Permethyl Derivatives Research Articles

Protective Effects of Marine Alkaloid Neolamellarin A Derivatives against Glutamate Induced PC12 Cell Apoptosis.

Marine alkaloids obtained from sponges possess a variety of biological activities and potential medicinal value. The pyrrole-derived lamellarin-like alkaloids, especially their permethyl derivatives, show low cytotoxicity and potent MDR reversing activity. Neolamellarin A is a novel lamellarin-like alkaloid which was extracted from marine animal sponges. We reported the synthetic method of permethylated Neolamellarin A and its derivatives by a convergent strategy in 2015. In 2018, we reported the synthesis and the neuroprotective activity in PC12 cells of 3,4-bisaryl-N-alkylated permethylated Neolamellarin A derivatives. In this report, another series of 15 different 3,4-bisaryl-N-acylated permethylated Neolamellarin A derivatives were synthesized, and the outstanding protective effects of these compounds against glutamate induced PC12 cell apoptosis were presented and discussed. These Neolamellarin A derivatives which possessed low cytotoxicity and superior neuroprotective activity may have the potential to be developed into antagonists against glutamate induced nerve cell apoptosis.

Epigallocatechin Gallate, a Green Tea Polyphenol Inhibits Mycobacterium smegmatis: In silico and In vitro Studies

Presence of multidrug resistant strains makes treatment of tuberculosis difficult. Conventional frontline antituberculosis therapy has high incidence of treatment failure due to non-adherence and severe side effects, and therefore warrants alternative approaches using safe, natural compounds. (-) Epigallocatechin gallate, a green tea catechin with significant antimicrobial properties, and its derivatives have been studied for their inhibitory effects on Mycobacterium smegmatis. Enoyl reductase (Inh A, PDB ID: 2NV6), an important drug target inhibited by the antituberculosis drug, isoniazid, was docked with geometrically optimized conformation of epigallocatechin gallate and two of its derivatives using AutoDock software. Perpropionate and permethyl derivatives of epigallocatechin gallate were synthesized and, along with epigallocatechin gallate, evaluated for their inhibitory and cytotoxic properties against M. smegmatis using microplate dilution method and alamarBlue® assay, respectively. Ames test was performed to assess the mutagenic potential of epigallocatechin gallate. It was docked successfully onto the InhA with docking energy of –9.38 kcal mol-1. Analysis of neighboring groups revealed 11 amino acid residues common to both isoniazid and epigallocatechin gallate. The minimum inhibitory concentrations for epigallocatechin gallate, perpropionate and permethyl were found to be 128 (58.2% inhibition), 8 (32.9%) and 4 (12.5%) µg/ml, respectively. Epigallocatechin gallate showed a cytotoxicity of 18.6% at a concentration of 8 µg/ ml, while the two derivatives did not show any toxicity, even at 64 µg/ml. Ames test revealed the absence of mutagenicity for epigallocatechin gallate, in presence and absence of metabolic activation. Non-mutagenic epigallocatechin gallate inhibits the growth of M. smegmatis and warrants further investigation as an adjunct therapy for pathogenic mycobacteria.

Efficient syntheses of permethylated derivatives of neolamellarin A, a pyrrolic marine natural product

The pyrrole-derived alkaloids with marine origin, especially their permethyl derivatives, have unique structures and promising biological activities. Marine natural product neolamellarins are a collection of lamellarin-like phenolic pyrrole compounds, which can inhibit hypoxia-induced HIF-1 activation. Many pyrrole-derived lamellarin-like alkaloids show potent MDR reversing activity. In this study, five permethylated derivatives of neolamellarin A were synthesized with their MDR reversing activity studied in order to identify new MDR reversal agents. A convergent strategy was adopted to synthesize the permethylated derivatives of neolamellarin A. Pyrrole was first converted into a corresponding N-trisisopropylsilyl (TIPS)-substituted derivative, then through iodination afforded 3,4-diiodinated pyrrole compound. The key intermediate, 3,4-disubstituent-1H-pyrrole, was obtained through desilylation of 3,4-disubstituent-1-TIPS pyrrole, which was prepared from 3,4-diiodinated pyrrole derivative and aryl boronic acid ester through Suzuki cross-coupling reaction between them. Then, the intermediate, 3,4-disubstituent-1H-pyrrole, reacted with fresh phenylacetyl chloride under n-BuLi/THF condition afforded the target compounds. Finally, we obtained five novel pyrrolic compounds, permethylated derivatives of neolamellarin A 16a–e, in 30%–37% yield through five step reactions. The bioactivity testing of these compounds are in process.

Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans

Sulfate modifications on terminal epitopes of N- and O-glycans have increasingly been implicated as critical determinants mediating a diverse range of biological recognition functions. To address these low abundance but important sulfated glycans, and the sulfoglycome in general, further development of enrichment strategies and enabling mass spectrometry (MS)-based mapping techniques are needed. In this report, we demonstrate that the sulfated glycans, with and without additional sialylation, can be successfully permethylated by the sodium hydroxide slurry method and be distinguished from phosphorylated glycans by virtue of this derivatization. In conjunction with simple microscale postderivatization fractionation steps, permethyl derivatives fully retaining the negatively charged sulfate moiety and separated from the nonsulfated ones, can be efficiently detected and sequenced de novo by advanced MS/MS in the positive-ion mode. In particular, we show that the highly sequence and linkage informative high energy collision induced dissociation (CID) MS/MS afforded by MALDI-TOF/TOF can be extended to sulfoglycomic applications. The sulfated parent ion selected for CID MS/MS was found to mostly retain the sulfate moiety and therefore allow efficient fragmentation via the usual array of glycosidic, cross ring, and concerted double cleavages. Collectively, the optimized strategy enables a high sensitivity detection and critical mapping of the sulfoglycome such as the one derived from lymph node tissues or cell lines in both negative and positive-ion modes. Novel sulfated epitopes were identified from a crude mouse lymph node preparation, which fully attested to the practical utility of the methodology developed.

New insights into the functions and N‐glycan structures of factor X activator from Russell’s viper venom

The coagulation factor X activator from Russell's viper venom (RVV-X) is a heterotrimeric glycoprotein. In this study, its three subunits were cloned and sequenced from the venom gland cDNAs of Daboia siamensis. The deduced heavy chain sequence contained a C-terminal extension with four additional residues to that published previously. Both light chains showed 77-81% identity to those of a homologous factor X activator from Vipera lebetina venom. Far-western analyses revealed that RVV-X could strongly bind protein S, in addition to factors X and IX. This might inactivate protein S and potentiate the disseminated intravascular coagulation syndrome elicited by Russell's viper envenomation. The N-glycans released from each subunit were profiled and sequenced by MALDI-MS and MS/MS analyses of the permethyl derivatives. All the glycans, one on each light chain and four on the heavy chain, showed a heterogeneous pattern, with a combination of variable terminal fucosylation and sialylation on multiantennary complex-type sugars. Amongst the notable features were the presence of terminal Lewis and sialyl-Lewis epitopes, as confirmed by western blotting analyses. As these glyco-epitopes have specific receptors in the vascular system, they possibly contribute to the rapid homing of RVV-X to the vascular system, as supported by the observation that slower and fewer fibrinogen degradation products are released by desialylated RVV-X than by native RVV-X.

Identification of Further Elongation and Branching of Dimeric Type 1 Chain on Lactosylceramides from Colonic Adenocarcinoma by Tandem Mass Spectrometry Sequencing Analyses

Mammalian glycan chain elongation is mostly based on extending the type 2 chain, Galbeta1-4GlcNAc, whereas the corresponding type 1 chain, Galbeta1-3GlcNAc, is not normally extended. In a broader context of developing high sensitivity mass spectrometry methodologies for glycomic identification of Le(a) versus Le(x) and linear versus branched poly-N-acetyllactosamine (polyLacNAc), we have now shown that the dimeric type 1 glycan chain, as carried on the lactosylceramides of a human colonic adenocarcinoma cell line, Colo205, not only can be further extended linearly but can likewise be branched at C6 of 3-linked Gal in a manner similar to polyLacNAc. A combination of chemical and enzymatic derivatization coupled with advanced mass spectrometry analyses afforded unambiguous identification of a complex mixture of type 1 and 2 hybrids as well as those fucosylated variants founded exclusively on linear and branched trimeric type 1 chain. We further showed by in vitro enzymatic synthesis that extended type 1 and the hybrid chains can be branched by all three forms of the human I branching enzymes (IGnT) currently identified but with lower efficiency and stringency with respect to branching site preference. Importantly, it was found that a better substrate is one that carries a Gal site for branching that is extended at the non-reducing end by a type 2 and not a type 1 unit, whereas the IGnTs are less discriminative with respect to whether the targeted Gal site is itself beta3- or beta4-linked to GlcNAc at the reducing end.

Glycomic mapping of O- and N-linked glycans from major rat sublingual mucin

Carbohydrate moieties of salivary mucins play various roles in life processes, especially as a microbial trapping agent. While structural details of the salivary O-glycans from several mammalian sources are well studied, very little information is currently available for the corresponding N-glycans. The existence of N-glycans alongside O-glycans on mucin isolated from rat sublingual gland has previously been implicated by total glycosyl compositional analysis but the respective structural data are both lacking. The advent of facile glycomic mapping and sequencing methods by mass spectrometry (MS) has enabled a structural reinvestigation into many previously unsolved issues. For the first time, high energy collision induced dissociation (CID) MALDI-MS/MS as implemented on a TOF/TOF instrument was applied to permethyl derivatives of mucin type O-glycans and N-glycans, from which the linkage specific fragmentation pattern could be established. The predominant O-glycans carried on the rat sublingual mucin were defined as sialylated core 3 and 4 types whereas the N-glycans were determined to be non-bisected hybrid types similarly carrying a sialylated type II chain. The masking effect of terminal sialylation on the tight binding of rat sublingual mucin to Galbeta1-->4GlcNAc specific lectins and three oligomannose specific lectins were clearly demonstrated in this study.

The Carboxy Terminus of EmbC from Mycobacterium smegmatis Mediates Chain Length Extension of the Arabinan in Lipoarabinomannan

D-Arabinofurans, attached to either a galactofuran or a lipomannan, are the primary constituents of mycobacterial cell wall, forming the unique arabinogalactan (AG) and lipoarabinomannan (LAM), respectively. Emerging data indicate that the arabinans of AG and LAM are distinguished by virtue of the additional presence of linear termini in LAM, which entails some unknown feature of the EmbC protein for proper synthesis. In common with the two paralogous EmbA and EmbB proteins functionally implicated for the arabinosylation of AG, EmbC is predicted to carry 13 transmembrane spanning helices in an integral N-terminal domain followed by a hydrophilic extracytoplasmic C-terminal domain. To delineate the function of this C-terminal domain, the embC knock-out mutant of Mycobacterium smegmatis was complemented with plasmids expressing truncated embC genes. The expression level of serially truncated EmbC protein thus induced was examined by EmbC-specific peptide antibody, and their functional implications were inferred from ensuing detailed structural analysis of the truncated LAM variants synthesized. Apart from critically showing that the smaller arabinans are mostly devoid of the linear terminal motif, beta-D-Araf(1-->2)-alpha-D-Araf(1-->5)-alpha-D-Araf(1-->5)-alpha-D-Araf, our studies clearly implicate the C-terminal domain of EmbC in the chain extension of LAM. For the first time a full range of arabinan chains as large as 18-22 Araf residues and beyond could be released intact by the use of an endogenous endo-D-arabinanase from M. smegmatis, profiled, and sequenced directly by tandem mass spectrometry. In conjunction with NMR studies, our results unequivocally show that the LAM-specific linear termini are an extension on a well defined inner branched Ara-(18-22) core. This hitherto unrecognized feature not only allows a significant revision of the structural model of LAM-arabinan since its first description a decade ago but also furnishes a probable molecular basis of selectivity in biosynthesis, as conferred by the EmbC protein.

Efficient Synthesis of Ningalin C.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Efficient synthesis of ningalin C.

[reaction: see text] A concise and efficient synthesis of the permethyl derivative of the marine alkaloid ningalin C (2) has been accomplished. The key step involves the formation of a pyrrolinone from an aminoquinone in one pot. An efficient route for the synthesis of the key aminoquinone has also been developed.

Single-, double-, and triple-charged cations based on iron-subgroup decamethylmetallocenes

Methods for the synthesis and structures of metallocenylmethyl cations are considered using stable permethyl derivatives as examples. The simplest and most convenient procedure for the preparation of mono-, di-, and trications involves oxidation of permethylmetallocenes (M = Ru or Os) with oxygen in the presence of strong protic acids. Based on the results of physicochemical studies (X-ray diffraction analysis, 1H and 13C NMR spectroscopy) and quantum-chemical calculations (density functional theory), a resonance hybrid with contributions of the metallonium (onium), carbocationic, and fulvene structures was proposed for the description of the structures of these cations.

Computed Properties of the CB9H10and CB11H12Free Radicals

Density functional calculations (B3LYP/6-31G(d)) are presented for the anion and radical of the mono-carbon carboranes, CBnHn+1, n = 4, 9, 11. The CB4H5-, CB9H10-, and CB11H12- anions, which are isoelectronic with the closo-dicarba species C2B3H5, C2B8H10, and C2B10H12, are predicted to have significant ionization potentials (2.88, 4.36, and 5.19 eV). The recently reported synthesis of the permethyl derivative of the CB11H12 radical is remarkable in view of the large predicted ionization potential of the CB11Me12- anion (4.32 eV). All calculated properties are consistent with the removal of an electron from a cage-bonding orbital with significant unpaired spin density on the boron opposite the carbon vertex.

Hydroxyakalone, a novel xanthine oxidase inhibitor produced by a marine bacterium, Agrobacterium aurantiacum.

A new xanthine oxidase inhibitor named hydroxyakalone was isolated from the culture broth of a marine bacterium Agrobacterium aurantiacum N-81106. Structure of hydroxyakalone was determined to be 4-amino-1H-pyrazolo[3,4-d]pyrimidine-3-one-6-ol by the spectral studies of hydroxyakalone and its permethyl derivative. The concentration to induce 50% inhibition (IC50) was 4.6 microM against xanthine oxidase.

Structural definition of acylated phosphatidylinositol mannosides from Mycobacterium tuberculosis: definition of a common anchor for lipomannan and lipoarabinomannan.

Based on chemical analysis, we have previously concluded that the biologically important lipoarabinomannan (LAM) and lipomannan (LM) from Mycobacterium are multiglycosylated forms of the phosphatidylinositol mannosides (PIMs), the characteristic cell envelope mannophosphoinositides of mycobacteria. Using definitive analytical techniques, we have now re-examined the reported multiacylated nature of PIMs in order to gain a better insight into their possible roles as biosynthetic precursors of LM and LAM. High-sensitivity fast atom bombardment-mass spectrometry analyses of the perdeuteroacetyl and permethyl derivatives of PIMs from Mycobacterium tuberculosis and Mycobacterium leprae enabled us to define the exact fatty acyl compositions of the multiacylated, heterogeneous PIM families, notably the dimannoside (PIM2) and the hexamannoside (PIM6). Specifically, in conjunction with other chemical and gas chromatography-mass spectrometry (GC-MS) analyses, the additional C16 fatty acyl substituent on PIM2 and its lyso form were defined as attached to the C6 position of mannose. We also present evidence for triacylated mannophosphoinositide as a common lipid anchor for both LM and LAM, and further postulate that acylation of PIM2 may constitute a key regulatory step in their biosynthesis.

Gibberellins and ?-amylase gene expression in germinating wheat grains

Gibberellins (GAs), GA8, GA17, GA19, GA20, GA29, and GA79 were identified by full-scan gas chromatography-mass spectrometry in a purified acidic fraction and GA8, GA20, GA79, and GA90 in a hydrolysed conjugate fraction from mature wheat grains. Gibberellin A20-13-O-glucoside was also quantified directly as the permethyl derivative in dry seed. The scutellum was identified as the major site of de novo GA biosynthesis by measuring ent-kaurene accumulation in vivo in grains treated with 2S,3S-paclobutrazol. Several GAs of the early 13-hydroxylation GA pathway began to accumulate in the axis and scutellum between 24 and 48 hours in untreated grains germinated at 15°C. Bioactive GA1 and GA3 also increased in the endosperm during this period, whereas abscisic acid contents of embryo and endosperm declined rapidly over 48 hours following imbibition. Treating grains with 2S,3S-paclobutrazol reduced GA1 plus GA3 content of scutella by 70–80% over 4 days without affecting significantly the steady-state pool of α-amylase mRNA transcripts. In contrast, a 50–80% reduction in the content of bioactive GAs in the endosperm was associated with a 70–78% decrease in transcripts for both α-amylase gene families in aleurones of paclobutrazol-treated grains. It was concluded that the initiation of α-amylase gene expression in wheat scutella was independent of de novo GA biosynthesis, whereas that in the aleurone was largely dependent on embryo-produced GAs.

Preferred conformation of C-glycosides. 12. Synthesis and conformational analysis of .alpha.,.alpha.-, .alpha.,.beta.-, and .beta.,.beta.-C-trehaloses

A single, unified strategy for the stereocontrolled synthesis of α,α-, α,β-, and β,β-C-trehaloses (1-3) was developed. The solution conformations of C-trehaloses 1-3, as well as their permethyl derivatives, were determined on the basis of vicinal coupling constants observed in the 1 H NMR spectra. The preferred conformations for α,α- and β,β-C-trehaloses (1 and 3), shown in Figure 1, were predicted and experimentally proven. A diamond-lattice analysis of α,β-C-trehalose (2), shown in Figure 2, revealed the relative stability of the three staggered conformers to be 2A> 2B>2C, and the experimental data were found to be consistent with this trend

PM3 study of organometallic radicals formed by elements in periodic Groups 13–16

Calculations have been made by the PM3 SCF method of the molecular and electronic structures of a range of neutral, cationic and anionic organometallic radicals derived from permethyl derivatives of elements from Groups 13–16. In Group 13, the planar methyls M(CH 3) 3 all yield pyramidal mononuclear radical-anions [M(CH 3) 3] −, but only for M = Al was a stable dinuclear radical-anion [Al 2(CH 3) 6] − found; this is a weak complex of [Al(CH 3) 2] . and [Al(CH 3) 4] −.In Group 14, dinuclear σ radical-cations [M 2(CH 3) 6] + of D 3 d symmetry are found for M = SiPb, and analogous * radical-cations are found for M = PBi in Group 15. The dinuclear [M 2(CH 3) 4] + formed by sulphur and selenium both have C 2 h symmetry.

Identification of the cytokinins in red pine seedlings

Zeatin-9-riboside was identified in shoots and roots of Pinus resinosa by GC-MS analysis of its permethyl derivative. Based on their chromatographic properties on Sephadex LH-20 and C18 HPLC, and their susceptibility to enzymatic degradation, several other cytokinins have been tentatively identified. The basic fraction of both the roots and shoots contained zeatin, whereas the shoots contained dihydrozeatin-O-glucoside and the roots contained zeatin-O-glucoside. Zeatin-9-riboside monophosphate, isopentenyladenosine monophosphate ([9R-5′P]iP) and glucosyl phosphate derivatives were detected in the acidic fractions from both roots and shoots. There were equivalent amounts of [9R-5′P]iP in both roots and shoots. The presence of equivalent amounts of [9R-5′P]iP in both the roots and shots suggests that cytokinin biosynthesis may be occurring in both locations.

FABMS/derivatisation strategies for the analysis of heparin-derived oligosaccharides

Derivatisation/FABMS strategies applicable to the structure analysis of low microgramme quantities of heparin-derived oligosaccharides are described. Negative and positive FAB data from permethyl derivatives and positive FAB data from the products of subsequent methanolysis are reported for sulfated tetrasaccharides prepared by nitropus acid degradation of heparin. The preparation and FAB behaviour of acetylated derivatives of sulfated oligosaccharides are described for the first time, and the stability of the sulfate groups to base-catalysed acetylation is demonstrated. The acetylation/FABMS methodology, which yields high quality data, shows promise for the characterisation of a wide range of sulfated glycoconjugates.

The formation of covalent adducts between benzo[a]pyrenediol epoxide and RNA: structural analysis by mass spectrometry

Racemic 7-r,8-t-dihydroxy-9-t,10-t-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene was reacted with yeast RNA. Modified nucleosides were isolated and resolved by high-performance liquid chromatography; nine adduct peaks were collected for analysis. The bases in these adducts were identified by comparing their retention times with those of adducts from poly(G), poly(A), and poly(C). These samples gave two major and two minor Guo adducts, four major Ado adducts, and at least four Cyd adducts. The relative efficiencies of adduct formation with the polyribonucleotides were poly(G) greater than yeast RNA greater than poly(A) greater than poly(C). Fluorescence measurements show that emission from Guo adducts is strongly quenched relative to that from Ado adducts. Liquid secondary ion mass spectrometry (LSIMS) of underivatized samples and electron-impact mass spectrometry (EIMS) of permethyl derivatives were used to confirm the base identities and establish the alkylation sites of the RNA adducts. Unique nitrogen-containing hydrocarbon fragments that were observed with all samples by EIMS establish that in each adduct analyzed the C-10 position of the hydrocarbon is linked to the exocyclic amino group of the base. This suggested that the multiple adducts formed with each base are diastereomers derived from cis/trans epoxide ring opening of the (+) and (-) enantiomers of the carcinogen. Several adducts exhibited molecular ions by both LSIMS and EIMS. Large fragments observed by EIMS usually resulted from the loss of CH3OH, CH3O., CH2O, CH3., and H. from the molecular ion. Major fragmentation pathways also resulted in formation of nucleoside, base, ribose, hydrocarbon, and base-hydrocarbon ions. Each of these major ions in turn resulted in further characteristic fragmentation patterns.