Azetidine-2-carboxylic acid derivatives from seeds of Fagus silvatica L. and a revised structure for nicotianamine

2(S),3′(S)-N-(3-Amino-3-carboxypropyl)azetidine-2-carboxylic acid and 2(S),3′(S),3″(S)-N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2-carboxylic acid have been isolated from seeds of Fagus silvatica L. (beechnuts). The structures have been established by PMR- and ¹³C-NMR-spectroscopy and by synthesis from l-azetidine-2-carboxylic acid. The second of the new amino acids is identical with nicotianamine. previously isolated from Nicotiana tabacum but assigned a different formula. The ring opening reactions of azetidine-2-carboxylic acid in neutral solution have been studied and the chemical and possibly biochemical precursor role of this amino acid for various amino acids including the two new ones described here, nicotianine [N-(3-amino-3-carboxypropyl)nicotinic acid] and methionine is discussed.     

Formation of (-⊃-1′,2′-epi-2-cis-xanthoxin acid from a precursor of abscisic acid

Tomato shoots and avocado mesocarp supplied with (±)-[2-¹⁴C]-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid metabolize it into (+)-abscisic acid and a more polar material that was isolated and identified as (?)-epi-1′(R),2′(R)-4′(S)-2-cis-xanthoxin acid. The (+)-1′(S),2′(S)-4′(S)-2-cis-xanthoxin acid recently synthesized from natural violaxanthin, has the 1′,2′-epoxy group on the opposite side of the ring to that of the 4′(S)-hydroxyl group and the compound is rapidly converted into (+)-abscisic acid. The 1′,2′-epoxy group of (?)-1′,2′-epi-2-cis-xanthoxin acid is on the same side of the ring as the 4′(S) hydroxyl group: the compound is not metabolized into abscisic acid. The configuration of the 1′,2′-epoxy group probably controls whether or not the 4′(S) hydroxyl group can be oxidized. (+)-2-cis-Xanthoxin acid is probably not a naturally occurring intermediate because a ‘cold trap’, added to avocado fruit forming [¹⁴C]-labelled abscisic acid from [2-¹⁴C]mevalonate, failed to retain [¹⁴C] label.     

Inhibition of the peptidyltransferase acceptor site by 2′(3′)-O-cycloleucyl- and α-aminoisobutyryl derivatives of cytidylyl-(3′–5′)adenosine

2′(3′)-O-(N-Benzyloxycarbonylcycloleucyl)adenosine (1a) was prepared by esterification of 5′-O-(4-methoxytrityl)adenosine with N-benzyloxycarbonylcycloleucine in the presence of dicyclohexylcarbodiimide and subsequent deprotection in acidic medium. The compound 1a was separated into pure 2′- and 3′-isomers using HPLC; these isomers were found to undergo an easy interconversion. Compound 1a was coupled with N-dimethylaminomethylene-2′,5′-di-O-tetrahydropyranylcytidine 3′-phosphate in the presence of dicyclohexylcarbodiimide to give, after subsequent deblocking, cytidylyl(3′→5′)2′(3′)-O-cycloleucyladenosine (1c). Compound 1c, as well as the related cytidylyl(3′→5′)2′(3′)-O-(α-aminoisobutyryl)adenosine (1d), inhibited the peptidyltransferase catalyzed transfer of an AcPhe residue to puromycin in the Ac[¹⁴C]Phe-tRNA·poly(U)·70 S E. coli ribosome system. A half of the maximum inhibition of AcPhe-puromycin formation (at 10^?5 M puromycin) was achieved at 9.5·10^?6 M of compound 1c and 9·10^?5 M of compound 1d, respectively. The inhibition of the puromycin reaction by compound 1d shows a mixed-type of inhibition kinetics. Further, none of the compounds 1c and 1d was an acceptor in the peptidyltransferase reaction. Both compounds 1c and 1d inhibited the binding of C-A-C-C-A[¹⁴C]Phe to the A site of peptidyltransferase in a system containing tRNA^Phe·poly(U)·70 S E. coli ribosomes, in which compound 1d was a much stronger inhibitor than 1c. These results indicate that the derivatives such as compounds 1c and 1d which contain an anomalous amino acid with a substituent in lieu of α-hydrogen can interfere with the peptidyltransferase A site; however, they are not acceptors in the peptidyltransferase reaction probably due to a misfit of the α-substituent.     

Phenolic lipids from related marine algae of the order dictyotales

2-(1′-Oxo-dodeca-5′, 8′, 11′, 14′, 17′(all Z)-pentaenyl)-5-methoxy-1, 3-dihydroxybenzene, 2- (1′-oxo-dodeca-5′, 8′, 11′, 14′, 17′(all Z)-pentaenyl)-1, 3, 5-trihydroxybenzene, 2-(17′-hydroxy-1′-oxo-dodeca-5′, 8′, 11′, 14′(all Z)-tetraenyl)-1, 3, 5-trihydroxybenzene and 2-(1′oxo-hexadecyl)-1, 3, 5-trihydroxybenzene have been isolated from the related brown algae Zonaria farlowii, Z diesingiana and Lobophora papenfussii. The structures of these new metabolites are based on extensive spectral analyses and comparisons with model compounds. The isolation of (+)-7, 8-dimethyltocol, from L. papenfussii, is also reported.     

THE NMR CONFORMATION STUDY OF THE COMPLEXES OF DEOXYCYTIDINE KINASE (dCK) AND 2′-DEOXYCYTIDINE/2′-DEOXYADENOSINE

The structures of the bound ¹³C/²H double-labelled 2′(R/S), 5′(R/S)-²H₂-1′,2′,3′,4′,5′-¹³C₅-2′-deoxyadenosine and the corresponding 2′-deoxycytidine moieties in the complexes with human deoxycytidine kinase (dCK) have been characterized for the first time by the solution NMR spectroscopy, using Transferred Dipole-Dipole Cross-correlated Relaxation and Transferred nOe experiments. It has been shown that the ligand adopts a South-type sugar conformation when bound to dCK.     

2′(3′)-O-l-phenylalanyl derivatives of N2,5′-anhydroformycin and N4,5′-anhydroformycin: new substrates for ribosomal peptidyltransferase with a fixed anti and syn conformation of the base

The $\text{2′(3′)-O-}\text{l}\text{-}\text{phenylalanyl}\text{-N}{}^2\text{,5′-anhydroformycin}$ (1c) and $\text{2′(3′)-O-}\text{l}\text{-}\text{phenylalanyl}\text{-N}{}^4\text{,5′-anhydroformycin}$ (2c), obtained by chemical synthesis, are substrates for ribosomal peptidyltransferase from Escherichia coli. Nucleoside 1c, which mimics an anti conformation of antibiotic formycin, has 80% of the acceptor activity of puromycin at 5 · 10^?4 M determined by the release of N-Ac-Phe residue from the 70 S $\text{ribosome}\text{-}\text{poly}\text{(}\text{U}\text{)-N-}\text{Ac}\text{-[}{}^{14}\text{C]}\text{Phe-tRNA}$ complex. The reaction product, $\text{2′(3′)-O-(N-}\text{acetyl}\text{)-}\text{l}\text{-}\text{phenylalanyl}\text{-}\text{l}\text{-}\text{phenylalanyl}\text{-N}{}^2\text{,5′-}\text{anhydroformycin}$ (1d), was characterized by paper electrophoresis before and after alkaline hydrolysis. By contrast, nucleoside 2c, which resembles a syn conformation of formycin, exhibited only 20% of the acceptor activity of puromycin at 5 · 10^∮4 M and essentially none in the concentration region between 1 · 10^?6 and 1 · 10^?4 M. The results which are in accord with previous models have shown that a substrate with its base in an anti conformation is preferable for the acceptor site of peptidyltransferase than the corresponding syn counterpart, Nevertheless, it is possible that an intermediate conformation, for example, high anti (amphi-minus), is an optimal arrangement for acceptor site substrates.     

Mapping messenger RNA methylations at single base resolution

The messenger RNA (mRNA) methylations in mammalian cells have been found to contain N⁶-methyladenosine (m⁶A), N⁶-2′-O-dimethyladenosine (m⁶Am), 7-methylguanosine (m⁷G), 1-methyladenosine (m¹A), 5-methylcytosine (m⁵C), and 2′-O-methylation (2′-OMe). Their regulatory functions in control of mRNA fate and gene expression are being increasingly uncovered. To unambiguously understand the critical roles of mRNA methylations in physiological and pathological processes, mapping these methylations at single base resolution is highly required. Here, we will review the progresses made in methylation sequencing methodologies developed mainly in recent two years, with an emphasis on chemical labeling-assisted single base resolution methods, and discuss the problems and prospects as well.     

2-Bromoadenosine-Substituted 2′,5′-Oligoadenylates Modulate Binding and Activation Abilities of Human Recombinant RNase L

Abstract

2-Bromoadenosine-substituted analogues of 2–5A, p5′A2′p-5′A2′p5′(br²A), p5′(br²A)2′p5′A2′p5′A, and p5′(br²A)2′p5′(br²A)2′p-S′(br²A), were prepared via a modification of a lead ion-catalyzed ligation reaction and were subsequently converted into the corresponding 5′-triphosphates. Both binding and activation of human recombinant RNase L by various 2-bromoadenosine-substituted 2–5A analogues were examined. Among the 2-bromoadenosine-substituted 2–5A analogues, the analogue with 2-bromoadenosine residing in the 2′-terminal position, p5′A2′p5′A2′p-5′(br²A), showed the strongest binding affinity and was as effective as 2–5A itself as an activator of RNase L. The CD spectrum of p5′A2′p-5′A2′p5′(br²A) was superimposable on that of p5′A2′p5′A2′p5′A, indicative of an anti orientation about the base-glycoside bonds as in naturally occurring 2–5A.     

Synthesis and urease inhibitory potential of benzophenone sulfonamide hybrid in vitro and in silico

This study deals with the synthesis of benzophenone sulfonamides hybrids (1–31) and screening against urease enzyme in vitro. Studies showed that several synthetic compounds were found to have good urease enzyme inhibitory activity. Compounds 1 (N′-((4′-hydroxyphenyl)(phenyl)methylene)-4′′-nitrobenzenesulfonohydrazide), 2 (N′-((4′-hydroxyphenyl)(phenyl)methylene)-3′′-nitrobenzenesulfonohydrazide), 3 (N′-((4′-hydroxyphenyl)(phenyl)methylene)-4′′-methoxybenzenesulfonohydrazide), 4 (3′′,5′′-dichloro-2′′-hydroxy-N′-((4′-hydroxyphenyl)(phenyl)methylene)benzenesulfonohydrazide), 6 (2′′,4′′-dichloro-N′-((4′-hydroxyphenyl)(phenyl)methylene)benzenesulfonohydrazide), 8 (5-(dimethylamino)-N′-((4-hydroxyphenyl)(phenyl)methylene)naphthalene-1-sulfono hydrazide), 10 (2′′-chloro-N′-((4′-hydroxyphenyl)(phenyl)methylene)benzenesulfonohydrazide), 12 (N′-((4′-hydroxyphenyl)(phenyl)methylene)benzenesulfonohydrazide) have found to be potently active having an IC₅₀ value in the range of 3.90–17.99?µM. These compounds showed superior activity than standard acetohydroxamic acid (IC₅₀?=?29.20?±?1.01?µM). Moreover, in silico studies on most active compounds were also performed to understand the binding interaction of most active compounds with active sites of urease enzyme. Structures of all the synthetic compounds were elucidated by ¹H NMR, ¹³C NMR, EI-MS and FAB-MS spectroscopic techniques.     

Synthesis of modified tryptophanyl-adenylates and of modified adenosine-triphosphates and their use as tools for elucidation of the mechanism of tryptophanyl-tRNA synthetase from yeast

Six analogs of tryptophanyl-adenylate, which is an important intermediate in the enzymatic synthesis of Trp-tRNA^Trp, have been prepared. Four compounds, tryptophanyl-8-bromoadenylate, tryptophanyl-2-chloroadenylate, tryptophanyl-7-deazaadenylate and tryptophanyl-(N⁶-methyl)adenylate, contain modifications in the nucleobase moiety, while tryptophanyl-2′ deoxyadenylate and tryptophanyl-3′-deoxyadenylate were modified in the carbohydrate part of the molecule. Three of these analogs (2-chloro, 7-deaza, 2′-deoxy analogs) as well as ATP analogs with the same modifications were substrates in the aminoacylation reaction; three analogs (8-bromo, N⁶-methyl, 3′-deoxy analogs) were inactive as well as the corresponding ATP analogs. In contrast, in the $\text{ATP}\text{PP}{}_{\mathrm{<mtext>i</mtext>}}$ pyrophosphate exchange in the absence of tRNA all ATP analogs except 8-bromo-ATP were substrates. However, the presence of tRNA reduced the number of ATP analogs being substrates to that number of substrates observed in the aminoacylation. Therefore, it can be concluded that the presence of tRNA is responsible for an increase of specificity. The diastereomers of adenosine 5′-O-(3-thiotriphosphate) (ATPαS), adenosine 5′-O-(2-thiotriphosphate) (ATPβS), and adenosine 5′-O-(3-thiotriphosphate) (ATPγS) were tested with various divalent metals as substrates in the pyrophosphate exchange reaction. The S_p diastereomer of ATPαS is a substrate with Mg²⁺, whereas the R_p diastereomer is inactive. Both diastereomers are inactive in the presence of Zn²⁺. Since Zn²⁺ binds preferentially to the sulfur atom, an explanation of these results is that the Mg²⁺ ion is not bound to the α-phosphate. Only the S_p isomer of the diastereomers of ATPβS acts as substrate in the presence of Mg²⁺. The stereospecificity becomes reversed in the presence of Zn²⁺. ATPγS acts as substrate with both Mg²⁺ and Zn²⁺. These results suggest that the Δ isomer of the β,γ-bidentate ATP-Mg²⁺ complex is the substrate for this enzyme. From these results a molecular model of the ATP-Mg²⁺ complex in the active site can be derived in which the nucleotide is attached to the enzyme by interactions in which the 3′-OH and 6-NH₂ group, one oxygen atom of the α-phosphorus atom, and the coordinated magnesium cation are all involved.     

Synthesis of 2′-O-(N-methylcarbamoylethyl) 5-methyl-2-thiouridine and its application to splice-switching oligonucleotides

The effect of 2′-O-(N-methylcarbamoyl)ethyl (MCE) modification on splice-switching oligonucleotides (SSO) was systematically evaluated. The incorporation of five MCE nucleotides at the 5′-termini of SSOs effectively improved the splice switching effect. In addition, the incorporation of 2′-O-(N-methylcarbamoylethyl)-5-methyl-2-thiouridine (s²T_MCE), a duplex-stabilizing nucleotide with an MCE modification, into SSOs further improved splice switching. These SSOs may be useful for the treatment of genetic diseases associated with splicing errors.     

Acylhydrazones of 20-keto steroids and their transformations: I. Synthesis and properties of 1′-acyl-substituted 3′-methylandrosteno[16,17-<Emphasis Type="Italic">d</Emphasis>]pyrazolines

Acetates of 3β-hydroxy-3′-methyl-1′(N)-acylandrost-5-eno[16,17-d]pyrazolines bearing monothiooxamide acyl groups were synthesized during the study of approaches to the synthesis of 3′-methylandrosteno[16,17-d]azoles, promising biologically active analogues of 20-keto pregnenanes, and their properties were investigated. The cyclization of Δ¹⁶-20-thiooxamidohydrazones to the corresponding heterocycles was shown to proceed under rigorous conditions and to depend partially on the nature of the oxamide grouping.     

The metabolism of [2-14C]indole in the rat

1. [2-¹⁴C]Indole has been synthesized from [¹⁴C]formate and o-toluidine via N[¹⁴C]-formyltoluidine. 2. When fed to rats, the ¹⁴C of [¹⁴C]indole (dose 70–80mg./kg. body wt.) is fairly rapidly excreted, and in 2 days an average of 81% appears in the urine, 11% in the faeces and 2·4% as carbon dioxide in the expired air. 3. Radioactivity is excreted in the urine as indoxyl sulphate (50% of the dose), indoxyl glucuronide (11%), oxindole (1·4%), isatin (5·8%), 5-hydroxyoxindole conjugates (3·1%), N-formylanthranilic acid (0·5%) and unchanged indole (0·07%). The faeces contain indoxyl sulphate (0·4% of the dose) and indole (0·2%), but the major metabolites have not been identified. 4. Fed to rats with biliary cannulae an average of 5·6% of a dose of [¹⁴C]indole (20–60mg./kg. body wt.) is excreted in the bile in 2 days. Radioactivity is present as indoxyl sulphate (0·8% dose) and 5-hydroxyoxindole conjugates (0·6%). 5. Rats further metabolize indoxyl into N-formylanthranilic acid and anthranilic acid, and oxindole into 5-hydroxyoxindole. 6. With rat-liver microsomes plus supernatant under aerobic conditions, indole gives indoxyl, oxindole, possibly isatin, N-formylanthranilic acid and anthranilic acid, but under anaerobic conditions gives only oxindole. Similarly, under aerobic conditions, oxindole gives 5-hydroxyoxindole, anthranilic acid and o-aminophenylacetic acid. 7. Indole is metabolized by two pathways, one via indoxyl to isatin, N-formylanthranilic acid and anthranilic acid, and the other via oxindole to 5-hydroxyoxindole and possibly to o-aminophenylacetic and anthranilic acid. 8. The following new compounds are described: 4-hydroxy-2-nitrophenylacetic acid, 3-, 4- and 5-benzyloxy-2-nitrophenylacetic acid, 5- and 7-hydroxyoxindole and 5-aminoacridine indoxyl sulphate.     

A simple method of the preparation of 2′-O-Methyladenosine Methylation of adenosine with methyl iodide in anhydrous alkaline medium

A simple and effective method of the methylation on the 2′-O position of adenosine is described. Adenosine is treated with CH₃I in an anhydrous alkaline medium at 0°C for 4 h. The major products of this reaction are monomethylated adenosine at either the 2′-O or 3′-O position (total of 64%) and the side products are dimethylated adenosine (2′,3′-O-dimethyladenosi, 21%, and N⁶-2′-O-dimethyladenosine, 11%). The ratio of 2′-O- and 3′-O-methyladenosine has been found to be 8 to 1. Therefore, this reaction preferentially favors the synthesis of 2′-O-methyladenosine. The monomethylated adenosine is isolated from reaction mixture by a silica gel column chromatography. Then the pure 2′-O-methyladenosine can be separated by crystallization in ethanol from the mixture of 2′-O and 3′-O-methylated isomers. The overall yield of 2′-O-methyladenosine is 42%.     

Synthesis of Chlorodideoxynucleosides Using Tris(2,4,6-tribromophenoxy)-dichlorophosphorane (BDCP)

Abstract

5′-Chloro-5′-deoxy-N,3′-O-dibenzoylthymidine (3a), 5′-chloro-5′-deoxy-N⁴, 3′-O-dibenzoyldeoxycytidine(3b), 5′-chloro-5′-deoxy-N⁶,3′-O-dibenzoyldeoxyadenosine(3c), N-benzoyl-1-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)thymine (5a) and N⁶-benzoyl-9-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)adenine (5b) have been synthesized in very high yields using a new efficient reagent, tris(2,4,6-tribrom-ophenoxy)dichlorophosphorane (BDCP). The reaction time was greatly reduced to 5–8 min. NOE data suggested an inversion of configuration at C₃-position and thus an SN2 mechanism has been proposed for the chlorination reaction.

     

New diacylglyceryltrimethylhomoserines from the marine microalga Nannochloropsis granulata and their nitric oxide inhibitory activity

Chemical investigation of polar lipids from the marine eustigmatophyte microalga Nannochloropsis granulata led to the isolation of six betaine lipid diacylglyceryltrimethylhomoserine (DGTS), namely, (2S)-1,2-bis-O-eicosapentaenoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (1), (2S)-1-O-eicosapentaenoyl-2-O-arachidonoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (2), (2S)-1-O-eicosapentaenoyl-2-O-myristoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (3), (2S)-1-O-eicosapentaenoyl-2-O-palmitoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (4), (2S)-1-O-eicosapentaenoyl-2-O-palmitoleoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (5), and (2S)-1-O-eicosapentaenoyl-2-O-linoleoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (6). Structures of the isolated DGTSs were elucidated based on both spectroscopic technique and degradation methods. This is the first report of isolation of 1 in pure state, and 2–6 are all new compounds. The isolated betaine lipids showed dose-dependent nitric oxide (NO) inhibitory activity against lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophage cells. Further study suggested that these betaine lipids (1–6) inhibit NO production in RAW264.7 macrophage cells through downregulation of inducible nitric oxide synthase expression, indicating the possible use as an anti-inflammatory agent. This is the first report of DGTS with anti-inflammatory activity.     

Alternative routes for the genesis of kinetin: A synthetic intramolecular route from 2′-deoxyadenosine to kinetin

We have tested the possible genesis of kinetin from a 2′-deoxyadenylate unit of DNA by a chemical route involving a head-to-tail transfer of deoxyribose from the 9 to the 3 position of the adenine nucleus via a cyclonucleoside, with subsequent elimination of 1′- and 3′-polar groups and 3 → N⁶ intramolecular rearrangement leading to kinetin. We have also determined quantitatively the per cent conversions to 3-furfuryladenine and/or kinetin of the following under autoclaving conditions at 120°, pH 4, 2 atm, and 4 hr: (1) adenine/furfury alcohol; (2) adenine/2-deoxy-d-ribose; (3) 2′-deoxyadenosine; (4) 3-furfuryladenine; (5) 3,5′-(3′-O-diethylphosphoryl-2′-deoxya-denosine)-cyclonucleoside p-toluenesulfonate. The sequence of reactions involving cyclonucleoside formation and rearrangement has been shown to be a chemically feasible route by which kinetin can be formed, although it is not the only way this cytokinin can be generated.     

Isolation and synthesis of trans- and cis-(−)-clovamides and their deoxy analogues from the bark of Dalbergia melanoxylon

N-(3′,4′-Dihydroxy-trans-cinnamoyl)-3-(3,4-dihydroxyphenyl)-L-alanine [(?)-clovamide], the major phenolic metabolite (0.1%) in the bark of Dalbergia melanoxylon, is associated with minor proportions of its cis-isomer, and similar pairs of geometrical isomers of their deoxy analogues N-(4′-hydroxycinnamoyl)-3-(3,4-dihydroxyphenyl)-L-alanine and N-(4′-hydroxycinnamoyl)-3-(4-hydroxyphenyl)-L-alanine. (?)-Trans-clovamide is synthesized by direct condensation of the acid chloride of caffeic acid with L-DOPA. Diagnostic CD spectra of these compounds and ¹³C spectra of (?)-trans- and (?)-cis-clovamides are recorded.     

4-N-Alkanoyl and 4-N-alkyl gemcitabine analogues with NOTA chelators for 68-gallium labelling

The conjugation of 4-N-(3-aminopropanyl)-2′-deoxy-2′,2′-difluorocytidine with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) ligand in 0.1?M Na₂CO₃ buffer (pH 11) at ambient temperature provided 4-N-alkylgemcitabine-NOTA chelator. Incubation of latter with excess of gallium(III) chloride (GaCl₃) (0.6?N AcONa/H₂O, pH?=?9.3) over 15?min gave gallium 4-N-alkylgemcitabine-NOTA complex which was characterized by HRMS. Analogous [⁶⁸Ga]-complexation of 4-N-alkylgemcitabine-NOTA conjugate proceeded with high labeling efficiency (94%–96%) with the radioligand almost exclusively found in the aqueous layer (～95%). The high polarity of the gallium 4-N-alkylgemctiabine-NOTA complex resulted in rapid renal clearance of the ⁶⁸Ga-labelled radioligand in BALB/c mice.