Mass spectrometry in the characterization of cereal seed proteins

In less then a decade, applications of matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) mass spectrometry to the investigation of prolamins have rapidly evolved from measurements of the molecular mass of isolated proteins to a proteomic approach attempting to characterise the complete protein pattern in the seed. Mass spectrometry is currently making significant contributions to the understanding of the composition and structure of the gluten proteins and, in turn, to the elucidation of structure-function relationships. Results obtained using mass spectrometry, including determination of the molecular masses of prolamins, direct verification of gene-derived sequences, determination of the number of cysteine residues and localisation of disulphide bonds, investigation of the gluten toxicity for celiac patients, qualitative and quantitative determination of gliadins in food and determination of the protein pattern and its modification during seed maturation by proteomic approaches, are summarised here, to illustrate current trends and individuate possible future perspectives.     

Investigation and correction of the gene-derived sequence of glutenin subunit 1Dx2 by matrix-assisted laser desorption/ionisation mass spectrometry

Direct matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) analysis of a mixture of tryptic peptides was used to verify the gene-derived amino acid sequence of the high molecular weight (HMW) subunit 1Dx2 of bread wheat. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, and optimising the matrix and the acquisition parameters for each mass range. This resulted in coverage of the whole sequence except for a short fragment T3 (3 amino acids), which was not detected. It also allowed the insertion of a Pro residue in position 59 to be identified. The results obtained provide evidence for the lack of a substantial level of glycosylation or other post-translational modifications of subunit 1Dx2, and demonstrate that MALDI-MS is the most useful method presently available for the direct verification of the gene-derived sequences of HMW glutenin subunits and similar proteins.     

Chelate ring closure kinetics in the reaction of a platinum(II) complex with diphosphines

The reactions of PPh2(CH2)nPPh2 (n=1–4, P-P) and PPh3 with cis- [PtPh2(CO)(SEt2)] have been studied in chloroform-d by 1H- and 31P{1H}-n.m.r. When n=2 or3 the first product observed is [PtPh2(P-P)], where the diphosphine is acting as a chelate, and ring closure is fast compared to the rate of entry of the phosphine into the complex. When n=1 or 4 the first observed product is [PtPh2(CO)(P-P)], with P-P acting as monodentate, and the second observable stage of reaction is ring closure. The rate constants and activation parameters kc at 298K (s–1), H (kJmol–1), S (JK–1mol–1) for the dppm and dppb complexes are 0.0198, 88±1, +17±3; and 0.00273, 38±2, –169±6, respectively. The formation of the large seven-membered ring is a strainless process, comparable to the intermolecular process. The increase in the enthalpy of activation as ring size decreases is due to ring strain and inter-atomic repulsions associated to the conformation of the four-membered chelate ring.     

Photochemical behavior of the drug atorvastatin in water

Atorvastatin undergoes a self-sensitized photooxygenation by sunlight in water. The main photoproducts, isolated by chromatographic techniques, have been identified by spectroscopic means. They present a lactam ring arising from an oxidation of pyrrole ring and an alkyl/aryl shift. A mechanism involving singlet oxygen addition and an epoxide intermediate is suggested.     

Synthesis of pyrido[2,1-b]- and thiazolo[2,3-b]purines

Some pyrido[2,1-b]- and thiazolo[2,3-b]purines, tricyclic compounds structurally related to [1,2,4]triazolo[1,5-c]quinazolines 1 have been synthesized with a view to study their possible adenosine and benzodiazepine receptors affinity.     

Thermodynamic studies on the addition of molecular oxygen to Co(II)-1,4,8,12 tetraazacyclopentadecane in aqueous solution at 25°C

Aqueous solutions containing Co²⁺ and the macrocyclic ligand 1,4,8,12-tetraazacyclopentadecane (L) are able to bind reversibly molecular oxygen to form the species Co₂L₂O ₂ ⁴⁺ between pH 5 and 7. This species at pH greater than 8 is replaced by the complex Co₂L₂O₂ (OH) ₂ ²⁺ whose concentration in the solution increases to about pH 10.5. At pH greater than 10.5, Co(II) exists totally in this -peroxobisydroxo complex. Both the above mentioned oxygenated forms suffer a relatively slow irreversible oxidation to Co(III). From oxygen binding curves, approximate values of the equilibrium constants were calculated for the addition of oxygen to CoL²⁺ to form the -peroxo and the -peroxo-bishydroxo complexes. The enthalpies of formation of this latter complex starting from both Co²⁺ or CoL²⁺ have been obtained by means of calorimetric measurements. The effects of the size of the macrocyclic ligand on the affinity and the enthalpy of oxygen binding have been considered.Presented at the sixth Italian meeting on Calorimetry and Thermal Analysis (AICAT) held in Naples, December 4–7, 1984.     

Model compounds of nylons—II : The crystal structure of N,N′-bis (β-chlorethyl)-glutaramide

The crystal structure of N,N′-bis(β-chloroethyl)-glutaramide (NNCEG) has been determined by X-ray diffraction analysis, as part of a research programme on simple model compounds for synthetic polyamides. It crystallizes in the monoclinic system, space group P2₁ Z = 2, with A = 4·941, B = 28·123, C = 4·835 Å and β = 113°53′. The structure was solved by the heavy atom method and refined to a final R value of 0·079. Each molecule forms hydrogen bonds along two directions (almost the a and the c directions with an angle close to 60°) giving rise to bidimensional layers (parallel to the ac plane with width b/2). A similar system of hydrogen bonds could be postulated for some nylons with odd number of CH₂'s between amide groups. The molecular conformation different from an all trans conformation is discussed in terms of the barriers to rotation around each bond considered by several authors. The twinning observed in most of the examined crystals is rationalized in terms of simple symmetry operations on molecular conformations of opposite chirality.     

Structural properties and dissociative fluxional motion of 2,9-dimethyl-1,10-phenanthroline in platinum(II) complexes

A dynamic 1H NMR study has been carried out on the fluxional motion of the symmetric chelating ligand 2,9-dimethyl-1,10-phenanthroline (Me2-phen) between nonequivalent exchanging sites in a variety of square-planar complexes of the type [Pt(Me)(Me2-phen)(PR3)]BArf, 1-14, (BArf = B[3,5-(CF3)2C6H3]4). In these compounds, the P-donor ligands PR3 encompass a wide range of steric and electronic characteristics [PR3 = P(4-XC6H4)3, X = H 1, F, 2, Cl 3, CF3 4, MeO 5, Me 6; PR3 = PMe(C6H5)2 7, PMe2(C6H5) 8, PMe3 9, PEt3 10, P(i-Pr)3 11, PCy(C6H5)2 12, PCy2(C6H5) 13, PCy3 14]. All complexes have been synthesized and fully characterized through elemental analysis, 1H and 31P{1H} NMR. X-ray crystal structures are reported for the compounds 8, 11, 14, and for [Pt(Me)(phen)(P(C6H5)3)]PF6 (15), all but the last showing loss of planarity and a significant rotation of the Me2-phen moiety around the N1-N2 vector. Steric congestion brought about by the P-donor ligands is responsible for tetrahedral distortion of the coordination plane and significant lengthening of the Pt-N2 (cis to phosphane) bond distances. Application of standard quantitative analysis of ligand effects (QALE) methodology enabled a quantitative separation of steric and electronic contributions of P-donor ligands to the values of the platinum-phosphorus 1J(PtP) coupling constants and of the free activation energies DeltaG++ of the fluxional motion of Me2-phen in 1-14. The steric profiles for both 1J(PtP) and DeltaG++ show the onset of steric thresholds (at cone angle values of 150 degrees and 148 degrees , respectively), that are associated with an overload of steric congestion already evidenced by the crystal structures of 11 and 14. The sharp increase of the fluxional rate of Me2-phen can be assumed as a perceptive kinetic tool for revealing ground-state destabilization produced by the P-donor ligands. The mechanism involves initial breaking of a metal-nitrogen bond, fast interconversion between two 14-electron three-coordinate T-shaped intermediates containing eta1-coordinated Me2-phen, and final ring closure. By use of the results from QALE regression analysis, a free-energy surface has been constructed that represents the way in which any single P-donor ligand can affect the energy of the transition state in the absence of aryl or pi-acidity effects.     

Mo(CO)6-Catalyzed oxidation of furan derivatives to E- and Z-enediones by cumyl hydroperoxide

E- and Z-Enediones are easily accessible by controlled oxidation of 2,5-disubstituted furans with Mo(CO)₆/cumyl hydroperoxide system. The use of t-butyl hydroperoxide, as oxygen donor, leads to the formation of 2H-pyran-3(6H)-one derivatives.     

Energy forms on conformal C1‐diffeomorphic images of the Sierpinski gasket

The energy form on a conformal C¹‐diffeomorphic image G of the Sierpinski gasket K is constructed by integrating the Lagrangian LG on G, which is given in terms of the pullback of the Lagrangian LK on K and of the differential of the diffeomorphism. The extension of this approach to the class of nested fractals is outlined. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)