Synthesis of positively charged galactosurfactants

An approach to synthesis of cationic carbohydrate surfactants with potential antimicrobial or transfected activities is described.     

Synthesis of positively charged galactose-bearing surfactants

An approach to the synthesis of cationic carbohydrate surfactants with potential antimicrobial and transfecting activities is proposed.     

Synthesis and characterization of digoxin-phospholipid conjugates

The preparation of immunoreactive derivatives of digoxin for analytical applications is most often carried out by periodate cleavage of the terminal sugar ring (digitoxose) followed by reaction with an enzyme, protein, carrier, or related biological molecules. Here we report an improved and more efficient synthesis which was developed to provide digoxin-phospholipid conjugates useful for liposome immunoassay. The approach used involved the linking of the cleaved digitoxose through a carboxymethyl oxime functionality, which provides much improved yields of readily purified products. The synthetic modification should be applicable to the preparation of analogous phospholipid conjugates involving linkage through a sugar ring (digitoxin, ouabain, and related cardiac glycosides) or to those involving steroids (i.e., 3-digoxigenone) which can be modified to form oxime derivatives remote from key functionalities important for immunorecognition by specific antibody. The characterization of the digoxin-phospholipid conjugates with high-resolution NMR and fast atom bombardment mass spectrophotometry will also be discussed.     

Synthesis and characterization of insulin-transferrin conjugates

Receptor-mediated endocytosis can be exploited for improving the transcellular delivery of therapeutic proteins. Insulin conjugated to transferrin by forming disulfide bonds has been shown to improve insulin oral bioavailability in diabetic rats. We are developing a combination strategy involving complexation hydrogels as delivery vehicles for insulin-transferrin conjugates. The complexation hydrogels developed in our laboratory have been shown to be promising carriers for oral delivery of proteins and peptides. Integrating the strategies based on the complexation hydrogels and insulin-transferrin conjugates may prove to be a novel approach for oral delivery of insulin and other therapeutic proteins. In this work, electrospray ionization mass spectrometry (ESI-MS) was used to study the modification of insulin during its reaction with transferrin. The stability of the conjugated insulin to enzymatic degradation was also studied. ESI-MS studies confirmed the site-specific modifications of insulin. The transferrin conjugation of insulin was also shown to increase the stability of insulin to enzymatic degradation.     

Synthesis and characterization of fluorescent Lucifer yellow-lipid conjugates

The syntheses of fluorescent lipid probes composed of Lucifer yellow dyes linked to either cholesterol or phospholipids are described. The spectral properties of these probes are characterized, and the probes are evaluated for use with model membranes and with live animal and plant cells. Of the probes synthesized, the cholesterol derivative is the easiest to prepare and appears to be the most useful because it readily labels the plasma membrane of live cells and maintains a high ratio of cell surface-to-cytoplasmic fluorescence.     

Synthesis and characterization of membrane-active GALA-OKT9 conjugates

Cellular processing of immunotoxins is inefficient, limiting the overall effectiveness of current immunotoxin therapies. Specifically, translocation of ribosome-inactivating toxins across intracellular membranes is agonizingly slow. In one strategy to improve immunotoxin efficacy, membrane-active peptides are attached to immunotoxins to facilitate transfer of the toxic moiety across a cellular membrane to the cytosol. pH-sensitive peptides are of particular interest, as the membrane activity can be localized to the endosomal/lysosomal pathway, reducing nonspecific interactions at the cell surface. In this study, GALA, a pH-sensitive peptide that forms multimeric pores in membranes, was chemically attached to OKT9, an anti-transferrin receptor mAb. Conjugates were tested by measuring release of encapsulated dyes from liposomes to determine the extent to which the membrane-lytic properties of GALA were retained. The most significant feature affecting the lytic properties of GALA-OKT9 conjugates was the number of attached GALA per OKT9. Conjugates with a single GALA per OKT9 caused almost no leakage while conjugates with two or three GALA per OKT9 caused significant leakage in a concentration-dependent manner. Invariably, GALA-OKT9 conjugates were significantly less active than unconjugated GALA, attributable to a decrease both in partitioning and in surface aggregation. No improvement in membrane-lytic activity was achieved by using a longer, more flexible poly(ethylene glycol) cross-linker. Attachment of GALA via C- versus N-terminal linkage had no effect on membrane-lytic properties. Size-selective release of high molecular weight dextrans was almost identical for conjugated and unconjugated GALA, suggesting that GALA forms the same pore structure regardless of conjugation state.     

Synthesis and characterization of oligonucleotide conjugates bearing electroactive labels

Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochemical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were prepared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides either at 3′ or 5′ end. Both labels were found to not disturb the recognition properties of the oligonucleotide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates with a disulfide at 3′ end and an electrochemical label at 5′ end.     

Polymerization of actin by positively charged liposomes

By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N-pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts.     

Synthesis and characterization of covalently linked single-stranded DNA oligonucleotide-dendron conjugates

A solution-phase synthesis and characterization of covalent DNA-dendron conjugates is presented. Thiol-terminated 12-base oligonucleotides were added to second- and third-generation triazine-based dendrons via thiol/disulfide exchange chemistry. Single-stranded DNA oligonucleotides were successfully attached to dendrons at the core, the periphery, and both. Proof of structure for these architectures is derived primarily from mass spectrometry and polyacrylamide gel electrophoresis and complemented by labeling analysis using Ellman's reagent and degradation analysis using a reducing agent.     

Detecting proteoglycans immobilized on positively charged nylon

Proteoglycans (PG) immobilized on positively charged Nylon 66 are detected readily by staining with Alcian blue. With the exception of hyaluronic acid, free glycosaminoglycans appear unreactive when treated similarly. Immobilization was performed by dot blotting or by electrophoretic transblotting from various gel supports. When transblotted to positively charged Nylon 66 from large-pore agarose-acrylamide gels, levels of 10-50 ng of PG could be detected by Alcian blue staining. This procedure appeared to be nearly 10(2) times more sensitive than staining of gels with toluidine blue. The transblot and staining procedure also appears to be effective with PG separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was applied to a preparation enriched in basement membrane components.     

Synthesis and characterization of poly(ethylene glycol)-insulin conjugates

Human insulin was modified by covalent attachment of short-chain (750 and 2000 Da) methoxypoly (ethylene glycol) (mPEG) to the amino groups of either residue PheB1 or LysB29, resulting in four distinct conjugates: mPEG(750)-PheB1-insulin, mPEG(2000)-PheB1-insulin, mPEG(750)-LysB29-insulin, and mPEG(2000)-LysB29-insulin. Characterization of the conjugates by MALDI-TOF mass spectrometry and N-terminal protein sequence analyses verified that only a single polymer chain (750 or 2000 Da) was attached to the selected residue of interest (PheB1 or LysB29). Equilibrium sedimentation experiments were performed using analytical ultracentrifugation to quantitatively determine the association state(s) of insulin derivatives. In the concentration range studied, all four of the conjugates and Zn-free insulin exist as stable dimers while Zn(2+)-insulin was exclusively hexameric and Lispro was monomeric. In addition, insulin (conjugate) self-association was evaluated by circular dichroism in the near-ultraviolet wavelength range (320-250 nm). This independent method qualitatively suggests that mPEG-insulin conjugates behave similarly to Zn-free insulin in the concentration range studied and complements results from ultracentrifugation studies. The physical stability/resistance to fibrillation of mPEG-insulin conjugates in aqueous solution were assessed. The data proves that mPEG(750 and 2000)-PheB1-insulin conjugates are substantially more stable than controls but the mPEG(750 and 2000)-LysB29-insulin conjugates were only slightly more stable than commercially available preparations. Circular dichroism studies done in the far ultraviolet region confirm insulin's tertiary structure in aqueous solution is essentially conserved after mPEG conjugation. In vivo pharmacodynamic assays reveal that there is no loss in biological activity after conjugation of mPEG(750) to either position on the insulin B-chain. However, attachment of mPEG(2000) decreased the bioactivity of the conjugates to about 85% of Lilly's HumulinR formulation. The characterization presented in this paper provides strong testimony to the fact that attachment of mPEG to specific amino acid residues of insulin's B-chain improves the conjugates' physical stability without appreciable perturbations to its tertiary structure, self-association behavior, or in vivo biological activity.     

Anion binding to neutral and positively charged lipid membranes

Aqueous anion binding to bilayer membranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated by using deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. Only those anions that exhibit chaotropic properties showed significant binding to POPC membranes. A detailed investigation of thiocyanate binding to neutral POPC and to positively charged mixed POPC/dihexadecyldimethylammonium bromide (DHDMAB) (8:2 mol/mol) membranes revealed changes in the 2H NMR quadrupole splittings from POPC specifically deuteriated at either the alpha-segment or the beta-segment of the choline head group which were consistent with a progressive accumulation of excess negative charge at the membrane surface with increasing SCN- concentration. Both the 2H and 31P NMR spectra indicated the presence of fluid lipids in a bilayer configuration up to at least 1.0 M NaSCN with no indication of any phase separation of lipid domains. Calibration of the relationship between the change in the 2H NMR quadrupole splitting and the amount of SCN- binding provided thiocyanate binding isotherms. At a given SCN- concentration the positively charged membranes bound levels of SCN- 3 times that of the neutral membranes. The binding isotherms were analyzed by considering both the electrostatic and the chemical equilibrium contributions to SCN- binding. Electrostatic considerations were accounted for by using the Gouy-Chapman theory. For 100% POPC membranes as well as for mixed POPC/DHDMAB (8:2 mol/mol) membranes the thiocyanate binding up to concentrations of 100 mM was characterized by a partition equilibrium with an association constant of K approximately 1.4 +/- 0.3 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis, characterization, and in vitro activity of dendrimer-streptokinase conjugates

Dendrimer conjugation with low molecular weight drugs has been of increasing interest recently for improving pharmacokinetics, targeting drugs to specific sites, and facilitating cellular uptake. Opportunities for increasing the performance of relatively large therapeutic proteins such as streptokinase (SK) using dendrimers are being explored in this study. Using the active ester method, a series of streptokinase-poly(amido amine) (PAMAM) G3.5 conjugates were synthesized with varying amounts of dendrimer-to-protein molar ratios. Characterization of these conjugates by GPC, IEC, and native-PAGE suggested that the conjugation reaction was successful, resulting in relatively pure SK-dendrimer conjugates. The conjugate made with an equimolar ratio of dendrimer to streptokinase (1:1) exhibited the highest enzymatic activity retention ( approximately 80% retained) that has been reported so far for conjugated streptokinase with macromolecules such as PEG or dextran. SK conjugates with higher streptokinase-to-dendrimer molar ratios (1:10 and 1:20) exhibited lower initial enzymatic activities. However, these conjugates showed sustained thrombolytic activity in plasma, perhaps due to the release of SK from the conjugate. All of the SK conjugates displayed significantly improved stability in phosphate buffer solution, compared to free SK. The high coupling reaction efficiencies and the resulting high enzymatic activity retention achieved in this study could enable a desirable way for modifying many bioactive macromolecules with dendrimers.     

Synthesis and characterization of water-soluble free-base, zinc and copper porphyrin-oligonucleotide conjugates

We describe the synthesis and characterization of a series of water-soluble free-base, zinc, and copper porphyrin-oligonucleotide (ODN) conjugates. A non-charged tetraarylporphyrin was directly attached to the 5'-position of thymine via a short amide linker. Such a linker should allow for rigid connection to the adjacent nucleobases, thus increasing the sensitivity for monitoring conformational changes of the ODNs by electronic circular dichroism due to capping effects or ligand binding. Two complementary synthetic approaches have been used to incorporate porphyrin chromophores into the DNA. In the first approach a porphyrin carboxylic acid is conjugated to 5'-amino-ODN. In the second approach the phosphoramidite of the porphyrin-amido-thymidine is coupled to 5'-hydroxy-ODN using standard automated phosphoramidite chemistry. In both cases a spontaneous metallation of the free-base porphyrin in porphyrin-DNA conjugates was observed during the porphyrin-DNA conjugate cleavage from the solid support and its consequent deprotection using concentrated aqueous ammonia. Zinc and copper porphyrin-DNA conjugates were isolated by HPLC and characterized together with the anticipated free-base porphyrin-DNA conjugate. Authentic zinc and copper porphyrin-DNA conjugates were intentionally prepared from intentionally metallated porphyrins to compare their spectroscopic and HPLC characteristics with isolated metallospecies and to confirm the spontaneous metallation.     

Cell surface negativity and the binding of positively charged particles

The binding of positively charged, colloidal ferric oxide particles to the surfaces of Ehrlich ascites tumour cells, before and after incubation with neuraminidase and/or ribonuclease, has been studied by electron microscopy. An attempt has been made to correlate the amount of binding observed, with the electrophoretic mobilities of the cells under similar conditions to those under which they were exposed to the ferric oxide. Although a progressive loss of staining was observed with progressive loss of cellular net surface negativity, neither property was predictable from knowledge of the other. Some of the difficulties inherent in quantitative staining techniques of this type are discussed.     

Synthesis and characterization of hapten-protein conjugates for antibody production against small molecules

For the generation of antibodies against small hapten molecules, the hapten is cross-linked with some carrier protein to make it immunogenic. However, the formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, resulting in large variations in the generation of the desired antibodies. In the study described here the hapten (mercaptopropionic acid derivative of atrazine) was coupled to carrier protein at five different molar ratios. The hapten-protein conjugates prepared were characterized thoroughly by spectrophotometric absorption, fluorescence, matrix-assisted laser desorption ionization (MALDI), and gel electrophoresis methods, before being used for the immunization and assay purposes. Electrophoresis and fluorescence methods were very useful in detecting hapten-protein cross-linking while MALDI-MS and spectrophotometric detection provided qualitatively comparable hapten density. The production of specific antibodies was sought following the generation of appropriate hapten-protein conjugates. A high antibody titer with moderate antibody specificity was obtained with hapten density around 15 molecules per carrier protein. The study proved useful for monitoring the course of hapten-protein conjugation for the production of specific antibodies against small molecules.     

Synthesis and physical characterization of poly(ethylene glycol)-gelatin conjugates

Poly(ethylene glycol) (PEG) with the terminal group of active ester was coupled to the amino group of gelatin to prepare PEG-grafted gelatin (PEG-gelatin). The affinity chromatographic study revealed that the PEG-gelatin with high degrees of PEGylation did not adsorb onto the gelatin affinity column, in remarked contrast to gelatin alone and the PEG-gelatin with low PEGylation degrees. The former PEG-gelatin showed a critical micelle concentration while it had the apparent molecular size of about 100 nm and a surface charge of almost zero. These findings indicate that the PEG-gelatin formed a micelle structure of which the surface is covered with PEG molecules grafted. When the body distribution of 125I-labeled gelatin and PEG-gelatin after intravenous injection was evaluated, the radioactivity of micellar PEG-gelatin was retained in the blood circulation compared with that of gelatin and the PEG-gelatin of no micelle formation. At the same PEGylation degree, the blood concentration was significantly higher for the PEG-gelatin prepared from PEG with a molecular weight of 12 000 than that of molecular weights of 2000 and 5000. It is concluded that the PEG-gelatin is a drug carrier with a micelle structure which retains in the blood circulation.     

Synthesis and characterization of two (111)In-labeled DTPA-peptide conjugates.

This report describes the synthesis and characterization of two (111)In-labeled DTPA-peptide conjugates (DTPA-MA and DTPA-BA). It is surprising to find that (111)In(DTPA-MA) and (111)In(DTPA-BA) are more hydrophilic than their corresponding (90)Y analogues, suggesting a different coordination sphere in (111)In and(90)Y complexes of the same DTPA-peptide conjugate. By a reversed phase HPLC method, both (111)In(DTPA-MA) and (111)In(DTPA-BA) showed only one radiometric peak in their respective HPLC chromatogram due to a rapid interconversion of different isomers (particularly cis and trans isomers for (111)In(DTPA-MA); cis-cis, cis-trans, trans-cis, and trans-trans isomers for (111)In(DTPA-BA)). The interconversion of different isomers involves the "wagging" of the diethylenetriamine backbone, "shuffling" of the NO or NO(2) donor sets, and a rapid inversion at the terminal amine-nitrogen atoms.