Synthesis, binding and bioactivity of gamma-methylene gamma-lactam ecdysone receptor ligands: advantages of QSAR models for flexible receptors.

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or 'attractors'. We describe the synthesis, in vitro binding and selected in vivo toxicity data for gamma-methylene gamma-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.

TGGE screening of the entire FBN1 coding sequence in 126 individuals with marfan syndrome and related fibrillinopathies.

Mutations in the gene for fibrillin-1 (FBN1) cause Marfan syndrome (MFS), an autosomal dominant heritable disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular system. FBN1 mutations have also been identified in a series of related disorders of connective tissue collectively termed type-1 fibrillinopathies. We have developed temperature-gradient gel electrophoresis (TGGE) assays for all 65 FBN1 exons, screened 126 individuals with MFS, other type-1 fibrillinopathies, and other potentially related disorders of connective tissue for FBN1 mutations, and identified a total of 53 mutations, of which 33 are described here for the first time. Several mutations were identified in individuals with fibrillinopathies other than classic Marfan syndrome, including aneurysm of the ascending aorta with only minor skeletal anomalies, and several individuals with only skeletal and ocular involvement. The mutation detection rate in this study was 42% overall, but was only 12% in individuals not fulfilling the diagnostic criteria for MFS, suggesting that clinical overdiagnosis is one reason for the low detection rate observed for FBN1 mutation analysis.

Protein-resistant self-assembled monolayers on gold with latent aldehyde functions.

In the present study, oligo(ethylene glycol) (OEG)-linked alkanethiols were synthesized which carry a vicinal diol on one end of the OEG chain. After self-assembled monolayer (SAM) formation on gold, the vicinal diols were converted into aldehyde functions by exposure to aqueous NaIO4, as previously used for SAMs with OEG chains buried in the center of the SAM [Jang et al. Nano Lett. 2003, 3, 691-694]. Mixed SAMs with latent aldehydes on 5% of the OEG termini showed high protein resistance, which greatly slowed the kinetics of protein coupling on the time scale of minutes. Small bioligands (such as biocytin hydrazide) or small heterobifunctional crosslinkers (maleimidopropionyl hydrazide, pyridyldithiopropionyl hydrazide) with hydrazide functions were efficiently bound to the aldehyde functions on the SAM, providing for specific capture of streptavidin or for fast covalent binding of proteins with free thiols or maleimide functions, respectively. In conclusion, OEG-terminated SAMs with latent aldehydes serve as protein-resistant sensor surfaces which are easily functionalized with small ligands or with heterobifunctional crosslinkers to which the bait molecule is attached in a subsequent step.

Self-assembled monolayers with latent aldehydes for protein immobilization.

Aldehyde functions are widely used for immobilization of biomolecules on glass surfaces but have found little attention for biofunctionalization of self-assembled monolayers (SAMs) on gold, due to interference between thiol and aldehyde functions. This problem was recently solved by synthesis of an alkanethiol that carried a vicinal diol group [Jang et al. (2003) Nano Lett. 3, 691-694]. The latter served as a latent aldehyde function that was unmasked by short exposure of the vicinal diol-terminated SAM to aqueous periodate. However, the synthesis of the new vicinal diol-terminated alkane thiol was time-consuming and had an overall yield of approximately 3.5%. In the present study, a general modular strategy was introduced by which SAM components with vicinal diol functions were rapidly synthesized with high yield: this was accomplished by amide bond formation between a SAM-forming carboxylic acid (exemplified by lipoic acid and 16-mercaptohexadecanoic acid) with 3-aminopropane-1,2-diol, using suitable protecting groups. The disulfide or free thiol group afforded SAM formation on gold and, after periodate oxidation of the vicinal diol functions, proteins were covalently bound via their lysine residues. At 1 mg/mL protein concentration, complete surface coverage was reached within minutes. No further protein was bound by nonspecific adsorption, but cognate proteins were specifically bound with high capacity. Pyrogallol-O-hexadecanoic acid and 10-undecenoic acid were also coupled with 3-aminopropane-1,2-diol by amide bond formation, thereby producing latent aldehyde-containing SAM components for metal oxides and hydrogen-terminated silicon, respectively, to show the general usefulness of the new synthetic design.