Multiple Bond Correlation Spectroscopy Research Articles

Microstructure determination of isobornyl methacrylate-styrene copolymer by NMR spectroscopy

An investigation of the microstructure of isobornyl methacrylate - styrene (I/S) copolymers prepared by the atom transfer radical polymerization (ATRP) using methyl-2-bromopropionate as an initiator and PMDETA copper complex as catalyst under nitrogen atmosphere at 70 °C has been done by two-dimensional NMR techniques. 2D- HSQC and TOCSY have been utilized to resolve the complex 1H NMR spectrum and to establish the compositional and configurational sequences of isobornyl methacrylate-styrene (I/S) copolymers. 2D HSQC and TOCSY spectra showed compositional and configurational sensitivity of α-methyl carbon of I unit and methine proton of S unit and are assigned up to the triad level. The methylene carbon (C10) also shows triad level of compositional sensitivity in 2D HSQC spectra. Heteronuclear multiple-bond correlation (HMBC) spectroscopy has been used to study carbonyl/quaternary carbon-proton coupling. The carbonyl and quaternary carbons showed compositional and configurational sensitivity upto the triad level. The values of reactivity ratios were determined by Kelen-Tudos (KT) and nonlinear error in variable method (RREVM) using copolymer composition data that were determined by 1H NMR spectrum. Reactivity ratios of co-monomers in I/S copolymer, determined from a linear Kelen-Tudos method (KT) and non-linear Error-in-Variable Method (EVM), are rI = 0.39 ± 0.09, rS = 0.44 ± 0.08 and rI = 0.42, rS = 0.47, respectively.

Isosakuranetin-5-O-rutinoside: A New Flavanone with Antidepressant Activity Isolated from Salvia elegans Vahl.

Ursolic acid (1) and a new flavanone, 5-O-(6-rhamnosylglucoside)-7-hydroxy-4'-methoxyflavanone (2), were isolated from the leaves of Salvia elegans Vahl. These natural products displayed antidepressant activity in mice as determined by means of a forced swimming test (FST) evaluation. Structural elucidation was carried out by chemical derivatization (acetylation) and spectroscopic analyses, such as 1H- and 13C-NMR and two-dimensional (2-D) COSY, heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple-bond correlation (HMBC) spectroscopy experiments.

Characterization of an Antibiotic Produced by Bacillus subtilis JW-1 that Suppresses Ralstonia solanacearum

Bacillus subtilis JW-1 was isolated from rhizosphere soil as a potential biocontrol agent of bacterial wilt caused by Ralstonia solanacearum. Seed treatment followed by a soil drench application with this strain resulted in >80% reduction in bacterial wilt disease compared with that in the untreated control under greenhouse conditions. The antibacterial compound produced by strain JW-1 was purified by bioactivity-guided fractionation. Based on mass spectroscopy and nuclear magnetic resonance spectral data ((1)H, (13)C, (1)H-(1)H correlation spectroscopies, rotating frame nuclear Overhauser effect spectroscopy, and heteronuclear multiple-bond correlation spectroscopy), the structure of this compound was elucidated as a cyclic lipopeptide composed of a heptapeptide (Gln-Leu-Leu-Val-Asp-Leu-Leu) bonded to a β-hydroxy-iso-hexadecanoic acid arranged in a lactone ring system.

Antiinflammatory and Antioxidant Flavonoids and Phenols from Cardiospermum halicacabum (倒地鈴 Dào Dì Líng)

Seventeen compounds, quercetin-3-O-α-l-rhamnoside (1), kaempferol-3-O-α-l-rhamnoside (2), apigenin-7-O-β-d-glucuronide (3), apigenin 7-O-β-d-glucuronide methyl ester (4), apigenin 7-O-β-d-glucuronide ethyl ester (5), chrysoeriol (6), apigenin (7), kaempferol (8), luteolin (9), quercetin (10), methyl 3,4-dihydroxybenzoate (11), p-coumaric acid (12), 4-hydroxybenzoic acid (13), hydroquinone (14), protocathehuic acid (15), gallic acid (16), and indole-3-carboxylic acid (17), were isolated from the ethanol extract of Taiwanese Cardiospermum halicabum. All chemical structures were determined by physical and extensive spectroscopic analyses such as 1H Nuclear Magnetic Resonance spectroscopy (NMR), 13C NMR, 1H-1H Correlation spectroscopy (1H-1H COSY), Heteronuclear Multiple Quantum Coherence spectroscopy (HMQC), Heteronuclear Multiple-bond Correlation spectroscopy (HMBC), and Nuclear Overhauser Effect spectroscopy (NOESY), as well as comparison with literature values. Furthermore, the High-Performance Liquid Chromatography-Photodiode Array Detector (HPLC-DAD) fingerprint profile was established for the determination of major constituents in the EtOAc extract and retention times of the isolated compounds. All isolated compounds were also evaluated for antiinflammatory and antioxidant activities.

Antiinflammatory and Antioxidant Flavonoids and Phenols from Cardiospermum halicacabum ( Dào Dì Líng).

Seventeen compounds, quercetin-3-O-α-l-rhamnoside (1), kaempferol-3-O-α-L-rhamnoside (2), apigenin-7-O-β-D-glucuronide (3), apigenin 7-O-β-D-glucuronide methyl ester (4), apigenin 7-O-β-D-glucuronide ethyl ester (5), chrysoeriol (6), apigenin (7), kaempferol (8), luteolin (9), quercetin (10), methyl 3,4-dihydroxybenzoate (11), p-coumaric acid (12), 4-hydroxybenzoic acid (13), hydroquinone (14), protocathehuic acid (15), gallic acid (16), and indole-3-carboxylic acid (17), were isolated from the ethanol extract of Taiwanese Cardiospermum halicabum. All chemical structures were determined by physical and extensive spectroscopic analyses such as 1 H Nuclear Magnetic Resonance spectroscopy (NMR), 13C NMR, 1H-1H Correlation spectroscopy (1H-1H COSY), Heteronuclear Multiple Quantum Coherence spectroscopy (HMQC), Heteronuclear Multiple-bond Correlation spectroscopy (HMBC), and Nuclear Overhauser Effect spectroscopy (NOESY), as well as comparison with literature values. Furthermore, the High-Performance Liquid Chromatography- Photodiode Array Detector (HPLC-DAD) fingerprint profile was established for the determination of major constituents in the EtOAc extract and retention times of the isolated compounds. All isolated compounds were also evaluated for antiinflammatory and antioxidant activities.

Complete NMR assignment of a sulfonated aromatic block copolymer via heteronuclear single-quantum correlation, heteronuclear multiple-bond correlation and heteronuclear single-quantum correlation total correlation spectroscopy

A sulfonated aromatic block copolymer (SABC), consisting of hydrophobic and hydrophilic blocks, was analyzed by heteronuclear single-quantum correlation (HSQC), heteronuclear multiple-bond correlation (HMBC) and HSQC total correlation spectroscopy (HSQC–TOCSY). Because of its complicated chemical structure with five different phenylene rings, 12 types of 1H signals and 24 types of 13C signals were observed in a narrow chemical shift range (7.0–8.0 p.p.m. for 1H and 118–162 p.p.m. for 13C). To improve the 1H signal separation, the temperature conditions for the 1H nuclear magnetic resonance (NMR) experiments were optimized. Moreover, 1H and 13C NMR signal assignments for the hydrophobic blocks were performed using HSQC and HMBC, with reference to the assignments of a model oligomer. For the hydrophilic blocks, furthermore, HSQC–TOCSY techniques were applied. As a result of these studies, complete 1H and 13C NMR signal assignments were made for the SABC. The ion-exchange capacity (IEC) and the copolymerization composition were calculated using the 1H NMR assignments for the SABC, and the IEC value obtained in this way was consistent with that obtained via titration. A sulfonated aromatic block copolymer (SABC), consisting of hydrophobic and hydrophilic blocks, was analyzed by 2D NMR techniques of HSQC, HMBC and HSQC–TOCSY. Because of its complicated chemical structure with five different phenylene rings, 12 types of 1H signals and 24 types of 13C signals were observed in a narrow chemical shift range (7.0–8.0 p.p.m. for 1H and 118–162 p.p.m. for 13C). As a result of these studies, complete 1H and 13C NMR signal assignment of SABC was performed.

STRUCTURAL CHARACTERIZATION AND ANTI-GLYCATION ACTIVITYIN VITROOF A WATER-SOLUBLE POLYSACCHARIDE FROMDENDROBIUM HUOSHANENSE

A water-soluble polysaccharide with anti-glycation activity, designated as DHP-W2, was isolated and purified from the fresh stems of Dendrobium huoshanense. DHP-W2 mainly consisted of glucose, xylose, galactose and trace of galacturonic acid, and its average molecular weight was approximately 73 kDa. Methylation analysis and one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, including 1H-NMR, 13C-NMR, 1H-1H correlation spectroscopy, heteronuclear single quantum correlation spectroscopy and heteronuclear multiple-bond correlation spectroscopy, revealed that the repeating units of DHP-W2 had a backbone consisting of 1,6-linked β-D-Glcp, 1,4-linked β-D-Glcp and 1,4,6-linked β-D-Glcp with branches at O-4/6. Sugar residues of branches were 1,2,4-linked α-D-Xylp, 1,4-linked α-D-Xylp, 1-linked α-D-Xylp, 1-linked α-D-Galp and 1-linked α-D-GalpA. Preliminary experiment in vitro indicated that DHP-W2 had an anti-glycation activity with dose- and time-dependent effects. PRACTICAL APPLICATIONS Because of potential biological and pharmacological functions, polysaccharides from edible and medicinal plants have gained special attention in the food industry. Dendrobium huoshanense C. Z. Tang and S. J. Cheng, 1984, which is a well-known edible and medicinal plant belonging to Orchidaceae family, has been used as a tonic to prevent cataractogenesis, throat inflammation and chronic superficial gastritis for centuries in China. This paper reports the structure features of a homogeneous polysaccharide obtained from D. huoshanense and its anti-glycation activity by a combination of chemical, instrumental and biochemical analysis approaches, including gas chromatography–mass spectrometry (GC-MS), methylation analysis, Fourier transform infrared, one- and two-dimensional nuclear magnetic resonance spectroscopy, and fluorescence spectrophotometry. The paper's purpose was to provide structural information not only for further elucidation of the relationship between structure and function but also for better understanding of pharmacological mechanism of D. huoshanense polysaccharides. It can be expected to develop the functions of the polysaccharides from D. huoshanense in food industries.

Simultaneous C7- and N1-prenylation of cyclo-l-Trp-l-Trp catalyzed by a prenyltransferase from Aspergillus oryzae

A putative prenyltransferase gene cTrpPT was amplified from Aspergillus oryzae DSM1147, cloned into pQE70 and overexpressed in Escherichia coli. The overproduced His(6)-CTrpPT was purified to near homogeneity and incubated with L-tryptophan or tryptophan-containing cyclic dipeptides in the presence of dimethylallyl diphosphate. The formation of the enzyme products was monitored with HPLC. It was shown that CTrpPT differed clearly from other known indole prenyltransferases in several aspects. This enzyme showed higher substrate specificity towards aromatic substrates, but lower regioselectivity regarding the prenylation position than other indole prenyltransferases. Cyclo-L-Trp-L-Trp was much better accepted than other cyclic dipeptides tested in this study. In comparison to other indole prenyltransferases with one dominant enzyme product, at least two product peaks were detected in the reaction mixtures of CTrpPT. (1)H- and (13)C-NMR analyses, including long-range (1)H-(13)C connectivities in Heteronuclear Multiple-Bond Correlation (HMBC) and Nuclear Overhauser Effect Spectroscopy (NOESY), proved the structures of the enzyme products as C7- and N1-prenylated derivatives with a ratio of 1:1.2 using cyclo-L-Trp-L-Trp as substrate. The K(M) values were determined at about 2.5 mM for dimethylallyl diphosphate and 0.3 mM for cyclo-L-Trp-L-Trp with a turnover number of 0.33 s(-1).

Hexa-, hepta- and nonaprenylhydroquinones isolated from marine sponges Sarcotragus muscarum and Ircinia fasciculata inhibit NF-k B signalling in H4IIE cells

Abstract Objectives Marine organisms have proven to be a rich source of potent pharmacologically active compounds. Three polyprenyl-1,4-hydroquinone derivates (hexaprenyl-1,4-hydroquinone, heptaprenyl-1,4-hydroquinone and nonaprenyl-1,4-hydroquinone) were isolated from the Zoobenthos-inhabiting sponges Sarcotragus muscarum and Ircinia fasciculata from the Eastern Mediterranean Sea (phylum: Porifera; class: Demospongiae). Methods Hexa-, hepta- and nonaprenylhydroquinone were identified by 1H-NMR, H,H-COSY, heteronuclear multiple bond correlation, FAB-MS and UV spectroscopy. The effects of the compounds on cell viability was determined using the MTT assay; anti-oxidative potential was measured using the Trolox equivalent antioxidative capacity assay. Inhibition of nuclear factor-k B activity was detected by secreted alkaline phosphatase assay. Activity against an array of protein kinases was determined in 96-well FlashPlates. Key findings All compounds had prominent antioxidative activity, comparable to that of the synthetic vitamin E derivate Trolox. Hexaprenylhydroquinone showed the greatest cytotoxicity in H4IIE hepatoma cells (EC50 2.5 μM). All three compounds inhibited NF-k B signalling in this cell line, with heptaprenylhydroquinone being the most active. Screening of 23 kinases involved in signal transduction pathways (cell proliferation, survival, angiogenesis and metastasis) showed that hexaprenylhydroquinone and heptaprenylhydroquinone inhibited the activity of the epidermal growth factor receptor (IC50 1.6 and 1.4 μg/ml, respectively), and heptaprenylhydroquinone also inhibited the activity of other kinases (Src tyrosine kinase, vascular endothelial growth factor receptor 3 and insulin-like growth factor 1 receptor). Conclusions The prenylated hydroquinones isolated from the marine sponges S. muscarum and I. fasciculata showed cytotoxic and antioxidative activities and inhibited NF-k B signalling in H4IIE hepatoma cells and protein kinases. These findings may result in the generation of new lead substances in cancer therapy.

Structural environments of carboxyl groups in natural organic molecules from terrestrial systems. Part 2: 2D NMR spectroscopy

Carboxyl groups are abundant in natural organic molecules (NOM) and play a major role in their reactivity. The structural environments of carboxyl groups in IHSS soil and river humic samples were investigated using 2D NMR (heteronuclear and homonuclear correlation) spectroscopy. Based on the 1H– 13C heteronuclear multiple-bond correlation (HMBC) spectroscopy results, the carboxyl environments in NOM were categorized as Type I (unsubstituted and alkyl-substituted aliphatic/alicyclic), Type II (functionalized carbon substituted), Type IIIa, b (heteroatom and olefin substituted), and Type IVa, b (5-membered heterocyclic aromatic and 6-membered aromatic). The most intense signal in the HMBC spectra comes from the Type I carboxyl groups, including the 2 J CH and 3 J CH couplings of unsubstituted aliphatic and alicyclic acids, though this spectral region also includes the 3 J CH couplings of Type II and III structures. Type II and III carboxyls have small but detectable 2 J CH correlations in all NOM samples except for the Suwannee River humic acid. Signals from carboxyls bonded to 5-membered aromatic heterocyclic fragments (Type IVa) are observed in the soil HA and Suwannee River FA, while correlations to 6-membered aromatics (Type IVb) are only observed in Suwannee River HA. In general, aromatic carboxylic acids may be present at concentrations lower than previously imagined in these samples. Vibrational spectroscopy results for these NOM samples, described in an accompanying paper [Hay M. B. and Myneni S. C. B. (2007) Structural environments of carboxyl groups in natural organic molecules from terrestrial systems. Part 1: Infrared spectroscopy. Geochim. Cosmochim. Acta (in press)], suggest that Type II and Type III carboxylic acids with α substituents (e.g., –OH, –OR, or –CO 2H) constitute the majority of carboxyl structures in all humic substances examined. Furoic and salicylic acid structures (Type IV) are also feasible fragments, albeit as minor constituents. The vibrational spectroscopy results also suggest that much of the “Type I” signal observed in the HMBC spectrum is due to carboxylic acid esters and possibly α-substituted alicyclic acids.

Comprehensive Sequence Distribution Analysis of Butyl methacrylate-Acrylonitrile Copolymers by Two-dimensional NMR Spectroscopy

The acrylonitrile-butyl methacrylate (A/B) copolymers were synthesized by solution polymerization utilizing 2,2′-azobisisobutyronitrile (AIBN) as free radical initiator. Reactivity ratios were assessed by Kelen-Tudos and non-linear error in variable method and were found to be rA=0.37±0.03, rB=1.44±0.16 (KT) and rA=0.36, rB=1.40 (RREVM) respectively which confirmed that butyl methacrylate monomer is more reactive than acrylonitirile towards propagating chain during radical polymerization. 2D HSQC spectroscopy in conjugation with TOCSY afforded explicit information about compositional and configurational sequences. Methine and β-methylene regions revealed compositional sensitivity up to pentad and tetrad level respectively with meso and racemic configurations. TOCSY demonstrated the coupling between the methine protons of A centered triad and pentad units with β-methylene protons of various tetrads. Heteronuclear multiple-bond correlation (HMBC) spectroscopy has been employed to study carbon (carbonyl/nitrile)-proton coupling. The carbonyl and nitrile carbons expressed compositional sensitivity up to the triad level.

Phosphorus–nitrogen compounds: Synthesis and spectral investigations on new spiro-cyclic phosphazene derivatives

The condensation reaction of { N-[(2-hydroxyphenylmethyl)amino]-4,6-dimethylpyridine} ( 2), which is a reduction product of 1, with trimer N 3P 3Cl 6 affords partially a substituted spiro-cyclic phosphazene derivative ( 3). The fully substituted phosphazenes ( 4 and 5) have also been obtained from the reactions of 3 with the excess of pyrrolidine and morpholine. The characterizations and spectral investigations of these compounds have been made by elemental analyses, FTIR, 1H-, 13C-, 31P NMR, correlation spectroscopy (COSY), heteronuclear chemical shift correlation (HETCOR), heteronuclear multiple-bond correlation (HMBC) and mass spectroscopy (MS). The salient features of spectral data of these compounds have been discussed.

Separation and characterization of alkyl phenol formaldehyde resins demulsifier by adsorption chromatography, gel permeation chromatography, infrared spectrometry and nuclear magnetic resonance spectroscopy

This paper deals with the separation and characterization of alkyl phenol formaldehyde resins demulsifier by infrared spectrometry and nuclear magnetic resonance spectroscopy after separation of the different surfactants and low molecular additives by adsorption chromatography. Firstly, the types of surfactants are identified by methylene blue chloride–chloroform test method and the elemental analysis such as Ca, K, Mg, Na, P, S and N. Then, the different surfactants and low molecular components are separated by adsorption chromatography after parts of low molecular components are dried in an oven, and the molecular weight distribution is measured by gel permeation chromatography also. Finally, the separated surfactants are determined by Fourier transform infrared (FTIR) spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The distortionless enhancement by polarization transfer (DEPT), H, C correlated spectroscopy (H, C–COSY), H, H correlated spectroscopy (H, H–COSY) and heteronuclear multiple-bond correlation (HMBC) spectroscopy are applied to determine the molecular structures.

CONFORMATIONAL ANALYSIS OF 2-(DIPHENYLPHOSPHANYL)-N, N-DIMETHYL-1-BENZ AMIDE AND 2-(DIPHENYLPHOSPHANYL)-PHENYL-PYRROLIDIN-1-YL-METHANONE BY NMR SPECTROSCOPY

We have synthesized 2-(Diphenylphosphanyl)-N, N-dimethyl-1-benzamide (1) and 2-(Diphenylphosphanyl)- phenyl-pyrrolidin-1-yl-methanone (2), and examined their conformations on the basis of NMR spectral data. Conformational analysis of the compounds is useful in deducing the structure in which they are active as a catalyst. In the present NMR measurements, 1H-X fg-JHMBC (field gradient J-Resolved Hetero-nuclear Multiple-Bond Correlation) spectroscopy was implemented as a tool for the determination of hetero-nuclear three bond, phosphorus and protons and carbon-protons coupling constants. By fitting a sine curve to the experimental data by the method of 3D J-resolved HMBC NMR measurements, accurate n J HX coupling constants were obtained. From the coupling constants, the corresponding dihedral angles, H3-C2-C3-P, H12-C12-C11-P, H16-C16-C11-P, H18-C18-C17-P, and H22-C22-C17-P, of compounds (1) and (2) were determined. The optimized structures of the compounds were obtained by molecular orbital calculations in which the dihedral angles experimentally determined were used.

Three new sesquiterpene hemiacetals from Achillea vermicularis

Three new sesquiterpene hemiacetals, tentatively named as achilleanone (1), vermiculone (2) and vermicularone (3) have been isolated from Achillea vermicularis, along with three other known compounds β-amyrin, oleonolic acid and β-sitosterol. The structure elucidation of new compounds was based primarily on two-dimensional (2D) NMR techniques including Nuclear Overhauser Effect/Enhancement (NOE), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC) and nuclear overhauser effect spectroscopy (NOESY) experiments. Compounds 1, 2 and 3 have displayed inhibitory potential against lipoxygenase enzyme in a concentration-dependent fashion with promising IC50 values.

2D NMR studies of acrylonitrile–methyl acrylate copolymers

The microstructure of acrylonitrile–methyl acrylate copolymers prepared by the solution polymerization using 2,2′-azobisisobutyronitrile (AIBN) as free radical initiator was investigated by two-dimensional NMR techniques. 2D-heteronuclear single quantum correlation (HSQC) and the total correlation spectroscopy (TOCSY) have been utilized to resolve the complex 1H NMR spectrum and to establish the compositional and configurational sequences of acrylonitrile–methyl acrylate copolymers. 2D HSQC and TOCSY showed compositional and configurational sensitivity of methine protons of A and M units upto the triad level. Heteronuclear multiple-bond correlation (HMBC) spectroscopy has been used to study carbon (carbonyl/nitrile)–proton coupling. The carbonyl and nitrile carbons showed compositional sensitivity upto the triad level. The values of reactivity ratios were determined by Kelen–Tudos (KT) and non-linear error in variable method (RREVM).

Secretion of an aluminum chelator, 2-isopropylmalic acid, by the budding yeast, Saccharomyces cerevisiae

An aluminum(III)-binding substance (ABS), that solubilizes Al(III) at neutral pH, was found to be secreted by Saccharomyces cerevisiae. A combination of anion exchange chromatography and preparative high performance liquid chromatography using an octadecylsilane (ODS) column separated ABS from the medium. The structure determination of ABS was performed using 1H and 13C NMR spectroscopy and heteronuclear multiple-bond correlation (HMBC) spectroscopy, and ABS was identified to be 2-isopropylmalic acid (2-iPMA). The structure was further confirmed using high resolution electrospray ionization mass spectrometry. Solubilization of otherwise sparingly soluble Al(III) by 2-iPMA at neutral pH indicated the binding of the compound with Al(III). This is supported by 27Al NMR spectrometry for a solution containing 10 mM Al(III) and 20 mM 2-iPMA at pH 6.6, where four Al(III) species were evident. Although the function of this compound is unclear, it might play a key role in Al detoxification.

Eurycomaoside: A New Quassinoid‐Type Glycoside from the Roots of Eurycoma longifolia.

A new C(19)-quassinoid-type glycoside has been isolated from the roots of Eurycoma longifolia. The structure elucidation of the compound was achieved by a combination of one- and two-dimensional NMR techniques, including (1)H-(1)H-correlation spectroscopy (COSY), (1)H-(13)C-heteronuclear correlation spectroscopy (HMQC), and (1)H-(13)C-heteronuclear multiple-bond correlation spectroscopy (HMBC), as well as high resolution electrospray ionization Fourier transformation mass spectrometry (HR-ESI-FT-MS) data. The C(1)-glycosidation site in the quassinoid framework is encountered for the first time.

Eurycomaoside: a new quassinoid-type glycoside from the roots of Eurycoma longifolia.

A new C(19)-quassinoid-type glycoside has been isolated from the roots of Eurycoma longifolia. The structure elucidation of the compound was achieved by a combination of one- and two-dimensional NMR techniques, including (1)H-(1)H-correlation spectroscopy (COSY), (1)H-(13)C-heteronuclear correlation spectroscopy (HMQC), and (1)H-(13)C-heteronuclear multiple-bond correlation spectroscopy (HMBC), as well as high resolution electrospray ionization Fourier transformation mass spectrometry (HR-ESI-FT-MS) data. The C(1)-glycosidation site in the quassinoid framework is encountered for the first time.

Structural Identification of 2′- and 3′-O-Acetyl-ADP-ribose as Novel Metabolites Derived from the Sir2 Family of β-NAD+-dependent Histone/Protein Deacetylases

The Sir2 (silent information regulator 2) family of histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life-span extension, and chromosomal stability. Their dependence on beta-NAD(+) for activity is unique among the known classes of histone/protein deacetylase. Sir2 enzymes have been shown to couple substrate deacetylation and beta-NAD(+) cleavage to the formation of O-acetyl-ADP-ribose, a newly described metabolite. To gain a better understanding of the catalytic mechanism and of the biological implications of producing this molecule, we have performed a detailed enzymatic and structural characterization of O-acetyl-ADP-ribose. Through the use of mass spectrometry, rapid quenching techniques, and NMR structural analyses, 2'-O-acetyl-ADP-ribose and 3'-O-acetyl-ADP-ribose were found to be the solution products produced by the Sir2 family of enzymes. Rapid quenching approaches under single-turnover conditions identified 2'-O-acetyl-ADP-ribose as the enzymatic product, whereas 3'-O-acetyl-ADP-ribose was formed by intramolecular transesterification after enzymatic release into bulk solvent, where 2'- and 3'-O-acetyl-ADP-ribose exist in equilibrium (48:52). In addition to (1)H and (13)C chemical shift assignments for each regioisomer, heteronuclear multiple-bond correlation spectroscopy was used to assign unambiguously the position of the acetyl group. These findings are highly significant, because they differ from the previous conclusion, which suggested that 1'-O-acetyl-ADP-ribose was the solution product of the reaction. Possible mechanisms for the generation of 2'-O-acetyl-ADP-ribose are discussed.