A fullerene-paclitaxel conjugate has been synthesized as a slow-release drug for aerosol liposome delivery of paclitaxel for lung cancer therapy. The conjugate was designed to release paclitaxel via enzymatic hydrolysis and subsequently has shown a half-life of release of 80 min in bovine plasma. A liposome formulation of the conjugate has been prepared using dilauroylphosphatidylcholine (DLPC), and its IC50 is virtually identical to the IC50 for a paclitaxel-DLPC formulation in human epithelial lung carcinoma A549 cells. With both clinically relevant kinetics of hydrolysis and significant cytotoxicity in tissue culture, the conjugate holds promise for enhanced therapeutic efficacy of paclitaxel in vivo.

A new class of water-soluble C60 transfecting agents has been prepared using Hirsch−Bingel chemistry and assessed for their ability to act as gene-delivery vectors in vitro. In an effort to elucidate the relationship between the hydrophobicity of the fullerene core, the hydrophilicity of the water-solubilizing groups, and the overall charge state of the C60 vectors in gene delivery and expression, several different C60 derivatives were synthesized to yield either positively charged, negatively charged, or neutral chemical functionalities under physiological conditions. These fullerene derivatives were then tested for their ability to transfect cells grown in culture with DNA carrying the green fluorescent protein (GFP) reporter gene. Statistically significant expression of GFP was observed for all forms of the C60 derivatives when used as DNA vectors and compared to the ability of naked DNA alone to transfect cells. However, efficient in vitro transfection was only achieved with the two positively charged C60 derivatives, namely, an octa-amino derivatized C60 and a dodeca-amino derivatized C60 vector. All C60 vectors showed an increase in toxicity in a dose-dependent manner. Increased levels of cellular toxicity were observed for positively charged C60 vectors relative to the negatively charged and neutral vectors. Structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized C60 compounds, the C60 vector/DNA complexes, their physical attributes (aggregation, charge) and their transfection efficiencies. Recently, similar Gd@C60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging (MRI). Thus, the successful demonstration of intracellular DNA uptake, intracellular transport, and gene expression from DNA using C60 vectors suggests the possibility of developing analogous Gd@C60-based vectors to serve simultaneously as both therapeutic and diagnostic agents.

The first fullerene (C60) immunoconjugates have been prepared and characterized as an initial step toward the development of fullerene immunotherapy (FIT).

Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the "aspartate-rich" motif D 100 DXX(D/E) that coordinates to Mg 2+ A and Mg 2+ C , and the "NSE/DTE" motif N 225 DXXSXXXE that chelates Mg 2+ B (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.Keywords enzyme kinetics; protein crystallography; terpenoid cyclase; farnesyl diphosphate Sesquiterpene synthases catalyze the metal ion-dependent cyclization of the universal acyclic substrate, farnesyl diphosphate (FPP), to form one of more than 300 known monocyclic, bicyclic, or tricyclic hydrocarbon or alcohol products of widely varied structure and stereochemistry [1][2][3][4][5]. These cyclic products represent branch points in terpenoid biosynthesis. Further modifications of cyclic sesquiterpenes and diterpenes involving downstream enzymes such as the cytochrome P450s of Artemisia annua and Taxus brevifolia, or the Fe(II)-α-I This work was supported by NIH grants GM56838 (D.W.C.) and GM30301 (D.E.C.).II Atomic coordinates and structure factors for N225D trichodiene synthase, the N225D trichodiene synthase-Mg 2+ 3 -PPi complex, N225D/S229T trichodiene synthase, Y295F trichodiene synthase, and the Y295F trichodiene synthase-Mg 2+ 3 -PPi complex have been deposited in the Research Collaboratory for Structural Bioinformatics (http://www.rcsb.org/pdb) with the following accession codes: 2PS4, 2PS5, 2PS6, 2PS7, and 2PS8, respectively. *Corresponding author. Fax: +1 215-573-2201. E-mail: chris@sas.upenn.edu. Fax: +1 401-863-9368. E-mail: David_Cane@Brown.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. To date, the dissection of the NSE/DTE-Mg 2+ B coordination motif in terpene cyclases by sitedirected mutagenesis has not been accompanied by X-ray crystal structure analysis. Accordingly, we now report the first exploration of structure-function relationships for this ...