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Eclosion hormone (EH) is an integral component in the cascade regulating the behaviors culminating in emergence of an insect from its old exoskeleton. Little is known regarding the EH solution structure; consequently, we utilized a computational approach to generate a hypothetical structure for Manduca sexta EH. The de novo algorithm exploited the restricted conformational space of disulfide bonds (Cys14-Cys38, Cys18-Cys34, and Cys21-Cys49) and predicted secondary structure elements to generate a thermodynamically stable structure characterized by 55% helical content, an unstructured N-terminus, a helical C-terminus, and a solvent-exposed loop containing Trp28 and Phe29. Both the strain and pseudo energies of the predicted peptide compare favorably with those of known structures. The 62-amino acid peptide was synthesized, folded, assayed for activity, and structurally characterized to confirm the validity of the model. The helical content is supported by circular dichroism and hydrogen-deuterium exchange mass spectrometry. Fluorescence emission spectra and acrylamide quenching are consistent with the solvent exposure predicted for Trp28, which is shielded by Phe29. Furthermore, thermodynamically stable conformations that deviated only slightly from the predicted Manduca EH structure were generated in silico for the Bombyx mori and Drosophila melanogaster EHs, indicating that the conformation is not species-dependent. In addition, the biological activities of known mutants and deletion peptides were rationalized with the predicted Manduca EH structure, and we found that, on the basis of sequence conservation, functionally important residues map to two conserved hydrophobic clusters incorporating the C-terminus and the first loop.

HIV attachment via the CD4 receptor is an important target for developing novel approaches to HIV chemotherapy. Cyclotriazadisulfonamide (CADA) inhibits HIV at submicromolar levels by specifically down-modulating cell-surface and intracellular CD4. An effective five-step synthesis of CADA in 30% overall yield is reported. This synthesis has also been modified to produce more than 50 analogues. Many tail-group analogues have been made by removing the benzyl tail of CADA and replacing it with various alkyl, acyl, alkoxycarbonyl and aminocarbonyl substituents. A series of sidearm analogues, including two unsymmetrical compounds, have also been prepared by modifying the CADA synthesis, replacing the toluenesulfonyl sidearms with other sulfonyl groups. Testing 30 of these compounds in MT-4 cells shows a wide range of CD4 down-modulation potency, which correlates with ability to inhibit HIV-1. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) approaches. The X-ray crystal structures of four compounds, including CADA, show the same major conformation of the central 12-membered ring. The solid-state structure of CADA was energy minimized and used to generate the remaining 29 structures, which were similarly minimized and aligned to produce the 3D-QSAR models. Both models indicate that steric bulk of the tail group, and, to a lesser extent, the sidearms mainly determine CD4 down-modulation potency in this series of compounds.

A water-soluble conjugate (1) with intact carboxyl groups was prepared by addition of poly(ethylene glycol) thiol (MPEG-SH) regiospecifically to the exo vinyl group of bilirubin. (1)H and (13)C NMR and absorbance spectroscopy in CDCl(3) and DMSO-d(6) confirmed the assigned structure and showed that pegylation did not disrupt the hydrogen-bonded ridge-tile conformation of the pigment moiety. Aqueous solutions of 1 were optically clear, but NMR signals were seen only from the MPEG portion and none from the tetrapyrrole, consistent with dissolved assemblies containing aggregated bilirubin cores within mobile polyether chains. On alkalinization (pH >12), signals from the pigment moiety reappeared. Titrimetric measurements on 1 in water showed the pK(a)'s of the two carboxyl groups to be similar (average 6.42). Control studies with pegylated half-esters of succinic, suberic, brassylic, thapsic, and 1,20-eicosanedioic acid showed that pegylation per se has little, if any, effect on carboxyl ionization. However, aggregation increases the apparent pK(a) by approximately 1-2 units. The molecularity of bilirubin in solution was further characterized by ultracentrifugation. Over the pH range 8.5-10 in buffer, bilirubin formed multimers with aggregation numbers ranging from approximately 2-7. Bilirubin is monomeric in DMSO or CHCl(3) at approximately 2 x 10(-)(5) M, but aggregation occurred when the CHCl(3) was contaminated with trace adventitious (perhaps lipoidal) impurities. These observations show that aggregation increases the pK(a)'s of aliphatic carboxylic acids relative to their monomer values in water. They are consistent with earlier (13)C NMR-based estimates of approximately 4.2 and approximately 4.9 for the aqueous pK(a)'s of bilirubin and similar studies of bilirubin in micellar bile-salt solutions. Together with earlier work, they confirm that the pK(a)'s of bilirubin are about normal for aliphatic carboxyls and suggest that the high (>7.5) values occasionally reported, including those based on CHCl(3) partitioning, are artifacts of aggregation or technique.