′-dialdehyde derivative of adenosine triphosphate (dialdehyde-ATP). When allowed to react with 0.6 mm dialdehyde-ATP in 0.1 m borate buffer (pH 8.6) containing 0.2 mm EDTA and 0.5 mm dithiothreitol, PFK loses essentially all activity (99%) in 30 min. The modified PFK remains inactive following dialysis of the reaction mixture against sodium borate (pH 8.0) containing fructose diphosphate, EDTA, and dithiothreitol. Experiments with [¹⁴C]dialdehyde-ATP show that 99% inactivation of PFK corresponds to incorporation of 3 to 4 mol of the ATP analog per PFK protomer. The inactivation of PFK with dialdehyde reagent is not caused by dissociation of the 340,000 M_r, tetramer to the 170,000 M_r dimer, as determined by analytical ultracentrifugation. Adenosine diphosphate or ATP protect PFK from inactivation by dialdehyde-ATP at pH 8.6, but fructose 6-phosphate, cyclic 3′,5t$\text{-}\text{?}$adenosine monophosphate, or fructose diphosphate, which protect PFK from modification by pyridoxal phosphate, provide little protection from inactivation. Amino acid analyses of dialdehyde-inactivated PFK and of a control sample of the enzyme were compared following reaction of each with 2,4-dinitrofluorobenzene. The results show that three or four lysine residues per PFK protomer are modified by dialdehyde-ATP. Additional data indicate that these lysine residues react with dialdehyde-ATP to form dihydroxymorpholine-like adducts rather than Schiff bases.     

PRODUCT INHIBITION OF RAT BRAIN HISTAMINE-N-METHYLTRANSFERASE

Abstract— The inhibition of S -adenosylmenthionine: histamine- N -methyltransferase (EC 2.1.1.8; HMT) by its products, 3-methylhistamine (3-MetHm) and S -adenosyl- l -homocysteine (SAH), was examined using a preparation of the enzyme which was partially purified from rat brains. SAH was found in in vitro experiments, to be a competative inhibitor of HMT in relation to S -adenosyl- l -methionine (SAM), with a K _i= 5.6 μM. SAH was shown to be a non-competitive inhibitor with respect to histamine (Hm) ( K _i= 5.0 μM). The K _m's for SAM and Hm were 10.2 and 3.0 μM respectively. On the other hand, 3-MetHm was determined to be a non-competitive inhibitor of HMT with respect to Hm ( K _i= 8.7 μM) and an uncompetitive inhibitor with respect to SAM ( K _i= 9.6 μM). These results suggest that the addition of the substrate to, and the release of products by, HMT occurs sequentially. In the nomenclature Of C leland (1963) the reaction is seemingly of the 'ordered Bi-Bi' type.     

Nucleosides VIII: 1 Synthesis of 2′, 3′-Dideoxy- and 2′, 3′-Didehydro-2′, 3′-Di Deoxyisoguanosine as Potential Antiretroviral Agents

Abstract

2′, 3′-Didehydro-2′, 3′-dideoxyisoguanosine (2) and 2′, 3′- dideoxyisoguanosine (3) have been synthesized by utilizing the Corey-Winter approach starting from isoguanosine. The 6-amino and 5′-hydroxy biprotected isoguanosine derivative was converted to the corresponding 2′, 3′- thionocarbonate, which was heated with triethyl phosphite to afford the 2′,3′- olefinic product. Either a tert-butyldimethylsilyl or a 4, 4′-dimethoxytrityl group was used in the protection of 5′-hydroxy function. Compounds 2 and 3 were found inactive against human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), and herpes simplex virus type 1 (HSV-1).

     

Inhibition of the activity of DNA polymerase alpha by 2',3'-dideoxythymidine 5'-triphosphate.

2′,3′-Dideoxythymidine 5′-triphosphate was found to strongly inhibit the activity of DNA polymerase α from mouse myeloma in the presence of manganese ion as divalent cation. The extent of inhibition by 2′,3′-dideoxythymidine 5′-triphosphate increased by raising pH of the reaction. The mode of inhibition by 2′,3′-dideoxythymidine 5′-triphosphate was competitive to the substrate, 2′-deoxythymidine 5′-triphosphate. Ki of the DNA polymerase α for 2′,3′-dideoxythymidine 5′-triphosphate (0.035 μM) was much lower than Km for 2′-deoxythymidine 5′-triphosphate (1.8 μM).     

Chemical Conversion of Uridine to 3′-Branched Sugar Nucleosides (Nucleosides and Nucleotides. 421.)

Abstract

Deamination of 1-(3-amino-3-deoxy-β-D-glucopyranosyl)-uracil gave a ring contracted nucleoside, 3′-deoxy-3′-formyluridine as a hemiacetal form, and uracil. Similar treatment of the 2′-deoxyderivative, 1-(3-amino-2,3-dideoxy-β-D-glucopyranosyl)uracil, gave the corresponding 2′,3′-dideoxy-3′-formyluridine in high yield. The 3′-epimerization of the 3′-formyluridine derivative was achieved and after reduction of the formyl groups, 2′,3′-dideoxy-3′(R and S)-hydroxymethyluridine were obtained.     

Identification of cytidine 2′,3′-cyclic monophosphate and uridine 2′,3′-cyclic monophosphate in Pseudomonas fluorescens pfo-1 culture

Cytidine 2′,3′-cyclic monophosphate (2′,3′-cCMP) and uridine 2′,3′-cyclic monophosphate (2′,3′-cUMP) were isolated from Pseudomonas fluorescens pfo-1 cell extracts by semi-preparative reverse phase HPLC. The structures of the two compounds were confirmed by NMR and mass spectroscopy against commercially available authentic samples. Concentrations of both intracellular and extracellular 2′,3′-cCMP and 2′,3′-cUMP were determined. Addition of 2′,3′-cCMP and 2′,3′-cUMP to P. fluorescens pfo-1 culture did not significantly affect the level of biofilm formation in static liquid cultures.     

Carotenoids of Anacystis nidulans,structures of caloxanthin and nostoxanthin

Reinvestigation of the carotenoids of Anacystis nidulans has confirmed the occurrence of β,β-carotene (β-carotene), β,β-caroten-3-ol (cryptoxanthin), β,β-carotene-3,3′-diol (zeaxanthin) and 2R,3R,3′R-β,β-carotene-2,3,3′-triol (absolute configuration assigned in the present work). In addition the previously unknown 2R,3R,2′R,3′R-β,β-carotene-2,3,2′,3′-tetrol has been isolated. The triol and the tetrol are considered identical with caloxanthin and nostoxanthin, respectively, for which allenic structures have been suggested by others. The chirality of these compounds followed from CD and ¹H NMR considerations.     

Biosynthesis of the isoflavan isomucronulatol: Origin of the 2′,3′,4′-oxygenation pattern

Feeding experiments with ¹⁴C-labelled isoflavones in seedlings and pods of bladder senna (Colutea arborescens) have demonstrated that 7-hydroxy-4′-methoxyisoflavone (formononetin), 7,3′-dihydroxy-4′-methoxyisoflavone (calycosin), 7,2′,3′-trihydroxy-4′-methoxyisoflavone (koparin) and 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone are excellent precursors of (3R)-isomucronulatol (7,2′-dihydroxy-3′,4′-dimethoxyisoflavan). 7,2′-Dihydroxy- 4′-methoxyisoflavone (2′-hydroxyformononetin) and 7-hydroxy-3′,4′-dimethoxyisoflavone (cladrin) were, however, poor substrates. Thus, the biosynthetic sequence to isomucronulatol from formononetin involves 3′-hydroxylation, 2′-hydroxylation and then 3′-O-methylation, followed presumably by stereospecific reduction of 7,2′-dihydroxy-3′,4′-dimethoxyisoflavone. Treatment of 2′,3′,4′-trimethoxyisoflavones with aluminium chloride in acetonitrile gives modest yields of 2′,3′-dihydroxy derivatives rather than 2′-monohydroxyisoflavones, and thus provides a convenient access to 2′,3′-dihydroxyisoflavones and related pterocarpans.     

Flavonoids from Merrillia caloxylon

Three chalcones and three flavones isolated from the fruit of Merrillia caloxylon (Rutaceae) have been characterised. Two of the flavones and two of the chalcones are related structurally, i.e. 3′,4′,5,7-tetramethoxyflavone with 2′- hydroxy-3,4,4′,6′-tetramethoxychalcone and 3′,4′,5,5′,7-pentamethoxyflavone with 2′,3-dihydroxy-4,4′,6′- trimethoxychalcone. A minor constituent was tentatively characterized as 5-hydroxy-3′,4′,5′,6,7-pentamethoxyflavone and this is accompanied by 2-hydroxy-3,4,4′,5,6′-pentamethoxychalcone and 5-hydroxy-3′,4′,6,7-tetramethoxyfiavone.     

Hydrolysis of 2',3'-cyclic adenosine monophosphate and 3',5'-cyclic adenosine monophosphate in subcellular fractions of normal and neoplastic mouse spleen

A comparison has been made between the capacity to hydrolyse 2′,3′-cyclic adenosine monophosphate and 3′,5′-cyclic adenosine monophosphate in subcellular fractions of normal and neoplastic (lymphosarcoma) spleen of C₅₇BL mice. The effect of X-irradiation on these activities was tested. Subcellular fractionation of normal and lymphosarcoma spleen points to a different overall localization of the enzymes. The 2′,3′-cyclic nucleotide phosphodiesterase (2′,3′-cAMPase) has its highest specific activity in the particulate fractions of the cell, while the data on 3′,5′-cyclic nucleotide phosphodiesterase (3′,5′-cAMPase) show the highest activity in the soluble fraction. The 2′,3′-cAMPase activity is higher in the tumor as compared to the normal tissue, while the opposite holds for 3′,5′-cAMPase. Total body irradiation of normal mice with a dose of 600 rads of X-rays, results in a clear drop in 2′,3′-cAMPase 48 hours after the exposure. The 3′,5′-cAMPase is hardly affected at this time. Neither imidazol nor Mg⁺⁺ has any influence on the 2′,3′-cAMPase. The pH optimum for 3′,5′-cAMPase and 2′,3′-cAMPase appears to be 7.7 and 6.2 respectively. This report suggests a no-identity of the two enzymes in mouse spleen, a situation different from that found in certain plants.     

NMR study of dideoxynucleotides with anti-human immunodeficiency virus (HIV) activity

The molecular structures of 3′-azido-2′,3′-dideoxyribosylthymine 5′-triphosphate (AZTTP), 2′,3′-dideoxyribosylinosine 5′-triphosphate (ddITP), 3′-azido-2′,3′-dideoxyribosylthymine 5′-monophosphate (AZTMP) and 2′,3′-dideoxyribosyladenine 5′-monophosphate (ddAMP) have been studied by NMR to understand their anti-HIV activity. For ddAMP and ddITP, conformations are almost identical with their nucleoside analogues with sugar ring pucker equilibriating between C3′-endo (∼75%) and C2′-endo (∼25%). AZTMP and AZTTP on the other hand show significant variations in the conformational behaviour compared with 3′-azido-2′,3′-dideoxyribo-sylthymine (AZT). The sugar rings for these nucleotides have a much larger population of C2′-endo (∼75%) conformers, like those observed for natural 2′-deoxynucleosides and nucleotides. The major conformers around C5′-O5′, C4′-C5′ and the glycosidic bonds are the β^t, γ⁺ and anti, respectively.     

Catalytic cycloalumination in steroid chemistry II: Selective functionalization of 2′-methylidene-2′,3′-ethano-(5α)-cholestane

The catalytic cycloalumination of 2′-methylidene-2′,3′-ethano-(5α)-cholestane with Et₃Al catalyzed by Cp₂ZrCl₂ was performed for the first time to give spiro[2′,3′-ethano-(5α)-cholestane-2′,3″-aluminacyclopentane] in a ～75% yield and with high stereoselectivity (>98%). The obtained cyclic organoaluminum compound was transformed in situ into heterocyclic spiran derivatives of 2′,3′-ethano-(5α)-cholestane.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca2+/Mg2+)-ATPase,and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.