Probing hemoglobin structure by means of traveling-wave ion mobility mass spectrometry

Hemoglobin (Hb) is a tetrameric noncovalent complex consisting of two α- and two β-globin chains each associated with a heme group. Its exact assembly pathway is a matter of debate. Disorders of hemoglobin are the most common inherited disorders and subsequently the molecule has been extensively studied. This work attempts to further elucidate the structural properties of the hemoglobin tetramer and its components. Gas-phase conformations of hemoglobin tetramers and their constituents were investigated by means of traveling-wave ion mobility mass spectrometry. Sickle (HbS) and normal (HbA) hemoglobin molecules were analyzed to determine whether conformational differences in their quaternary structure could be observed. Rotationally averaged collision cross sections were estimated for tetramer, dimer, apo-, and holo-monomers with reference to a protein standard with known cross sections. Estimates of cross section obtained for the tetramers were compared to values calculated from X-ray crystallographic structures. HbS was consistently estimated to have a larger cross section than that of HbA, comparable with values obtained from X-ray crystallographic structures. Nontetrameric species observed included apo- and holo- forms of α- and β-monomers and heterodimers; α- and β-monomers in both apo- and holo- forms were found to have similar cross sections, suggesting they maintain a similar fold in the gas phase in both the presence and the absence of heme. Heme-deficient dimer, observed in the spectrum when analyzing commercially prepared Hb, was not observed when analyzing fresh blood. This implies that holo-α-apo-β is not an essential intermediate within the Hb assembly pathway, as previously proposed.     

MALDI-MS/MS with Traveling Wave Ion Mobility for the Structural Analysis of N-Linked Glycans

The preference for singly charged ion formation by MALDI makes it a better choice than electrospray ionization for profiling mixtures of N-glycans. For structural analysis, fragmentation of negative ions often yields more informative spectra than fragmentation of positive ones but such ions are more difficult to produce from neutral glycans under MALDI conditions. This work investigates conditions for the formation of both positive and negative ions by MALDI from N-linked glycans released from glycoproteins and their subsequent MS/MS and ion mobility behaviour. 2,4,6-Trihydroxyacetophenone (THAP) doped with ammonium nitrate was found to give optimal ion yields in negative ion mode. Ammonium chloride or phosphate also yielded prominent adducts but anionic carbohydrates such as sulfated N-glycans tended to ionize preferentially. Carbohydrates adducted with all three adducts (phosphate, chloride, and nitrate) produced good negative ion CID spectra but those adducted with iodide and sulfate did not yield fragment ions although they gave stronger signals. Fragmentation paralleled that seen following electrospray ionization providing superior spectra than could be obtained by PSD on MALDI-TOF instruments or with ion traps. In addition, ion mobility drift times of the adducted glycans and the ability of this technique to separate isomers also mirrored those obtained following ESI sample introduction. Ion mobility also allowed profiles to be obtained from samples whose MALDI spectra showed no evidence of such ions allowing the technique to be used in conditions where sample amounts were limiting. The method was applied to N-glycans released from the recombinant human immunodeficiency virus glycoprotein, gp120.     

Controlling switching in bistable [2]catenanes by combining donor-acceptor and radical-radical interactions

Two redox-active bistable [2]catenanes composed of macrocyclic polyethers of different sizes incorporating both electron-rich 1,5-dioxynaphthalene (DNP) and electron-deficient 4,4'-bipyridinium (BIPY(2+)) units, interlocked mechanically with the tetracationic cyclophane cyclobis(paraquat-p-phenylene) (CBPQT(4+)), were obtained by donor-acceptor template-directed syntheses in a threading-followed-by-cyclization protocol employing Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloadditions in the final mechanical-bond forming steps. These bistable [2]catenanes exemplify a design strategy for achieving redox-active switching between two translational isomers, which are driven (i) by donor-acceptor interactions between the CBPQT(4+) ring and DNP, or (ii) radical-radical interactions between CBPQT(2(?+)) and BIPY(?+), respectively. The switching processes, as well as the nature of the donor-acceptor interactions in the ground states and the radical-radical interactions in the reduced states, were investigated by single-crystal X-ray crystallography, dynamic (1)H NMR spectroscopy, cyclic voltammetry, UV/vis spectroelectrochemistry, and electron paramagnetic resonance (EPR) spectroscopy. The crystal structure of one of the [2]catenanes in its trisradical tricationic redox state provides direct evidence for the radical-radical interactions which drive the switching processes for these types of mechanically interlocked molecules (MIMs). Variable-temperature (1)H NMR spectroscopy reveals a degenerate rotational motion of the BIPY(2+) units in the CBPQT(4+) ring for both of the two [2]catenanes, that is governed by a free energy barrier of 14.4 kcal mol(-1) for the larger catenane and 17.0 kcal mol(-1) for the smaller one. Cyclic voltammetry provides evidence for the reversibility of the switching processes which occurs following a three-electron reduction of the three BIPY(2+) units to their radical cationic forms. UV/vis spectroscopy confirms that the processes driving the switching are (i) of the donor-acceptor type, by the observation of a 530 nm charge-transfer band in the ground state, and (ii) of the radical-radical ilk in the switched state as indicated by an intense visible absorption (ca. 530 nm) and near-infrared (ca. 1100 nm) bands. EPR spectroscopic data reveal that, in the switched state, the interacting BIPY(?+) radical cations are in a fast exchange regime. In general, the findings lay the foundations for future investigations where this radical-radical recognition motif is harnessed in bistable redox-active MIMs in order to achieve close to homogeneous populations of co-conformations in both the ground and switched states.     

Nonionic diethanolamide amphiphiles with unsaturated C18 hydrocarbon chains: thermotropic and lyotropic liquid crystalline phase behavior

The neat and lyotropic liquid crystalline phase behavior of three nonionic diethanolamide amphiphiles with C18 hydrocarbon chains containing one, two or three unsaturated bonds has been examined. This has allowed the effect of degree of unsaturation on the phase behavior of diethanolamide amphiphiles to be investigated. Neat linoleoyl and linolenoyl diethanolamide undergo a transition from a glassy liquid crystal to a liquid crystal at ～-85 °C, while neat oleoyl diethanolamide undergoes a transition at ～-60 °C to a liquid crystalline material before re-crystallizing at -34 °C. Oleoyl diethanolamide then undergoes a third transition from a crystalline phase to a smectic liquid crystalline phase at ～5 °C. In the absence of water, the transition temperature from a smectic liquid crystal to an isotropic liquid decreases with increasing unsaturation. The addition of water results in the formation of a lamellar phase (L(α)) for all three amphiphiles. The lamellar phase is stable under excess water conditions up to temperatures of at least 70 °C. Approximate partial binary amphiphile-water phase diagrams generated for the three unsaturated C18 amphiphiles indicate that the excess water point for each amphiphile occurs at ～60% (w/w) amphiphile.     

Complete family of mono-, bi-, and trinuclear Re(I)(CO)3Cl complexes of the bridging polypyridyl ligand 2,3,8,9,14,15-hexamethyl-5,6,11,12,17,18-hexaazatrinapthalene: syn/anti isomer separation, characterization, and photophysics

The syn and anti isomers of the bi- and trinuclear Re(CO)(3)Cl complexes of 2,3,8,9,14,15-hexamethyl-5,6,11,12,17,18-hexaazatrinapthalene (HATN-Me(6)) are reported. The isomers are characterized by (1)H NMR spectroscopy and X-ray crystallography. The formation of the binuclear complex from the reaction of HATN-Me(6) with 2 equiv of Re(CO)(5)Cl in chloroform results in a 1:1 ratio of the syn and anti isomers. However, synthesis of the trinuclear complex from the reaction of HATN-Me(6) with 3 equiv of Re(CO)(5)Cl in chloroform produces only the anti isomer. syn-{(Re(CO)(3)Cl)(3)(μ-HATN-Me(6))} can be synthesized by reacting 1 equiv of Re(CO)(5)Cl with syn-{(Re(CO)(3)Cl)(2)(μ-HATN-Me(6))} in refluxing toluene. The product is isolated by subsequent chromatography. The X-ray crystal structures of syn-{(Re(CO)(3)Cl)(2)(μ-HATN-Me(6))} and anti-{(Re(CO)(3)Cl)(3)(μ-HATN-Me(6))} are presented both showing severe distortions of the HATN ligand unit and intermolecular π stacking. The complexes show intense absorptions in the visible region, comprising strong π → π* and metal-to-ligand charge-transfer (MLCT) transitions, which are modeled using time-dependent density functional theory (TD-DFT). The energy of the MLCT absorption decreases from mono- to bi- to trinuclear complexes. The first reduction potentials of the complexes become more positive upon binding of subsequent Re(CO)(3)Cl fragments, consistent with changes in the energy of the MLCT bands and lowering of the energy of relevant lowest unoccupied molecular orbitals, and this is supported by TD-DFT. The nature of the excited states of all of the complexes is also studied using both resonance Raman and picosecond time-resolved IR spectroscopy, where it is shown that MLCT excitation results in the oxidation of one rhenium center. The patterns of the shifts in the carbonyl bands upon excitation reveal that the MLCT state is localized on one rhenium center on the IR time scale.     

Encapsulation of phthalocyanine supramolecular stacks into virus-like particles

We report herein the encapsulation of a water-soluble phthalocyanine (Pc) into virus-like particles (VLPs) of two different sizes, depending on the conditions. At neutral pH, the cooperative encapsulation/templated assembly of the particles induces the formation of Pc stacks instead of Pc dimers, due to an increased confinement concentration. The Pc-containing VLPs may potentially be used as photosensitizer/vehicle systems for biomedical applications such as photodynamic therapy.     

Switching the Switch: Ligand Induced Disulfide Formation in HDAC8

Human histone deacetylase 8 is a well-recognized target for T-cell lymphoma and particularly childhood neuroblastoma. PD-404,182 was shown to be a selective covalent inhibitor of HDAC8 that forms mixed disulfides with several cysteine residues and is also able to transform thiol groups to thiocyanates. Moreover, HDAC8 was shown to be regulated by a redox switch based on the reversible formation of a disulfide bond between cysteines Cys₁₀₂ and Cys₁₅₃. This study on the distinct effects of PD-404,182 on HDAC8 reveals that this compound induces the dose-dependent formation of intramolecular disulfide bridges. Therefore, the inhibition mechanism of HDAC8 by PD-404,182 involves both, covalent modification of thiols as well as ligand mediated disulfide formation. Moreover, this study provides a deep molecular insight into the regulation mechanism of HDAC8 involving several cysteines with graduated capability to form reversible disulfide bridges.