Water-soluble carbosilane dendrimers: synthesis biocompatibility and complexation with oligonucleotides; evaluation for medical applications

Novel amine- or ammonium-terminated carbosilane dendrimers of type nG-[Si{OCH2(C6H3)-3,5-(OCH2CH2NMe2)2}]x, nG-[Si{O(CH2)2N(Me)(CH2)2NMe2}]x and nG-[Si{(CH2)3NH2}]x or nG-[Si{OCH2(C6H3)-3,5-(OCH2CH2NMe3 +I-)2}]x, nG-[Si{O(CH2)2N(Me)(CH2)2NMe3 +I-}]x, and nG-[Si{(CH2)3NH3 +Cl-}]x have been synthesized and characterized up to the third generation by two strategies: 1) alcoholysis of Si--Cl bonds with amino alcohols and subsequent quaternization with MeI, and 2) hydrosilylation of allylamine with Si--H bonds of the dendritic systems and subsequent quaternization with HCl. Quaternized carbosilane dendrimers are soluble in water, although degradation is apparent due to hydrolysis of Si--O bonds. However, dendrimers containing Si--C bonds are water-stable. The biocompatibility of the second-generation dendrimers in primary cell cultures of peripheral blood mononuclear cells (PBMCs) and erythrocytes have been analyzed, and they show good toxicity profiles over extended periods. In addition, we describe a study on the interactions between the different carbosilane dendrimers and DNA oligodeoxynucleotides (ODNs) and plasmids along with a comparative analysis of their toxicity. They can form complexes with DNA ODNs and plasmids at biocompatible doses via electrostatic interaction. Also a preliminary transfection assay has been accomplished. These results demonstrate that the new ammonium-terminated carbosilane dendrimers are good base molecules to be considered for biomedical applications.     

Synthesis of carbosilane dendrons and dendrimers derived from 1,3,5-trihydroxybenzene

Several carbosilane wedges of generations 1-3 have been synthesized, following the divergent method, containing at the focal point a C-Br bond and as peripheral functional groups SiMeCl₂, SiMe(C₃H₅)₂, SiMe₂Cl, SiMe₂H, and ester units SiMe₂{(C₃H₆)N(C₂H₄CO₂Me)₂}. The dendrons functionalized with SiMe(C₃H₅)₂ and SiMe₂{(C₃H₆)N(C₂H₄CO₂Me)₂} groups were used to synthesize spherical dendrimers derived from 1,3,5-(HO)₃C₆H₃, leaving the outer groups unchanged. The allyl dendrimers thus obtained were used as precursors to prepare new dendrimers functionalized with SiMeCl₂, SiMe₂Cl, SiMe₂H, amine units SiMe₂{(C₃H₆)NH₂} and also ester units SiMe₂{(C₃H₆)N(C₂H₄CO₂Me)₂}.     

Unprecedented hybrid scorpionate/phosphine ligand able to be anchored to carbosilane dendrimers

The synthesis of a novel hybrid pyrazolate/phosphine anionic ligand [CH2=CHCH2B(CH2PPh2)(pz)2]- is described. Coordination of this ligand to metals in a fac tridentate fashion occurs in the complexes [CH2=CHCH2B(CH2PPh2)(pz)2M(cod)], prepared by reactions of the lithium salt of the ligand with [M(mu-Cl)(cod)]2 (M=Rh, Ir). They are pentacoordinated, with the rhodium complex showing a distorted trigonal-bipyramidal structure in the solid state, as determined by X-ray diffraction methods. Furthermore, the ligand has been linked to the periphery of a carbosilane dendrimer, resulting in the polyanionic dendrimer [Li(TMED)]4[Si{(CH2)3SiMe2(CH2)3B(CH2PPh2)(pz)2}4], which leads further to the corresponding metallodendrimer with four rhodium atoms.     

Rhodium or ruthenium units peripherally coordinated to carbosilane dendrimers functionalized with P-stereogenic monophosphines

Carbosilane dendrimers containing P-stereogenic monophosphines as terminal groups, Dend-{CH₂PPhR}_n (R = 2-biphenylyl or 9-phenanthryl), were reacted with [RhCl(COD)]₂ or [RuCl₂(p-cymene)]₂ to afford the corresponding chiral metalladendrimers Dend-{CH₂PPhR(RhCl(COD))}_n or Dend-{CH₂PPhR(RuCl₂(p-cymene))}_n, respectively. Attempts to obtain the first generation Ru-dendrimer for R = 2-biphenylyl proved unsuccessful, probably due to the steric hindrance of R. Complete characterization of these species was achieved by multinuclear NMR spectra, including 2D experiments, mass spectrometry, and optical rotation determinations. The catalytic properties of the rhodium dendrimers were tested in the hydrogenation of dimethylitaconate and those of the ruthenium derivatives in the asymmetric hydrogen transfer of acetophenone. The following model chiral compounds, (CH₃)₃Si{CH₂PPhR(RhCl(COD))} and (CH₃)₃Si{CH₂PPhR(RuCl₂(p-cymene))}, were prepared in order to detect potential dendritic effects. All compounds were active in the catalytic conditions tested, but low or null e.e. were found.     

Thermodynamic properties of the first to fifth generations of carbosilane dendrimers with allyl terminal groups

Temperature dependences of the specific heats, characteristic temperatures, and enthalpies of physical transformations of the first to fifth generations of carbosilane dendrimers with allyl terminal groups were studied using an adiabatic vacuum calorimeter in the temperature range 6—340 K. The error of measurements was, as a rule, about 0.2%. Thermodynamic characteristics of physical transformations of the dendrimers were determined and their thermodynamic functions C _p°(T), H°(T)—H°(0), S°(T)—S°(0), and G°(T)—H°(0) were calculated for the temperature range 0—340 K. The thermodynamic functions of the dendrimers are linearly related to their molecular weights, the number of allyl groups on their outer spheres, and the number of moles of diallylmethylsilane per mole of the dendrimers formed. Additive dependence of the properties of the dendrimers on their chemical composition and structure indicates that the energy of interaction between structural fragments of the dendrimers is independent of the dendrimer generation number. The fractal dimensions, D, of all dendrimers studied in this work are 1.2—1.3 in the temperature range 30—50 K, thus indicating a chain-layered structure of the dendrimer glasses.     

A series of carbosilane dendrimers with acetyl end-group:From synthesis to their unique optical characteristics

A series of carbosilane dendrimers with acetyl end-group were synthesized.Their structures were characterized by ~1H NMR,IR, and MS,respectively.Then dendrimers were coordinated with lanthanide ions(Eu~(3+) and Tb~(3+),respectively).The luminescence spectra of the complexes show narrow-width emissions in visible light region.     

Determination of theophylline binding to human serum proteins by isotachophoresis

Free theophylline was isolated from human serum by ultrafiltration and analysed in a leading electrolyte of 7.5 mM morpholinoethanesulphonic acid with ammediol as a counter ion at pH 8.90 and alpha-alanine as a terminator. The UV (280 nm) absorbance of the theophylline spike between serine and bicine as spacers was integrated. Binding percentages to human pool serum, human albumin and alpha 1-acid glycoprotein (orosomucoid) were determined at physiological concentrations, and found to be 55, 44 and 12%, respectively. The calibration lines were straight from 0 to 30 mg/l, with a standard deviation of 0.2 mg/l. The detection limit was 1 mg/l. The time of analysis was 12 min at 40 microA in a 0.2 mm I.D. capillary.     

Cyanovirin-N binding to Manalpha1-2Man functionalized dendrimers

Manalpha1-2Man functionalized G(3) and G(4)-PAMAM dendrimers have been synthesized and characterized by MALDI-TOF MS and NMR spectroscopy. Precipitation assays to assess the binding of the dimannose-functionalized dendrimers to Cyanovirin-N, a HIV-inactivating protein that blocks virus-to-cell fusion through high mannose mediated interactions, are presented.     

Mode of binding of camptothecins to double helix oligonucleotides

We report an NMR study on the interaction of topotecan (Tpt) and other camptothecins (Cpts) with several double helix and single strand oligonucleotides. The results obtained by (31)P NMR spectroscopy, nuclear Overhauser experiments (NOE) and molecular dynamics (MD) simulations show that Cpt drugs do not intercalate into the double helix, as suggested by many authors. Phosphorus NMR spectra indicated that no deformation occurs at any level of the phosphodiester backbone, while 2D NOESY experiments allowed the detection of several contacts between the aromatic protons of Cpts and those of the double helix. Models of the drug/oligonucleotide complexes, built on the basis of NOE data, show that the drug is located at the end of the double helix, by stacking the A and B rings with the guanine or cytidine of the terminal CG base pairs, with a preference for the 3[prime or minute]-terminal end sites. Cpts interact with double strand, as well as with single strand oligomers, as can be seen from the NMR shift variation observed on the drug protons; but this shielding effect cannot be an evidence of intercalation, as it is largely due to external non-specific interactions of the positively charged drug with the negatively charged ionic surface of the oligonucleotide. The molecular weight of one of the complexes was obtained from the correlation time value. The conformational behaviour of the DNA fragment d(CGTACG)(2) was studied by MD simulations on a ns time scale in the presence of water molecules and Na(+) ions. Different models were examined and the deformations induced on the phosphodiester backbone by molecules that are known to intercalate, were monitored by MD simulations.     

A versatile reagent to synthesize diverse ionic liquids ranging from small molecules and dendrimers to functionalized proteins

An ionic liquid "reagent" bearing a succinimidyl activated ester is reported that can be used to synthesize a variety of small molecule and macromolecular ionic liquids. In addition, the ionic liquid reagent was used to modify lysozyme, and the protein retained its structure and function after modification. This study describes a facile and reliable route to new ionic liquid compositions.     

Phosphorus dendrimers: from synthesis to applications

Various methods of synthesis of phosphorus dendrimers are presented, in particular a fast method which allows creating each generation in only one step, while multiplying by 5 the number of end-groups. These methods of synthesis can easily be modified to place selectively on the surface, inside, within the branches or at the core, functional groups chosen according to the properties or the applications which they can bring. In many cases, the size of the dendrimer (its generation) has an influence on these properties. Various applications in the fields of catalysis, materials, and biology are presented. To cite this article: Anne-Marie Caminade et al., C. R. Chimie 6 (2003).     

Contributions of various serum proteins to the binding of prasterone sulfate in humans.

The binding of prasterone sulfate (PS) in human plasma was investigated. Binding percentages of PS to human plasma, human serum albumin (HSA), human alpha 1-acid glycoprotein (AGP) and human gamma-globulin (GGL) were independent of the PS concentration between 0.1 and 8.0 micrograms/ml. The mean binding percentages were 99.1% for human plasma, 98.3% for HSA, 12.6% for AGP and 8.1% for GGL. Though PS is an acidic drug, binding of PS to AGP was observed. From the binding index, it was found that PS mainly bound to HSA in human plasma and that the contributions of AGP and GGL to PS in plasma were negligible.     

Cellular binding of nanoparticles in the presence of serum proteins

Cellular binding of cationic nanoparticles in the presence of serum proteins was probed with two-colour fluorescence microscopy. Cationic nanoparticles associate with serum proteins in solution and bind to the cell surface as a single anionic complex. Displacement of serum proteins from the nanoparticles was found to be protein dependent.     

High-performance affinity chromatography of human serum concanavalin A binding proteins

A high-performance concanavalin A (Con A) affinity column Gelpack GL-L55C (Hitachi Kasei Industries) was successfully used for the fractionation of human serum Con A-binding proteins. Serum proteins that have strong affinity to Con A (ca. 11% of the recovered proteins) could be fractionated within 80 min. By analysing the eluates from the column by micro two-dimensional electrophoresis, followed by blotting and Con A staining, the specificity of the column was effectively visualized. Although the protein-binding capacity of the column gradually decreased during repeated loading of serum or tissue extracts, the specificity of the column to Con A-binding proteins did not change. Serum lipoproteins have been eluted from the column with 6 M urea, suggesting that the capacity decrease is caused by the binding of lipids or lipoproteins to the column.     

Water-soluble poly(aryl ether) dendrimers as a potential fluorescent detergent to form micelles at very low CMC

The self-assembly of amphiphilic pyrene-cored poly(aryl ether) dendrimers has been studied. Pyrene excimer emission at 500 nm from the higher generation 6 is observed in KOH aqueous solution at the concentration as low as 1.8 × 10⁻⁵ M, while the excimer emission from lower generation 5 could not be detected at 3.5 × 10⁻⁵ M. The results indicate that the self-aggregation in higher generation dendrimer takes place more efficiently than in lower generation in aqueous solution.     

Supramolecular complex induced by the binding of sodium dodecyl sulfate to PAMAM dendrimers

Isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) were employed to study the spontaneous supramolecular complexation of amine terminated PAMAM dendrimer (G3[EDA] PAMAM-NH2) induced by the binding of an anionic surfactant, sodium dodecyl sulfate (SDS). At pHor=10, the electrostatic binding ceased because the deprotonated PAMAM dendrimer was uncharged, and hence the surfactant-induced supramolecular assembly could not be formed.     

New approach to the determination phosphorothioate oligonucleotides by ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry

This text presents a novel method for the separation and detection of phosphorothioate oligonucleotides with the use of ion pair ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry The research showed that hexafluoroisopropanol/triethylamine based mobile phases may be successfully used when liquid chromatography is coupled with such elemental detection. However, the concentration of both HFIP and TEA influences the final result. The lower concentration of HFIP, the lower the background in ICP-MS and the greater the sensitivity. The method applied for the analysis of serum samples was based on high resolution inductively coupled plasma mass spectrometry. Utilization of this method allows determination of fifty times lower quantity of phosphorothioate oligonucleotides than in the case of quadrupole mass analyzer. Monitoring of ³¹P may be used to quantify these compounds at the level of 80 μg L⁻¹, while simultaneous determination of sulfur is very useful for qualitative analysis. Moreover, the results presented in this paper demonstrate the practical applicability of coupling LC with ICP-MS in determining phosphorothioate oligonucleotides and their metabolites in serum within 7 min with a very good sensitivity. The method was linear in the concentration range between 0.2 and 3 mg L⁻¹. The limit of detection was in the range of 0.07 and 0.13 mg L⁻¹. Accuracy varied with concentration, but was in the range of 3%.