Atmospheric solids analysis probe mass spectrometry: An easy bolt-on for the synthetic undergraduate teaching laboratory.

RATIONALE: High costs and student numbers can often hinder implementation of mass spectrometry (MS) in the undergraduate teaching laboratory, often with technicians running samples on students' behalf, and the implementation of MS only in discrete or isolated experiments. This study explores the use of atmospheric solids analysis probe MS (ASAP-MS) as a relatively low-cost, benchtop instrument, and its potential for application as a 'bolt-on' to existing undergraduate organic chemistry experiments. METHODS: Thirteen products synthesised in undergraduate laboratory experiments were analysed by ASAP-MS, along with their starting materials. Analysis was carried out with a Waters RADIAN ASAP mass spectrometer, at four different cone voltages simultaneously to provide fragmentation information. RESULTS: Out of the 13 undergraduate experiments, ASAP-MS was shown to be complementary in 11 of these, either through simple analysis of the precursor ion or by a more complex analysis of the fragments. CONCLUSIONS: ASAP-MS provided spectra that both complement and enhance intended learning outcomes in existing organic chemistry experiments, showing its versatility as a bolt-on technique. Moving forward, ASAP-MS will be integrated into the University of Surrey's undergraduate teaching laboratory.

The Essence in Selectivity of Copper-Mediated Intermolecular Nucleophilic Substitution of a meta C-H Bond in 2-Methyl-N-methoxyaniline: A Theoretical Study.

The detailed mechanism for NHC-Cu(I)-catalyzed intermolecular nucleophilic substitution of the C-H bonds at aniline (2-methyl-N-methoxyaniline) was studied via DFT methods to reveal the essence of the selectivity. Calculations revealed that the meta C-H functionalization proceeds via two nucleophilic attacks on the aromatic ring rather than a one-step meta C-H substitution to give the experimentally observed major product. The reaction is initiated by activation of the substrate via oxidative addition with an NHC-Cu(I) catalyst, through which an umpolung occurs at the ring. From the activated intermediate, methoxyl group transfer to benzyl forms a resting state, while a nucleophile can attack the ortho position of benzyl to form a more stable intermediate. The nucleophile group can then transfer to the meta position by a 1,2-Wagner-Meerwein rearrangement to form the final product through a proton shuttle. In contrast, other transfer processes affording ortho- or para-substituted products encounter higher activation barriers. This work investigates the relationship of product selectivity with the umpolung of the aromatic ring, as well as the priority of a nucleophilic attack at the ortho position of the aromatic, 1,2-Wagner-Meerwein rearrangement from the ortho-substituted intermediate, and proton shuttle from the meta-substituted intermediate.

Scalable Electrochemical Aerobic Oxygenation of Indoles to Isatins without Electron Transfer Mediators by Merging with an Oxygen Reduction Reaction.

An approach to electrochemical oxygenation of indoles leading to isatins was developed by merging with a complementary cathode oxygen reduction reaction. The features of this green protocol include the use of molecular oxygen as the sole oxidant, it being free of an electron transfer mediator, and gram-scale preparation. Mechanistic studies suggested a radical process, and the two oxygen atoms in the isatins were both most likely from molecular oxygen. A detailed mechanism of the reaction utilizing density functional theory calculations was elucidated.

Metal-ligand Lability and Ligand Mobility Enables Framework Transformation via Ligand Release in a Family of Crystalline 2D Coordination Polymers.

A family of seven silver(I)-perfluorocarboxylate-quinoxaline coordination polymers, [Ag4 (O2 CRF )4 (quin)4 ] 1-5 (RF =(CF2 )n-1 CF3 )4 , n=1 to 5); [Ag4 (O2 C(CF2 )2 CO2 )2 (quin)4 ] 6; [Ag4 (O2 CC6 F5 )4 (quin)4 ] 7 (quin=quinoxaline), denoted by composition as 4 : 4 : 4 phases, was synthesised from reaction of the corresponding silver(I) perfluorocarboxylate with excess quinoxaline. Compounds 1-7 adopt a common 2D layered structure in which 1D silver-perfluorcarboxylate chains are crosslinked by ditopic quinoxaline ligands. Solid-state reaction upon heating, involving loss of one equivalent of quinoxaline, yielding new crystalline 4 : 4 : 3 phases [Ag4 (O2 C(CF2 )n-1 CF3 )4 (quin)3 ]n (8-10, n=1 to 3), was followed in situ by PXRD and TGA studies. Crystal structures were confirmed by direct syntheses and structure determination. The solid-state reaction converting 4 : 4 : 4 to 4 : 4 : 3 phase materials involves cleavage and formation of Ag-N and Ag-O bonds to enable the structural rearrangement. One of the 4 : 4 : 3 phase coordination polymers (10) shows the remarkably high dielectric constant in the low electric field frequency range.

Hypervalent iodine promoted the synthesis of cycloheptatrienes and cyclopropanes.

A new strategy is reported for intramolecular Buchner-type reactions using PIDA as a promotor. Traditionally, the Buchner reaction is achieved via Rh-carbenoids derived from RhII catalysts with diazo compounds. Herein, the first metal-free Buchner-type reaction to construct highly strained cycloheptatriene- and cyclopropane-fused lactams is presented. The advantage of these transformations is in their mild reaction conditions, simple operation, broad functional group compatibility and rapid synthetic protocol. In addition, scaled-up experiments and a series of follow-up synthetic procedures were performed to clarify the flexibility and practicability of this method. DFT calculations were carried out to clarify the mechanism.

Hydrogen bonding vs. halogen bonding: the solvent decides.

Control of intermolecular interactions is integral to harnessing self-assembly in nature. Here we demonstrate that control of the competition between hydrogen bonds and halogen bonds, the two most highly studied directional intermolecular interactions, can be exerted by choice of solvent (polarity) to direct the self-assembly of co-crystals. Competitive co-crystal formation has been investigated for three pairs of hydrogen bond and halogen bond donors, which can compete for a common acceptor group. These competitions have been examined in seven different solvents. Product formation has been determined and phase purity has been examined by analysis of powder X-ray diffraction patterns. Formation of hydrogen-bonded co-crystals is favoured from less polar solvents and halogen-bonded co-crystals from more polar solvents. The solvent polarity at which the crystal formation switches from hydrogen-bond to halogen-bond dominance depends on the relative strengths of the interactions, but is not a function of the solution-phase interactions alone. The results clearly establish that an appreciation of solvent effects is critical to obtain control of the intermolecular interactions.

Highly selective CO2vs. N2 adsorption in the cavity of a molecular coordination cage.

Two M8L12 cubic coordination cages, as desolvated crystalline powders, preferentially adsorb CO2 over N2 with ideal selectivity CO2/N2 constants of 49 and 30 at 298 K. A binding site for CO2 is suggested by crystallographic location of CS2 within the cage cavity at an electropositive hydrogen-bond donor site, potentially explaining the high CO2/N2 selectivity compared to other materials with this level of porosity.

Arene Selectivity by a Flexible Coordination Polymer Host.

The coordination polymers [Ag4 (O2 CCF3 )4 (phen)3 ]⋅ phen⋅arene (1⋅phen⋅arene) (phen=phenazine; arene=toluene, p-xylene or benzene) have been synthesised from the solution phase in a series of arene solvents and crystallographically characterised. By contrast, analogous syntheses from o-xylene and m-xylene as the solvent yield the solvent-free coordination polymer [Ag4 (O2 CCF3 )4 (phen)2 ] (2). Toluene, p-xylene and benzene have been successfully used in mixed-arene syntheses to template the formation of coordination polymers 1⋅phen⋅arene, which incorporate o- or m-xylene. The selectivity of 1⋅phen⋅arene for the arene guests was determined, through pairwise competition experiments, to be p-xylene>toluene≈benzene>o-xylene>m-xylene. The largest selectivity coefficient was determined as 14.2 for p-xylene:m-xylene and the smallest was 1.0 for toluene:benzene.

Solvent-vapour-assisted pathways and the role of pre-organization in solid-state transformations of coordination polymers.

A family of one-dimensional coordination polymers, [Ag4(O2C(CF2)2CF3)4(phenazine)2(arene) n ]·m(arene), 1 (arene = toluene or xylene), have been synthesized and crystallographically characterized. Arene guest loss invokes structural transformations to yield a pair of polymorphic coordination polymers [Ag4(O2C(CF2)2CF3)4(phenazine)2], 2a and/or 2b , with one- and two-dimensional architectures, respectively. The role of pre-organization of the polymer chains of 1 in the selectivity for formation of either polymorph is explored, and the templating effect of toluene and p-xylene over o-xylene or m-xylene in the formation of arene-containing architecture 1 is also demonstrated. The formation of arene-free phase 2b , not accessible in a phase-pure form through other means, is shown to be the sole product of loss of toluene from 1-tol·tol [Ag4(O2C(CF2)2CF3)4(phenazine)2(toluene)]·2(toluene), a phase containing toluene coordinated to Ag(I) in an unusual µ:η(1),η(1) manner. Solvent-vapour-assisted conversion between the polymorphic coordination polymers and solvent-vapour influence on the conversion of coordination polymers 1 to 2a and 2b is also explored. The transformations have been examined and confirmed by X-ray diffraction, NMR spectroscopy and thermal analyses, including in situ diffraction studies of some transformations.

Synthesis and receptor binding in trans-CD ring-fused A-CD estrogens: comparison with the cis-fused isomers.

Ligands which selectively activate only one of the estrogen receptors, ERα or ERß, are current pharmaceutical targets. Previously, we have reported on substituted cis A-CD ligands in which the B-ring of the steroidal structure has been removed and cis refers the stereochemistry of the CD ring junction as compared to trans in estradiol. These compounds often showed good potency and selectivity for ERß. Here we report the synthesis and binding affinities for a similar series of trans A-CD ligands, and compare them to the cis-series. Counterintuitively, trans A-CD ligands, which are structurally more closely related to the natural ligand estradiol, show weaker binding and less ß-selectivity than their cis-counterparts.

Novel ligands balance estrogen receptor ß and α agonism for safe and effective suppression of the vasomotor response in the ovariectomized female rat model of menopause.

Vasomotor thermo-dysregulation (hot flashes) are an often debilitating symptom of menopause. Effective treatment is achieved primarily through activation of the estrogen receptor (ER)α with estrogens but is also associated with increased risk for breast and uterine cancer. In this study, we have tested novel compounds lacking the B ring of 17-hydroxy-ß-estradiol (E2) (A-CD compounds) with differing ratios of ERα:ERß binding affinities for the ability to reduce diurnal/nocturnal tail-skin temperatures (TSTs) in the ovariectomized female rat menopausal hot flash model. Normal mammary tissue expresses the predominantly antiproliferative ERß. Therefore, we hypothesized that a preferential ERß agonist with fractional ERα activity would safely reduce TSTs. The A-CD compound, L17, is a preferential ERß agonist that has a ratio of ERß:ERα binding affinity relative to E2 of 9.3 (where ERß:ERα for E2, 1.0). In the ovariectomized rat, daily administration of low doses (1 mg/kg) of the A-CD compound TD81 (ERα:ERß relative affinity, 15.2) was ineffective in temperature regulation, whereas L17 showed a trend toward TST reduction. Both E2 and the A-CD compound, TD3 (ERß:ERα relative affinity, 5.0), also reduced TSTs but had marked proliferative effects on mammary and uterine tissues. At 2 mg/kg, L17 strongly reduced TSTs even more effectively than E2 but, importantly, had only minimal effect on uterine weight and mammary tissues. Both E2- and L17-treated rats showed similar weight reduction over the treatment period. E2 is rapidly metabolized to highly reactive quinones, and we show that L17 has 2-fold greater metabolic stability than E2. Finally, L17 and E2 similarly mediated induction of c-fos expression in neurons within the rat thermoregulatory hypothalamic median preoptic nucleus. Thus, the A-CD compound, L17, may represent a safe and effective approach to the treatment of menopausal hot flashes.

Preferential estrogen receptor ß ligands reduce Bcl-2 expression in hormone-resistant breast cancer cells to increase autophagy.

Acquired resistance to selective estrogen receptor (ER) modulators (SERM) and downregulators (SERD) is a significant clinical problem in the treatment of estrogen (E2) receptor-positive (ER(+)) breast cancers. There are two ER subtypes, ERα and ERß, which promote and inhibit breast cancer cell proliferation, respectively. Although ER(+) breast cancers typically express a high ratio of ERα to ERß, the acquisition of SERM resistance in vitro and in vivo is associated with increased relative expression of the ERß. On some gene enhancers, ERß has been shown to function in opposition to the ERα in the presence of E2. Here, we demonstrate that two different ERß agonists, WAY-20070 and a novel "A-CD" estrogen called L17, produce a marked reduction in G(2)-M phase correlated with effects on cyclin D1 and cyclin E expression in a SERM/SERD-resistant breast cancer cell line. ERß agonists recruited both the ERα and ERß to the Bcl-2 E2-response element strongly reducing Bcl-2 mRNA and protein in an ERß-dependent manner. L17 recruited RIP140 to the Bcl-2 promoter in cells overexpressing ERß. Exposure to the ERß ligands also resulted in increased processing of LC3-I to LC3-II, indicative of enhanced autophagic flux. The coaddition of ERß agonist and the autophagy inhibitor chloroquine resulted in a significant accumulation of sub-G1 DNA which was completely prevented by the addition of the caspase inhibitor Z-VAD-FMK. We propose that combined therapies with an ERß agonist and an inhibitor of autophagy may provide the basis for a novel approach to the treatment of SERM/SERD-resistant breast cancers.

Experimental versus predicted affinities for ligand binding to estrogen receptor: iterative selection and rescoring of docked poses systematically improves the correlation.

The computational determination of binding modes for a ligand into a protein receptor is much more successful than the prediction of relative binding affinities (RBAs) for a set of ligands. Here we consider the binding of a set of 26 synthetic A-CD ligands into the estrogen receptor ERα. We show that the MOE default scoring function (London dG) used to rank the docked poses leads to a negligible correlation with experimental RBAs. However, switching to an energy-based scoring function, using a multiple linear regression to fit experimental RBAs, selecting top-ranked poses and then iteratively repeating this process leads to exponential convergence in 4-7 iterations and a very strong correlation. The method is robust, as shown by various validation tests. This approach may be of general use in improving the quality of predicted binding affinities.

A-CD estrogens. I. Substituent effects, hormone potency, and receptor subtype selectivity in a new family of flexible estrogenic compounds.

Long-term use of estrogen supplements by women leads to an increased risk of breast and uterine cancers. Possible mechanisms include metabolism of estradiol and compounds related to tumor-initiating quinones, and ligand-induced activation of the estrogen receptors ERα and ERß which can cause cancer cell proliferation, depending on the ratio of receptors present. One therapeutic goal would be to create a spectrum of compounds of variable potency for ERα and ERß, which are resistant to quinone formation, and to determine an optimum point in this spectrum. We describe the synthesis, modeling, binding affinities, hormone potency, and a measure of quinone formation for a new family of A-CD estrogens, where the A-C bond is formed by ring coupling. Some substituents on the A-ring increase hormone potency, and one compound is much less quinone-forming than estradiol. These compounds span a wide range of receptor subtype selectivities and may be useful in hormone replacement therapy.

Oxidative modification of citrate synthase by peroxyl radicals and protection with novel antioxidants.

In mammals, aging is linked to a decline in the activity of citrate synthase (CS; E.C. 2.3.3.1), the first enzyme of the citric acid cycle. We used 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), a water-soluble generator of peroxyl and alkoxyl radicals, to investigate the susceptibility of CS to oxidative damage. Treatment of isolated mitochondria with AAPH for 8-24 h led to CS inactivation; however, the activity of aconitase, a mitochondrial enzyme routinely used as an oxidative stress marker, was unaffected. In addition to enzyme inactivation, AAPH treatment of purified CS resulted in dityrosine formation, increased protein surface hydrophobicity, and loss of tryptophan fluorescence. Propyl gallate, 1,8-naphthalenediol, 2,3-naphthalenediol, ascorbic acid, glutathione, and oxaloacetate protected CS from AAPH-mediated inactivation, with IC(50) values of 9, 14, 34, 37, 150, and 160 muM, respectively. Surprisingly, the antioxidant epigallocatechin gallate offered no protection against AAPH, but instead caused CS inactivation. Our results suggest that the current practice of using the enzymatic activity of CS as an index of mitochondrial abundance and the use of aconitase activity as an oxidative stress marker may be inappropriate, especially in oxidative stress-related studies, during which alkyl peroxyl and alkoxyl radicals can be generated.

Rational design, synthesis, and optical properties of film-forming, near-infrared absorbing, and fluorescent chromophores with multidonors and large heterocyclic acceptors.

A new series of film-forming, low-bandgap chromophores (1 a,b and 2 a,b) were rationally designed with aid of a computational study, and then synthesized and characterized. To realize absorption and emission above the 1000 nm wavelength, the molecular design focuses on lowering the LUMO level by fusing common heterocyclic units into a large conjugated core that acts an electron acceptor and increasing the charge transfer by attaching the multiple electron-donating groups at the appropriate positions of the acceptor core. The chromophores have bandgap levels of 1.27-0.71 eV, and accordingly absorb at 746-1003 nm and emit at 1035-1290 nm in solution. By design, the relatively high molecular weight (up to 2400 g mol(-1)) and non-coplanar structure allow these near-infrared (NIR) chromophores to be readily spin-coated as uniform thin films and doped with other organic semiconductors for potential device applications. Doping with [6,6]-phenyl-C(61) butyric acid methyl ester leads to a red shift in the absorption only for 1 a and 2 a. An interesting NIR electrochromism was found for 2 a, with absorption being turned on at 1034 nm when electrochemically switched (at 1000 mV) from its neutral state to a radical cation state. Furthermore, a large Stokes shift (256-318 nm) is also unique for this multidonor-acceptor type of chromophore, indicating a significant structural difference between the ground state and the excited state. Photoluminescence of the film of 2 a was further probed at variable temperatures and the results strongly suggest that the restriction of bond rotations certainly helps to diminish non-radiative decay and thus enhance the luminescence of these large chromophores.

Investigation of residues Lys112, Glu136, His138, Gly247, Tyr248, and Asp249 in the active site of yeast cystathionine beta-synthase.

Cystathionine beta-synthase (CBS), the first enzyme of the reverse transsulfuration pathway, catalyzes the pyridoxal 5'-phosphate-dependent condensation of l-serine and l-homocysteine to form l-cystathionine (l-Cth). A model of the l-Cth complex of the truncated form of yeast CBS (ytCBS), comprising the catalytic core, was constructed to identify residues involved in the binding of l-homocysteine and the distal portion of l-Cth. Residue K112 was selected for site-directed mutagenesis based on the results of the in silico docking of l-Cth to the modeled structure of ytCBS. Residues E136, H138, Y248, and D249 of ytCBS were also targeted as they correspond to identical polar residues lining the mouth of the active site in the structure of human CBS. A series of 8 site-directed mutants was constructed, and their order of impact on the ability of ytCBS to catalyze the beta-replacement reaction is G247S asymptotically equal to K112Q > K112L asymptotically equal to K112R >> Y248F > D249A asymptotically equal to H138F > E136A. The beta-replacement activity of G247S, which corresponds to the homocystinuria-associated G307S mutant of human CBS, is undetectable. The Kml-Ser of the K112L and K112R mutants is increased by 50- and 90-fold, respectively, while Kml-Hcys increases by only 2- and 4-fold, respectively. The Kml-Hcys of H138F and Y248F is increased by 8- and 18-fold, respectively. These results indicate that, while the targeted residues are not direct determinants of l-Hcys binding, G307, Y248, and K112 play essential roles in the maintenance of appropriate active-site conformation.

Deconstructing estradiol: removal of B-ring generates compounds which are potent and subtype-selective estrogen receptor agonists.

Estradiol and related estrogens have been widely used as supplements to relieve menopausal symptoms, but they lead to an increased risk of breast and endometrial cancer. Here we report the synthesis of a new family of compounds where we have removed the B-ring from the steroid ABCD structure, and functionalized the A-ring. These A-CD compounds show a preferential affinity for the estrogen receptor subtype ERbeta. Some show binding affinities which are greater than estradiol. The presence of electron-withdrawing substituents on the A-ring should reduce the tendency of these compounds to form carcinogenic metabolites, so they might lead to a safer approach to hormone replacement therapy.

Interaction force diagrams: new insight into ligand-receptor binding.

A method is described to calculate and visualize the interaction forces of ligand-receptor complexes. Starting from an X-ray crystallographic structure, a "thawing" procedure results in a force-field energy-minimized geometry which is close to the crystallographic starting point. By subtracting non-bonded interactions of the ligand with each amino acid residue and using the resulting force vectors to describe the slope of the remaining potential, two types of interaction force diagrams are created; the first shows the direction of the force vectors in 3D and the second shows the magnitude of the force vectors. The latter representation leads to definition of an 'Interaction Force Fingerprint' (IFFP) which is characteristic of the ligand-receptor binding. IFFPs are used to discuss ligand binding in the human estrogen receptors ERalpha and ERbeta, and provide new insight into ligand selectivity between receptor isoforms.

Stability of carbon-centered radicals: effect of functional groups on the energetics of addition of molecular oxygen.

In this paper we examine a series of hydrocarbons with structural features which cause a weakening of the C-H bond. We use theoretical calculations to explore whether the carbon-centered radicals R(*) which are created after breaking the bond can be stabilized enough so that they resist the addition of molecular oxygen, i.e. where the reaction R(*) + O(2) --> ROO(*) becomes energetically unfavorable. Calculations using a B3LYP-based method provide accurate bond dissociation enthalpies (BDEs) for R-H and R-OO(*) bonds, as well as Gibbs free energy changes for the addition reaction. The data show strong correlations between R-OO(*) and R-H BDEs for a wide variety of structures. They also show an equally strong correlation between the R-OO(*) BDE and the unpaired spin density at the site of addition. Using these data we examine the major functional group categories proposed in several experimental studies, and assess their relative importance. Finally, we combine effects to try to optimize resistance to the addition of molecular oxygen, an important factor in designing carbon-based antioxidants.