Journal articles on the topic 'Mass spectrometers; structure elucidation; ion-molecular reactions'

Gas chromatography–high-resolution mass spectrometry (GC–HRMS) is a powerful nontargeted screening technique that promises to accelerate the identification of environmental pollutants. Currently, most GC–HRMS instruments are equipped with electron ionization (EI), but atmospheric pressure ionization (API) ion sources have attracted renewed interest because: (i) collisional cooling at atmospheric pressure minimizes fragmentation, resulting in an increased yield of molecular ions for elemental composition determination and improved detection limits; (ii) a wide range of sophisticated tandem (ion mobility) mass spectrometers can be easily adapted for operation with GC–API; and (iii) the conditions of an atmospheric pressure ion source can promote structure diagnostic ion–molecule reactions that are otherwise difficult to perform using conventional GC–MS instrumentation. This literature review addresses the merits of GC–API for nontargeted screening while summarizing recent applications using various GC–API techniques. One perceived drawback of GC–API is the paucity of spectral libraries that can be used to guide structure elucidation. Herein, novel data acquisition, deconvolution and spectral prediction tools will be reviewed. With continued development, it is anticipated that API may eventually supplant EI as the de facto GC–MS ion source used to identify unknowns.

Lipid A, the membrane-bound phosphoglycolipid component of bacteria, is held responsible for the clinical syndrome of gram-negative sepsis. In this study, the fragmentation behavior of a set of synthetic lipid A derivatives was studied by electrospray ionization multistage mass spectrometry (ESI-MSn), in conjunction with tandem mass spectrometry (MS/MS), using low-energy collision-induced dissociation (CID). Genealogical insight about the fragmentation pathways of the deprotonated 4’-monophosphoryl lipid A structural analogs led to proposals of a number of alternative dissociation routes that have not been reported previously. Each of the fragment ions was interpreted using various possible mechanisms, consistent with the principles of reactions described in organic chemistry. Specifically, the hypothesized mechanisms are: (i) cleavage of the C-3 primary fatty acid leaves behind an epoxide group attached to the reducing sugar; (ii) cleavage of the C-3’ primary fatty acid (as an acid) generates a cyclic phosphate connected to the nonreducing sugar; (iii) cleavage of the C-2’ secondary fatty acid occurs both in acid and ketene forms; iv) the C-2 and C-2’ primary fatty acids are eliminated as an amide and ketene, respectively; (v) the 0,2A2 cross-ring fragment contains a four-membered ring (oxetanose); (vi) the 0,4A2 ion is consecutively formed from the 0,2A2 ion by retro-aldol, retro-cycloaddition, and transesterification; and (vii) formations of H2PO4− and PO3− are associated with the formation of sugar epoxide. An understanding of the relation between 0,2A2 and 0,4A2-type sugar fragments and the different cleavage mechanisms of the two ester-linked primary fatty acids is invaluable for distinguishing lipid A isomers with different locations of a single ester-linked fatty acid (i.e., at C-3 or C-3’). Thus, in addition to a better comprehension of lipid A fragmentation processes in mass spectrometers, our observations can be applied for a more precise elucidation of naturally occurring lipid A structures.

Abstract A reversed-phase liquid chromatographic method combined with fluorescence and multiple mass spectrometric detection in series is presented for the separation and structure elucidation of bisphenol A diglycidyl ether (BADGE) and novolac glycidyl ether (NOGE) derivatives. Atmospheric pressure chemical ionization in the positive ion mode and collision induced fragmentation in the ion trap allowed identification of BADGE- and NOGE-related compounds originating from reactions of the glycidyl ethers with bisphenols, solvents, and chain stoppers. Two extracts from food-can coatings were investigated in detail. It was possible to elucidate the structures of many substances and consequently to draw conclusions about the production of the lacquers.

The energy barrier for the keto–enol isomerization of the isolated acetone ion to its distonic (enol) isomer lies above its lowest dissociation limit and so the spontaneous isomerization can never be observed. Keto–enol isomerizations can be catalyzed within appropriate ion–molecule complexes. The present study involved two systems, [(CH3)2C=O···H+···O(H)CH2·] (ion 1) and [(CH3)2C=O···H+····OCH3] (ion 2), in both stable and metastable adducts. When acetone is bound to ·CH2OH though a proton bridge, shown as ion 1, an enol acetone ion is produced. This reaction results from a proton attaching to the acetone, which then gives an H· atom back to the radical site by a 1,6-H transfer, involving a transition state of low energy requirement. In contrast, when the acetone is protonated and bound to the radical CH3O· (ion 2), the above rearrangement does not take place. The metastable complex ion 2 loses a methyl radical, producing a new [C3H7O2]+ isomer of structure [CH3C+(O)···(H)OCH3]. Tandem mass spectrometry combined with ab initio calculations were used to investigate the two systems. Potential energy surface diagrams were obtained by calculations at the MP2/6-31+G(d) level of theory to aid further elucidation of the reaction mechanisms. Key words: ion–molecule complexes, keto–enol mechanisms, ion rearrangements and structures.

The stereochemistry of organic ions in the gas phase can be regarded from two different points of view: (i) stereoselectivity in ion formation and (ii) stereospecifity of ion fragmentations. Fast ionization by electron or photon impact shows little stereoselection. Differences in the ionization energies and cross sections between stereoisomers are generally small, save for a few exceptions. Proton or larger ion transfer, as employed in chemical ionization mass spectrometry, gives more possibilities for stereoselection. Bi- or polyfunctional molecules can capture the proton in a hydrogen-bond stabilized [M + H]+ ion, which is feasible only with a favourable spatial orientation of the chelating groups. Adduct ions [M + R]+ can also be formed stereoselectively. The use of a chiral ionizing medium adds a new dimension, since enantiomers can be distinguished, or even independently identified. The stereochemistry of even-electron cations in the gas-phase is most pronounced with polyfunctional species. The stereochemical behaviour is ruled by two reactivity principles, i.e. the geometry-dependent stabilization of [M + H]+ ions by chelation, and the anchimeric assistance by neighbouring groups in elimination of small molecules (water, ammonia, alcohols, acetic acid, etc.). The stereochemistry of odd-electron cations seems to be governed by three principles, i.e. the thermochemistry of decompositions proceeding with simple-bond cleavage, stereoelectronic effects on bond dissociations in the presence of a control orbital, and long-range interactions resulting in transfer of a hydrogen atom or a larger group. All these three reaction classes have limited areas of application. The stereochemistry of even-electron anions has been developing rapidly. The reactivity of gas-phase anions finds numerous analogies in their chemistry in solution, e.g. hydride transfer reactions and nucleophilic substitution. The applications of mass spectrometry to configurational assignment and structure elucidation remain restricted to selected classes of organic compounds.

A structural investigation of perfluorocarboxylic acid derivatives formed in the reaction with N,N-dimethylformamide dialkylacetals employing several techniques of mass spectrometry (MS) is described. Two derivatizing reagents, dimethylformamide dimethyl acetal (DMF-DMA) and dimethylformamide diethylacetal (DMF-DEA) were used. In contrast to carboxylic acids, perfluorocarboxylic acids are not able to form alkyl esters as the main product in this reaction. We found that perfluorooctanoic acid (PFOA) forms a salt with N,N-dimethylformamide dialkylacetals. This salt undergoes a further reaction inside the injection block of a gas chromatograph (GC) by loss of CO2 and then forms 1,1-perfluorooctane-(N,N,N,N-tetramethyl)-diamine. The GC-MS experiments using both electron ionization (EI) and positive-ion chemical ionization (PCI) revealed that the same reaction products are formed with either derivatizing reagent. Subjecting the perfluorocarboxylic acid derivative to electrospray ionization (ESI) and direct analysis in real time (DART), both positive- and negative-ion modes indicated that cluster ions are formed. In the positive-ion mode, this cluster ion consists of two iminium cations and one PFOA anion, while in the negative-ion mode, it comprises two PFOA anions and one cation. The salt structure was further confirmed by liquid injection field desorption/ionization (LIFDI) as well as infrared (IR) spectroscopy. We propose a simple mechanism of N,N,N′,N′-tetramethylformamidinium cation formation. The structure elucidation is supported by specific fragment ions as obtained by GC-EI-MS and GC-PCI-MS analyses.

Abstract. Guaiacol (2-methoxyphenol) and its derivatives can be emitted into the atmosphere by thermal degradation (i.e., burning) of wood lignins. Due to its volatility, guaiacol is predominantly distributed atmospherically in the gaseous phase. Recent studies have shown the importance of aqueous-phase reactions in addition to the dominant gas-phase and heterogeneous reactions of guaiacol, in the formation of secondary organic aerosol (SOA) in the atmosphere. The main objectives of the present study were to chemically characterize the main products of the aqueous-phase photonitration of guaiacol and examine their possible presence in urban atmospheric aerosols. The aqueous-phase reactions were carried out under simulated sunlight and in the presence of hydrogen peroxide and nitrite. The formed guaiacol reaction products were concentrated by solid-phase extraction and then purified with semi-preparative high-performance liquid chromatography (HPLC). The fractionated individual compounds were isolated as pure solids and further analyzed with liquid-state proton, carbon-13 and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, and direct infusion negative ion electrospray ionization tandem mass spectrometry ((−)ESI-MS/MS). The NMR and product ion (MS2) spectra were used for unambiguous product structure elucidation. The main products of guaiacol photonitration are 4-nitroguaiacol (4NG), 6-nitroguaiacol (6NG), and 4,6-dinitroguaiacol (4,6DNG). Using the isolated compounds as standards, 4NG and 4,6DNG were unambiguously identified in winter PM10 aerosols from the city of Ljubljana (Slovenia) by means of HPLC/(−)ESI-MS/MS. Owing to the strong absorption of ultraviolet and visible light, 4,6DNG could be an important constituent of atmospheric "brown" carbon, especially in regions affected by biomass burning.

Abstract. Guaiacol (2-methoxyphenol) and its derivatives can be emitted into the atmosphere by thermal degradation (i.e. burning) of wood lignins. Due to its volatility, guaiacol is predominantly distributed in the atmospheric gaseous phase. Recent studies have shown the importance of aqueous-phase reactions in addition to the dominant gas-phase and heterogeneous reactions of guaiacol, in the formation of secondary organic aerosol (SOA) in the atmosphere. The main objectives of the present study were to chemically characterize the low-volatility SOA products of the aqueous-phase photonitration of guaiacol and examine their possible presence in urban atmospheric aerosols. The aqueous-phase reactions were carried out under simulated sunlight and in the presence of H2O2 and nitrite. The formed guaiacol reaction products were concentrated by using solid-phase extraction (SPE) and then purified by means of semi-preparative high-performance liquid chromatography (HPLC). The fractionated individual compounds were isolated as pure solids and further analyzed with liquid-state 1H, 13C and 2D nuclear magnetic resonance (NMR) spectroscopy and direct infusion negative ion electrospray ionization tandem mass spectrometry ((–)ESI-MS/MS). The NMR and product ion (MS2) spectra were used for unambiguous product structure elucidation. The main products of guaiacol photonitration are 4-nitroguaiacol (4NG), 6-nitroguaiacol (6NG), and 4,6-dinitroguaiacol (4,6DNG). Using the isolated compounds as standards, 4NG and 4,6DNG were unambiguously identified in winter PM10 aerosols from the city of Ljubljana (Slovenia) by means of HPLC/(–)ESI-MS/MS. Owing to the strong absorption of UV and visible light, 4,6DNG could be an important constituent of atmospheric "brown" carbon, especially in regions affected by biomass burning.

Abstract. Chemical ionization mass spectrometry with the nitrate reagent ion (NO3- CIMS) was used to investigate the products of the nitrate radical (NO3) initiated oxidation of four monoterpenes in laboratory chamber experiments. α-Pinene, β-pinene, Δ-3-carene, and α-thujene were studied. The major gas-phase species produced in each system were distinctly different, showing the effect of monoterpene structure on the oxidation mechanism and further elucidating the contributions of these species to particle formation and growth. By comparing groupings of products based on the ratios of elements in the general formula CwHxNyOz, the relative importance of specific mechanistic pathways (fragmentation, termination, and radical rearrangement) can be assessed for each system. Additionally, the measured time series of the highly oxidized reaction products provide insights into the ratio of relative production and loss rates of the high-molecular-weight products of the Δ-3-carene system. The measured effective O:C ratios of reaction products were anticorrelated with new particle formation intensity and number concentration for each system; however, the monomer : dimer ratios of products had a small positive trend. Gas-phase yields of oxidation products measured by NO3- CIMS correlated with particle number concentrations for each monoterpene system, with the exception of α-thujene, which produced a considerable amount of low-volatility products but no particles. Species-resolved wall loss was measured with NO3- CIMS and found to be highly variable among oxidized reaction products in our stainless steel chamber.

Acanthus ilicifolius herb (AIH), the dry plant of Acanthus ilicifolius L., has long been used as a folk medicine for treating acute and chronic hepatitis. Phenylethanoid glycosides (PhGs) are one family of the main components in AIH with hepatoprotective, antioxidant, and anti-inflammatory activities. In this study, the pharmacokinetics of AIH was investigated preliminarily by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS). A simultaneously quantitative determination method for four PhGs (acteoside, isoacteoside, martynoside, and crenatoside) in rat plasma was first established by UPLC-MS/MS. These four PhGs were separated with an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) by gradient elution (mobile phase: MeCN and 0.1% formic acid in water, 0.4 mL/min). The mass spectrometry detection was performed using negative electrospray ionization (ESI−) in multiple reaction monitoring (MRM) mode. By the established method, the preliminary pharmacokinetics of AIH was elucidated using the kinetic parameters of the four PhGs in rat plasma after intragastric administration of AIH ethanol extract. All four PhGs showed double peaks on concentration-time curves, approximately at 0.5 h and 6 h, respectively. Their elimination half-lives (t1/2) were different, ranging from 3.42 h to 8.99 h, although they shared similar molecular structures. This work may provide a basis for the elucidation of the pharmacokinetic characteristics of bioactive components from AIH.

Biotransformation reactions that xenobiotics undergo during their metabolism are crucial for their proper excretion from the body, but can also be a source of toxicity, especially in the case of reactive metabolite formation. Unstable, reactive metabolites are capable of covalent binding to proteins, and have often been linked to liver damage and other undesired side effects. A common technique to assess the formation of reactive metabolites employs trapping them in vitro with glutathione and characterizing the resulting adducts by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Some endogenous compounds, however, can interfere with xenobiotic metabolites of interest, making the analysis more difficult. This study demonstrates the usefulness of isotope-labeled compounds to detect and elucidate the structures of both stable metabolites and trapped adducts of three estrogen analogs using an untargeted LC-MS/MS workflow. The metabolism of estradiol, estrone and ethinyl estradiol was investigated. Unlabeled and deuterated versions of these three compounds were incubated with human or rat liver microsomes in the presence of two different trapping agents, namely glutathione and N-acetylcysteine. The detection of closely eluting deuterated peaks allowed us to confirm the formation of several known metabolites, as well as many previously uncharacterized ones. The structure of each adduct was elucidated by the detailed analysis of high-resolution MS/MS spectra for elucidating fragmentation pathways with accurate mass measurements. The use of isotopic labeling was crucial in helping confirm many metabolites and adduct structures, as well as removing endogenous interferences.

Sesquiterpene lactones (STLs) from the cocklebur Xanthium sibiricum exhibit significant anti-tumor activity. Although germacrene A oxidase (GAO), which catalyzes the production of Germacrene A acid (GAA) from germacrene A, an important precursor of germacrene-type STLs, has been reported, the remaining GAOs corresponding to various STLs’ biosynthesis pathways remain unidentified. In this study, 68,199 unigenes were studied in a de novo transcriptome assembly of X. sibiricum fruits. By comparison with previously published GAO sequences, two candidate X. sibiricum GAO gene sequences, XsGAO1 (1467 bp) and XsGAO2 (1527 bp), were identified, cloned, and predicted to encode 488 and 508 amino acids, respectively. Their protein structure, motifs, sequence similarity, and phylogenetic position were similar to those of other GAO proteins. They were most strongly expressed in fruits, according to a quantitative real-time polymerase chain reaction (qRT-PCR), and both XsGAO proteins were localized in the mitochondria of tobacco leaf epidermal cells. The two XsGAO genes were cloned into the expression vector for eukaryotic expression in Saccharomyces cerevisiae, and the enzyme reaction products were detected by gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) methods. The results indicated that both XsGAO1 and XsGAO2 catalyzed the two-step conversion of germacrene A (GA) to GAA, meaning they are unlike classical GAO enzymes, which catalyze a three-step conversion of GA to GAA. This cloning and functional study of two GAO genes from X. sibiricum provides a useful basis for further elucidation of the STL biosynthesis pathway in X. sibiricum.

The lithium ion battery (LIB) electrolyte is a delicate system susceptible to a manifold of influences both from the outside and inside. Thermal stress and overcharging of the LIB can result in unwanted side reactions such as rapid electrolyte degradation and the so-called thermal runaway, respectively. Here, the degradation of the conducting salt, namely lithium hexafluorophosphate (LiPF6 )poses many risks to the LIB and the environment. Hydrolysis of LiPF6 leads to the formation of hydrofluoric acid (HF) and POF3 which can react with the organic compounds within the electrolyte to form highly toxic organofluorophosphates (OFPs). The formation of OFPs in LIBs due to thermal stress has been covered in literature. In this study the electrochemically induced formation of OFPs is discussed. Electrolytes containing fluoroethylene carbonate (FEC) as a film-forming additive indicated the presence of OFPs forming at operation voltages of 4.8 V in self-assembled LIB coin cells. Cycling of the coin cells with cut-off voltages of 4.8 V gave rise to 15 non-acidic and two acidic OFPs after 10 charge/discharge steps. The quantity of FEC had an impact on the amount of OFPs formed which gives reason to incorporate FEC in the formation pathways proposed in this study. The formation pathway of OFPs through EC-polymerization proposed in literature is evaluated and an alternative mechanism with FEC as the carbonyl-carbon donor is presented. Structure elucidation and separation of the formed OFPs is achieved by utilization of a) hydrophilic interaction liquid chromatography (HILIC) for acidic OFPs and b) reversed-phase (RP) chromatography for non-acidic OFPs hyphenated to a high-resolution ion trap time-of-flight mass spectrometer (IT-TOF-MS). Based on the findings in this study and due to the highly toxic nature of OFPs further investigation of the formation of OFPs in FEC-containing electrolytes is necessary. Further, the impact of the operation voltages needs to be assessed to ensure for safe and efficient LIBs.

Stevia rebaudiana (Bertoni) Bertoni is a plant species native to Brazil and Paraguay well-known by the sweet taste of their leaves. Since the recognition of rebaudioside A and other steviol glycosides as generally recognized as safe by the United States Food and Drug Administration in 2008 and grant of marketing approval by the European Union in 2011, the species has been widely cultivated and studied in several countries. Several efforts have been dedicated to the isolation and structure elucidation of minor components searching for novel non-caloric sugar substitutes with improved organoleptic properties. The present review provides an overview of the main chemical approaches found in the literature for identification and structural differentiation of diterpene glycosides from Stevia rebaudiana: High-performance Thin-Layer Chromatography, High-Performance Liquid Chromatography, Electrospray Ionization Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy. Modification of diterpene glycosides by chemical and enzymatic reactions together with some strategies to scale up of the purification process saving costs are also discussed. A list of natural diterpene glycosides, some examples of chemically modified and of enzymatically modified diterpene glycosides reported from 1931 to February 2021 were compiled using the scientific databases Google Scholar, ScienceDirect and PubMed.

A new screening technology that combines biochemical analysis with the resolution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capability of the HRS technology to analyze biologically active compounds in complex mixtures is demonstrated by screening a plant natural product extract library for estrogen receptor (ER) a and fi binding activity. The simultaneous structure elucidation of biologically active components in crude extracts was achieved by operating the HRS system in combination with mass spectrometry (MS). In contrast to conventional microtiter-type bioassays, the interactions of the extracts with the ER and the employed label, coumestrol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a HPLC separation column. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flow-through fluorescence detector. Primary screening of the extract library was performed in the fast-flow injection analysis mode (FlowScreening) wherein the chromatographic separation system was bypassed. The library was screened at high speed, using two assay lines in parallel. A total of 98% of the identified hits were confirmed in a traditional 96-well microplate-based fluorescence polarization assay, indicating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the functional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in combination with MS. Information on the number of active compounds, their retention times, the molecular masses, and the MS/MS-fingerprints as a function of their biological activity was obtained from 50% of the functional active extracts in real time. This dramatically enhances the speed of biologically active compound characterization in natural product extracts compared to traditional fractionation approaches.

Isolation, characterization of natural products dimeric amide alkaloids from roots of the Piper chaba Hunter. The synthesis of these products using intermolecular [4+2] cycloaddition reaction has been described. Obtained products were characterized using IR, 1HNMR, 13CNMR and Mass Spectroscopy. Introduction The awesome structural diversity and complexity of natural products inspire many chemists to consider how nature creates these molecules. Nature’s biosynthetic enzymes offer a powerful and practical route to many organic compounds, and synthetic chemists sometimes seek to imitate the efficiency and elegance of the biosynthetic machinery by designing biomimetic reactions that approximate natural reaction pathways. Probably the most astonishing biomimetic reactions1 are tandem processes that combine several transformations in sequence and produce complicated structures from comparably simple starting materials in a single laboratory operation. Biosynthesis is described as “the reaction or reaction sequence occurred in organism or its immediate environment will be viewed as biosynthesis” where as biomimetic synthesis describes as “A specific reaction or a sequence of reactions that mimic a proposed biological pathway is defined as bimimetic synthesis. An early example is Sir Robert Robinson’s landmark synthesis of tropinone in 1917.2 Forty-two years later, Gilbert Stork and Albert Eschenmoser independently proposed that the steroid ring system could be formed by tandem cation-π cyclizations of a polyene in an ordered transition state.3 A non-enzymatic version of this reaction type was demonstrated in W. S. Johnson’s classic synthesis of progesterone in 1971.4 Chapman’s synthesis of carpanone is a striking example of the power of biomimetic strategies.5 In 1980, Black proposed that the endiandric acids could arise biosynthetically from linear polyenes.6 In 1982, K. C. Nicolaou gave chemical support to Black’s hypothesis by chemically synthesizing endiandric acids A-G.7 Biomimetic Synthesis of Natural Products which involves, The biomimetic polyene carbocyclizations reaction, The biomimetic cycloaddition reaction, The biomimetic electrocyclization reaction, The polyether biomimetic synthesis, The biomimetic oxidative coupling of phenol, Some other interesting biomimetic synthesis, The present biomimetic synthesis of chabamides or dimeric amide alkaloids involves cycloaddition reactions. The Diels Alder reaction In the Diels-Alder reaction a six membered ring is formed through fusion of a 4 π component, usually a diene and a 2 π component which is commonly referred to as the Figure 1. dienophile. The Diels Alder reaction has proven to be great synthetic value, forming a key-step in the construction of compounds containing six-membered rings. Cyclohexene ring generated all the way through the formation of two new σ-bonds and one π bond with four adjacent stereocenters. The reaction is named after Otto Diels and Kurt Alder, two German chemists who studied the synthetic and theoretical aspects of this reaction in great detail.8 Their efforts have been rewarded with the 1950 Noble prize. Figure 2 Schematic representation of the Diels-Alder reaction. Cis principle In Diels-Alder reactions, the stereoselectivity is generally high due to the “cis principle”, which states that Diels-Alder reactions require a cisoid conformation for the diene and suprafacial-suprafacial mode of reaction, meaning that both ends of the diene attack from the same face of the dienophile in a syn fashion. Frontier Molecular Orbital (FMO) Approach Diels-Alder rections can be devided into, normal electron demand and inverse electron demand additions. This difference is based on the way the rate of the reaction responds to the introduction of electron withdrawing and electron donating substituents. Normal electron demand Diels-Alder reactions are promoted by electron donating substituents on the diene and electron withdrawing substituents on the dienophile. In contrast, inverse electron demand reactions are accelerated by electron withdrawing substituents on the diene and electron donating ones on the dienophile. There also exists an intermediate class, the neutral Diels-alder reaction, which is accelerated by both electron withdrawing and donating substitutents. The way the substituents affect the rate of the reaction can be rationalized with aid of Frontier Molecular Orbital (FMO) theory. This theory was developed during a study of the role of orbital asymmetry in pericyclic reactions by Woodward and Hoffmann9 and, independently, by fukui10 Later, Houk contributed significantly to the understanding of the reactivity and selectivity of these processes.11 The FMO theory states that a reaction between two compounds is controlled by the efficiency with which the molecular orbitals of the individual reaction partners interact. The interaction is most efficient for the reactivity is completely determined by interactions of the electrons that are highest in energy of the of the reaction partners (those in the Highest Occupied Molecular Orbital, the HOMO) with the Lowest Unoccupied Molecular Orbital (LUMO) of the other partner, applied to the Diels-alder reactions, two modes of interaction are possible. The reaction can be controlled by the interaction of the HOMO of the diene and the LUMO of the Dienophile (normal electron demand), or by the interaction between the LUMO of the diene and the HOMO of the dienophile (inverse electron demand), as illustrated in Fig-B. In the former case, a reduction of the diene-HOMO and dienophile-LUMO energy gap can be realized by either raising the energy of the HOMO of the diene by introducing electron donating substituents or lowering the energy of the dienophile LUMO by the introduction of electron donating substituents or lowering the energy of the dienophile LUMO by the introduction of electron withdrawing substituents. A glance at Fig-A confirms that in the formation of two new bonds, orbital symmetry is conserved so that, according to Woodward and Hoffmann, the reaction is concerted. In other words, no intermediate is involved in the pericyclic process such as the Diels-Alder reaction.12 This conclusion is consistent with a number of experimental observations. The cis or trans conformation of the dienophile is fully conserved in the configuration of the cycloadduct, which proves that there is no intermediate involved with a lifetime long enough to allow rotation around C-C bond. Selectivity can arise when substituted dienes and dienophiles are employed in the Diels-Alder reaction. Two different cycloadducts denoted as endo and exo are possible. Under the usual conditions their ratio is kinetically controlled. Alder and Stein already discerned that there usually exists a preference for formation of the endo isomer i.e formulated as tendency of maximum accumulation of unsaturation, (the Alder-Stein rule)13 Indeed, there are only very few examples of Diels-Alder reactions where the exo isomer is major product.14 The interactions underlying this behavior have been subject of intensive research. Since the reactions leading to endo and exo product share the same initial state, the difference between the respective transition-state energies fully account for the observed selectivity. These differences are typically in the range of 10-15 kJ per mole.15 Woodward and Katz16 suggested that secondary orbital interactions are of primary importance. These interactions are illustrated in fig-B for the normal electron demand (HOMO-diene, LUMO-dienophile controlled). The symmetry allowed overlap between π-orbital of the carbonyl group of the dienophile and the diene-HOMO is only possible in the endo activiated complex. Hence, only the endo transition state is stabilized so that the reaction forming the endo adduct is faster than that yielding exo product. This interpretation has been criticized by Mellor, who attributed the endo selectivity to steric interactions. Steric effects are frequently suggested as important in determining the selectivity of Diels-Alder reactions, particularly of α-subsituted dienophiles, and may ultimately lead to exo-selectivity. 17 For other systems, steric effects in the exo activated complex can enhance endo selectivity. 18 In summary, it seems for most Diels-Alder reactions secondary orbital interactions afford a satisfactory rationalization of the endo-exo selectivity. However, since the endo-exo ratio is determined by small differences in transition state energies, the influence of other interactions, most often steric in origin and different for each particular reaction is likely to be felt. The compact character of the Diels-Alder activated complex (the activation volume of the retro Diels-Alder reaction is negative) will attenuate these effects.19 Results and Discussions Chabamides F & G as dimeric amide alkaloids were isolated from this plant Piper chaba Hunter. These two dimers were formed by Diels-Alder reaction employing monomer trichostachine. This hypothesis was further confirmed by the mass spectrum, which showed a significant peak at m/z 294.113 [M++Na], assigned to the trichostachine ion arising by the Retro-Diels–Alder cleavage of molecular ion into two halves. Finally, to confirm the existence of the compounds F and G, we extracted the roots of P.chaba with MeOH at room temperature followed HPLC/electron spray ionization (ESI) MS experiments. In HPLC/ESIMS of the MeOH extract showed the presence of peaks at m/z 563 [M++Na] and 543 [M++1] at about 8.8 min and 10.6 min of LC retention time, respectively. To prove this biosynthetic hypothesis we have carried out the intermolecular [4+2] cycloaddition reaction with the trichostachine under solvent free conditions (Scheme 1). Reaction mixture was analysed by the LC-MS, which clearly indicted the presence of the compounds 1 and 2 (retention time and mass). In HPLC analysis, retention times of the synthetic 1 and 2 were identical to those of chabamide F and G, confirming the structure and stereochemistry are same as that of isolated alkaloids. Based on above result during Diels-Alder reaction of trichostachine, we developed two kinds of methodologies for this biomimetic synthesis of dimeric amide alkaloids based on catalytic. On the basis of a biosynthetic hypothesis (described in Chapter I) by the intermolecular Diels-Alder reaction, we chosen piperine (1a), pellitorine (1c) and trans-fagaramide (1c) as substrates to perform the biomimetic synthesis of the dimeric chabamides (Compound H-K) and this study also identified plausible products between piperine (1a) and pellitorine (1c). This study not only explains formation of cyclo adducts but also explains the different mechanistic aspects in Diels-Alder reaction (endo and exo products) of copper salts in aqueous medium. Under normal conditions only combinations of dienes and dienophiles that have FMO’s of similar energy can be transformed into a Diels-Alder adduct. When the gap between the FMO’s large, forcing conditions are required, and undesired side reactions and retro Diels-Alder reactions can easily take over. These cases challenge the creativity of the organic chemist and have led to the invention of a number of methods for promoting reluctant Diels-Alder reactions under mild conditions.20 Plausible mechanism for Diels-Alder reaction: Sijbren Otto. et. al studied extensively on copper (II) catalyzed Diels-Alder reactions on various moieties. 25, 26 Based on these reports we proposed plausible mechanism for this copper catalyzed Diels-Alder reaction. The first step in the cycle comprises rapid coordination of the lewis acid to the dienophile leading to a complex in which the dienophile is activated for reaction with the diene. The cycloadduct has dissociated from the lewis acid in order to make the catalyst available for another cycle. However we didn’t carry any kinetic studies to prove this mechanism. Plausible mechanism of Diels-Alder reaction catalyzed by copper (II) salts Use of lewis acids in Diels-Alder reaction is to lower LUMO dienophile energy to result in the decrease of the LUMO dienophile-HOMO diene gap (normal electron demand) or reduce LUMO diene energy to result in the decrease of the LUMO diene-HOMO dienophile gap (inverse electron demand). The presence of Lewis acids, the Diels-Alder dimerization of piperine, pellitorine, piperine with fagaramide, peperine with pellitorine, gave much lower combined yields in neat conditions. Wie et al. previously reported 21, 22 Diels-Alder reaction of piperine and in both thermal and by lewis acid of Co(II) Cl2.6H2O/P(Ph)3/Zn (1:10:10 mol %) in 3-octanol at 170oC with isomerised product (24 %) and 77 % over all yield. To find the optimum conditions towards the catalyst, piperine (1a) was taken to perform the Diels-Alder reaction in presence of variety of lewis acids and metal salts (Table 1). The highest catalytic activity was attained for the reaction using 10 mol % of Cu (II) salts. The role of copper salts in this reaction can be attributed to its Lewis acid ability, which enhances both the electron donating capacity of diene and electron withdrawing capacity of the dienophile (required for normal electron demand for Diels-Alder reaction). The The catalytic activitiy of Lewis acids like Cu+2 mainly relies on their coordinating character to assemble both dienophile and diene to such a way that promote the reaction to wards the reaction barrier. To find the optimum conditions towards the solvent several reactions were carried out under the solvents like benzene, toluene, xylene, water and results were tabulated (Table 2). Among organic solvents xylene is better to get considerable yield with copper salts. Later water was found to be the best for both yield and selectivity of this cycloaddition. Cycloaddition reactions of piperine (1a): Lewis acids catalyzed cycloaddition reactions (Scheme 2) of piperine (1a) under organic and aqueous solvent conditions to give resultant cycloadducts 2a, 3a, 4a, 5a and 6a, among them 2a is major ortho-exo cyclohexene type dimeric amide alkaloid and also known as chabamide, which is previously isolated23 from this plant, isomer 3a is previously isolated from Piper nigrum21 Remaining isomers (4a-6a) were synthesized from piperine by Diels-Alder reaction by Kun Wei. et al. its physical and spectroscopic data were identical with reported data22 (1H-NMR, 13C-NMR & Mass spectra). In the cycloaddition of piperine (1a), solvents toluene, xylene and water were used in presence of cuper (II) salts. Reaction showed good overall yield and more exo selectivity in organic solvent like xylene. Water catalyzed reactions were ended with good overall yield and minute decrease in exo selectivity, infinitesimal increase in endo selectivity (Table 2). This reaction showed completely regioselectivity (yield of 2a+3a>4a+5a+6a) due to maximum involvement of α-double bond rather than γ-double bond of 1a during Diels-Alder reaction. Cycloaddition reactions of pellitorine (1b): Same catalytic and solvent conditions were employed for pellitorine (1b) as used in piperine (1a) for the biomimetic synthesis (Scheme 3) of chabamide J & K (Chapter-II). These dimers were plausibly generated by monomer pellitorine by cycloaddtion reactions in biosynthesis. During cycloaddition of pellitorine (1b), solvents like toluene xylene and water were used in presence of cupper (II) salts. In former catalyzed reaction showed good overall yield and more endo selectivity in both organic (xylene) and water. Increase in endo selectivity is more in aqueous medium rather than organic solvent like xylene (Table 2). Cycloaddition of pellitorine under above said catalytic conditions gave corresponding cycloadducts 2b, 3b, 4b and 5b. Physical and spectral data of adducts 2b & 3b are identical with compound J & K (chabamide J & K mentioned in Chapter-II) and all physical and spectral data of adduct 4b is identical with nigramide O which is isolated previously from piper nigrum.21 The structure of 5b a new cycloadduct formed during this biomoimetic synthesis employ pellitorine as monomer, its structure was elucidated by 1D and 2D spectral data. This reaction showed completely regioselectivity (yield of 2b+5b≈3b+4b) due to maximum involvement of α-double bond rather than γ-double bond of 1b during Diels-Alder reaction. Structure elucidation of compound 5b: Compound 5b was obtained as a pale yellow oil, had the molecular formula of C28H50N2O2, as deduced from the HRESIMS (Fig-9) m/z, 447.3958 [M++H]. IR spectrum (Fig-1) implied the presence of carbonyl (1648 cm-1) and NH (3304 cm-1). The 1H NMR spectrum of 5b revealed the presence of a trans double bond at δ 5.28 (dd, J = 15.0, 10.0 Hz, H-4"), 5.63 (m, H-5"), two isobutylamide groups at δ 3.15 (m), 3.17 (m), 3.17 (m, H2-1'), 1.74 (m, H-2'), 0.91 (d, J = 6.7 Hz, H-3'), 0.90 (d, J = 6.7, H- 3'), 5.53 (br t, J = 5.7 Hz, NH) and δ 2.96 (m, H1-1'''), 2.97 (m, H2-1'''), 1.73 (m, H-2'''), 0.87 (d, J = 6.7 Hz, H-3'''), 0.86 (d, J = 6.7 Hz, H-3'''), 3.15 (br t, J = 6.0 Hz, NH), n-amyl group and 1-heptene unit at δ 1.96 (m, H-6), 1.40 (m, H-7), 1.20 (m, H-8), 1.27 (m, H-9), 0.86 ( t, J = 6.5 Hz, H-10) and δ 5.28 (dd, J = 15.0, 10.0 Hz, H-4"), 5.63 (m, H-5"), 1.89 (m, H-6"), 1.30 (m, H-7"), 1.28 (m, H-8"), 1.27 (m, H-9"), 0.88 (t, J = 6.5 Hz, H-10"), respectively (Table 3). The 13C NMR spectrum (Fig-3) displayed the presence of 28 carbon atoms and were further classified by DEPT experiments (Fig-4) into categories of 6 methyls, 10 methylenes, 10 methines and 2 quaternary carbons including two carbonyls (δ 173.80 and 173.04). ' The analyses of the 1H and 13C NMR spectral data of 5b showed a high degree of similarity to dimeric alkaloid, compound J naturally isolated from this plant (Chapter-II) compound is meta-endo while 5b is meta-exo product. Furthermore, the detailed elucidation of the 2D NMR data (COSY, HSQC and HMBC) had determined the planar structure of 5b. The 1H homodecoupling NMR (Fig-7) experiments of 5b revealed the connectivities H-2 (δ 2.45, m) to H-3 (δ 5.56, ddd, J = 10.0, 4.3, 2.6 Hz) to H-4 (δ 5.98, dt, J = 10.0, 1.8 Hz) to H-5 (δ 2.41, m) to H-2"( δ 2.68, dd, J = 11.3, 10.0 Hz) to H-3" (δ 2.82, ddd, J = 10.1, 10.0, 5.0 Hz ) via cyclohexene ring protons. The meta-orientation of the carbonyl and isobutylamide groups were established by HMBC (Fig-6) correlations for δ 2.45 (m, H-2), 5.56 (ddd, J = 10.0, 4.3, 2.6 Hz, H-3), 2.82 (ddd, J = 10.3, 10.0, 5.0 Hz, H-3")/δ 173.80 (C-1) and δ 2.68 (dd, J = 10.3, 10.0 Hz, H-2"), 2.41 (m, H-5), 2.82 (ddd, J = 10.3, 10.0, 5.0 Hz, H-3")/δ 173.04 (C-1"). Furthermore, the 1H-1H COSY (Fig-7) cross-peaks between δ 2.82 (ddd, J = 10.3, 10.0, 5.0 Hz, H-3") and δ 5.28 (dd, J = 15.0, 10.0 Hz, H-4"), and δ 5.63 (m, H-5") and δ 2.41 (m, H-5), 1.96 (m, H-6), 1.40 (m, H-7), coupled with the HMBC correlation for δ 5.63 (m, H-5'') to δ 28.35 (C-7"), δ 1.40 (m, H-7) to δ 37.04 (C-5) established the attachment of the 1-heptene and n-amyl groups at C-3" and C-5, respectively. The analysis of the 1H-1H coupling constants and NOESY (Fig- 8) data allowed us to determine the relative stereochemistry of compound 5b. The coupling constants of H-2"/H-5 and H-2"/H-3" (10.3 Hz) indicated anti relations of H-2"/H-5 and H-2"/H-3". In the NOESY spectrum correlations were observed at δ 2.45 (H-2) δ 2.82 (H-3") and δ 2.41 (H-5) and correlations were not observed at δ 2.68 (H-2") with δ 2.82 (H-3") and δ 2.68 (H-2") with δ 2.41 (H-5). These data were in agreement with the β-orientation for H-2" and α-orientation for H-3" and H-5. Thus, based on these spectral data the stereostructure of 5b was confirmed and trivially named as chabamide L. Cycloaddition reaction between piperine (1a) and pellitorine (1b): Our aim of this cycloaddition reaction is to explain to study different cycloadducts and selectivity of diene among piperine and pellitorine (Scheme 4). This biomimetic synthesis will explain the probability of diene, which participated in Diels-Alder reaction between piperine (1a) and pellitorine (1b) both were isolated from same plant (P. chaba). Nigramide N, which is formed biosynthetically via cycloaddition reaction between piperine and pellitorine, this adduct previously isolated from roots of P. nigrum 21 by Wei. et. al. Lewis acid catalyzed cycloaddition reactions of piperine (1a) and pellitorine (1b) under organic and aqueous solvent conditions to give resultant cycloadducts 2c, 3c, 4c, 2a and 3b. Cycloadduct 2c and 3c is new cycloadducts and their structures were illustrated by 1D and 2D spectral data. Structure elucidation of compound 2c: Compound 2c was obtained as pale yellow liquid. The molecular formula of 2c was established as C31H44N2O4 by HRESIMS (Fig-18), which provided a molecular ion peak at m/z 509.3381 [M++H], in conjunction with its 13C NMR spectrum (Fig-12). The IR spectrum displayed absorption bands diagnostic of carbonyl (1640 cm-1) (Fig-10). The 300 MHz 1H NMR spectrum (in CDCl3) indicated the presence of two signals at δ 5.86 (dd, J = 15.6, 10.1 Hz) and 6.27 (d, J = 15.6 Hz), which were assigned to trans-olefinic protons by the coupling constant of 15.6 Hz. It also displayed aromatic protons due to two 1, 3, 4-trisubstituted aromatic rings at δ 6.82 (1H, br s), 6.76 (1H, dd, J = 7.8, 1.4 Hz), 6.75 (1H, d, J = 7.8 Hz) (Fig-11), (Table 4). In addition to the above-mentioned moieties, combined inspection of 1H NMR and 1H–1H COSY revealed the presence of cyclohexene ring, one isobutylamide and one pyrrolidine ring. The 13C NMR spectrum displayed the presence of 31 carbon atoms and were further confirmed by DEPT experiments into categories of 11 methylenes, 12 methines and 5 quaternary carbons including two carbonyls (δ 173.01 and 172.50). On the basis of these characteristic features, database and literature search led the skeleton of compound 2c as a dimeric alkaloidal framework. A comprehensive analysis of the 2D NMR data of compound 2c facilitated the proton and carbon assignments. 1H–1H COSY spectrum suggested the sequential correlations of δ 3.51 (dq, J = 5.0, 2.6 Hz)/5.62 (dt, J = 9.8, 2.6 Hz)/6.10 (ddd, J = 9.8, 1.5 Hz)/2.20 (m)/2.72 (ddd, J = 11.1, 10.1, 5.2 Hz)/3.35 (dd, J = 11.1, 9.8 Hz) assignable to H-2-H-3-H-4-H-5-H-3"-H-2" of the cyclohexene ring. Concerning the connections of the n-amyl and 3, 4-methylenedioxy styryl groups, HMBC spectrum (Fig-15) showed correlations of H-4, H-6, H-7/C-5; H-5", H-4"/C-3", which implies that these units were bonded to the cyclohexene ring at C-5 and C-3". Further, HMBC correlations of two methylene protons at δ 5.95 with 147.91 (C-8"), 146.87 (C-9"), confirmed the location of methylenedioxy group at C-8", and C-9". Remaining units, isobutylamine and pyrrolidine (rings) were connected through carbonyl groups at C-2 and C-2", which was confirmed by HMBC correlations of H-2 and H-1' to C-1 (δ 173.01) and H-2" and H-1''' to C-1" (δ 172.50). The assignment of the relative configuration of compound 2c, and confirmation of overall structure were achieved by the interpretation of the NOESY spectral data and by analysis of 1H NMR coupling constants. The large vicinal coupling constants of H-2"/H-2 (11.1 Hz) and H-2"/H-3" (11.1 Hz) indicated anti-relations of H-2"/H-2 and H-2"/H-3" and the axial orientations for these protons. In the NOESY spectrum (Fig-17), the occurrence of the correlations between H-2/H-3" and the absence of NOE effects between H-2/H-2" and H-2"/H-3" supported the above result. This data indicated β-orientation for H-2" and α-orientation for H-2 and H-3". The α-orientation of H-5 was suggested by the coupling constant of H-5/H-3" (5.2 Hz) and the absence of the NOESY correlations between H-3" and H-2". On the basis of these spectral data, the structure of compound 2c was unambiguously established and trivially named as chabamide M. Structure elucidation of compound 3c: Compound 3c was obtained as pale yellow liquid. The molecular formula of 3c was established as C31H44N2O4 by HRESIMS (Fig-27), which provided a molecular ion peak at m/z 509.3391 [M++H], in conjunction with its 13C NMR spectrum (Fig-21). The IR spectrum displayed absorption bands diagnostic of carbonyl (1624 cm-1) moiety (Fig-19). The 300 MHz 1H NMR spectrum (in CDCl3) indicated the presence of two signals at δ 4.63 (dd, J = 15.6, 10.0 Hz) and 5.46 (dt, J = 15.6, 6.8 Hz), which were assigned to trans-olefinic protons by the coupling constant of 15.6 Hz. It also displayed aromatic protons due to two 1, 3, 4-trisubstituted aromatic ring at δ 6.75 (1H, br s), 6.73 (1H, d, J = 7.8, 1.4 Hz), 6.71 (1H, d, J = 7.8 Hz) (Fig-20). In addition to the above-mentioned moieties, combined inspection of 1H NMR and 1H–1H COSY revealed the presence of cyclohexene ring, one isobutylamide and one pyrrolidine ring. The 13C NMR spectrum displayed the presence of 31 carbon atoms (Table 5), and were further classified by DEPT experiments (Fig-22) into categories of 11 methylenes, 12 methines and 5 quaternary carbons including two carbonyls (δ 173.34 and 173.88). On the basis of these characteristic features, database and literature searches led the skeleton of compound 3c as a dimeric alkaloidal framework. A comprehensive analysis of the 2D NMR data of compound 3c facilitated the proton and carbon assignments. 1H–1H COSY spectrum (Fig-25) suggested the sequential correlations of δ 2.82 (m)/5.63 (dt, J = 9.7, 1.9 Hz)/5.82 (ddd, J = 9.7, 4.8, 1.9 Hz)/3.94 (dq, J =.10.0, 1.9 Hz)/2.76 (ddd, J = 11.7, 10.0 Hz)/3.36 (dt, J = 11.7, 4.8 Hz) assignable to H-2-H-3-H-4-H-5-H-3"-H-2" of the cyclohexene ring. Concerning the connections of the 3, 4-methylenedioxyphenyl and 1-heptene groups, HMBC spectrum (Fig-24) showed correlations of H-7, H-11, H-3"/C-5; H-5", H-4", H-5/C-3", which implies that these units were bonded to the cyclohexene ring at C-5 and C-3". Further, HMBC correlations of two methylene protons at δ 5.92 with 147.42 (C-8"), 146.49 (C-9"), confirmed the location of methylenedioxy group at C-8", and C-9". Remaining units, pyrrolidine and isobutylamine were connected through carbonyl groups at C-2 and C-2", which was confirmed by HMBC correlations of H-2 and H-1' to C-1 (δ 171.34) and H-2" and H-1''' to C-1" (δ 173.88). The assignment of the relative configuration of compound 3c, and confirmation of overall structure were achieved by the interpretation of the NOESY spectral data and by analysis of 1H NMR coupling constants. The large vicinal coupling constants of H-3"/H-2" (11.7 Hz) and H-5/H-3" (10.0 Hz), indicated anti-relations of H-3"/H-5 and H-3"/H-2" and the axial orientations for these protons. In the NOESY spectrum (Fig-26), the occurrence of the correlations between H-2"/H-5 and the absence of NOE effects between H-3"/H-2" and H-3"/H-5 supported the above result. These data indicated β-orientation for H-2" and α-orientation for H-2 and H-3". The α-orientation of H-2 was suggested by the coupling constant of H-2/H-2" (4.8 Hz) and the occurrence of the NOESY correlations between H-2" and H-2. On the basis of this spectral data, the structure of compound 3c was unambiguously established and trivially named as Chabamide N. Cycloaddition reaction between piperine (1a) and E-fagaramide (1c) Lewis acid catalyzed cycloaddition reactions (Scheme 5) of piperine (1a) and trans-fagaramide (1c) under aqueous solvent conditions to give resultant cycloadducts 2d, 3d and 2a. To carry this biomimetic synthesis to explain compound H and I (mentioned in chapter-II), we taken piperine (1a) which is isolated from same plant and trans fagaramide was synthesized by reported method.24 Cycloaddition reaction between 1a and 1c end up with overall yield 70% in xylene and 75% in water. In both solvents ortho products were formed dominantly compared with meta products. Spectral data 1D and 2D of cycloadducts 2d & 3d were identical with compound H & I (see chapter I, compound H & K). Cycloadduct 2a is identical with chabamide. This cycloaddition reaction practically proved as biomimetic synthesis for compound H and I. Acknowledgements: The authors are thankful to Director IICT for his constant encouragement and CSIR New Delhi for providing the fellowship References and Notes Braun, M. Synth. Highlights 1991, 232 Robinson, R. Chem. Soc. 1917, 762. Stork, G.; Burgstahler, A. W. Am. Chem. Soc. 1955, 38, 1890. Johnson, W. S.; Gravestock, M. B.; McCarry, B. E. Am. Chem. Soc. 1971, 93, 4332. Chapman, O. L.; Engel, M. R.; Springer, J. P.; Clardy, J. C. Am. Chem. Soc. 1971, 93, 6696. Bandaranayake, W. M.; Banfield, J. E.; Black, D. St. C. Chem. Soc., Chem Commun. 1980, 902. Nicolaou, K. C.; Zipkin, R. E.; Petasis, N. A. Am. Chem. Soc. 1982, 104, 5558. O.; Alder, K. Ann. 1928, 460, 98. Woodward, R. B.; Hoffmann, R. Chem. 1969, 81, 797. Fakui, K. Chem. Res. 1971, 4, 57. Houk, K. N. Chem.. Res. 1975, 8, 361. Houk, K. N.; Li, Y.; Evanseck, D. Angew Chem., Ed. Engl. 1992, 31, 682. Alder, K.; Stein, G. Chem. 1937, 50, 510. Fotiadu, F.; Michel, F.; Buono, G. Tetraheron Lett. 1990, 34, 4863. Gleiter, R.; Bohm, M. C. Pure Appl. Chem. 1983, 55, 237. Woodward, R. B.; Katz, T. J. Terahedron 1958, 5, 70. Kakushima, M. J. Chem. 1979, 57, 2564. Houk, K. N. Tetrahedron Lett. 1970, 30, 2621. Houk, K. N.; Luskus, L. J. Am. Chem. Soc. 1971, 93, 4606. Otto, S.; Bertoncin, F.; Engberts, J. F. N. Am. Chem. Soc., 1996, 118, 7702–7707. Wei, K.; Li, W.; Koike, K.; Chen, Y-J.; Nikaido, T. Org. Chem. 2005, 70, 1164. Wei, K.; Li, W.; Koike, K.; Chen, Y-J.; Nikaido, T. Lett. 2005, 7, 2833–2835. Rukachaisirikul, T.; Prabpai, S.; Champung, P.; Suksamrarn, A. Planta Med. 2002, 68, 850-853. Nagao, Y.; Seno, K.; Kawabata, K.; Miyasaka, T.; Takao, S.; Fujita, Tetrahedron Lett. 1980, 21, 841. Otto, S.; Boccaletti, G.; Engberts, J. B. F. N. Am. Chem. Soc. 1998, 120, 4238–4239. Otto, S.; Bertoncin, F.; Engberts, J. B. F. N. Am. Chem. Soc. 1996, 118, 7702–7707. O.; Alder, K. Ann. 1931, 490, 243. Woodward, R. B.; Baer, H. Am. Chem. Soc. 1948, 70, 1161. Breslow, R.; Rideout, D. C. Am. Chem. Soc. 1980, 102, 7816. Breslow, R.; Guo, T. Am. Chem. Soc. 1988, 110, 5613. Grieco, P.A.; Nunes, J. J.; Gaul, M. D. Am. Chem. Soc. 1990, 112, 4595.

The pursuit of science is a wonderful journey of discovery along which there are a myriad of avenues to be explored. There have always been so many objects of fascination, so many questions to ask along the way, so many possibilities to understand new principles, that making the decision about which problem to address and then having the self-discipline to explore it in depth challenge all who practice the art. How then are we, as cell biologists, to cope with the mountain of information that is accumulating as we enter the twenty-first century? We now have the potential to decipher the primary sequences of every single cellular protein for several model organisms. Just how are we to put this information into an intelligible framework for understanding cell physiology? The turn of a century is a time at which we can permit ourselves the luxury of looking backwards as well as forwards. Where were we a century ago, what were the challenges that faced us then and how do these questions relate to our future goals? As a cell biologist standing on the threshold of the twentieth century, one must have had a similar feeling of elation and expectation to that which we have at the present time. The Theory of Cells had been established by Schleiden and Schwan in 1838–1839, and in the following fifty years it had led to unifying ideas about the nature of plants and animals, an understanding of embryonic development, and the mysteries of the fertilisation of the egg and genetic continuity in terms of ‘cellular immortality’. These were truly halcyon days. By the end of the nineteenth century many of the central principles of cell biology were firmly established. Virchow had maintained in 1855 that every cell is the offspring of a pre-existing parent cell, but the realisation that the cell nucleus is essential for this continuity had to wait another 30 years. By this time, Miecher had already made in 1871 his famous discovery of nuclein, a phosphorus-rich substance extracted from preparations of nuclei from sperm and pus cells, and over the next twenty years a spectrum of sophisticated dyes became available that facilitated the visualisation of not only nuclein but also asters, spindle fibres, and microsomal components of cytoplasm in fixed preparations of cells. The centrosome, discovered independently by Flemming in 1875 and Van Beneden in 1876, and named by Boveri in 1888, was already considered to be an autonomous organelle with a central role in cell division. The behaviour of chromosomes, centrosomes, astral fibres and spindle fibres throughout mitosis and meiosis had been described in exquisite detail. Galeotti had even concluded by 1893 that the unequal distribution of chromatin in cancer cells correlates with an inequality of the centrosomes and the development of abnormal spindles - a conclusion reinforced by others over a century later (Pihan et al., 1998; Lingle et al., 1998). It had taken 200 years following Leuwenhoek's first observation of sperm to Hertwig's demonstration in 1875 that fertilisation of the egg is accomplished by its union with one spermatozoon. This demonstration was rapidly followed by Van Beneden's discovery - eventually to unify genetics and cell biology - that the nuclei of germ cells each contain one half the number of chromosomes characteristic of body cells. By 1902, both Sutton and Boveri had realised that the behaviour of chromosomes in meiosis precisely parallels the behaviour of Mendel's genetic particles described some 35 years earlier. In many ways we have witnessed during the past 50 years, and particularly in the last quarter century, a series of exciting breakthroughs in establishing an understanding of genetic function and continuity that are comparable to those of the previous century in demonstrating cellular function and continuity. The determination of the structure of DNA in 1953 and the elucidation of the genetic code throughout the 1960s led to the rapid realisation of the code's universality. The parallel development of sophisticated techniques for studying the genetics of the model bacterium Escherichia coli and its plasmids and viruses paved the way for a new era in biology. We were soon to construct recombinant DNA molecules in vitro, propagate them and eventually express them in E. coli, taking full advantage of the universality of the code. The principles of cloning DNA molecules had been clearly enunciated by Berg and Hogness in the early 1970s, and I myself had the great fortune as a young post-doc to share in this excitement and participate in putting some of these principles into their early practice. By the end of that decade, genes had been cloned from a multitude of eukaryotes and, moreover, technologies had been developed by Maxam and Gilbert and by Sanger that enabled these cloned genes to be sequenced. The accelerating accumulation of knowledge enabled by these simple technical breakthroughs has been astounding, leading to the determination of the complete genome sequences of budding yeast, the nematode Caenorhabditis elegans and the fruit fly, Drosophila melanogaster, and the prospect of the complete human sequence within a few years. To date we have managed this accumulating wealth reasonably well. Cloned genes have allowed cell biologists access to the encoded proteins, and as a consequence we have a working knowledge of many cellular processes. The sub-cellular meanderings of molecules have been charted with increasing accuracy, and gene products have been positioned in regulatory pathways. The concerted application of genetic and molecular approaches has given new insights into cell biology. This is particularly evident from work on the yeasts, which have come into their own as model systems with our realisation of the extent to which cell biological processes have been conserved. Nevertheless, the resulting regulatory pathways that emerge from our current ways of looking at the cell are rather unidimensional, gene products being placed into linear pathways as a result of either molecular or genetic analyses. Our current views are often blind to the fact that the cell is a multidimensional structure whose components are arranged in space, have multiple contacts that change with time and can respond simultaneously to a multitude of signals. Glimpses of such complexity are emerging from studies in which microarrays of all the identified open reading frames (ORFs) from the complete budding yeast genome have been screened for changes in patterns of gene expression throughout the cell cycle or upon sporulation. Cell-cycle-dependent periodicity was found for 416 of the 6220 monitored ORFs, and over 25% of these genes were found to be clustered at particular chromosomal sites, which suggesting there are global chromosomal responses in transcriptional control (Cho et al., 1998). The study of sporulation is perhaps the first example of the application of this type of technology to a developmental process. It revealed that, of the 6220 genes, about 500 undergo repression and 500 induction in seven temporally distinct patterns during the sporulation process, identifying potential functions for many previously uncharacterised genes (Chu et al., 1998). These studies already reveal layers of complexity in the regulation of the levels of transcripts as cells prepare for and pass through the different stages of meiosis. How much more complex are these patterns likely to be when viewed in terms of proteins, and their interactions, locations and functions within the cell? It seems clear, however, that a wonderful molecular description of the events of meiosis that can match the cytological understanding revealed by the work of Van Beneden and Boveri one hundred years ago is within our grasp. The cataloguing of all cellular proteins is now feasible through a combination of 2D-gel analysis and mass spectrometry, from which molecular mass data can be correlated with the fragment sizes of peptides predicted from whole genome sequence data (the emerging field of proteomics). It is not an easy task, but it seems just a matter of time before we have all this information at our fingertips. Yet how can we know the functions of all these proteins and have a full 3D picture of how they interact within a cell and the dynamics with which they do so? Yeast may be the first eukaryote for which some of these problems can be approached. Its genome is six-times smaller than that of C. elegans and 200 times smaller than the human genome, and has the further advantage that the genes can be easily disrupted through homologous recombination. Thus the prospect of systematic gene deletion to study the function of the 3700 novel ORFs identified in the whole genome sequence is feasible for this organism (Winzeler et al., 1999). One group in particular has devised a multifaceted approach for doing this: the affected gene is simultaneously tagged with an in-frame transcriptional reporter and further modified to epitope tag the affected protein, which thus allows the latter to be immunolocalised within cells (Ross-MacDonald et al., 1999). We can thus see the glimmerings of a holistic, genome-wide, cell-wide unravelling of cellular physiology. Some of these approaches will be easily adaptable to higher organisms. We will soon have read-outs of RNA expression patterns in cells undergoing a variety of developmental and physiological programmes in normal and diseased states. The analysis of function and the identification of ORFs in higher eukaryotes are likely to be more problematic. However, solutions for the rapid assessment of the functions of novel genes are already emerging. New insights are coming from labs using double-stranded RNA to interfere with cellular processes in C. elegans. It was originally found in this organism that the injection of double-stranded RNA corresponding to part of the mRNA of a gene prevents the expression of that gene through a mechanism that currently remains mysterious (Fire, 1999). The technique works extremely well in the nematode and even in the fruit fly, but doubts had been cast as to whether it would ever be valuable in mammals. The recent finding that the technique does indeed work in the mouse may well accelerate programmes to identify gene function by circumventing the particularly lengthy procedures for disruption of mouse genes (Wianny and Zernicka-Goetz, 2000). The multiple layers of complexity revealed by these emerging studies give some indication of the computational power that will be needed to model the cell. Is it now time for a new breed of mathematical biologists to emerge? Our present generation of cellular and molecular biologists have lost sight of some of the basic principles of physical chemistry, and quantitative analyses are done poorly if at all. Should the quantification of reaction kinetics now come out of the traditional domain of enzymology and be applied to multiple cellular processes - if we are truly to understand the dynamics of the living cell? If the yeast cell is complex, then how much greater complexity will we find in multicellular eukaryotes, given all the potential for cell-cell interactions? These problems are perhaps most alluring in the field of development, in which many phenomena are now demanding attention at the cellular level. In recent decades we have seen classical embryological approaches supplemented by genetic analyses to define the components of many developmental signalling pathways. This has demonstrated the existence of a conserved collection of molecular switches that can be used in a variety of different developmental circumstances. We are perhaps reaching the limits at which conventional genetic analyses can interpret these processes: often the precise relationships between components of regulatory pathways is not clear. We require a better grasp of how the molecules within the pathways interact, which will require the concerted application of sub-cellular fractionation, to identify molecular complexes, and proteomics. This has to be achieved in a way that allows us to interpret the consequences of multiple signalling events between different cell types. In the introduction to his famous text The Cell in Development and Inheritance, E. B. Wilson wrote almost a century ago: ‘It has only recently become possible adequately to formulate the great problems of development and heredity in terms of cellular biology - indeed we can as yet do little more than so formulate them.’ Has our perspective changed during the past one hundred years? Are not these the same challenges that lie ahead for the twenty-first century? It is now rather like being Alice in Wonderland in a room with many doors, each of which marks the onset of a new journey. Undoubtedly, any of the doors will lead to remarkable opportunities, but to what extent can we, as Alice, rely upon drinking from the bottle, or eating the biscuit, that happens to be at hand? We will have to use the existing resources, but it will be fascinating to see what new ingenuities we can bring to bear to help us on our journey through Wonderland. I have the feeling that we are to witness conceptual challenges to the way we think about cell biology that we cannot yet begin to appreciate…but what I would give to be around in one hundred years time to witness the progress we have made on our journeys!

V. Rama Subbaraoa* aNatural Products Laboratory, Organic Chemistry Division-I, Indian Institute of Chemical Technology, Habsiguda, Hyderabad 500007, India. Study and optimization of Diels-Alder reaction of piperine in aqueous ionic solutions using Gn.HCl as a catalyst. The semi-synthesis of these products using intermolecular [4+2] cycloaddition reaction has been described. Obtained products were characterized using IR, HNMR, CNMR and Mass Spectroscopy. Introduction An outsized number of phenomena concern to and are conducted in liquid phase involving ionic species (Millions of years ago, Mother Nature discovered the secrets of water molecule) in different biological and other natural processes. Salt present in the oceans, a striking example from Nature, is a multi component salt solution reflecting the distant marine origin of life on earth together with the composition of physiological fluids. In general the ionic solutions play roles in several industrial and geological processes in addition to their deep impact on the biological molecules. This enormous power of ionic solutions is based on the interactions of ion with solvent. In this work, we present some interesting results with comprehensive implications on the application of ion-solvent (i-s) interactions on organic reactions. Ion-Solvent interactions Cohesion among molecules in the liquid phase results from intermolecular forces. These forces include hydrogen-bonding, dipole-dipole, multi polar, dispersion interactions and also interactions emerging from the repulsion between two molecules. The cohesion due to intermolecular forces gives rise to a 'pressure' which is experienced by the solvent molecules. A liquid undergoing a small, isothermal volume expansion does work against the cohesive forces which causes a change in the internal energy, U. The function (∂U/∂V)T, is called as internal pressure (Pi) of a liquid and is supported by the equation of state. Internal pressure increases upon the addition of some solutes like NaCl, KCI, etc. and decreases by salts like of guanidinium salts. Diels-Alder Reaction in aqueous medium For long time water was not a popular solvent for the Diels-Alder reaction. Before 1980 its use had been reported only incidentally. Diels and Alder themselves performed the reaction between furan and maleic acid in an aqueous medium in 1931,27 an experiment which was repeated by Woodward and Baer in 1948. 28 They noticed a change in endo-exo selectivity when comparing the reaction in water with ether. The extreme influence of water can exert on the Diels-Alder reaction was rediscovered by Breslow in 1980, much by coincidence 29,30 while studying the effect of β-cyclodextrin on the rate of a Diels-Alder reaction in water, accidentally. Schem 1. Alternatively, Grieco et al., have repeatedly invoked the internal pressure of water as an explanation of the rate enhancement of Diels-Alder reactions in these solvents. 31 They probably inspired by the well known large effects of the external pressure on rates of cycloadditions. However the internal pressure of water is very low and offers no valid explanation for its effects on the Diels-Alder reaction. The internal pressure is defined as the energy required bringing about an infinitesimal change in the volume of the solvent at constant temperature. Due to the open and relatively flexible hydrogen-bond network of water, a small change in volume of these solvents does not require much energy. A related, but much more applicable solvent parameter is the cohesive energy density. This quantity is a measure of energy required for evaporation of the solvent per unit volume. The reactions in water were less accelerated by pressure than those in organic solvents, which is in line with notion that pressure diminishes hydrophobic interactions. The effect of water on the selectivity of Diels-Alder reactions Three years after the Breslow report on the large effects of water on the rate of the Diels-Alder reaction, he also demonstrated that the endo-exo selectivity of this reaction benefits markedly from employing aqueous media. Based on the influence of salting-in and salting-out agents, Breslow pinpoints hydrophobic effects as the most important contributor to the enhanced endo-exo selectivity. Hydrophobic effects are assured to stabilize the more compact endo transition state more than the extended exo transition state. In Breslow option the polarity of water significantly enhances the endo-exo selectivity. In conclusion, the special influence of water on the endo-exo selectivity seems to be a result of the fact that this solvent combines in it three characteristics that all favors formation of the endo/exo adduct. 1. water is strong hydrogen bond donor 2. water is polar and water induces hydrophobic interactions. Study of salting-out and salting-in reagents towards the Diels-Alder reaction of piperine (1): The special effects of water as solvent for valuable Diels-Alder reaction (Scheme 1) of piperine (1), greatly altered by the addition of ionic solutes (Table 1) such as LiCl, LiBr, LiClO4,- NaCl, NaBr, KF, KCl, KBr, MgCl2, CaCl2, guanidinium chloride, guanidinium carbonate, guanidinium nitrate. Aqueous salts solutions accelerated cycloaddition reactions (Scheme 1) of piperine (1) to give resultant cycloadducts 2, 3 and 4 among them 2 is major ortho-exo cyclohexene type dimeric amide alkaloid and also known as chabamide, which is previously isolated from this plant, isomer 3 is also known adduct and previously isolated from Piper nigrum. 21 Cycloadduct 4 was synthesized from piperine by Diels-Alder reaction by Wei. et al. its physical and spectroscopic data were identical with reported data22 (1H-NMR & Mass spectra). Table 1: Study of different salts towards the Diels-Alder reaction of piperine (1). aOverall yield of adducts after HPLC, un-reacted piperine was recovered in all reactions. Reaction showed good overall yield and more exo selectivity. This reaction showed completely regioselectivity (yield of 2+3>4) due to maximum involvement of α-double bond rather than γ-double bond of 1 during Diels-Alder reaction. Table 2: Comparision of salting-out and salting-in reagents towards the Diels-Alder reaction of piperine (1). Study of Salting-out reagents Increased rate in Diels-Alder reaction (over all yield up to 79 %) of piperine (1) has been attributed to the hydrophobic effect. Owing to the difference in polarity between water and the reactants, water molecules tend to associate amongst themselves, excluding the organic reagents and forcing them to associate together forming small drops surrounded by water. A further method of increasing the rate of Diels-Alder reaction in water is so called ‘salting-out’ effect. Among the salting-out reagents used (Table 1) in this methodology CaCl2 is the best reagent and gave 79 % over all yield. If anion size increases, reaction yield decreases, where as cation size increases, reaction yield increases. Here a salt such as calcium chloride is added to the aqueous solution. In this case water molecules attracted to the polar ions, increasing the internal pressure and reducing the volume. This has the effect of further excluding the organic reagents. For reactions such as Diels-Alder, which have negative activation volumes, the rates are enhanced by this increase in internal pressure in much the same way as expected for an increase in external pressure. This salting-out reagent showed good exo selectivity, due to formation of cycloadduct 2 (ortho-exo) is major up to 69 % (cycloadduct ratio) compare to cycloadducts 3 (21 %, meta-exo) and 4 (10%, meta-exo) are poor in yield. Schem 2. Plausible mechanism of Diels-Alder reaction catalyzed by Gn.HCl. Study of Salting-in reagents Among the tested salting-in reagents used in this methodology (Table 1) guanidinium chloride (Gn.HCL) is the best reagent and gave 81 % overall yield, where as LiClO4 end up with only 15 % overall yield. Gn.HCL reagent exhibited well selectivity towards the Diels-Alder reaction of piperine in given conditions (scheme 1). Formation of cycloadduct 2 in 80 %, 3 in 15 % and 4 in 5 % ratio is clearly indicates this methodology received good attention towards the exo selectivity in Diels-Alder reaction of piperine. Overall yield is also high with salting-in reagents when compare to salting-out reagents. Procedure for aqueous ionic salts catalyzed Diels–Alder reactions of piperine (1): To a stirred mixture of piperine (1) (50.0 mg, 0.175 mmol), 6M aqueous guanidinium. Hydrochloride (2 mL) in a round bottom flask fitted with condenser and refluxed for 70 h in an oil bath. After completion of the reaction, monitored by TLC (dipped in 5% solution of phosphomolybdic acid in methanol and heating), the reaction mixture was cooled to room temperature and diluted with water (3 mL). Then extracted with EtOAc (2x5 mL), the combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue obtained was then purified by reversed-phase (RP) HPLC (column: Phenomenex Luna C18, 250 x 10 mm, 10µ), solvent system: 80% acetonitrile in water, flow rate: 1.5 mL/min, to give pure compounds of adducts (2) 0.065 g, (3) 0.012 g and (4) 0.004 g. Cycloaddition reaction between piperine (1a) and pellitorine (1b): Our aim of this cycloaddition reaction is to explain to study different cycloadducts and selectivity of diene among piperine and pellitorine (Scheme 4). This biomimetic synthesis will explain the probability of diene, which participated in Diels-Alder reaction between piperine (1a) and pellitorine (1b) both were isolated from same plant (P. chaba). Nigramide N, which is formed biosynthetically via cycloaddition reaction between piperine and pellitorine, this adduct previously isolated from roots of P. nigrum 21 by Wei. et. al. Lewis acid catalyzed cycloaddition reactions of piperine (1a) and pellitorine (1b) under organic and aqueous solvent conditions to give resultant cycloadducts 2c, 3c, 4c, 2a and 3b. Cycloadduct 2c and 3c is new cycloadducts and their structures were illustrated by 1D and 2D spectral data. Structure elucidation of compound 2c: Compound 2c was obtained as pale yellow liquid. The molecular formula of 2c was established as C31H44N2O4 by HRESIMS (Fig-18), which provided a molecular ion peak at m/z 509.3381 [M++H], in conjunction with its 13C NMR spectrum (Fig-12). The IR spectrum displayed absorption bands diagnostic of carbonyl (1640 cm-1) (Fig-10). The 300 MHz 1H NMR spectrum (in CDCl3) indicated the presence of two signals at δ 5.86 (dd, J = 15.6, 10.1 Hz) and 6.27 (d, J = 15.6 Hz), which were assigned to trans-olefinic protons by the coupling constant of 15.6 Hz. It also displayed aromatic protons due to two 1, 3, 4-trisubstituted aromatic rings at δ 6.82 (1H, br s), 6.76 (1H, dd, J = 7.8, 1.4 Hz), 6.75 (1H, d, J = 7.8 Hz) (Fig-11), (Table 4). In addition to the above-mentioned moieties, combined inspection of 1H NMR and 1H–1H COSY revealed the presence of cyclohexene ring, one isobutylamide and one pyrrolidine ring. The 13C NMR spectrum (Fig-12) displayed the presence of 31 carbon atoms and were further confirmed by DEPT experiments (Fig-13) into categories of 11 methylenes, 12 methines and 5 quaternary carbons including two carbonyls (δ 173.01 and 172.50). On the basis of these characteristic features, database and literature search led the skeleton of compound 2c as a dimeric alkaloidal framework. A comprehensive analysis of the 2D NMR data of compound 2c facilitated the proton and carbon assignments. 1H–1H COSY spectrum (Fig-16) suggested the sequential correlations of δ 3.51 (dq, J = 5.0, 2.6 Hz)/5.62 (dt, J = 9.8, 2.6 Hz)/6.10 (ddd, J = 9.8, 1.5 Hz)/2.20 (m)/2.72 (ddd, J = 11.1, 10.1, 5.2 Hz)/3.35 (dd, J = 11.1, 9.8 Hz) assignable to H-2-H-3-H-4-H-5-H-3"-H-2" of the cyclohexene ring. Concerning the connections of the n-amyl and 3, 4-methylenedioxy styryl groups, HMBC spectrum (Fig-15) showed correlations of H-4, H-6, H-7/C-5; H-5", H-4"/C-3", which implies that these units were bonded to the cyclohexene ring at C-5 and C-3". Further, HMBC correlations of two methylene protons at δ 5.95 with 147.91 (C-8"), 146.87 (C-9"), confirmed the location of methylenedioxy group at C-8", and C-9". Remaining units, isobutylamine and pyrrolidine (rings) were connected through carbonyl groups at C-2 and C-2", which was confirmed by HMBC correlations of H-2 and H-1' to C-1 (δ 173.01) and H-2" and H-1''' to C-1" (δ 172.50). The assignment of the relative configuration of compound 2c, and confirmation of overall structure were achieved by the interpretation of the NOESY spectral data and by analysis of 1H NMR coupling constants. The large vicinal coupling constants of H-2"/H-2 (11.1 Hz) and H-2"/H-3" (11.1 Hz) indicated anti-relations of H-2"/H-2 and H-2"/H-3" and the axial orientations for these protons. In the NOESY spectrum (Fig-17), the occurrence of the correlations between H-2/H-3" and the absence of NOE effects between H-2/H-2" and H-2"/H-3" supported the above result. This data indicated β-orientation for H-2" and α-orientation for H-2 and H-3". The α-orientation of H-5 was suggested by the coupling constant of H-5/H-3" (5.2 Hz) and the absence of the NOESY correlations between H-3" and H-2". On the basis of these spectral data, the structure of compound 2c was unambiguously established and trivially named as chabamide M. Compound 3a: IR (KBr) nmax: 2923, 2855,1628, 1489, 1242, 1128, 1035 cm-1 d ppm 0.69 & 1.25 (2H, m, H-2'"), 1.15 & 1.23 (2H, m, H-4'"), 1.31 & 1.40 (2H, m, H-3'"), 1.52 (2H, m, H-2'), 1.56 (2H, m, 4'), 1.61 (2H, m, H-3'), 2.94 (1H, td, J = 10.1, 10.1, 5.5 Hz, H-3"), 3.02 & 3.60 (2H, m, H-5'"), 3.09 & 3.32 (2H, m, H-1'"), 3.51 (2H, m, H-1'), 3.61 (1H, m, H-2), 3.61 (2H, m, H-5'), 3.78 (1H, dq, J = 10.0, 2.3 Hz, H-5), 4.07 (1H, t, J = 10.1, H-2"), 5.72 (1H, ddd, J = 9.8, 5.0, 2.7 Hz, H-3), 5.88 (2H, s, H-12), 5.89 (1H, dt, 10.3, 1.8 Hz, H-4), 5.90 (1H, J =15.8, 9.8 Hz, H-4"), 5.92 (1H, s, H-12"), 6.37 (1H, d, J = 15.8 Hz, H-5"), 6.68 (1H, brs, H-7), 6.69 (1H, d, J = 8.0 Hz, H-10"), 6.70 (1H, dd, J = 8.0, 1.4 Hz, H-11), 6.69 (1H, d, J = 8.0 Hz, H-10), 6.74 (1H, dd, J = 8.0, 1.6 Hz, H-11"), 6.79 (1H, brs, H-7"). ESIMS (m/z): 571 [M+ +H] Table 4: 1H & 13C NMR data of cycloadduct 2c in CDCl3 (300 MHz, δ in ppm, mult, J in Hz) Compound 4a: IR (KBr) nmax: 2926, 2857,1627, 1484, 1440, 1240, 1034 cm-1 1H NMR (300 MHz, CDCl3): d ppm 0.81 & 1.35 (2H, m, H-2'), 1.29 & 1.47 (2H, m, H-4'), 1.35 (2H, m, H-2"'), 1.36 & 1.51 (1H, m, H-3'), 1.47 (2H, m, H-4"'), 1.51 (2H, m, H-3"'), 2.92 (2H, m, H-1"'), 2.99 (1H, ddd, J = 12.5, 9.7, 5.5 Hz, H-4"), 3.22 (2H, m, H-1'), 3.29 & 3.71 (2H, m, H-5'), 3.38 (1H, m, H-4"'), 3.44 (1H, dd, J = 12.1, 10.1 Hz, H-5"), 3.59 (1H, t, J = 5.3 Hz, H-5), 3.70 (1H, dq, J = 12.1, 2.1, H-2), 5.65 (1H, dd, J = 15.6, 9.5 Hz, H-3"), 5.70 (1H, dt, J = 9.9, 1.6, H-3), 5.81 (1H, d, J = 15.6 Hz, H-2"), 5.84 (1H, s, H-12"), 5.90-5.92 (2H, brs, H-12), 5.96 (1H, ddd, J = 9.2, 5.8, 2.6 Hz, H-4), 6.55 (1H, dd, J =7.9, 1.5 Hz, H-11"), 6.61 (1H, d, J = 8.2 Hz, H-10"), 6.62 (1H, d, J = 1.4 Hz, H-7"), 6.79 (1H, d, J = 7.9 Hz, H-10), 6.92 (1H, dd, J = 8.0, 1.5 Hz, H-11), 7.01 (1H, d, J = 1.5 Hz, H-7). 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Behcet’s disease (BD) is an immune disease characterized by chronic and relapsing systemic vasculitis of unknown etiology, which can lead to blindness and even death. Despite continuous efforts to discover biomarkers for accurate and rapid diagnosis and optimal treatment of BD, there is still no signature marker with high sensitivity and high specificity. As the link between glycosylation and the immune system has been revealed, research on the immunological function of glycans is being actively conducted. In particular, sialic acids at the terminus of glycoconjugates are directly implicated in immune responses, cell–cell/pathogen interactions, and tumor progression. Therefore, changes in sialic acid epitope in the human body are spotlighted as a new indicator to monitor the onset and progression of immune diseases. Here, we performed global profiling of N-glycan compositions derived from the sera of 47 healthy donors and 47 BD patients using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to preferentially determine BD target glycans. Then, three sialylated biantennary N-glycans were further subjected to the separation of linkage isomers and quantification using porous graphitized carbon-liquid chromatography (PGC-LC)/multiple reaction monitoring (MRM)-MS. We were able to successfully identify 11 isomers with sialic acid epitopes from the three glycan compositions consisting of Hex5HexNAc4NeuAc1, Hex5HexNAc4Fuc1NeuAc1, and Hex5HexNAc4NeuAc2. Among them, three isomers almost completely distinguished BD from control with high sensitivity and specificity with an area under the curve (AUC) of 0.945, suggesting the potential as novel BD biomarkers. In particular, it was confirmed that α2,3-sialic acid at the terminus of biantennary N-glycan was the epitope associated with BD. In this study, we present a novel approach to elucidating the association between BD and glycosylation by tracing isomeric structures containing sialic acid epitopes. Isomer-specific glycan profiling is suitable for analysis of large clinical cohorts and may facilitate the introduction of diagnostic assays for other immune diseases.