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Inspired by the role of intracellular liquid-liquid phase separation (LLPS) in formation of membraneless organelles, there is great interest in developing dynamic compartments formed by LLPS of intrinsically disordered proteins (IDPs) or short peptides. However, the molecular mechanisms underlying the formation of biomolecular condensates have not been fully elucidated, rendering on-demand design of synthetic condensates with tailored physico-chemical functionalities a significant challenge. To address this need, here we design a library of LLPS-promoting peptide building blocks composed of various assembly domains. We show that the LLPS propensity, dynamics, and encapsulation efficiency of compartments can be tuned by changes to the peptide composition. Specifically, with the aid of Raman and NMR spectroscopy, we show that interactions between arginine and aromatic amino acids underlie droplet formation, and that both intra- and intermolecular interactions dictate droplet dynamics. The resulting sequence-structure-function correlation could support the future development of compartments for a variety of applications., (© 2023. The Author(s).)

Uridine (U) mimetics are sought after as tools for biochemical and pharmacological studies. Previously, we have identified recognition patterns of U by proteins. Here, we targeted the characterization of uridine mimetics-cyanuryl-ribose (CR), barbituryl-ribose (BR), and 6-azauridine (AU)-with a view to identify analogs with potentially more binding interactions than U with target biomolecules. We found that CR, BR, and AU retain selective U's natural H-bonds with adenosine vs guanosine. CR/AU and BR were 100- and 10,000-fold more acidic, respectively, than U. Under physiological pH, 54, 51, and 77% of CR, AU, and BR molecules, respectively, are ionized vs 13% for U. The electron-rich nature of CR and BR vs U was reflected by their 13 C NMR chemical shifts and ε values. CR/AU and BR prefer N conformation (up to 73%) vs U (56%). Unlike U that prefers gg conformation around exocyclic methylol (48%), CR/AU and BR prefer both gt and gg rotamers. In conclusion, replacement of uridine's C6 by N or carbonyl, or C5-C6 by an amide, results in significant changes in U's ionization, electron density, conformation, base-stacking, etc., leading to potentially tighter binding than U with a target protein or nucleic acid and potential use for various biochemical and pharmacological applications., Competing Interests: The authors declare no competing financial interest., (© 2020 The Authors. Published by American Chemical Society.)

The method developed and introduced here enables for the first time (to the authors' knowledge), a quantitative assessment of the interaction of SmI 2 with substrates prior to the electron transfer stage. As a proof of concept, equilibrium constants for some model substrates including carbonyl compounds and aromatic nuclei are reported here. In addition, the first equilibrium constants with some common ligands were also determined. The equilibrium constants range from approximately 0.07 m -1 for diisopropyl ketone to 2500 m -1 for hexamethylphosphoramide (HMPA). It is shown that the data acquired by this method, which is based on the concept of shift reagents, can shed light on the most intimate details of the reaction mechanism, and this method is a useful tool for planning a synthetic process., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ornithogalum is an ornamental flowering species that grows from a bulb and is highly susceptible to soft-rot disease caused by Pectobacterium carotovorum (Pc). Interspecific hybridization between O. thyrsoides and O. dubium yielded hybrids with enhanced resistance to that pathogen. The hybrids displayed distinct phenolic-compound profiles with several peaks that were specifically heightened following Pc infection. Three of these compounds were isolated and identified as novel kaempferol O-tri -glycosides . The structures of these compounds were elucidated using reversed phase high-performance liquid chromatography (RP-LC), RP-LC coupled to high-resolution mass spectrometry (RP-LC-MS), and nuclear magnetic resonance (NMR) (1D 1 H and 13 C, DEPT, HMQC, HMBC, COSY, and NOE), in order to achieve pure and defined compounds data. The new compounds were finally identified as kaempferol 3- O -[4- O -α-l-(3- O -acetic)-rhamnopyranosyl-6- O -β-d-xylopyranosyl]-β-d-glucopyranoside, kaempferol 3- O -[4- O -α-l-(2- O -acetic)-rhamnopyranosyl-6- O -β-d-xylopyranosyl]-β-d-glucopyranoside and kaempferol 3- O -[4- O -α-l-(2,3- O -diacetic)-rhamnopyranosyl-6- O -β-d-xylopyranosyl]-β-d-glucopyranoside., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Smart materials are created in nature at interfaces between biomolecules and solid materials. The ability to probe the structure of functional peptides that engineer biogenic materials at this heterogeneous setting can be facilitated tremendously by use of DNP-enhanced solid-state NMR spectroscopy. This sensitive NMR technique allows simple and quick measurements, often without the need for isotope enrichment. Here, it is used to characterize a pentalysine peptide, derived from a diatom's silaffin protein. The peptide accelerates the formation of bioinspired silica and gets embedded inside the material as it is formed. Two-dimensional DNP MAS NMR of the silica-bound peptide and solution NMR of the free peptide are used to derive its secondary structure in the two states and to pinpoint some subtle conformational changes that the peptide undergoes in order to adapt to the silica environment. In addition, interactions between abundant lysine residues and silica surface are identified, and proximity of other side chains to silica and to neighboring peptide molecules is discussed.

Hybrids of methylenediphosphonotetrathioate and crown-ether (MDPT-CE) were synthesized forming 7-,8-,9-,10- and 13-membered rings. Both 7- and 13-membered ring-containing compounds were found to be highly stable to air-oxidation for at least four weeks. These hybrids bind Zn(II) by both MDPT and CE moieties, forming a 2 : 1 L : Zn(II) complex. Interestingly, the 13-membered ring MDPT-CE showing a high affinity to Zn(II) (Ka 3 ± 0.5 × 10(6) mol(-2) L(2)) does not bind Li(I) or Na(I). The 13-Membered MDPT-CE hybrid is a promising water-soluble, air-stable, high-affinity Zn(II)-chelator, exhibiting selectivity to Zn(II) vs. Mg(II), Na(I), and Li(I).

Pyridine and its derivatives are rather difficult to reduce, and the products often undergo a very fast reoxidation to regain aromaticity. The reduction of quinoline by SmI2 results in an instantaneous [2 + 3] cyclization reaction, forming a bridged seven-membered ring within a polycyclic system.

Methionine motifs are methionine-rich metal-binding segments found in many human, yeast, and bacterial proteins involved in the transportation of copper ion to other cellular pathways, and in protecting copper from oxidation. Methionine motifs are found to bind Ag(I) and Cu(I) ions. Proteins or peptides that can bind different metal ions should have the ability to differentiate between them, to be able to shuttle them to various pathways in the cell. This study utilizes electron paramagnetic resonance spectroscopy together with circular dichroism and nuclear magnetic resonance to probe structural changes in the methionine segment upon coordinating Cu(I) and Ag(I) metal ions. The data collected here indicate that methionine segments experience structural changes while coordinating Cu(I) and Ag(I), however, the differences between the coordination of Cu(I) vs. Ag(I) to the methionine segment are mild. Since Cu(I) and Ag(I) metal ions are pretty similar in their nature and charge, the minor structural changes reported here are significant towards the understanding of the differences in the transport mechanism of these two metal ions in prokaryotic and eukaryotic cells.

DNA molecules were recently converted using ultrasonic irradiation into microcapsules that can trap hydrophobic molecules in aqueous solution. These DNA microcapsules are capable of penetrating prokaryotic and eukaryotic cells, delivering drugs and transferring genetic information e.g. for protein expression into the host cells. DNA molecules of different sizes and structures can be assembled into spherical capsules, but to date, the interactions that hold them together in these large structural constructs are unknown. In the current study, capsules prepared from a 12 base double helix DNA were investigated using NMR spectroscopy. Solution NMR studies of the DNA emulsion reveal DNA molecules with a perturbed structure with a size similar to the precursor DNA based on diffusion NMR measurements. 2D NMR correlation measurements and chemical shift perturbation analysis show partial unzipping of AT base pairs in the centre of the modified duplex, freeing nucleoside bases to interact with other bases on other precursor molecules thereby facilitating aggregation. Slow tumbling of the microspheres renders them invisible in solution NMR spectra; therefore magic angle spinning NMR measurements are performed which provide limited evidence of the DNA in the microcapsule state.

The nitration of tetraiodoethylene (7) yields 1,1-diiodo-2,2-dinitroethylene (8). The latter reacts with alkylamines 9 or alkyldiamines 11 to give the corresponding acyclic 1,1-diamino-2,2-dinitroethylenes 10 or their cyclic analogs 12, respectively. On the basis of liquid and solid-state (13)C and (15)N NMR data, x-ray analysis and ab initio calculations, we suggest that the title compounds are always zwitterionic and that the C(A)-C(N) bond is not a true double bond., (Copyright © 2012 John Wiley & Sons, Ltd.)

Dinucleotides (Np(n)N'; N and N' are A, U, G, or C, n = 2-7) are naturally occurring physiologically active compounds. Despite the interest in dinucleotides, the composition of their complexes with metal ions as well as their conformations and species distribution in living systems are understudied. Therefore, we investigated a series of Mg(2+) and Ca(2+) complexes of Np(n)N's. Potentiometric titrations indicated that a longer dinucleotide polyphosphate (N is A or G, n = 3-5) linker yields more stable complexes (e.g., log K of 2.70, 3.27, and 3.73 for Ap(n)A-Mg(2+), n = 3, 4, 5, respectively). The base (A or G) or ion (Mg(2+) or Ca(2+)) has a minor effect on K(M)(ML) values. In a physiological medium, the longer Ap(n)As (n = 4, 5) are predicted to occur mostly as the Mg(2+)/Ca(2+) complexes. (31)P NMR monitored titrations of Np(n)N's with Mg(2+)/Ca(2+) ions showed that the middle phosphates of the dinucleotides coordinate with Mg(2+)/Ca(2+). Multidimensional potential of mean force (PMF) molecular dynamics (MD) simulations suggest that Ap(2)A and Ap(4)A coordinate Mg(2+) and Ca(2+) ions in both inner-sphere and outer-sphere modes. The PMF MD simulations additionally provide a detailed picture of the possible coordination sites, as well as the cation binding process. Moreover, both NMR and MD simulations showed that the conformation of the nucleoside moieties in Np(n)N'-Mg(2+)/Ca(2+) complexes remains the same as that of free mononucleotides.

Plants are an infinite source of bioactive compounds. We screened the Israeli flora for compounds that interfere with the organization of the actin cytoskeleton. We found an activity in lipidic extract from Iris germanica that was able to increase HeLa cell area and adhesion and augment the formation of actin stress fibers. This effect was not observed when Ref52 fibroblasts were tested and was not the result of disruption of microtubules. Further, the increase in cell area was Rac1-dependent, and the iris extract led to slight Rac activation. Inhibitor of RhoA kinase did not interfere with the ability of the iris extract to increase HeLa cell area. The increase in HeLa cell area in the presence of iris extract was accompanied by impairment of cell migration and arrest of the cell cycle at G1 although the involvement of Rac1 in these processes is not clear. Biochemical verification of the extract based on activity-mediated fractionation and nuclear magnetic resonance analysis revealed that the active compounds belong to the group of iridals, a known group of triterpenoid. Purified iripallidal was able to increase cell area of both HeLa and SW480 cells.

The 24 h exposure of water plants (etiolated duckweed) to RF-EMF between 7.8 V m(-1) and 1.8 V m(-1), generated by AM 1.287 MHz transmitting antennas, resulted in alanine accumulation in the plant cells, a phenomenon we have previously shown to be a universal stress signal. The magnitude of the effect corresponds qualitatively to the level of RF-EMF exposure. In the presence of 10 mM vitamin C, alanine accumulation is completely suppressed, suggesting the involvement of free radicals in the process. A unique biological connection has thus been made between exposure to RF-EMF and cell stress, in the vicinity of RF transmitting antennas. This simple test, which lasts only 24 h, constitutes a useful bioassay for the quick detection of biological cell stress caused in the vicinity of RF irradiating antennas.

Dinucleoside polyphosphates, or dinucleotides (Np(n)N'; N, N' = A, U, G, C; n = 2-7), are naturally occurring ubiquitous physiologically active compounds. Despite the interest in dinucleotides, and the relevance of their conformation to their biological function, the conformation of dinucleotides has been insufficiently studied. Therefore, here we performed conformational analysis of a series of Np(n)N' Na(+) salts (N = A, G, U, C; N' = A, G, U, C; n = 2-5) by various NMR techniques. All studied dinucleotides, except for Up(4/5)U, formed intramolecular base stacking interactions in aqueous solutions as indicated by NMR. The conformation around the glycosidic angle in Np(n)N's was found to be anti/high anti and the preferred conformation around the C4'-C5', C5'-O5' bonds was found to be gauche-gauche (gg). The ribose moiety in Np(n)N's showed a small preference for the S conformation, but when attached to cytosine the ribose ring preferred the N conformation. However, no predominant conformation was observed for the ribose moiety in any of the dinucleotides. Molecular dynamics simulations of Ap(2)A and Ap(4)A Na(+) salts supported the experimental results. In addition, three modes of base-stacking were found for Ap(2/4)A: α-α, β-β and α-β, which exist in equilibrium, while none is dominant. We conclude that natural, free Np(n)N's (n = 2-5) at physiological pH exist mostly in a folded (stacked), rather than extended conformation, in several interconverting stacking modes. Intramolecular base stacking of Np(n)N's does not alter the conformation of each of the nucleotide moieties, which remains the same as that of the mononucleotides in solution.

We report on a rather unknown feature of oligonucleotides, namely, their potent antioxidant activity. Previously, we showed that nucleotides are potent antioxidants in Fe(II)/Cu(I/II)-H(2)O(2) systems. Here, we explored the potential of 2'-deoxyoligonucleotides as inhibitors of the Fe(II)/Cu(I/II)-induced *OH formation from H(2)O(2). The oligonucleotides [d(A)(5,7,20); d(T)(20); (2'-OMe-A)(5)] proved to be highly potent antioxidants with IC(50) values of 5-17 or 48-85 microM in inhibiting Fe(II)/Cu(I)- or Cu(II)-induced H(2)O(2) decomposition, respectively, thus representing a 40-215-fold increase in potency as compared with Trolox, a standard antioxidant. The antioxidant activity is only weakly dependent on the oligonucleotides' length or base identity. We analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry and (1)H-NMR spectroscopy the composition of the d(A)(5) solution exposed to the aforementioned oxidative conditions for 4 min or 24 h. We concluded that the primary (rapid) inhibition mechanism by oligonucleotides is metal ion chelation and the secondary (slow) mechanism is radical scavenging. We characterized the Cu(I)-d(A)(5) and Cu(II)-d(A)(7) complexes by (1)H-NMR and (31)P-NMR or frozen-solution ESR spectroscopy, respectively. Cu(I) is probably coordinated to d(A)(5) via N1 and N7 of two adenine residues and possibly also via two phosphate/bridging water molecules. The ESR data suggest Cu(II) chelation through two nitrogen atoms of the adenine bases and two oxygen atoms (phosphates or water molecules). We conclude that oligonucleotides at micromolar concentrations prevent Fe(II)/Cu(I/II)-induced oxidative damage, primarily through metal ion chelation. Furthermore, we propose the use of a short, metabolically stable oligonucleotide, (2'-OMe-A)(5), as a highly potent and relatively long lived (t(1/2) approximately 20 h) antioxidant.

Our aim was to study the effects of cucurbitacin glucosides extracted from Citrullus colocynthis leaves on human breast cancer cell growth. Leaves were extracted, resulting in the identification of cucurbitacin B/E glucosides. The cucurbitacin glucoside combination (1:1) inhibited growth of ER(+) MCF-7 and ER(-) MDA-MB-231 human breast cancer cell lines. Cell-cycle analysis showed that treatment with isolated cucurbitacin glucoside combination resulted in accumulation of cells at the G(2)/M phase of the cell cycle. Treated cells showed rapid reduction in the level of the key protein complex necessary to the regulation of G(2) exit and initiation of mitosis, namely the p34(CDC2)/cyclin B1 complex. cucurbitacin glucoside treatment also caused changes in the overall cell morphology from an elongated form to a round-shaped cell, which indicates that cucurbitacin treatment caused impairment of actin filament organization. This profound morphological change might also influence intracellular signaling by molecules such as PKB, resulting in inhibition in the transmission of survival signals. Reduction in PKB phosphorylation and inhibition of survivin, an anti-apoptosis family member, was observed. The treatment caused elevation in p-STAT3 and in p21(WAF), proven to be a STAT3 positive target in absence of survival signals. Cucurbitacin glucoside treatment also induced apoptosis, as measured by Annexin V/propidium iodide staining and by changes in mitochondrial membrane potential (DeltaPsi) using a fluorescent dye, JC-1. We suggest that cucurbitacin glucosides exhibit pleiotropic effects on cells, causing both cell cycle arrest and apoptosis. These results suggest that cucurbitacin glucosides might have therapeutic value against breast cancer cells.

The elucidation of the molecular recognition of adenosine deaminase (ADA), the interpretation of the catalytic mechanism, and the design of novel inhibitors are based mostly on data obtained for the crystalline state of the enzyme. To obtain evidence for molecular recognition of the physiologically relevant soluble enzyme, we studied its interactions with the in situ formed inhibitor, 6-OH-purine riboside (HDPR), by 1D-15N- and 2D-(1H-15N)- NMR using the labeled primary inhibitor [15N4]-PR. We synthesized both [15N4]-PR and an [15N4]-HDPR model, from relatively inexpensive 15N sources. The [15N4]-HDPR model was used to simulate H-bonding and possible Zn2+-coordination of HDPR with ADA. We also explored possible ionic interactions between PR and ADA by 15N-NMR monitored pH-titrations of [15N4]-PR. Finally, we investigated the [15N4]-PR-ADA 1:1 complex by 2D-(1H-15N) NMR. We found that HDPR recognition determinants in ADA do not include any ionic-interactions. HDPR N1 H is an H-bond acceptor, and not an H-bond donor. Despite the proximity of N7 to the Zn2+-ion, no coordination occurs; instead, N7 is an H-bond acceptor. We found an overall agreement between the crystallographic data for the crystallized ADA:HDPR complex and the 15N-NMR signals for the corresponding soluble complex. This finding justifies the use of ADA's crystallographic data for the design of novel inhibitors.

A molecular mechanics force field has been developed which accurately reproduces experimental solid state structures and conformer interconversion barriers for a series of sterically congested diaryl and triaryl phosphines and some of their chalcogenide and Cr(CO)5 derivatives.

Fig fruit has been a typical component in the health-promoting Mediterranean diet for millennia. To study the potential health-promoting constituents of fig fruits, six commercial fig varieties differing in color (black, red, yellow, and green) were analyzed for total polyphenols, total flavonoids, antioxidant capacity, and amount and profile of anthocyanins. Using reversed-phase liquid chromatography (RP-LC), various concentrations of anthocyanins but a similar profile was found in all varieties studied. Hydrolysis revealed cyanidin as the major aglycon. Proton and carbon NMR confirmed cyanidin-3-O-rhamnoglucoside (cyanidin-3-O-rutinoside; C3R) as the main anthocyanin in all fruits. Color appearance of fig extract correlated well with total polyphenols, flavonoids, anthocyanins, and antioxidant capacity. Extracts of darker varieties showed higher contents of phytochemicals compared to lighter colored varieties. Fruit skins contributed most of the above phytochemicals and antioxidant activity compared to the fruit pulp. Antioxidant capacity correlated well with the amounts of polyphenols and anthocyanins (R2 = 0.985 and 0.992, respectively). In the dark-colored Mission and the red Brown-Turkey varieties, the anthocyanin fraction contributed 36 and 28% of the total antioxidant capacity, respectively. C3R contributed 92% of the total antioxidant capacity of the anthocyanin fraction. Fruits of the Mission variety contained the highest levels of polyphenols, flavonoids, and anthocyanins and exhibited the highest antioxidant capacity.

5-Dimethylamino-1-aminoindan undergoes thermal decomposition and reacts with 6-chlorouracil to give 5-indanyl-6-chlorouracil derivative 9. The formation of 9 may be rationalized by a putative mechanism based on the intermediacy of the imminium methide species 8a.

The chemical stability and reactivity of hydroxy-1-aminoindans and their N-propargyl derivatives are strongly affected by the position of the OH group and its orientation relative to that of the amino moiety. Thus, the 4- and 6-OH regioisomers were found to be stable, while the 5-OH analogues were found to be inherently unstable as the free bases. The latter, having a para orientation between the OH and the amino moieties, could be isolated only as their hydrochloride salts. 7-Hydroxy-1-aminoindans and 7-hydroxy-1-propargylaminoindans represent an intermediate case; while sufficiently stable even as free bases, they exhibit, under certain experimental conditions, unexpected reactivity. The instability of the 5- and 7-hydroxy-aminoindans is attributed to their facile conversion to the corresponding, reactive quinone methide (QM) intermediates. The o-QM obtained from 7-hydroxy-aminoindans was successfully trapped with ethyl vinyl ether via a Diels-Alder reaction to give tricyclic acetals 32a,b.

[reaction: see text] In a recent article, we reported on the base-catalyzed rearrangements of dipropargyl selenides, -sulfides, -sulfoxides, and -sulfones that eventually lead to polycyclic aromatic products. In the present work, we report on the first isolation and characterization of the thiophene dioxide intermediates 5b,c from a mild tandem isomerization/cyclization/aromatization of bis(pi-conjugated propargyl) sulfones. Monoallene 2b,c and diallene 3b intermediates were also identified by NMR. A kinetic study of the rearrangement of 1a-c revealed that the unusual facile tandem process is highly dependent on the nature of gamma-substitution.

(13)C DNMR studies of codeine and sinomenine (derivatives of N-Me morphinan) indicated that N-inversion-C-N rotation (NIR) is unusually fast for these substituted piperidines when compared with other N-Me piperidines. Since only broadening, but no signal splitting, was reached at low temperatures and the difference of chemical shifts (Delta delta) for individual conformers with the equatorially and axially oriented N-Me substituent was unavailable, the limits of the NIR barrier for these amines were determined by line shape analysis using Delta delta values provided by ab initio calculations. On the basis of the comparison of experimentally determined (13)C NMR chemical shifts for tropane conformers with the ones calculated at different theory levels for this N-Me piperidine, the B3LYP/6-31G(p)/GIAO level was chosen as a sufficiently accurate method for calculations of Delta delta. By this new "semiempirical" procedure of line shape analysis the NIR barrier for the studied morphinans lies within a 25-27 kJ mol(-1) (6.0-6.5 kcal mol(-1)) range. A low NIR barrier for morphine alkaloids is supposed to be an important factor in the activation of morphine receptor.

Sundry dipropargyloxy disulfides have been successfully prepared, and their rearrangement paths and products have been found to be dependent upon their substitution pattern. Inter alia compounds of two heretofore unknown structure types-10 and 11 on one hand and 14 and 15 on the other-have been obtained and characterized.

A detailed high-field NMR analysis of several acylated tetrahydrobenzazepines, supported by molecular mechanics calculations, indicates that the heterocyclic ring in these compounds exists in a chair conformation, with the carbonyl oriented anti to the aryl moiety in the dominant rotamer. Surprisingly, ring methylenes are typically diastereotopic at room temperature, as the barriers for the process of enantiomerization of the seven-membered ring are much higher than expected. It is shown that ring inversion is correlated (but not concerted) with rotation of the amide moiety, as the carbonyl is forced out of conjugation with the nitrogen in the transition state.

Octakis(bromomethyl)naphthalene (4) adopts in the crystal a chiral conformation with a helical central naphthalene core and the bromomethyl groups disposed in an alternate up-down "in" arrangement. According to MM3 calculations, this conformation is less stable than the corresponding all alternated "out" form, while B3LYP/LANL2DZ calculations suggest the opposite stability order. The topomerization barrier (16.0 kcal mol(-1)) is ascribed to an enantiomerization process requiring 180 degrees rotation of all the bromomethyl groups and reversal of the helical sense of the naphthalene core.

The hitherto unsolved problem of the origin of the unusually high nitrogen inversion-rotation (NIR) barriers in 7-azabicyclo[2.2.1]heptanes (the bicyclic effect) was examined using the natural bond orbital (NBO) approach. Reinvestigating the NIR barrier for tropane by DNMR, we found that NIR barriers increase smoothly on going from nitrogen-bridged bicyclic systems of a larger ring size to the smaller ring homologous systems. The experimental NIR barriers are reproduced with good accuracy using the MP2/6-31G level of theory. The NBO analysis for these and other azabicycles led to the conclusion that the height of these barriers is mostly determined by the energy of the sigma-orbitals of the C(alpha)(-)C(beta) bonds as well as the nitrogen lone pair. Thus, the bicyclic effect is actually an extreme case of a common C(alpha-)N-C(alpha) tripyramid geometry-NIR barrier dependence for N-bridged bicyclic amines. By establishing the rate-determining role of the C(alpha-)N-C(alpha) tripyramid fragment for NIR, we have derived the first sufficiently accurate quantitative correlations amine geometry-NIR barrier for monocyclic as well as bicyclic N-H and N-Me amines (i.e., for an amine set which also includes the bicyclic effect systems).

The octaethyl triptycenes 7 and 8 were synthesized by Diels-Alder reaction of the octaethylanthracene 4with the corresponding benzynes. Low-temperature NMR spectra of 7 and 8 are consistent with the presence of the fully alternated conformation "a" of C(2) symmetry. However, in the determined crystal structures, the compounds adopt a higher energy conformation with a pair of vicinal ethyls oriented in the same direction.

2,2,6,6-Tetramethyl substituted piperidines with a beta-branched N-alkyl substituent were synthesized by the photoreaction of N-Me precursors with ketones. The main conformation features of these sterically-hindered amines (established by NMR and IR spectroscopy) are a ring in the chair form, an eclipsed conformation for the N-substituent and an intramolecular OH...N bond. High barriers for the geminal substituent topomerization were measured for these piperidines at different temperatures by means of line-shape analysis of the temperature-dependent 13C and 1H NMR spectra. An MM3-derived conformation scheme indicated that, for one of the studied analogues, the rotation of the N-substituent determines a slow topomerization rate. A new mechanism of nitrogen inversion--a concerted hydrogen-bond dissociation/nitrogen inversion process--is considered for hydrogen-bonded amines.

In a recent paper, we reported on the base-catalyzed rearrangement of bis-propargylic sulfoxides that eventually leads to polycyclic products featuring an unsaturated, cyclic substituent such as cyclohexenyl or phenyl. Due to steric constraints, the latter is positioned roughly perpendicularly to the tricyclic core, and in most cases, two rotamers can be observed in the ground state. In the present work, we report on the synthesis and the products of both symmetrical and asymmetrical starting materials. We also measure, by NMR techniques, the rotation rate of the side chain for several such polycyclic sulfoxides. The barriers for this process, which is similar to a biphenyl rotation, are very strongly dependent on the nature of the substrate, ranging between <7 and 21.0 kcal.mol(-1) for sulfoxides with two five-membered rings and two seven-membered rings, respectively. These barriers can be successfully simulated by molecular-mechanics calculations, and the geometries of the transition states are discussed.

Coumarin ester derivatives 1, substituted at C-4 and/or C-12 with alkyl chains, were synthesized and intercalated within DMPC liposomal bilayers. By correlating the 13C chemical shift with medium polarity [E(T)(30)], the relative location of these substrates within the liposomal bilayer was determined. The length of the alkyl chain substituents clearly influences the lipophilicity of the substrates and their location and orientation within the liposome: Superoxide readily saponifies the C-12 esteric linkage of 1, when this reaction site lies in a polar region of the liposome (E(T)(30) > 45 kcal/mol), to give the corresponding 7-hydroxy coumarin derivatives 2. However, when C-12 lies deeper and is hence less available to O(2)(*-), the lactonic carbon C-2, which lies in a shallower region (E(T)(30) = 43-49), is the preferred site for superoxide-mediated cleavage. When coumarin 1 is disubstituted with long chains at both C-12 and C-4, these derivatives lie deep within the bilayer and react only slowly with O(2)(*-). These results indicate there is indeed a correlation between location within the bilayer and substrate reactivity. Contrary to the suggestion of Dix and Aikens (Chem. Res. Toxicol.6:2-18; 1993) superoxide can penetrate deep within the liposomal bilayer. Nevertheless, its concentration drops precipitously (to approximately 16% of what it is near the interface) below E(T) values of 38, thereby precluding substantial reaction with many highly lipophilic substrates. This work also confirms the findings of others that reactions of small oxy-radicals occur within cellular membranes and appear to be of significant biological importance.

The presence of betacyanin pigments and their isoforms has been detected in the fruit of Hylocereus polyrhizus, a vine cactus native to South America. Along with the known betanin and phyllocactin (6'-O-malonylbetanin), a new betacyanin was structurally elucidated as betanidin 5-O-[6'-O-(3"-hydroxy-3"-methyl-glutaryl)-beta-D-glucopyranoside] (proposed trivial name hylocerenin) by means of electrospray MS/MS, HPLC, and NMR techniques.

Conformational preferences in alkyl- as well as Ph-substituted 3-piperideines (1,2,3,6-tetrahydropyridines) have been characterized by ab initio and molecular mechanics calculations. A set of rules and subrules for estimation of the conformational equilibrium (in terms of preferred substituent orientation) in these systems. with differently positioned ring substituent (-s), is presented. Examples of the revision of some previous stereochemical assignments demonstrate the reliability of these rules.

1,2,3,4,5,6,7,8-Octaethylanthracene (5), 1,2,3,4,6,7,8-heptaethylfluorene (7), and 1,2,3,4,5,6,7,8-octaethylfluorene (8) were synthesized by Friedel-Crafts ethylations of 9,10-dihydroanthracene and fluorene. MM3 calculations indicate that the two ethylated six-membered rings of 5 and 7 are conformationally independent. According to the calculations, two low-energy conformers of each compound are possible with the ethyl groups attached to the external aryl rings arranged in an alternated "up-down" orientation. MM3 calculations indicate that in the lowest energy conformation the central fluorene core of 8 adopts a twisted conformation to avoid repulsive steric interactions between the ethyls at the bay region. Two fully alternated up-down conformations are possible for 8, differing in the orientation ("in" or "out") of the ethyls in the bay region. MM3 calculations predict that the lowest energy conformer is the fully alternated "out" form of C(2)() symmetry. The rotational barriers of 5, 7, and 8 are in the 8.7-11.3 kcal mol(-1) range, the largest barrier corresponding to the more crowded octaethylfluorene 8. Anthracene 5 adopts in the crystal a conformation of approximate C(2)(h) symmetry with pairs of peri groups on the same edge of the molecule oriented syn. The conformations adopted in the crystal by 7 and 8 do not correspond to the calculated lowest energy form. In the conformation of 7 in the crystal the ethyl groups on the trisubstituted ring adopt the unusual all syn arrangement. Octaethylfluorene 8 adopts a conformation with a twisted central fluorene core but with a syn arrangement of a pair of vicinal ethyl groups.

In previous studies we have elucidated the presence of powerful, natural antioxidants (NAO) in water extracts of spinach leaves and demonstrated their biological activity in both in vitro and in vivo systems. In the present study, the chemical identity of several of these antioxidant components is presented. Spinach leaves were extracted with water and the 20,000 g supernatant which contained the antioxidant activity was extracted with a water:acetone (1:9) solution. The 20,000 g supernatant obtained was further purified on reverse phase HPLC using C-8 semi-preparative column. Elution with 0.1% TFA resulted in five hydrophilic peaks. Elution with acetonitrile in TFA resulted in seven additional hydrophobic peaks. All the peaks were detected at 250 nm. All the fractions obtained showed antioxidant activity when tested using three different assays. Based on 1H and 13C NMR spectroscopy four of the hydrophobic fractions were identified as glucuronic acid derivatives of flavonoids and three additional fractions as trans and cis isomers of p-coumaric acid and others as meso-tartarate derivatives of p-coumaric acid. The present study demonstrates for the first time the presence of both flavonoids and p-coumaric acid derivatives as antioxidant components of the aqueous extract of spinach leaves.

Since the nitrogens of nucleosides and nucleotides play an important role in the molecular recognition of these compounds, (15)N NMR became a method of choice in this field. Fully (15)N-labeled adenine, required in the latter studies, was obtained in four synthetic steps, in a good yield. Likewise, ((15)N(5))-2-hexylthioether-adenine and ((15)N(5))-8-Br-adenine were obtained in five synthetic steps from the relatively inexpensive (15)N sources: (15)N-NH(4)Cl, (15)N-NH(4)OH, (15)N-NaNO(2). Full (15)N labeling of these adenine prototypes enabled to obtain high-resolution (15)N NMR spectra of these bases at 60.8 MHz. Furthermore, the spectra suggested the existence of the N3-H species in the tautomeric mixtures of these compounds in solution, in addition to the well-reported N9-H (major) and N7-H (minor) tautomers. These observations were also supported by quantum mechanical calculations of the tautomeric equilibria in the gas phase and in solution of the above-mentioned adenine compounds. The gas-phase tautomeric equilibria were estimated using density functional theory and second-order perturbation theory methods. Solvent effects were included by means of both continuum and discrete solvation models. The observation of the existence of the N3-H tautomer has a clear impact on the possible H-bonding patterns of these adenine prototypes and on their molecular recognition by various biological macromolecules. The above(15)N-labeled analogues are expected to find use as (15)N NMR probes for numerous biochemical studies.

A perfluorinated derivative of phthalocyanine was synthesized as the free base, hexadeca-(2,2,2-trifluoroethoxy) phthalocyanine (H2F48Pc), and as a zinc complex, hexadeca-(2,2,2-trifluoroethoxy)-phthalocyaninatozinc (ZnF48Pc), and their spectroscopic and photochemical properties were studied. The absorption bands are shifted bathochromically relative to simple phthalocyanines, exhibiting the longest wavelength band near 735 nm (H2F48Pc) and 705 (ZnF48Pc). The solvatochromism of both compounds was modeled by Reichardt's ET(30) parameter and Kamlet, Abboud and Taft multiparameter approach. The former, simpler, model was found to be adequate. We found that H2F48Pc undergoes unique basic and acidic titrations in organic solvents. These titration processes are accompanied by spectral changes that are explained on the basis of the chromophore's symmetry. Singular value decomposition was employed to resolve the spectra into the contributions of the species at various stages of protonation and to obtain the equilibrium constants. Nuclear magnetic resonance spectra (1H, 19F and 13C) for the free base were obtained in a tetrahydrofurand8 solution. The carbon spectrum, taken as a function of temperature, provided evidence for the presence of a tautomerization process, which switches the two internal hydrogens between the four central nitrogen atoms. As far as we know, this is the first report of the measurement of the free energy of activation for such process (delta G = 10.6-11.4 kcal mol-1 between 217 and 330 K) for a phthalocyanine, in solution. Like most other phthalocyanines these two compounds also act as photosensitizers and as generators of singlet molecular oxygen. The absolute quantum yields (phi delta) for ZnF48Pc was 0.58 +/- 0.01 in benzene and 0.35 +/- 0.01 in lipid vesicles. H2F48Pc had lower yields, 0.16 and 0.005, respectively. Either protonation or deprotonation of the pyrrole nitrogens in H2F48Pc lowered the phi delta.

Dimerization of tetraethylbenzyne (generated by reaction of 1, 2-dibromo-3,4,5,6-tetraethylbenzene (8) with 1 equiv of BuLi) afforded in low yield octaethylbiphenylene (3), together with a major product which was characterized as 2,3,4,5,3',4', 5'-heptaethyl-2'-vinylbiphenyl (9). X-ray diffraction indicates that biphenylene 3 adopts in the crystal a conformation of approximate C(2)(h )()symmetry with the ethyl groups within each phenylene ring arranged in an alternated up-down fashion. Notably, pairs of vicinal ethyl groups located at peri positions are oriented in a syn arrangement in the crystal. Low temperature NMR spectroscopy is consistent with the presence in solution of either the crystal conformation or a fully alternated conformation lacking any syn interaction. Molecular mechanics (MM3), semiempirical (AM1, PM3), and ab initio calculations indicate that the crystal conformation is a high energy form, and that the lowest energy conformation is the fully alternated form. The topomerization barrier of the methylene protons of the ethyl groups of 3 is 9.4 +/- 0.1 kcal mol(-)(1), which is between the rotational barriers of 8 and 1,2,3, 4-tetraethylbenzene 7 (9.9 +/- 0.1 and 8.2 +/- 0.1 kcal mol(-)(1), respectively). The similarity in rotational barriers suggests that a given tetraethylphenylene subunit does not markedly affect the rotational barrier of the ethyl groups of the other subunit.

From the leaves of Renealmia chrysotrycha (Zingiberaceae), we isolated a sesquiterpene alcohol for which spectral data suggested the structure of a 10-aromadendranol. A meticulous NMR investigation, based mainly on vicinal proton-proton coupling constants and NOE interactions, and confirmed by a molecular mechanics calculation, established its relative stereochemistry as that of ledol. This finding resolves several recent literature ambiguities. Three known compounds (aromadendrene, cis-calamenene and palustrol) were also isolated.

In the context of our high-throughput organic synthesis program, we have studied the reactivity of special beta-keto esters toward the Biginelli reaction. We have found that a cyclic beta-keto ester reacts with one molecule of urea and two molecules of aldehyde to give a new family of spiro heterobicyclic aliphatic rings in good yields. Interestingly, the Biginelli product was not detected. After analysis of products using HPLC, 1H NMR, and 13C NMR, we have found that the reaction is driven by a regio-specific condensation of two molecules of aldehyde with the other reagents to afford only products harboring substituents exclusively in cis configuration. Monte Carlo minimization studies using MM2 force field suggest that cis products are energetically more stable than the trans counterparts. Together with previously reported data, these results suggest that the trans products were not obtained as result of steric hindrance produced by the equatorial position of one of the ring substituents. This new reaction is useful for high-throughput organic synthesis. Indeed, the new scaffold can be used to introduce additional groups in the molecules through remaining functional groups by a "domino strategy".

Bicyclic cyclopropenes 14a, 14b, and 26 were prepared by various synthetic routes. Polymer rose Bengal (p-RB) photosensitized oxygenation of bicyclooctenes 14a,b in CDCl(3) proceeded sluggishly (variable O(2) uptake of ca. 0.35-0.75 equiv in 8 h) and was accompanied by sensitizer bleaching. Preparative gas chromatography of the complex product mixtures from 14a and 14b yielded both dienes (Z- and E-29, 30, and 31) and enones (E- and Z-12, 32, 34). By contrast, p-RB photosensitized oxidation of bicyclononene 26 in CDCl(3) proceeded somewhat more rapidly (O(2) uptake of ca. 1 equiv in 2.5 h) yielding enones (20, 42-45) exclusively upon GC separation. The diene products, observed in the case of 14, result from the thermolysis of the remaining unreacted cyclopropenes, while the enones are the oxygenation products. The oxygenation was slowed by radical inhibitors, but not by (1)O(2) quenchers; nor were any oxidation products observed when these cyclopropenes were reacted with triphenylphosphine ozonide, a chemical (1)O(2) source. The data indicates that a photosensitizer-initiated free radical autoxidative process is involved. Likely intermediates in this oxygenation are epoxide 27 or 37 and hydroperoxide 28 or 38, for the bicyclooctene (14) and bicyclononene (26) systems, respectively. The absence of (1)O(2) product in these cyclopropene systems, in contradistinction to their higher homologues, may be attributable to either the relatively long C(alpha)-H(allylic) distance in alkylcyclopropenes, which places the abstractable allylic hydrogen "out of reach", or their relatively high IP. Either, or both, of these factors may have slowed the rate of the singlet oxygenation of the cyclopropenes to a point where free radical processes compete favorably. In the course of this study, we also explored the singlet oxygenation (DABCO inhibited) of enones 12a,b and 20. These generated, respectively, a mixture of peroxides identified as alpha-keto hydroperoxides 51/54 and hemiperketals 52/55 (the cyclic form of beta-keto hydroperoxides 53/56). Phosphine reduction of these peroxides yields the corresponding alcohols 33/43 and 32/42.