A solvent-free matrix application method for matrix-assisted laser desorption/ionization imaging of small molecules

Matrix application continues to be a critical step in sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Imaging of small molecules such as drugs and metabolites is particularly problematic because the commonly used washing steps to remove salts are usually omitted as they may also remove the analyte, and analyte spreading is more likely with conventional wet matrix application methods. We have developed a method which uses the application of matrix as a dry, finely divided powder, here referred to as dry matrix application, for the imaging of drug compounds. This appears to offer a complementary method to wet matrix application for the MALDI-MSI of small molecules, with the alternative matrix application techniques producing different ion profiles, and allows the visualization of compounds not observed using wet matrix application methods. We demonstrate its value in imaging clozapine from rat kidney and 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylic acid from rat brain. In addition, exposure of the dry matrix coated sample to a saturated moist atmosphere appears to enhance the visualization of a different set of molecules.

The chemistry of some organotellurium compounds

Tbe formation of Pd(TeR)n and (CuTeR)n from the reaction between telluroesters and Pd(II)or Cu(II) suggested that these organa­tellurium reagents may be useful precursors of RTe- ligands in reactions with transition-metal substrates. Also the formation of telluronium salts Me2RTeI- from the reaction between telluroesters and methyl iodide, together with the above, confirm the cleavage of -cõ-Te bonds rather than -C-Te bonds. The formation of a carboxylic acid from the toluene solution of a ditelluride d palladium(O) complex in the presence of light oxygen (from air) is demonstrated. When the solvent employed is p-xylene an aldehyde is formed.The reaction proceeds via the free radical, RTeO, with Pd(PPh3)4 as a catalyst.It has also been shown that the oxidation of aldehydes to carboxylic acids is catalysed by ditelluride. Spin trapping experiments with PhCH=N(O)But (phenyl-t-butyl-nitrone) have provided evidence that the oxidative addition of an alkyl halide (RX=Mei, BunBr, BusecBr, ButBr, BrCH2-CH=CHCH2Br, and Br(CH2)4Br) to diphenyltelluride and reductive elimination of CH3SCN from Ph2(CH3)Te(NCS) proceeds via radical pathways. A mechanism is proposed for oxidative addition which involves the preformation of a charge transfer complex of alkyl halide and diphenyltelluride.The first step is the formation of a charge transfer complex, and the initial product of the oxidative addition is a "covalent" form of the tellurium(IV)compound. When the radical R is more stable, Ph2TeX2 may be the major tellurium(IV)product. The reaction of RTeNa (R=p-EtOC6H4, Ph) with organic dihalides X2(CH2)n (n=1,2,3,4) affords telluronium salts (n=3,4; X=Cl, Br) the nature of which is discussed.For n=l (X=Br, I)the products are formulated as charge transfer complexes of stoichiometry (RTe)2(CH2).CH2X2• For n=2, elimination of ditelluride occurs with the formation of an alkene. Some 125’Te Mõssbauer data are discussed and it is suggested that the unusually low value of 6 (7.58 mm.s-1 ) for  p-EtO.C6H4.Te)2(cH2)cH2Br2 relates to removal of 5's electronsfrom the spare pair orbltal via the charge transfer interaction. 125Te Mossbauer data for (p-EtO.C6H4)Te(CH2)4Br are typical of a tellurium (IV) compound and in particular ∇ is in the expected range for a telluronium salt. The product of the reaction of Na Te (C6H4.OEt), with 1,3-dibromopropane is, from the Mössbauer data, also a telluronium salt.

Free radicals as potential antitumour agents

The aim of this work was to use extremely low concentrations of free radical generating compounds as a 'catalyst' to trigger endogenous free radical chain reactions in the host and to selectively eliminate neoplastic cells in the host. To test the hypothesis, a number of free radical generating compounds were screened on several malignant cell lines in vitro to select model compounds that were used against tumour models in vivo. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and its derivatives were selected at the model compounds for in vivo experiments in view of their high cytotoxic potency against several malignant cell lines in vitro. The water soluble derivative, 2,2-diphenyl-1-(2', 4'-dinitro-6'-sulphophenyl) hydrazyl (DDSH) given by subcutaneous injections demonstrated significant antitumour activities against the MAC 16 murine colon adenocarcinoma implanted subcutaneously in male NMRI mice at nanomolar concentration range. 40-60% of long term survival of over 60 days was achieved (compared with control survival of 20 days) with total tumour elimination. This compound was also active against both P388 leukaemia in male BDF1 mice and TLX5 lymphoid tumour in male CBA/CA mice at a similar concentration range. However, some of these animals died suddenly after treatment with no evidence of disease present at post mortem. The cause of death was unknown but thought to be related to the treatment. There was significant increase in serum level of malondialdehyde (MDA) following treatment, but did not correlate to the antitumour activities of these compounds. Induction of supcroxide dismutase (SOD), and glutathione peroxidase (GPx) occurred around day 8 after the administration of DDSH. Histological sections of MAC16 tumours showed areas of extensive massive haemorrhagic necrosis and vascular collapse associated with perivascular cell death following the administration of nanomolar concentration of DDSH which was probably compatible with the effects of free radicals. It was concluded that the antitumour activities of these compounds may be related to free radical and cytokine production.

Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.