Su-Ching Lin

New Chromogenic and Fluorescent Probes for Anion Detection: Formation of a [2 + 2] Supramolecular Complex on Addition of Fluoride with Positive Homotropic Cooperativity

Two new chromogenic and fluorescent probes for anions have been designed, synthesized, and characterized. These probes contain multiple hydrogen bonding donors including hydrazine, hydrazone, and hydroxyl functional groups for potential anion interacting sites. Despite the possible flexible structural framework due to the presence of sp3 carbon linkage, X-ray structure analysis of probe 2 displayed an essentially planar conformation in the solid state owing to strong crystal packing interactions comprising a combination of favorable pi-pi stacking effect and hydrogen bonding to cocrystallized CH3OH molecules. Both probes 1 and 2 display orange color in DMSO solution and show fairly weak fluorescence at room temperature. Binding studies revealed that both probes 1 and 2 show noticeable colorimetric and fluorescent responses only to F-, OAc-, and H2PO4- among the nine anions tested (F-, Cl-, Br-, I-, OAc-, H2PO4-, HSO4-, ClO4-, and NO3-). The general trend of the sensitivity to anions follows the order of F- > OAc- > H2PO4- > Cl- > Br- approximately I- approximately HSO4- approximately ClO4- approximately NO3-. A 1:2 (probe to anion) binding stoichiometry was found for probe 1 with OAc- and H2PO4- and probe 2 with F-, OAc-, and H2PO4-. The binding isotherm of probe 1 to F- was found to be complicated with apparent multiple equilibria occurring in solution. The formation of an aggregated supramolecular complex upon addition of fluoride is proposed to rationalize the observed optical responses and is supported by ESI mass spectrometry and pulsed-field gradient NMR spectroscopy. Data analysis suggests that the binding of probe 1 to F- shows positive homotropic cooperativity.

A turn-like structure "KKPE" segment mediates the specific binding of viral protein A27 to heparin and heparan sulfate on cell surfaces.

Vaccinia viral envelope protein A27 (110 amino acids) specifically interacts with heparin (HP) or heparan sulfate (HS) proteoglycans for cell surface attachment. To examine the binding mechanism, a truncated soluble form of A27 (sA27-aa; residues 21–84 of A27) with Cys71 and Cys72 mutated to Ala was used as the parent molecule. sA27-aa consists of two structurally distinct domains, a flexible Arg/Lys-rich heparin-binding site (HBS) (residues 21–32; 21STKAAKKPEAKR32) and a rigid coiled-coil domain (residues 43–84), both essential for the specific binding. As shown by surface plasmon resonance (SPR), the binding affinity of sA27-aa for HP (KA = 1.25 × 108 m−1) was approximately 3 orders of magnitude stronger than that for nonspecific binding, such as to chondroitin sulfate (KA = 1.65 × 105 m−1). Using site-directed mutagenesis of HBS and solution NMR, we identified a “KKPE” segment with a turn-like conformation that mediates specific HP binding. In addition, a double mutant T22K/A25K in which the KKPE segment remained intact showed an extremely high affinity for HP (KA = 1.9 × 1011 m−1). Importantly, T22K/A25K retained the binding specificity for HP and HS but not chondroitin sulfate, as shown by in vitro SPR and in vivo cell adhesion and competitive binding assays. Molecular modeling of the HBS was performed by dynamics simulations and provides an explanation of the specific binding mechanism in good agreement with the site-directed mutagenesis and SPR results. We conclude that a turn-like structure introduced by the KKPE segment in vaccinia viral envelope protein A27 is responsible for its specific binding to HP and to HS on cell surfaces.

Vaccinia Viral Protein A27 Is Anchored to the Viral Membrane via a Cooperative Interaction with Viral Membrane Protein A17

Background: Vaccinia viral protein A27 associates with viral membrane protein A17 for anchoring to the viral membrane. Results: A27 specifically interacts with two binding regions within the N-terminal domain of A17. Conclusion: The A27-A17 interaction is mediated through a specific and cooperative binding mechanism. Significance: As demonstrated, the F1 and F2 bindings are critical for A27 anchoring to the viral membrane and virion assembly. The vaccinia viral protein A27 in mature viruses specifically interacts with heparan sulfate for cell surface attachment. In addition, A27 associates with the viral membrane protein A17 to anchor to the viral membrane; however, the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and the underlying binding mechanism, we expressed and purified the N-terminal (18–50 residues) and C-terminal (162–203 residues) fragments of A17, which are denoted A17-N and A17-C. Through surface plasmon resonance, the binding affinity of A27/A17-N (KA = 3.40 × 108 m−1) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (KA = 3.40 × 105 m−1), indicating that A27 prefers to interact with A17-N rather than A17-C. Despite the disordered nature of A17-N, the A27-A17 interaction is mediated by a specific and cooperative binding mechanism that includes two active binding sites, namely 32SFMPK36 (denoted as F1 binding) and 20LDKDLFTEEQ29 (F2). Further analysis showed that F1 has stronger binding affinity and is more resistant to acidic conditions than is F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of the A17 protein were packaged into mature virus particles at a reduced level, demonstrating that the F1/F2 interaction plays a critical role in vivo. Using these results in combination with site-directed mutagenesis data, we established a computer model to explain the specific A27-A17 binding mechanism.

Solid-state NMR study of fluorinated steroids.

Solid-state {(1)H}(13)C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy was performed to analyze two fluorinated steroids, i.e., betamethasone (BMS) and fludrocortisone acetate (FCA), that have fluorine attached to C9, as well as two non-fluorinated analogs, i.e., prednisolone (PRD) and hydrocortisone 21-acetate (HCA). The (13)C signals of BMS revealed multiplet patterns with splittings of 16-215Hz, indicating multiple ring conformations, whereas the (13)C signals of FCA, HCA, and PRD exhibited only singlet patterns, implying a unique conformation. In addition, BMS and FCA exhibited substantial deviation (>3.5ppm) in approximately half of the (13)C signals and significant deviation (>45ppm) in the (13)C9 signal compared to PRD and HCA, respectively. In this study, we demonstrate that fluorinated steroids, such as BMS and FCA, have steroidal ring conformation(s) that are distinct from non-fluorinated analogs, such as PRD and HCA.

Self-assembly of tetrametallic square [Re4(CO)12Br4(μ-pz)4] (pz = pyrazine) from [Re(CO)4Br(pz)]. A mechanistic approach

Self-assembly of the tetranuclear square [Re4(CO)12Br4(μ-pz)4] (pz = pyrazine) from the monometallic complex [Re(CO)4Br(pz)] in acetone at room temperature has been investigated. The mechanistic pathway is examined and proved by both in-situ1H NMR and ES-MS studies. Both techniques are helpful to identify three species, Re2(CO)8Br2(μ-pz), Re(CO)3Br(pz)2 and Re2(CO)7Br2(μ-pz)(pz), as intermediates. The other intermediates, Re3(CO)11Br3(μ-pz)2, Re3(CO)10Br3(μ-pz)2(pz), Re4(CO)14Br4(μ-pz)3 and Re4(CO)13Br4(μ-pz)3(pz), detected by electrospray mass spectral techniques also support the proposed mechanistic pathway.

Hindered rotation along carbon–nitrogen bonds. The effect of carbonyliron in complexes of 7-azanorbornadiene derivatives

Hindered rotations have been observed for certain N-substituted 7-azanorbornadiene (7-azabicyclo[2.2.1]hepta-2,5-diene) derivatives and their iron complexes. The free energies of rotation (ΔG‡) have been estimated from the coalescence of NMR signals. The average values found for acetyl amides (Ib and IIb), benzoyl amides (IIc and IIIc), and carbamates (Ia and IIIa) are 17.8, 16.0 and 14.3 kcal mol–1 respectively, in accord with electronic effects. For the iron complexes 1–4 however, steric factors predominate where the rotations are hindered mostly by the crowding by the ligands. The barriers found for the tetracarbonyliron complexes (3a–3c) of ortho-substituted anilines are proportional to the size of the phenyl substituents, i.e. 12.7 kcal mol–1 for Me (3a), 13.6 kcal mor–1 for CN (3b), and 14.0 kcal mol–1 for l (3c). Tricarbonyliron complexes (2 and 4) experience a lower barrier to rotation with respect to the corresponding tetracarbonyl ones (1 and 3) due to a variation in co-ordination geometry. The energies estimated for the iron carbonyl complexes decrease in the orders 1( > Ia) > 2 as well as 3a > 4a. The crystal structures of tetracarbonyl- and tricarbonyl-[1,4-dihydro-1,4-(o-tolylimino)naphthalene]iron 3a and 4a have been determined.

NMR investigation of magnesium chelation and cation-induced signal shift effect of testosterone.

We have previously reported that testosterone (Tes) is able to interact with magnesium chloride dissolved in methanol. In this study, we have applied 1H and 13C NMR spectroscopies to a series of Tes solutions containing Mg2+ at various concentrations. High-resolution 13C NMR spectra of Tes/Mg2+ revealed well-resolved 13C signals, and the intensities of those arising from C3, C5, C16, and C17 decreased linearly with increasing Mg2+ concentration. The magnitude of the chelation affinity could be deduced from the slopes of the 13C intensity variations; typically, the greater the slope the higher the chelation affinity. The results revealed Tes/Mg2+ chelation to be mediated by the oxygen atom attached to C3 in ring A, and the hydroxyl group attached to C17 in ring D. With regard to the chelation specificity, we showed that Tes chelates Mg2+, but not Ca2+ or Zn2+. We also explored the cation-induced signal shift effects of Tes in the presence of Mg2+, Ca2+, or Zn2+. We demonstrate that high-resolution 13C NMR spectroscopy provides a better probe than 1H NMR for the detection of cation chelation and cation-induced signal shift effects for steroid compounds such as Tes.