A potential drug delivery vehicle based on a melamine dendrimer with twelve free thiols on the periphery for constructing bio-labile disulfides has been synthesized. Under ideal conditions for the native chemical ligation reaction, attempts for attaching the cell penetrating peptide TAT, via native chemical ligation proved difficult due to the low solubility of the dendrimer. A camptothecin derivative containing a reactive disulfide was prepared for disulfide exchange with the melamine dendrimer. Up to 7 exchange reactions were achieved as determined by mass spectroscopy. NMR and mass spectroscopy was used to characterize all of the intermediates. Capping groups to replace the hydrophobic piperidine with more water-soluble groups to aid the ligation reaction and optimization of the disulfide exchange step to give 12 substitutions have been proposed for future studies. The end target is a peptide dendrimer containing a cell penetrating peptide to mediate endocytosis and a bio-labile linker connecting an anti-tumor drug to the dendrimer, which would ultimately be released inside the cancerous cell.

A potential drug delivery vehicle based on a melamine dendrimer with twelve free thiols on the periphery for constructing bio-labile disulfides has been synthesized. Under ideal conditions for the native chemical ligation reaction, attempts for attaching the cell penetrating peptide TAT, via native chemical ligation proved difficult due to the low solubility of the dendrimer. A camptothecin derivative containing a reactive disulfide was prepared for disulfide exchange with the melamine dendrimer. Up to 7 exchange reactions were achieved as determined by mass spectroscopy. NMR and mass spectroscopy was used to characterize all of the intermediates. Capping groups to replace the hydrophobic piperidine with more water-soluble groups to aid the ligation reaction and optimization of the disulfide exchange step to give 12 substitutions have been proposed for future studies. The end target is a peptide dendrimer containing a cell penetrating peptide to mediate endocytosis and a bio-labile linker connecting an anti-tumor drug to the dendrimer, which would ultimately be released inside the cancerous cell.

A general picture of dendrimer conformation has appeared through studies of various dendrimer systems. Though the studies define some conformational abilities of a dendrimer, the studies are only able to examine one portion of the general picture. NMR studies of a generation three melamine dendrimer with unique NMR signatures from core to periphery describes most, if not all, of the general concepts of dendrimers in one system. A generation three melamine dendrimer was synthesized by a convergent route using diamines identified from competition reactions towards a monochlorotriazine. The cyclic monoamines surveyed displayed a relative reactivity range of 40x, expanding the previously identified series to a range of 320x. Azetidine is 40x more reactive than the cyclic, nine-membered ring (C8H17N), and 320x more reactive than benzyl amine. Sterics and pKa values explain the differences in reactivity of the cyclic monoamines. Differences in the nucleophilicity of the amine groups consisting of 2-aminoazetidine, 2-aminopyrrolidine, and 4-aminopiperidine are 180x, 70x and 20x, respectively. One-dimensional NMR spectra of the exchangeable NH region show that the dendrimer supports a rich rotamer population. Observations of the data show that the rotamer populations change from a preferred extended conformation to a more closed conformation, indicative of sterics being a driving force of conformational architecture. Variable temperature NOESY studies show that the peripheral groups backfold into the interior of the dendrimer in DMSO- d6. The backfolding can be removed by changing the solvent to either CDCl3 or CD3OD. Variable temperature (VT) coefficients measured for the exchangeable NH protons implies that solvent may be excluded from the interior of the dendrimer. Proton relaxation studies provide evidence that the dendrimer tumbles slowly in solution, and the periphery moves more freely than the interior. Synthesis towards the attachment of carboplatin-like peripheral groups on a generation three dendrimer was unsuccessful. A diethyl malonate unit was attached to the periphery of the dendrimer followed by capping with 4-aminomethylpiperidine. Hydrolysis of the esters and treatment with activated platinum led to a black precipitate product. Two alternate routes of achieving the desired platinated dendrimer are described.

A general picture of dendrimer conformation has appeared through studies of various dendrimer systems. Though the studies define some conformational abilities of a dendrimer, the studies are only able to examine one portion of the general picture. NMR studies of a generation three melamine dendrimer with unique NMR signatures from core to periphery describes most, if not all, of the general concepts of dendrimers in one system. A generation three melamine dendrimer was synthesized by a convergent route using diamines identified from competition reactions towards a monochlorotriazine. The cyclic monoamines surveyed displayed a relative reactivity range of 40x, expanding the previously identified series to a range of 320x. Azetidine is 40x more reactive than the cyclic, nine-membered ring (C8H17N), and 320x more reactive than benzyl amine. Sterics and pKa values explain the differences in reactivity of the cyclic monoamines. Differences in the nucleophilicity of the amine groups consisting of 2-aminoazetidine, 2-aminopyrrolidine, and 4-aminopiperidine are 180x, 70x and 20x, respectively. One-dimensional NMR spectra of the exchangeable NH region show that the dendrimer supports a rich rotamer population. Observations of the data show that the rotamer populations change from a preferred extended conformation to a more closed conformation, indicative of sterics being a driving force of conformational architecture. Variable temperature NOESY studies show that the peripheral groups backfold into the interior of the dendrimer in DMSO- d6. The backfolding can be removed by changing the solvent to either CDCl3 or CD3OD. Variable temperature (VT) coefficients measured for the exchangeable NH protons implies that solvent may be excluded from the interior of the dendrimer. Proton relaxation studies provide evidence that the dendrimer tumbles slowly in solution, and the periphery moves more freely than the interior. Synthesis towards the attachment of carboplatin-like peripheral groups on a generation three dendrimer was unsuccessful. A diethyl malonate unit was attached to the periphery of the dendrimer followed by capping with 4-aminomethylpiperidine. Hydrolysis of the esters and treatment with activated platinum led to a black precipitate product. Two alternate routes of achieving the desired platinated dendrimer are described.

1 / Brittany K Casey

A variety of dendrimers based on melamine are designed, synthesized, and evaluated for drug delivery systems. The synthesis of a dendrimer, including multiple copies of four orthogonally reactive groups, is described. The three groups on the surface are nucleophilic and include four free hydroxyl groups, four tert-butyldiphenylsilyl (TBDPS) ether groups, and sixteen amines masked as tert-butoxycarbonyl (BOC) groups. The core of the dendrimer displays two electrophilic monochlorotriazines. The dendrimer above is further manipulated for in vivo biodistribution: incorporation of the reporting groups Bolton-Hunter and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid); PEGylation for biocompatibility and size tuning. In preliminary biodistribution studies, dendrimers circulate in the blood for a longer time as the molecular weight increases, which is important to passively target tumor tissues via the EPR effect. Also, high uptake by the tumor tissues was observed in mice bearing prostate cancer xenografts. A drug delivery vehicle for the anticancer agent paclitaxel is described. This drug delivery vehicle contains sixteen molecules of paclitaxel via acid-labile ester linkage, two Bolton-Hunter groups, and sixteen monochlorotriazine groups for PEGylation. The in vitro drug release studies shows faster release of paclitaxel at lower pH in PBS.

The convergent strategy towards dendrimer synthesis is well-suited to generate macromolecules with a diverse periphery, at the expense of time and effort, while the divergent strategy has historically been effective at yielding higher generation dendrimers, although they are often plagued by impurities. Both the convergent and divergent routes were applied to the synthesis of melamine-based dendrimers, offering a comparison of the routes within a system. Generation-1 dendrons heterogeneously functionalized with Boc-protected amines and hydrazones were synthesized convergently and coupled to a generation-1 tris(piperazine) core to yield a generation-2 dendrimer bearing 18 Boc-amines and three hydrazones. Although the yield for the final coupling step was rather low (56%), the yields for all intermediate steps were quite high. Attempts toward obtaining a generation-3 dendrimer through this route were unsuccessful due presumably to steric hindrance. The materials obtained showed no impurities in their 1H and 13C NMR and mass spectra, although several chromatographic purifications were necessary throughout the synthesis. A divergent strategy based on addition of a dichlorotriazine monomer to polyamine cores was used to synthesize dendrimers of generations 1-5. All intermediates and dendrimers were either purified by precipitation, or did not need purification. 1H NMR spectroscopy indicated that reactions were complete up to G4-NH2 by integration, and mass spectroscopy confirmed that assignment. HPLC and GPC of Gn-Cl dendrimers showed sharp peaks for G1-G3, but G4-Cl appeared to have a small amount of impurities that are similar in size and polarity to the fully-substituted dendrimer. The G1-G3 dendrimers were confidently assigned as pure by conventional organic chemistry standards, but the assignment of purity to higher generations remained tentative. A G1-Cl dendrimer was functionalized with imidazole, and then deprotected and PEGylated with PEG5000 to yield a water soluble dendrimer. The imidazole-capped, Boc-protected dendrimer and the deprotected dendrimer were characterized by 1H and 13C NMR spectroscopy and mass spectrometry. The degree of PEGylation on the PEGylated material could not be definitively ascertained; however, the material is capable of solubilizing very hydrophobic Zn-phthalocyanines in water.

The convergent strategy towards dendrimer synthesis is well-suited to generate macromolecules with a diverse periphery, at the expense of time and effort, while the divergent strategy has historically been effective at yielding higher generation dendrimers, although they are often plagued by impurities. Both the convergent and divergent routes were applied to the synthesis of melamine-based dendrimers, offering a comparison of the routes within a system. Generation-1 dendrons heterogeneously functionalized with Boc-protected amines and hydrazones were synthesized convergently and coupled to a generation-1 tris(piperazine) core to yield a generation-2 dendrimer bearing 18 Boc-amines and three hydrazones. Although the yield for the final coupling step was rather low (56%), the yields for all intermediate steps were quite high. Attempts toward obtaining a generation-3 dendrimer through this route were unsuccessful due presumably to steric hindrance. The materials obtained showed no impurities in their 1H and 13C NMR and mass spectra, although several chromatographic purifications were necessary throughout the synthesis. A divergent strategy based on addition of a dichlorotriazine monomer to polyamine cores was used to synthesize dendrimers of generations 1-5. All intermediates and dendrimers were either purified by precipitation, or did not need purification. 1H NMR spectroscopy indicated that reactions were complete up to G4-NH2 by integration, and mass spectroscopy confirmed that assignment. HPLC and GPC of Gn-Cl dendrimers showed sharp peaks for G1-G3, but G4-Cl appeared to have a small amount of impurities that are similar in size and polarity to the fully-substituted dendrimer. The G1-G3 dendrimers were confidently assigned as pure by conventional organic chemistry standards, but the assignment of purity to higher generations remained tentative. A G1-Cl dendrimer was functionalized with imidazole, and then deprotected and PEGylated with PEG5000 to yield a water soluble dendrimer. The imidazole-capped, Boc-protected dendrimer and the deprotected dendrimer were characterized by 1H and 13C NMR spectroscopy and mass spectrometry. The degree of PEGylation on the PEGylated material could not be definitively ascertained; however, the material is capable of solubilizing very hydrophobic Zn-phthalocyanines in water.

A variety of dendrimers based on melamine are designed, synthesized, and evaluated for drug delivery systems. The synthesis of a dendrimer, including multiple copies of four orthogonally reactive groups, is described. The three groups on the surface are nucleophilic and include four free hydroxyl groups, four tert-butyldiphenylsilyl (TBDPS) ether groups, and sixteen amines masked as tert-butoxycarbonyl (BOC) groups. The core of the dendrimer displays two electrophilic monochlorotriazines. The dendrimer above is further manipulated for in vivo biodistribution: incorporation of the reporting groups Bolton-Hunter and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid); PEGylation for biocompatibility and size tuning. In preliminary biodistribution studies, dendrimers circulate in the blood for a longer time as the molecular weight increases, which is important to passively target tumor tissues via the EPR effect. Also, high uptake by the tumor tissues was observed in mice bearing prostate cancer xenografts. A drug delivery vehicle for the anticancer agent paclitaxel is described. This drug delivery vehicle contains sixteen molecules of paclitaxel via acid-labile ester linkage, two Bolton-Hunter groups, and sixteen monochlorotriazine groups for PEGylation. The in vitro drug release studies shows faster release of paclitaxel at lower pH in PBS.

The interest in using gene therapy to target a variety of both inherited and acquired diseases has intensified over the last two decades. Because free DNA is easily degraded by serum nucleases in the bloodstream, the need for developing carrier systems that can compact and protect the DNA was quickly realized. Viral vector systems were some of the earliest carriers used, primarily because of the ease with which such systems can infect host cells. However, difficulties experienced when using viral vectors, including immunogenicity and the potential for genetic recombinations, forced researchers to design alternative delivery strategies. Non-viral vectors offer one alternative to overcome this dilemma. In addition to avoiding the biological problems experienced using viral carriers, non-viral vectors also offer the potential for large-scale production. Dendrimers are one non-viral carrier that has shown appreciable ability to deliver DNA into cells, a process called transfection. In the past, triazine dendrimers have shown biocompatibility, and the ability to synthesize these structures to contain cationic charges on the surface makes these structures potentially suitable for transfection studies. In this study, a small library of triazine dendrimers was synthesized in an attempt to understand how variations to both the periphery and core of triazine dendrimers affect the transfection efficiency of these dendriplexes. In the first subset of structures, a common core was used and various peripheral groups were appended to the dendrimer surface. The physicochemical and biological data, obtained in collaboration with Thomas Kissel at Philipps-Universitat Marburg, showed that the surface groups have a notable affect on transfection efficiency. Dendrimers with a higher amine number and neutral surface groups show high DNA binding affinity and higher transfection efficiency. In the second subset of dendrimers, variations to the core showed that transfection efficiency is improved both by increasing generation number and dendrimer flexibility. With this data in hand, triazine dendrimers with both higher generation number and higher flexibility have been synthesized. Two different triazine linker groups, trimethylene-dipiperidine and polyglycoldiamine, have been used. These structures will be evaluated to determine if increasing both flexibility and generation number together can further improve transfection efficiency.