High-throughput discovery of organic cages and catenanes using computational screening fused with robotic synthesis.

Supramolecular synthesis is a powerful strategy for assembling complex molecules, but to do this by targeted design is challenging. This is because multicomponent assembly reactions have the potential to form a wide variety of products. High-throughput screening can explore a broad synthetic space, but this is inefficient and inelegant when applied blindly. Here we fuse computation with robotic synthesis to create a hybrid discovery workflow for discovering new organic cage molecules, and by extension, other supramolecular systems. A total of 78 precursor combinations were investigated by computation and experiment, leading to 33 cages that were formed cleanly in one-pot syntheses. Comparison of calculations with experimental outcomes across this broad library shows that computation has the power to focus experiments, for example by identifying linkers that are less likely to be reliable for cage formation. Screening also led to the unplanned discovery of a new cage topology-doubly bridged, triply interlocked cage catenanes.

Nanoprecipitation technique for the encapsulation of agrochemical active ingredients.

In 1997, a research programme was initiated to assess the ability of nanospheres (NS) to improve the biodelivery of a new insecticide to plants. Stable polymeric NS, with a size near 135 nm and an encapsulation rate in the range of 3.5%, have been obtained using a nanoprecipitation method with Eudragit S100 polymer. Biological studies have been performed on cotton plants infested with aphid, to estimate the direct contact efficacy of NS formulations on the insects and the systemicity of the encapsulated active ingredient and its level of penetration through the plant, compared to a classical suspension used as a reference. Results indicate that NS formulations are not so good as the reference in terms of speed of action and sustained release. Nevertheless, NS formulation performed better than the reference to enhance the systemicity of the AI and improve its penetration through the plant. It is concluded that the NS do not provide a controlled release of AI but, due to their small size, they enhance the penetration in the plant compared to the classical suspension.

Interactions of lipid-oligonucleotide conjugates with low-density lipoprotein.

The ability of antisense oligonucleotides to interdict, sequence-specifically, the expression of pathogenic genes affords an exciting new strategy for therapeutic intervention (1-3). Oligonucleotides with physiological phosphodiester internucleotide bonds are rapidly degraded, predominantly by exonucleases. Numerous oligonucleotide analogs have therefore been synthesized to confer resistance toward nuclease activity (3). The phosphorothioate analog is the most extensively studied, and phosphorothioate oligodeoxynucleotides have been shown to be potent inhibitors of the expression of their target genes in vitro and in vivo (1,3). However, phosphorothioate oligodeoxynucleotides also bind avidly and nonspecifically to proteins, thus provoking a variety of non-antisense effects (4). Oligonucleotide analogs that do not bind to proteins are therefore expected to display less nonantisense side effects. However, protein binding also affects the in vivo disposition of oligonucleotides. Nonphosphorothioate oligonucelotide analogs generally do not bind to serum proteins, and are therefore rapidly cleared from the circulation, protein-bound phosphorothioate oligodeoxynucelotides circulate much longer (5,6).

Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation.

Several studies have shown improved efficacy of cholesteryl-conjugated phosphorothioate antisense oligodeoxynucleotides. To gain insight into the mechanisms of the improved efficacy in vivo, we investigated the disposition of ISIS-9388, the 3'-cholesterol analog of the ICAM-1-specific phosphorothioate oligodeoxynucleotide ISIS-3082, in rats. Intravenously injected [(3)H]ISIS-9388 was cleared from the circulation with a half-life of 49.9 +/- 2.2 min (ISIS-3082, 23.3 +/- 3.8 min). At 3 h after injection, the liver contained 63.7 +/- 3. 3% of the dose. Compared to ISIS-3082, the hepatic uptake of ISIS-9388 is approximately 2-fold higher. Endothelial, Kupffer and parenchymal cells accounted for 45.7 +/- 5.7, 33.0 +/- 5.9 and 21.3 +/- 2.6% of the liver uptake of [(3)H]ISIS-9388, respectively, and intracellular concentrations of approximately 2, 75 and 50 microM, respectively, could be reached in these cells (1 mg/kg dose). Preinjection with polyinosinic acid or poly-adenylic acid reduced the hepatic uptake of [(3)H]ISIS-9388, which suggests the involvement of (multiple) scavenger receptors. Size exclusion chromatography of mixtures of the oligonucleotides and rat plasma indicated that ISIS-9388 binds to a larger extent to high molecular weight proteins than ISIS-3082. Analysis by agarose gel electrophoresis indicated that ISIS-9388 binds more tightly to plasma proteins than ISIS-3082. The different interaction of the oligonucleotides with plasma proteins possibly explains their different dispositions. We conclude that cholesterol conjugation results in high accumulation of phosphorothioate oligodeoxynucleotides in various liver cell types, which is likely to be beneficial for antisense therapy of liver-associated diseases.

Carrier-mediated delivery of 9-(2-phosphonylmethoxyethyl)adenine to parenchymal liver cells: a novel therapeutic approach for hepatitis B.

Our aim is to selectively deliver 9-(2-phosphonylmethoxyethyl)adenine (PMEA) to parenchymal liver cells, the primary site of hepatitis B virus (HBV) infection. Selective delivery is necessary because PMEA, which is effective against HBV in vitro, is hardly taken up by the liver in vivo. Lactosylated reconstituted high-density lipoprotein (LacNeoHDL), a lipid particle that is specifically internalized by parenchymal liver cells via the asialoglycoprotein receptor, was used as the carrier. PMEA could be incorporated into the lipid moiety of LacNeoHDL by attaching, via an acid-labile bond, lithocholic acid-3alpha-oleate to the drug. The uptake of the lipophilic prodrug (PMEA-LO) by the liver was substantially increased after incorporation into LacNeoHDL. Thirty minutes after injection of [(3)H]PMEA-LO-loaded LacNeoHDL into rats, the liver contained 68.9% +/- 7.7% of the dose (free [(3)H]PMEA, <5%). Concomitantly, the uptake by the kidney was reduced to <2% of the dose (free [(3)H]PMEA, >45%). The hepatic uptake of PMEA-LO-loaded LacNeoHDL occurred mainly by parenchymal cells (88.5% +/- 8.2% of the hepatic uptake). Moreover, asialofetuin inhibited the liver association by >75%, indicating uptake via the asialoglycoprotein receptor. The acid-labile linkage in PMEA-LO, designed to release PMEA during lysosomal processing of the prodrug-loaded carrier, was stable at physiological pH but was hydrolyzed at lysosomal pH (half-life, 60 to 70 min). Finally, subcellular fractionation indicates that the released PMEA is translocated to the cytosol, where it is converted into its active diphosphorylated metabolite. In conclusion, lipophilic modification and incorporation of PMEA into LacNeoHDL improves the biological fate of the drug and may lead to an enhanced therapeutic efficacy against chronic hepatitis B.

Modification of the plasma clearance and liver uptake of steroid ester-conjugated oligodeoxynucleotides by association with (lactosylated) low-density lipoprotein.

Low-density lipoprotein (LDL) has been proposed as carrier for the selective delivery of anticancer drugs to tumor cells. We reported earlier the association of several lipidic steroid-conjugated anticancer oligodeoxynucleotides (ODNs) with LDL. In the present study, we determined the stability of these complexes. When the complexes were incubated with a mixture of high-density lipoprotein and albumin, or with rat plasma, the oleoyl steroid-conjugated ODNs appeared to be more stably associated with LDL than the cholesteryl-conjugated ODN. Intravenously injected free lipid-ODNs were very rapidly cleared from the circulation of rats. The area under the curve (AUC) of the lipid-ODNs in plasma was <0.4 microg x min/mL. After complexation with LDL, plasma clearance of the lipid-ODNs was delayed. This was most evident for ODN-5, the ODN conjugated with the oleoyl ester of lithocholic acid (AUC = 6.82 +/- 1.34 microg x min/mL). The AUC of ODN-4, a cholesteryl-conjugated ODN, was 1.49 +/- 0.37 microg x min/mL. In addition, the liver uptake of the LDL-complexed lipid-ODNs was reduced. The lipid-ODNs were also administered as a complex with lactosylated LDL, a modified LDL particle that is selectively taken up by the liver. A high proportion of ODN-5 was transported to the liver along with lactosylated LDL (69.1 +/- 8.1% of the dose at 15 min after injection), whereas much less ODN-4 was transported (36.6 +/- 0.1% of the dose at 15 min after injection). We conclude that the oleoyl ester of lithocholic acid is a more potent lipid anchor than the other steroid lipid anchors. Because of the stable association, the oleoyl ester of lithocholic acid is an interesting candidate for tumor targeting of anticancer ODNs with lipoproteins.

Introduction of the closed cranial window technique in gerbils and verification by observation of the effects of specific drugs.

The exact mechanisms of cerebral arterial hypoxia are not perfectly defined. Our purpose is to adapt and validate, with drugs well known in rats and rabbits, a closed cranial window technique in gerbils. The method was used with seventeen gerbils to measure diameter changes of the pial arterioles under normoxia (after the topical application of agonists and antagonists of ATP-sensitive and Ca2+-dependent potassium channels), as well as under hypoxia. In normoxia, aprikalim (10(-6) M), a direct activator of ATP-sensitive potassium channels, increases the diameter of pial arterioles by 10+/-2% (N = 17). This effect is inhibited by glibenclamide (10(-6) M), but not affected by iberiotoxin (10(-6) M), a specific inhibitor of Ca2+-dependent potassium channels. The adenosine-induced dilation by 19+/-5% (N = 17) is reduced by 59+/-16% with iberiotoxin, by 33+/-23% with glibenclamide and inhibited by theophylline (10(-5) M). In hypoxia (15% O2), pial arteriole diameters are increased by 24+/-5% (N = 17) and partially decreased by the application of glibenclamide and iberiotoxin to 59+/-11% and 54+/-5%, respectively. These data are similar to those obtained in other species and validate the closed cranial window technique on gerbils. They indicate that, as for rats and rabbits, both ATP-sensitive and Ca2+-dependent potassium channels are present in gerbil pial vessels and play a role in hypoxia.

Synthesis of a lipophilic prodrug of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and its incorporation into a hepatocyte-specific lipidic carrier.

PURPOSE: 9-(2-Phosphonylmethoxyethyl)adenine (PMEA), a potent inhibitor of Hepatitis B virus replication, is in vivo hardly taken up by parenchymal liver cells (the site of infection). Our aim is to examine whether lactosylated reconstituted HDL (LacNeoHDL), a lipidic particle that is specifically internalized by parenchymal liver cells, is a suitable carrier for the selective delivery of PMEA to this cell type. METHODS: To incorporate PMEA into LacNeoHDL, we synthesized a lipophilic prodrug (PMEA-LO) by coupling PMEA via an acid-labile phosphonamidate bond to lithocholic acid-3alpha-oleate. RESULTS: The yield of the synthesis was 52% ([3H]PMEA-LO: 24%). [3H]PMEA-LO readily incorporated into LacNeoHDL (13 molecules/particle) without affecting the size and net negative charge of the carrier. Further, incubation studies at lysosomal pH showed [3H]PMEA was completely released from the carrier whereas, at neutral pH or in plasma, appreciable release was not observed. CONCLUSIONS: The conjugation of PMEA with lithocholic acid-3alpha-oleate results in a lipophilic prodrug that readily associates with Lac-NeoHDL. The association of the prodrug does not affect the physicochemical properties of the particle, and PMEA is released from the carrier at lysosomal pH. These findings indicate that by using the prodrug approach, LacNeoHDL is a suitable carrier to deliver PMEA to parenchymal liver cells.

Targeted delivery of oligodeoxynucleotides to parenchymal liver cells in vivo.

Anti-sense oligodeoxynucleotides (ODNs) hold great promise for correcting the biosynthesis of clinically relevant proteins. The potential of ODNs for modulating liver-specific genes might be increased by preventing untimely elimination and by improving the local bioavailability of ODNs in the target tissue. In the present study we have assessed whether the local ODN concentration can be enhanced by the targeted delivery of ODNs through conjugation to a ligand for the parenchymal liver cell-specific asialoglycoprotein receptor. A capped ODN (miscellaneous 20-mer sequence) was derivatized with a ligand with high affinity for this receptor, N2-[N2-(N2,N6-bis{N-[p-(beta-d-galactopyranosyloxy) anilino] thiocarbamyl}-L-lysyl)-N6-(N-{p-[beta-D -galactopyranosyloxy] anilino} thiocarbamyl)-L-lysyl]-N6-[N- (p-{beta-D-galactopyranosyloxy}anilino)thiocarbamyl]-L-lysine (L3G4) (Kd 6.5+/-0.2 nM, mean+/-S.D.). Both the uptake studies in vitro and the confocal laser scan microscopy studies demonstrated that L3G4-ODN was far more efficiently bound to and taken up by parenchymal liver cells than underivatized ODN. Studies in vivo in rats showed that hepatic uptake could be greatly enhanced from 19+/-1% to 77+/-6% of the injected dose after glycoconjugation. Importantly, specific ODN accumulation of ODN into parenchymal liver cells was improved almost 60-fold after derivatization with L3G4, and could be attributed to the asialoglycoprotein receptor. In conclusion, the scavenger receptor-mediated elimination pathway for miscellaneous ODN sequences can be circumvented by direct conjugation to a synthetic tag for the asialoglycoprotein receptor. In this manner a crucial requisite is met towards the application of ODNs in vivo to modulate the biosynthesis of parenchymal liver cell-specific genes such as those for apolipoprotein (a), cholesterol ester transfer protein and viral proteins.

Stable incorporation of a lipophilic daunorubicin prodrug into apolipoprotein E-exposing liposomes induces uptake of prodrug via low-density lipoprotein receptor in vivo.

Many tumors express elevated levels of low-density lipoprotein (LDL) receptors. Therefore, native LDL and synthetic LDL-like particles have been proposed as carriers for antineoplastic drugs. We demonstrated earlier that small apolipoprotein E (apoE)-exposing liposomes were specifically recognized by the LDL receptor. In this study, we incorporated a lipophilic derivative of daunorubicin (LAD) into the apoE liposomes. Up to 11 molecules of LAD could be incorporated per particle without significantly changing the size, lipid composition, and ability to bind apoE of the liposomes. The biological fate of the prodrug was largely determined by its carrier (70% of the initially incorporated LAD was still associated to the liposomes after 4 h of circulation in mice). Compared with free daunorubicin, the circulation half-life of the liposome-associated prodrug was substantially prolonged and undesired tissue disposition was reduced. The role of the LDL receptor in the metabolism of LAD-loaded apoE liposomes was demonstrated in rats with up-regulated hepatic LDL receptors. In these rats, the liver uptake of the prodrug and carrier was increased 5-fold. The addition of apoE was essential for LDL receptor-mediated uptake of the drug-carrier complex. In LDL receptor-deficient mice, the circulation time of both the prodrug and the carrier increased approximately 2-fold compared with wild-type mice. We conclude that LAD-loaded apoE liposomes constitute a stable drug-carrier complex that is well suited for LDL receptor-mediated selective drug delivery to tumors.

Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of liposomes to the hepatic asialoglycoprotein receptor.

A series of glycolipids have been prepared which contain a cluster galactoside moiety with high affinity for the hepatic asialoglycoprotein receptor and a bile acid ester moiety which mediates stable incorporation into liposomes. Loading of liposomes with these glycolipids at a ratio of 5% (w/w) resulted in efficient recognition and uptake of the liposomes by the liver. Preinjection with asialofetuin almost completely inhibited the uptake, establishing that the liposomes were selectively recognized and processed by the asialoglycoprotein receptor on liver parenchymal cells. In contrast, a glycolipid content of 50% (w/w) led to a liver uptake that could not be inhibited by preinjection with asialofetuin, indicating that the liposomes were now processed by the Gal/Fuc-recognizing receptor on liver macrophages. The results presented in this study are important for future targeting of water-soluble and amphiphilic drugs, enveloped in these glycolipid-laden liposomes, to parenchymal liver cells.

Phosphorylcholine Coating of Iron Oxide Nanoparticles.

In order to develop thin-walled superparamagnetic nanoparticle suspensions as a contrast agent for magnetic resonance imaging, phosphorylcholine PC was used to coat iron oxide cores of 5 nm. Weak stable positively charged suspensions can be obtained at concentration greater than 3 mmol.l-1 (corresponding to about 3.2 molecules per nm2), while the addition of phosphorylglycerol PG decreases the electrophoretic mobility. Raising the pH over 6 leads to flocculation: the binding of PC on iron oxides as a function of pH appears to be reversible. By Langmuir analysis, two adsorption domains may be observed with a maximal density of 3.48 and 6.55 mol.nm-2, interpreted as a multilayer formation. Copyright 1999 Academic Press.

Low-density lipoprotein receptor-mediated delivery of a lipophilic daunorubicin derivative to B16 tumours in mice using apolipoprotein E-enriched liposomes.

Many tumours express relatively high levels of low-density lipoprotein (LDL) receptors on their membranes. The LDL receptor is, therefore, an attractive target for the selective delivery of antineoplastic drugs to tumour cells. We reported previously on the synthesis of small apolipoprotein E (apoE)-containing liposomes that behave in vivo in a very similar way to native LDL. In this study, we examined the interaction of this liposomal carrier with cultured B16 melanoma cells. Binding of apoE liposomes to the cells is saturable, with a maximum binding of approximately 90000 particles per cell. Cross-competition studies indicated that apoE liposomes are bound by the LDL receptor. Association of apoE liposomes to B16 cells is strictly Ca2+ dependent, which forms additional evidence for a role of the LDL receptor. The affinity of apoE liposomes for the LDL receptor on B16 cells is 15-fold higher than that of LDL (0.77 vs 11.5 nM respectively). ApoE is essential for the LDL receptor recognition because liposomes lacking apoE were, in competition studies, 20- to 50-fold less effective than apoE-containing liposomes. We examined in B16 tumour-bearing mice the tumour-localizing properties of apoE liposomes and the disposition of an incorporated lipophilic derivative of daunorubicin (LAD). Tissue distribution studies showed that LAD-loaded apoE liposomes were taken up and processed by the major LDL receptor-expressing organs (i.e. adrenals, liver and spleen). Of all other tissues, the tumour showed the highest uptake. The distribution patterns of LAD-loaded apoE liposomes and native LDL in the tumour-bearing mice were very similar, which supports the role of the LDL receptor in the disposition of the prodrug-loaded particles. The disposition of LAD followed the pattern of the liposomal carrier. We conclude that apoE liposomes enable LDL receptor-mediated specific delivery of antineoplastic (pro)drugs to tumours, and, therefore, constitute an attractive novel option for anti-tumour chemotherapy.

Preparation of conjugates of oligodeoxynucleotides and lipid structures and their interaction with low-density lipoprotein.

The high expression level of receptors for low-density lipoprotein (LDL) on tumor cells makes LDL an attractive carrier for selective delivery of drugs to these cells. The aim of this study is to allow incorporation of oncogene-directed antisense oligodeoxynucleotides (ODNs) into the lipid moiety of LDL. Therefore, ODNs were conjugated with oleic acid, cholesterol, and several other steroid lipids. These latter steroid lipids were synthesized starting from bile acids and were varied in lipophilicity by attaching oleic acid ester structures. The lipid structures, activated as pentafluorophenyl esters, were conjugated in solution phase to 3'-amino-tailed ODNs. The ODNs conjugated with lithocholic acid, oleic acid, and cholesterol could easily be purified by reversed phase (RP)-HPLC. However, the ODNs conjugated with the oleoyl steroid ester structures irreversibly bound to the column material. These highly lipidic ODNs were separated from the nonconjugated ODN by electrophoresis in a 1% low-melting agarose gel containing 0.1% Tween 20. This method was found to be very effective in isolating the ODNs conjugated to the oleoyl steroid ester structures. The ODNs conjugated with cholesterol and the oleoyl esters of lithocholic and cholenic acid associated readily and nearly completely with LDL. However, the less lipidic ODNs and the ODN conjugated with the dioleoyl ester of chenodeoxycholic acid did not and did incompletely associate, respectively. Lithocholic acid and oleic acid are probably not sufficiently lipophilic to induce association with LDL, whereas the dioleoyl ester structure is probably too bulky and extended to allow partitioning into the lipid moiety of LDL. We conclude that several of the lipid-ODNs can associate readily with LDL, enabling delivery of oncogene-directed antisense ODNs via the LDL receptor pathway.

Synthesis of a lipophilic daunorubicin derivative and its incorporation into lipidic carriers developed for LDL receptor-mediated tumor therapy.

PURPOSE: Many tumors express elevated levels of LDL receptors (apoB, E receptors) on their membranes. Selective delivery of anti-neoplastic drugs to tumors by incorporation of these drugs into LDL or LDL-resembling particles should improve the efficacy of tumor therapy and minimize the severe side-effects. Since the apolipoproteins on the particles are essential for the LDL receptor recognition, drugs should preferably be incorporated into the lipid moiety. Most anti-tumor agents are too hydrophilic for incorporation into these carriers. METHODS: We synthesized LAD, a lipophilic prodrug of daunorubicin, by coupling the drug via a lysosomally degradable peptide spacer to a cholesteryl oleate analog. RESULTS: The overall yield of the synthesis was 50% with a purity of > 90%. Radioactively ([3H]) labeled LAD was obtained via a slightly modified procedure (yield 40%). The octanol/water partition coefficient of LAD is 30-fold higher than that of daunorubicin. LAD could be incorporated into triglyceride-rich lipid emulsions and small liposomes, which, if provided with apoE, have been demonstrated earlier to be cleared in vivo via the LDL receptor. The liposomes contained approximately 10 molecules of LAD per liposomal particle. Analysis of differently sized LAD-containing emulsions suggests that LAD associates with the surface of lipidic particles. In the presence of human serum, LAD did not dissociate from the emulsion particles, indicating a firm association of LAD with the carrier. CONCLUSIONS: The coupling of a cholesterol ester analog to daunorubicin results in a lipophilic prodrug that can be firmly anchored into lipidic carries. LAD-loaded emulsions and liposomes provided with recombinant apoE will be tested in the near future for their ability to deliver LAD to tumor tissue in vivo via the LDL receptor.

In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells.

Systemically administered phosphorothioate antisense oligodeoxynucleotides can specifically affect the expression of their target genes, which affords an exciting new strategy for therapeutic intervention. Earlier studies point to a major role of the liver in the disposition of these oligonucleotides. The aim of the present study was to identify the cell type(s) responsible for the liver uptake of phosphorothioate oligodeoxynucleotides and to examine the mechanisms involved. In our study we used ISIS-3082, a phosphorothioate antisense oligodeoxynucleotide specific for murine ICAM-1. Intravenously injected [3H]ISIS-3082 (dose: 1 mg/kg) was cleared from the circulation of rats with a half-life of 23.3+/-3.8 min. At 90 min after injection (>90% of [3H]ISIS-3082 cleared), the liver contained the most radioactivity, whereas the second-highest amount was recovered in the kidneys (40.5+/-1.4% and 17.9+/-1.3% of the dose, respectively). Of the remaining tissues, only spleen and bone marrow actively accumulated [3H]ISIS-3082. By injecting different doses of [3H]ISIS-3082, it was found that uptake by liver, spleen, bone marrow, and kidneys is saturable, which points to a receptor-mediated process. Subcellular fractionation of the liver indicates that ISIS-3082 is internalized and delivered to the lysosomes. Liver uptake occurs mainly (for 56.1+/-3.0%) by endothelial cells, whereas parenchymal and Kupffer cells account for 39.6+/-4.5 and 4.3+/-1.7% of the total liver uptake, respectively. Preinjection of polyinosinic acid substantially reduced uptake by liver and bone marrow, whereas polyadenylic acid was ineffective, which indicates that in these tissues scavenger receptors are involved in uptake. Polyadenylic acid, but not polyinosinic acid, reduced uptake by kidneys, which suggests renal uptake by scavenger receptors different from those in the liver. We conclude that scavenger receptors on rat liver endothelial cells play a predominant role in the plasma clearance of ISIS-3082. As scavenger receptors are also expressed on human endothelial liver cells, our findings are probably highly relevant for the therapeutic application of phosphorothioate oligodeoxynucleotides in humans. If the target gene is not localized in endothelial liver cells, the therapeutic effectiveness might be improved by developing delivery strategies that redirect the oligonucleotides to the actual target cells.

Supramolecular assembly using helical peptides.

We investigated supramolecular assemblies of various hydrophobic helical peptides. The assemblies were formed at the air/water interface or in aqueous medium. The hexadecapeptide, Boc-(Ala-Aib)s-OMe (BA16M), was reported to take alpha-helical structure by X-ray analysis. Several derivatives were prepared, which have the repeating sequence of Ala-Aib, Lys(Z)-Aib or Leu-Aib, or have the terminal chemically modified. CD spectra of the peptides indicated helical conformation in ethanol solution. The surface pressure-area isotherms of the peptide monolayers showed an inflection at the surface area corresponding to the cross section along the helix axis, and the monolayers were collapsed by further compression. All the helical peptides oriented their helix axis parallel to the air/water interface on the basis of the results of transmission IR spectra and RAS of the monolayers transferred onto substrates. A small mound was observed in the isotherm of BA16M and other derivatives, which was ascribed to the phase transition from the liquid state to the solid state. One mol% of FITC-labeled peptide was mixed into the monolayers to visualize the phase separation of the solid and liquid states at the surface pressure of the coexisting region. Various shapes of the dark domain were observed at the top of the mound in the isotherms by fluorescence microscopy. The helical peptides formed two-dimensional crystals at the air/water interface when they were compressed to the solid state. An amino-terminated helical peptide, HA16B, was suspended in an aqueous medium by a sonication method and transparent dispersion was obtained. The dynamic light scattering measurement of the dispersion revealed the particle size of 75 nm with a narrow size distribution. The molecular assembly of the helical peptide in water was called "Peptosome", because it takes a vesicular structure.

Formation of protein multilayers and their competitive replacement based on self-assembled biotinylated phospholipids.

Based on specific recognition processes the build-up of protein multilayers was achieved using streptavidin layers as a docking matrix. For this purpose, streptavidin was organized at biotin-containing monolayers, liposomes, and self-assembled layers on gold. Thus, mixed double and triple layers of streptavidin, Con A, Fab fragments, and hormones were prepared and characterized by fluorescence microscopy and plasmon spectroscopy. Using biotin analogues with lower binding constants several cycles of multilayer formation followed by competitive replacement could be achieved.

Attempts to mimic docking processes of the immune system: recognition-induced formation of protein multilayers.

The assemblage of protein multilayers induced by molecular recognition, as seen, for example, in the immune cascade, has been mimicked by using streptavidin as a docking matrix. For these experiments, this protein matrix was organized on liposomes, monolayers at the air-water interface, and self-assembled layers on gold, all three containing biotin lipids. The docking of streptavidin to biotin at liposomal surfaces was confirmed by circular dichroism. Mixed double and triple layers of streptavidin, concanavalin A, antibody Fab fragments, and hormones are prepared at the air-water interface and on gold surfaces and were characterized by fluorescence microscopy and plasmon spectroscopy. With the use of biotin analogs that have lower binding constants it has been possible to achieve multiple formation and competitive replacement of the oriented protein assemblages.