Christophe Saudan

Dendrimers with a cyclam core. Absorption spectra, multiple luminescence, and effect of protonation

Abstract We have synthesized two dendrimers ( 4 and 5 ) consisting of a 1,4,8,11-tetraazacyclotetradecane (cyclam) core appended with four dimethoxybenzene and eight naphthyl units ( 4 ) and 12 dimethoxybenzene and 16 naphthyl units ( 5 ). The absorption and luminescence spectra of these compounds and the changes taking place upon protonation of their cyclam core have been investigated. In acetonitrile-dichloromethane 1:1 v/v solution they exhibit three types of emission bands, assigned to naphthyl localized excited states ( λ max =337 nm), naphthyl excimers ( λ max ca 390 nm), and naphthyl-amine exciplexes ( λ max =480 nm). The tetraamine cyclam core undergoes only two protonation reactions, whose constants have been obtained by fitting the spectral changes. Protonation not only prevents exciplex formation for electronic reasons, but also causes strong nuclear rearrangements in the cyclam structure which affect excimer formation between the peripheral naphthyl units of the dendrimers.

Water contributes actively to the rapid crossing of a protein unfolding barrier

The cold-shock protein CspB folds rapidly in a N <= => U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive (Delta V(++)(NU)=16(+/-4) ml/mol) and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well (Delta V(++)(NU)=53(+/-6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, Delta V(++)(UN) decreases significantly, suggesting that compact unfolded forms become populated under these conditions.

Cyclam-based dendrimers as ligands for lanthanide ions

We have investigated the complexation of lanthanide ions (Nd3+, Eu3+, Gd3+, Tb3+, Dy3+) with three cyclam-based ligands (cyclam = 1,4,8,11-tetraazacyclotetradecane), namely 1,4,8,11-tetrakis(naphthylmethyl)cyclam (1), and two dendrimers consisting of a cyclam core appended with four dimethoxybenzene and eight naphthyl units (2) and twelve dimethoxybenzene and sixteen naphthyl units (3). In the free ligands the fluorescence of the naphthyl units is strongly quenched by exciplex formation with the cyclam nitrogens. Complexation with the metal ions prevents exciplex formation and revives the intense naphthyl fluorescence. Fluorescence and NMR titration experiments have revealed the formation of complexes with different metal/ligand stoichiometries in the case of 1, 2 and 3. Surprisingly, the large dendrimer 3 gives rise to a stable [M(3)3]3+ species. Energy transfer from the lowest singlet and triplet excited states of the peripheral naphthyl units to the lower lying excited states of Nd3+, Eu3+, Tb3+, Dy3+ coordinated to the cyclam core does not take place.

Examining reactivity and specificity of cytochrome c peroxidase by using combinatorial mutagenesis.

Combinatorial mutagenesis was used to investigate the role of three key residues in cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae, Arg48, Trp51, and Trp191, in control of the reactivity and selectivity of the heme‐containing enzyme. Libraries were prepared by randomization of these residues and were subsequently screened for activity against the phenolic substrate guaiacol. Screening conditions were employed that favor either mutants with high activity or those with both high activity and stability of the reactive enzyme intermediates. The results obtained suggest a dual role for Arg48 of CCP: in addition to stabilizing reactive enzyme intermediates, the distal arginine residue plays a major role in restriction of access to the ferryl oxygen atom by small molecules and thereby controls reactivity and substrate specificity of the peroxidase. At position 51 of CCP, either a phenylalanine or a tryptophan residue is required both for catalytic and structural reasons. In contrast, either polar or positively charged residues are accepted at the position of Trp191, which is located inside the core of the protein. The variability at position 191 can be interpreted as a reflection of the mechanism of cytochrome c peroxidase, which transforms the nonpolar Trp191 into a transient cation radical.

Simple and Dendritic Cyclam Derivatives. Photophysical Properties, Effect of Protonation and Zn2+ Coordination, Preliminary Screening as Inhibitors of Tumour Cell Growth

We have synthesized two novel dendrimers (BG1 and BG2) consisting of a 1,4,8,11-tetraazacyclotetradecane (cyclam, 1) core with appended four dimethoxybenzene and eight benzyl units (BG1) and twelve dimethoxybenzene and sixteen benzyl units (BG2). The absorption and luminescence spectra of these compounds and the changes taking place upon protonation and Zn2+ coordination of their cyclam core have been investigated in acetonitrile-dichloromethane 1:1 v/v solution. For comparison purposes, the absorption and luminescence spectra of 1,4,8,11-tetrabenzyl-cyclam (2), and dendrons BD1 and BD2, model compounds of the branches of BG1 and BG2 respectively, have also been studied. BD1, BD2, BG1, and BG2 exhibit the absorption and emission spectra of their 1,3-dimethoxybenzene unit, but in the two dendrimers the emission intensity is quenched by the cyclam amine groups and increases upon protonation and metal coordination. In order to test if these cyclam derivatives have an antitumour effect, we have studied their action on proliferation in the human neuroblastoma TS12 cell line. Screening experiments have shown that cell proliferation was (i) strongly reduced by the tetrabenzyl substituted cyclam 2, and (ii) unaffected by cyclam and the benzo dendrimers BG1 and BG2. Antitumour screening experiments have also been performed on the tetranaphthyl substituted cyclam 3 and the naphtho-dendrimer NG2, whose photophysical properties have been previously studied. Cell proliferation came out to be moderately reduced by 3, whereas dendrimer NG2 had no effect, similar to dendrimers BG1 and BG2.

High-Pressure Stopped-Flow Studies to Characterize Transient Conformational Transitions of a Membrane Enzyme: Na, K-ATPase

The kinetics of ATP-induced phosphorylation and K+ binding of Na,K-ATPase has been investigated at different pressures by applying the fluorescence stopped-flow method. For both type of reactions, a conformational transition is considered to act as rate-limiting reaction step. These transitions are characterized by large activation volumes up to +100 ml mol−1. The significance of the determined values in terms of Kramers’ theory is discussed. A molecular interpretation related to solvation changes and cavity formation in the transmembrane domain of the protein is suggested. A large negative reaction volume is found upon ATP binding (—100 ml mol−1). The selective binding of Na+ and K+ leads to smaller, but positive values.