Jean-Claude Blais

Synthesis, Cellular Internalization and Photodynamic Activity of Glucoconjugated Derivatives of Tri and Tetra(meta-hydroxyphenyl)chlorins

Glucoconjugated tri and tetra(meta-hydroxyphenyl)chlorins have been synthesized in order to explore how glucoconjugation of the macrocycle affects the photoactivity of the molecule. Internalization processes, photosensitizing efficacy of TPC(m-O-GluOH)(3) and TPC(m-O-GluOH)(4), in HT29 human adenocarcinoma cells have been compared to those of tetra(meta-hydroxyphenyl) chlorin (m-THPC, Foscan). The tetra glucoconjugated chlorin, TPC(m-O-GluOH)(4), was found to be poorly internalized and weakly photoactive. In contrast, the asymmetric and more amphiphilic compound TPC(m-O-GluOH)(3), exhibited superior phototoxicity compared to m-THPC. Drug concentration, temperature and sodium azide effects indicated that TPC(m-O-GluOH)(3) internalization partly proceeds via an active receptor-mediated endocytosis mechanism. Cellular uptake appeared as a saturable process and remained 30% lower than for mTHPC. However, a maximum phototoxicity in HT29 cells (survival fraction of 2+/-0.6%) were observed for concentration as low as 2 microM. A 4-fold higher concentration of m-THPC was necessary to observe the same level of photoactivity. This higher phototoxicity has been correlated to a greater mitochondrial affinity. On the basis of these results, work is in progress to further evaluate the potential of glycosylated chlorins in photodynamic therapy (PDT).

Synthesis and Photochemical Behavior of Phosphorus Dendrimers Containing Azobenzene Units within the Branches and/or on the Surface

We describe the synthesis of three series of phosphorus-containing dendrimers having azobenzene derivatives specifically placed at some generations in the interior and/or on the surface. The largest compound obtained possesses 48 azobenzene groups on the surface. Irradiation at 350 nm induces isomerization of the azobenzene groups from the E form to the Z form, whatever their location. The thermal back-isomerization to the E form in the dark at room temperature was observed in all cases. The kinetics of this Z-->E back-isomerization was studied in several cases; the rate is not dependent on the number of azobenzene units or of the generation, when the azobenzene groups are linked to the surface of the dendrimer. A different behavior was observed when the azobenzene groups were located within the framework of the dendrimer.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry of synthetic polyesters

Abstract We report the analysis of aliphatic and aromatic polyesters by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) using 2,5-dihydroxybenzoic acid and t-3-indoleacrylic acid as matrices. Broad asymmetrical distributions of linear oligomer related peaks (protonated and cationised molecules) were observed with both matrices. Peaks corresponding to cyclic oligomers were observed for the aliphatic polyester. The oligomer molecular weight distribution derived from the MALDI spectra exhibits a certain variability with both laser fluence and matrix material. This effect probably originates from different solubilities of the various oligomers. In addition, for those polycondensates which are characterised by a very broad distribution with a tail in the high mass range, a discrimination against the higher mass oligomers is likely to exist; the agreement between the average molecular weights determined by MALDI and size exclusion chromatography is not as good as for poly(ethylene glycol) or poly(methyl methacrylate).

Formation of Non-Specific Protein Cluster Ions in Matrix-Assisted Laser Desorption/Ionization: Abundances and Dynamical Aspects

Intact non-covalent biological complexes resulting from interactions in solutions have been observed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). However, their analysis is hampered by the presence of non-specific, non-covalent complexes (clusters) produced under MALDI conditions. A study of the influence of laser energy and ion extraction conditions allows information to be obtained on (i) the collision regime in the plume and (ii) ion stability, through the abundances and time-of-flight (ttof) deviations of bovine insulin clusters using a sinapinic acid matrix. In the linear mode, it is shown that (i) the neutral contribution to quasi-molecular peaks is below 10% and (ii) for related cluster species, the ttof difference between neutrals and ions is less than 10 ns. Although the abundance of cluster ions increases relatively to the abundance of the monomeric ions with laser energy, heavier cluster ions are unfavored at higher laser energy. On the other hand, the abundance of clusters is, to a first approximation, independent of the electric field in the first acceleration region (750–32,500 V cm−1) and of the extraction delay (up to 4000 ns). Gaussian-like distribution profiles are observed, whatever the acceleration field, for all cluster ion signals. This is in good agreement with the natural isotopic distribution. There is no significant difference between monomeric and cluster ion initial velocities (420 m s−1 ± 18 m s−1). Our results suggest a single origin for bovine insulin cluster ions that are observed by using a sinapinic acid matrix. The clusters should be formed promptly after the laser pulse (< 10 ns), in the selvedge of the surface. Smaller cluster ions could be formed by consecutive fragmentations of higher mass molecular clusters occurring after plume formation and before a significant ion acceleration.

Identification by mass spectroscopy of three major early proteins associated with virosomes in vaccinia virus-infected cells

Virosomes are cytoplasmic sites of replication of vaccinia virus DNA and were prepared from virus-infected HeLa cells. The early virosomal proteins were 35S-labelled and SDS polyacrylamide gel electrophoresis revealed the presence of three major early 35S-labelled proteins of 34, 24 and 45 kDa. The masses of molecules present in the 34 and 24 kDa proteins were measured by the convenient and sensitive MALDI TOF mass spectroscopy technique. Identification of the three virosomal proteins was carried out by MALDI mass spectroscopy of corresponding tryptic digests. For each protein at least 13 measured masses matched, within less than 0.1 Da, calculated tryptic peptides of the vaccinia virus proteins H5R (34 kDa), E3L (24 kDa) and E5R (45 kDa). In addition, virosomes contained several structural proteins from the infecting virus and a 45 kDa keratin-related protein. This work demonstrates directly that the abundant early vaccinia virus proteins H5R, E3L and E5R are associated with the virosomes.

Solution- and crystal-phase covalent modification of lysozyme by a purpose-designed organoruthenium complex. A MALDI-TOF MS study of its metal binding sites.

Study of the reaction between the transition organometallic complex 4‐ruthenocenyl 2,6‐dimethylpyrylium tetrafluoroborate and the enzyme hen egg white lysozyme (HEWL) in solution and by diffusion in crystals was performed by use of a combination of spectroscopic and chromatographic methods. Conjugation involving the lysine residues of lysozyme appeared to occur readily, yielding very stable ruthenocenyl pyridinium adducts with average degrees of incorporation ranging from 0.2 to 1.8 metal complexes per protein molecule, depending on reaction conditions. Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) revealed that the protein conjugates were in fact mixtures of unmodified, mono‐, di‐ and sometimes tripyridinium adducts. In combination with reversed‐phased HPLC, we were able to show that six different monoruthenocenyl pyridinium adducts were formed in solution. This result was confirmed by trypsin digestion of a ruthenocenyl pyridinium conjugate and MALDI‐TOF MS analysis of the peptide mixture, which showed that lysines 1, 13, 33, 96, 97 and 116 were involved in the reaction with the pyrylium complex, lysines 13, 33 and 116 being the major binding sites. In the tetragonal crystal state, no binding of the ruthenium complex was shown to occur at lysine 116, owing to steric hindrance at this particular position.

Organic film thickness effect in secondary ion mass spectrometry and plasma desorption mass spectrometry

Abstract The secondary ion emission of superposed Langmuir—Blodgett films of fatty acids has been studied using kiloelectronvolt (secondary ion mass spectrometry; SIMS) and megaelectronvolt (plasma desorption mass spectrometry; PDMS) particle bombardment. It is shown that for thick films (10–12 monolayers), the composition of the ejected volume and the escape depth of the secondary ions are similar using kiloelectronvolt and megaelectronvolt primary ions. A complementary study has been developed to investigate the film thickness effect in SIMS. Contrary to what was observed in PDMS, the molecular ion emission of surface layers decreases when the number of underlayers increases. This decrease most likely results from differences in energy exchanges in the film before ion emission. This indicates that the secondary ion emission originates from an energetically homogeneous region.

Molecular sputtering and damage induced by kiloelectron ions in organic monolayer—metal systems

Abstract Irradiation experiments, using kiloelectronvolt Cs + primary ions, have been carried out on Langmuir-Blodgett monolayers adsorbed on Au substrates. From these experiments, disappearance cross-section, σ, of the ejected molecular ions have been deduced; σ corresponds to the monolayer surface area from which molecules are ejected or damaged by the impact of the primary ion. The influence of many parameters—energy and angle of incidence of the primary ion, thickness and density of the monolayer—on the disappearance cross-section has been investigated. The results clearly show that σ linearly depends on the energy lost by the primary ion passing through the monolayer. The energy lost in the substrate is not involved in the ejection and the damage of molecules. These results are not consistent with an ion ejection directly induced by collision cascades in the organic monolayer. They rather suggest the formation of a vibrationally excited region prior to ion ejection.

Secondary ion mass spectrometry of glycosylated porphyrins

Abstract Solid secondary ion mass spectrometry (SIMS) has been used to study a series of acetylated precursors of glycosylated porphyrins in the course of their synthesis. Characteristic positive and negative spectra have been readily obtained for all the compounds up to more than 2000 Da. In addition to the protonated or deprotonated molecules, fragment ions arising from cleavages between the oxygen atom of the ether bond and the acetylated sugar units were observed; these fragments were more abundant in the negative spectra. The solid SIMS spectra are not complicated by chemical noise or chemical reactions occurring in the presence of a matrix which makes the method well suited for rapid screening during synthesis.

Target environment and energy deposition in particle induced desorption: 252Cf plasma desorption mass spectrometry, secondary ions mass spectrometry and fast atom bombardment mass spectrometry

Abstract The “static” secondary ion mass spectrometry (SIMS), 252Cf plasma desorption mass spectrometry (PDMS) and fast atom bombardment mass spectrometry (FABMS) (thioglycerol) spectra recorded in the absence and in the presence of lithium iodide have been compared for pyrimidine derivatives. For the three particle-induced desorption methods, an evaluation of the energy transfer during desorption to protonated and cationized molecules has been made on the basis of previous information concerning the relative kinetics and thermochemistry of competitive decomposition processes obtained using MSMS. It is shown that abundances of metal-containing parent ions are drastically reduced in the presence of a liquid matrix compared with what is observed with solid targets. This effect is particularly pronounced for parent ions resulting from lithium—cation/proton exchanges. On the other hand, the primary particle energy (MeV vs. keV) does not significantly change the abundance distributions of parent ions. For the SIMS and PDMS techniques where solid targets are bombarded, a high yield of fragment ions is observed. In PDMS, however, the distribution of internal energies appears to be narrower than in SIMS because higher energy processes are heavily disfavored. For the FABMS technique, a significantly lower fraction of detected ions is present as fragments compared with SIMS or PDMS. Nevertheless, a tail of the distribution of internal energies towards high values exists, as among the fragment ions a large fraction represents high energy processes, i.e., consecutive decompositions.