Comparison of various silica‐based monoliths for the analysis of large biomolecules†

In the present study, three types of silica‐based monoliths, i.e. the first and second generations of commercial silica monolithic columns and a wide‐pore prototype monolith were compared for the analysis of large biomolecules. These molecules possess molecular weights between 1 and 66 kDa. The gradient kinetic performance of the first‐generation monolith was lower than that of the second generation, for large biomolecules (>14 kDa) but very close with smaller ones (1.3–5.8 kDa). In contrast, the wide‐pore prototype column was particularly attractive with proteins larger than 19 kDa (higher peak capacity). Among these three columns, the selectivity and retention remained quite similar but a possible larger number of accessible and charged residual silanols was noticed on the wide‐pore prototype material, which led to unpredicted small changes in selectivity and slightly broader peaks than expected. The peak shapes attained with the addition of 0.1% formic acid in the mobile phase remained acceptable for MS coupling, particularly for biomolecules of less than 6 kDa. It was found that one of the major issues with all of these silica‐based monoliths is the possible poor recovery of large biomolecules (principally with monoclonal antibody fragments of more than 25 kDa).     

Synthesis of stable analogues of thiamine di- and triphosphate as tools for probing a new phosphorylation pathway

Thiamine (vitamin B1) is an essential nutritional factor metabolized inside the body in its mono-, di-, and triphosphate forms. Although the action of thiamine and thiamine diphosphate have been intensely investigated, many questions remain unanswered and the role of thiamine triphosphate is still especially unknown. To probe recent hypotheses on the implication of thiamine triphosphate in a new phosphorylation pathway involving synaptic proteins, we synthesized a series of thiamine di- and triphosphate analogues that are resistant to both enzymatic and chemical hydrolyses. The key step in the preparation of the title compounds is the coupling of thiamine propyl disulfide with adequately protected methylenebis-phosphonic acid, the corresponding triphosphate analogue, and difluoromethylenebisphosphonic acid.     

A preliminary 3D model for cytochrome P450 2D6 constructed by homology model building

Summary A homology model building study of cytochrome P450 2D6 has been carried out based on the crystal structure of cytochrome P450 101. The primary sequences of P450 101 and P450 2D6 were aligned by making use of an automated alignment procedure. This alignment was adjusted manually by matching -helices (C, D, G, I, J, K and L) and -sheets (3/4) of P450 101 that are proposed to be conserved in membrane-bound P450s (Ouzounis and Melvin [Eur. J. Biochem., 198 (1991) 307]) to the corresponding regions in the primary amino acid sequence of P450 2D6. Furthermore, -helices B, B and F were found to be conserved in P450 2D6. No significant homology between the remaining regions of P450 101 and P450 2D6 could be found and these regions were therefore deleted. A 3D model of P450 2D6 was constructed by copying the coordinates of the residues from the crystal structure of P450 101 to the corresponding residues in P450 2D6. The regions without a significant homology with P450 101 were not incorporated into the model. After energy-minimization of the resulting 3D model of P450 2D6, possible active site residues were identified by fitting the substrates debrisoquine and dextrometorphan into the proposed active site. Both substrates could be positioned into a planar pocket near the heme region formed by residues Val³⁷⁰, Pro³⁷¹, Leu³⁷², Trp³¹⁶, and part of the oxygen binding site of P450 2D6. Furthermore, the carboxylate group of either Asp¹⁰⁰ or Asp³⁰¹ was identified as a possible candidate for the proposed interaction with basic nitrogen atom(s) of the substrates. These findings are in accordance with a recently published predictive model for substrates of P450 2D6 [Koymans et al., Chem. Res. Toxicol., 5 (1992) 211].     

Exo-metal coordination by a tricyclic [(P(mu-N-2-NC5H4))2(mu-O)]2 dimer in [(P(mu-N-2-NC5H4))2(mu-O)]2(CuCl x (C5H5N)2)4 (2-NC5H4 = 2-pyridyl, C5H5N = pyridine)

The in situ reaction of the phosphazane dimer [CIP(mu-N-2-NC5H4)]2 (2) with CuCl in the presence of CsH5N/H2O gives the title complex [(P(mu-N-2-NC5H4))2(mu-O)]2(CuCl x (C5H5N)2)4 (1), containing a tricyclic [(P(mu-N-2-NC5H4))2(mu-O)]2 ligand which is isoelectronic with species of the type [(P(mu-NR))2NR]2.     

Isotope shift measurements in titanium I

The use of an effusive beam of titanium atoms crossed with a CW single-mode tunable dye laser has allowed the high-resolution, Doppler-free study of the isotope shifts between⁵⁰Ti,⁴⁸Ti and⁴⁶Ti, for seven 3d ² 4s ² a³ F _J 3d ² 4s 4p z ⁵ D _J , visible transitions of Ti I. The measurements show without ambiguity the existence of a non-negligible field shift. Using the values of the nuclear radii of titanium (coming from muonic X-ray measurements), it is possible to determine the respective values of the field and mass shifts.     

Base Hydrolysis of Pentaaminecobalt (III) Complexes: The [CoX(dien) (dapo)]n+ system. Part 3. The internal conjugate base mechanism

Azide anation and racemization of optically pure mer-exo(H)- and mer-endo(H)-[Co(OH)(dien)(dapo)]²⁺ ( A and B (X = OH), resp.; dien = N-(2-aminoethyl)ethane-1,2-diamine; dapo = 1,3-diaminopropan-2-ol) involve the same symmetrical pentacoordinate intermediate as the base hydrolyses of the corresponding mer-exo(H)- and mer-endo(H)-[CoX(dien)(dapo)]²⁺ species A and B , respectively, where X = Cl, Br, or N₃. The kinetic parameters of the anation process are fully compatible with the independently measured competition ratio. The rate data reveal that substitution of OH^? is unexpectedly fast, viz. it is not consistent with the usual sequence Br^? > Cl^? > H₂O > N > OH^?. This behavior is interpreted on the basis of an internal conjugate base mechanism which involves an amino-hydroxo/aminato-aqua tautomerism, viz. the reaction is actually an OH^? -catalyzed substitution of [CoH₂O(dien)(dapo)]³⁺ where deprotonation occurs effectively at the secondary-amine site NH of dien.     

Influence of the coordination number of nitrogen on the structure of 6aλ4-thia-1,3,4,6-tetraazapentalenic systems. Crystal structure analyses of 3-(2-pyridylimino)-3H-[1,2,4]thiadiazolo[4,3-a]pyridine and its 1-methylated salt

Structural data were obtained by X-ray crystallography for the title compounds which show that they are essentially planar and exhibit an approximately linear N2-S1-N8 arrangement. In compound 3 the separation between the sulfur atom and the pyridine nitrogen atom (2.61 Å) is larger than the Huggins constant energy distance (2.58 Å), suggesting that there is little or no bonding between them. The methylated salt 4 , on the contrary, has a closer S…N(pyridine) distance (2.19 Å) with an estimated bond dissociation energy of 6 kcal/mole.     

Cucurbituril‐mediated immobilization of fluorescent proteins on supramolecular biomaterials

The reversible introduction of functionality at material surfaces is of interest for the development of functional biomaterials. In particular, the use of supramolecular immobilization strategies facilitates mild reaction and processing conditions, as compared to other covalent analogues. Here, the engineering of multicomponent supramolecular materials, beyond the use of a single supramolecular entity is proposed. Cucurbit[8]uril (Q8) mediated host–guest chemistry is combined with hydrogen bonding supramolecular 2‐ureido‐4‐pyrimidinone (UPy)‐based materials. The modular incorporation of a UPy‐additive that presents one guest to incorporate into the Q8 host allows for selective supramolecular functionalization at the water–polymer material interface. Supramolecular ternary complex formation at the material surface was studied by X‐ray photoelectron spectroscopy, which as a result of large overlap in atomic composition of the different components showed minor changes is surface composition upon complex formation. Surface MALDI‐ToF MS measurements revealed useful insights in the formation of complexes. Protein immobilization was monitored using both fluorescence spectroscopy and quartz crystal microbalance with dissipation monitoring, which successfully demonstrated ternary complex formation. Although proteins could selectively be immobilized onto the surfaces, control of the system's stability remains a challenge as a result of the dynamicity of the host–guest assembly. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 3607–3616     

Synthesis, Conformational Studies, X-ray Structures, and Complexation Properties of Semirigid Macrocyclic Phosphonamides

The semirigid phosphonamide ligands 1-5 have been synthesized from the macrocyclic precursors 6-9 by reaction with 1,3-propanediol ditosylate or 1,2-dichloroethane. For the thiophosphoryl compounds 1 and 2, and the phosphoryl derivative 5, the reactions were carried out in biphasic aqueous NaOH solutions. The phosphoryl derivatives 3 and 4 were better obtained from NaH in anhydrous tetrahydrofuran. The conformations of the hosts in solution were deduced from low-temperature NMR and NOE difference experiments. Conformational equilibria between exo and endo forms are observed for the 18-membered macrocycles 1 and 3. The exo conformer predominates in solution for the 21-membered macrocycle 2, whereas 4 exists as rapidly exchanging conformers. The X-ray crystal structures of macrocycles 1, 2, and 5 have been determined as well as the complexes 1.Hg(SCN)(2) and 5.LiNO(3). In the Hg(2+) complex the metal ion is located out of the macrocyclic cavity and is coordinated to the thiophosphoryl unit. In 5.LiNO(3)()()the Li(+) cation is located inside the macrocyclic cavity and is coordinated to a tetrahedral array of oxygen donors. Free energies of complexation (DeltaG degrees ) of the phosphorylated ligands 3-5 with alkali metal and ammonium cations were determined in CHCl(3) saturated with H(2)O by picrate extraction experiments. The -DeltaG degrees values are greatest for 4 complexing K(+) and NH(4)(+) (7.3 and 8.0 kcal/mol, respectively). The relationships between structure and binding are discussed.     

Supercritical fluid chromatography of carbohydrates

Supercritical fluid chromatography (SFC) is a relatively new technique applied to polar solutes such as carbohydrates about 10 years ago. The developments in the SFC of carbohydrates are summarized and a comparison between capillary and packed column SFC is presented. High-efficiency capillary columns are suitable only for derivatized carbohydrates since various packed columns are well adapted for non derivatized mono-, di- and trisaccharides and provide complementary selectivities.