Non-natural glycosphingolipids and structurally simpler analogues bind HIV-1 recombinant Gp120

Interactions of recombinant gp120 (rgp120) with non-natural glycosphingolipids (GSLs) and structurally simpler analogues have been studied using a competitive adhesion assay. Conjugates of cellobiosyl ceramide and melibiosyl ceramide were synthetically prepared as water-soluble GSL analogues. These ligands were screened against a panel of biologically relevant analogues, and the results show that their interactions with rgp120 are comparable to natural cellular receptors. Glycolipid interactions with rgp120 were probed further by the synthesis and testing of structurally simpler analogues that were obtained by reductive amination of lactose, cellobiose, and melibiose with a biotinylated amino ethylene glycol moiety. RGp120 did not recognize conjugates lacking a lipid component. However, palmitoylation of the secondary amino alditols yielded compounds with comparable rgp120 affinity to the natural cellular receptor, galactosyl ceramide (GalCer). Taken together, the SAR showed that both a hydrophobic and a hydrophilic component are required for rgp120 recognition. Moreover, structural variability in the carbohydrate headgroup did not significantly alter rgp120 recognition indicating that this interaction is not highly specific.     

Hormonotoxins. I. Strategy for synthesis of ovine luteinizing hormone--gelonin conjugate bearing the toxin in the beta-subunit

The amino groups in the beta-subunit of ovine luteinizing hormone (oLH) were modified by thiolation using N-succinimidyl-3-(2-pyridyldithio) propionate so that it may be coupled in a disulfide linkage to similarly modified ribosome inactivating protein, gelonin. The modified beta-subunit was able to hybridize with free LH alpha-subunit and the complex retained full biological activity. However, when gelonin was coupled to the beta-subunit, the resulting conformational changes masked or eliminated the sites necessary for intersubunit recognition of the free alpha-subunit. This has important implications for the design in the synthesis of gonadotropin-toxin/drug conjugates.     

Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity

Arginine-rich cell-penetrating peptides (CPPs) are promising transporters for intracellular delivery of antisense morpholino oligomers (PMO). Here, we determined the effect of L-arginine, D-arginine and non-alpha amino acids on cellular uptake, splice-correction activity, cellular toxicity and serum binding for 24 CPP-PMOs. Insertion of 6-aminohexanoic acid (X) or beta-alanine (B) residues into oligoarginine R8 decreased the cellular uptake but increased the splice-correction activity of the resulting compound, with a greater increase for the sequences containing more X residues. Cellular toxicity was not observed for any of the conjugates up to 10 microM. Up to 60 microM, only the conjugates with > or = 5 Xs exhibited time- and concentration-dependent toxicity. Substitution of L-arginine with D-arginine did not increase uptake or splice-correction activity. High concentration of serum significantly decreased the uptake and splice-correction activity of oligoarginine conjugates, but had much less effect on the conjugates containing X or B. In summary, incorporation of X/B into oligoarginine enhanced the antisense activity and serum-binding profile of CPP-PMO. Toxicity of X/B-containing conjugates was affected by the number of Xs, treatment time and concentration. More active, stable and less toxic CPPs can be designed by optimizing the position and number of R, D-R, X and B residues.     

Amino acid substitution in the lactose carrier protein with the use of amber suppressors.

Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains. The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside. Most of the amino acid substitutions in the regions of the putative loops (between transmembrane alpha helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity. In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found. The substitution of proline for Trp-33 (in the region of the first alpha helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity. Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine. It is concluded that Trp-33 may be involved directly or indirectly in sugar recognition.     

Conjugates of superoxide dismutase with the Fc fragment of immunoglobulin G.

We constructed conjugates of superoxide dismutase (SOD) and the Fc fragment of human immunoglobulin G. The lysyl residues of bovine erythrocyte Cu,Zn-SOD were covalently linked with cysteine residues of the Fc fragment using N-succinimidyl 4-(N-maleimido)-butylate as a crosslinking agent. Analysis by gel filtration and SDS-PAGE revealed that the conjugates were composed of one molecule of SOD linked with one molecule of Fc [SOD-(Fc)1] and one SOD molecule linked with several Fc molecule [SOD-(Fc)n]. The resulting SOD-Fc conjugates retained more than 90% of the enzyme activity of SOD. When those conjugates were administered intravenously to mice, the half-lives of SOD activity in the circulation were 29 and 42 h for SOD-(Fc)1 and SOD-(Fc)n, respectively, while free SOD had a half-life of 5 min. Intravenous administration of the conjugates to mice markedly repressed the increase in serum glutamic-oxaloacetic transaminase (GOT) activity induced by paraquat. These results suggest that SOD-Fc conjugates, which have long half-lives, effectively perform dismutation of superoxide radicals and may be useful for preventing tissue injury caused by hazardous oxygen metabolites.     

Facile synthesis of stable and lectin-recognizable DNA-carbohydrate conjugates via diazo coupling

Stable and lectin-recognizable DNA-carbohydrate conjugates were prepared by diazo coupling of lactose and cellobiose derivatives to fragmented salmon testes DNA. The diazo coupling is suggested to take place selectively to guanine bases since the amount of lactose moiety introduced was directly proportional to the G content of various DNAs with different G contents. According to the CD spectra, the conjugates bearing carbohydrate less than 25% content kept a typical B-type conformation similar to native DNA. The conjugates possessed higher melting temperature and stronger nuclease resistance both to exo- and endonucleases than native DNA. Gel shift assay and fluorescence binding assay showed that the DNA-lactose conjugates were specifically bound to galactose-specific lectin RCA(120) with strong binding affinity (Ka = 10(4)-10(5) M(-1)) due to glycoside cluster effect. This facile method will be a useful protocol of molecular design for cell-targeted gene therapy.     

A uniform approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A uniform approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with an aglycone moiety amino group can be coupled with PBC carboxyl derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a coupling agent. Both N- and O-glycosides differing in the conformational mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides. For example, not only oligosaccharides obtained by chemical synthesis but also reducing oligosaccharides isolated from natural sources can be transformed into N-glycosides. The approach was tested by the example of conjugation of the carboxyl derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand for lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand.

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system.     

An efficient method for conjugation of a lipopolysaccharide from Salmonella enterica sv. Minnesota with probes bearing hydrazine or amino functional groups

A conjugation method for coupling probes bearing hydrazine or primary amino groups to a lipopolysaccharide (LPS) is described. LPS is modified through the hydroxyl groups present in its O-antigen moiety by activation with cyanogen bromide in aqueous acetone using triethylamine to enhance the electrophilicity of CNBr. The method yields conjugates with good labeling ratios, preserving the endotoxic activity of the lipid A moiety, which in blood exerts pleiotropic effects on many tissues and organs, resulting in multiple-organ damage, circulatory collapse, and death. Conjugation of smooth-form LPS from Salmonella enterica sv. Minnesota to dansyl hydrazine yielded a labeling ratio of 330 nmol dansyl/mg LPS, with nearly no loss of the original endotoxic activity. In the case of horseradish peroxidase, in which a spacer was introduced, the ratio was 28 nmol HRP/mg of LPS, preserving 65% of the original endotoxic activity. This work shows that under these conditions of CNBr activation, the labeling process has practically no effect on the endotoxic behavior of LPS. The method can be used effectively for the conjugation of LPS to probes bearing primary amino, hydrazine, or hydrazide functional groups.     

Multiwalled Carbon Nanotube-Superoxide Dismutase Conjugate Towards Alleviating Induced Oxidative Stress

Superoxide dismutase (SOD), an antioxidative enzyme, was covalently immobilized onto oxidized multi-walled carbon nanotubes (MwCNTs) using diimide activated amidation reaction. MwCNT(SOD) conjugates with high aqueous dispersible nature were obtained and characterized further using field emission scanning electron microscopy and Fourier transformed infrared spectroscopy. The synthesized conjugates even after 96 h incubation time period were found to retain significant superoxide anion scavenging activity. The synthesized MwCNT(SOD) conjugate were found to no significant toxicity in limited doses. Conditions for oxidative stress in human skin HaCat cells were optimized using H₂O₂ (1 mM) as an external stress factor and subsequently anti-oxidative response of synthesized MwCNT(SOD) conjugate was analyzed. Upon incubation with oxidative stress induced HaCat cells, MwCNT(SOD) conjugates were found to alleviate the increased oxidative stress indicating its enhanced uptake and efficient antioxidative activity. Low level of reactive oxygen species and increased SOD activity in HaCat cells post treatment with MwCNT(SOD) conjugate further confirms the antioxidative activity of SOD enzyme in conjugation with MwCNTs which acts as an efficient intracellular drug delivery vehicles.     

Isolation and characterization of thiodigalactoside-resistant mutants of the lactose permease which possess an enhanced recognition for maltose

In the current study, lactose permease mutants were isolated which exhibited an enhanced recognition for maltose (an alpha-glucoside) but a diminished recognition for thiodigalactoside, TDG (a beta-galactoside). Maltose/TDGR mutants were obtained from four different parental strains encoding either a wild-type permease (pTE18), a mutant lactose permease which recognizes maltose (pB15) or mutant lactose permeases which recognize maltose but are resistant to inhibition by cellobiose (pTG and pBA). A total of 27 independent mutants were isolated: 12 from pTE18, 10 from pB15, 3 from pTG, and 2 from pBA. DNA sequencing of the 27 mutants revealed that the mutants contain single base pair substitutions within the lac Y gene which result in single amino acid substitutions within the lactose permease. All of the mutants obtained from pTE18, pTG, and pBA involved a change of Tyr-236 to histidine, phenylalanine, or asparagine. From pB15, three different types of mutants were obtained: Tyr-236 to histidine, Ile-303 to phenylalanine, or His-322 to asparagine. When assayed for [14C]maltose transport, the maltose/TDGR mutants were seen to transport maltose significantly faster than the wild type. Furthermore, although TDG was shown to inhibit the uptake of maltose in the four parental strains, all of the mutant strains exhibited a dramatic resistance to TDG inhibition. Most of the maltose/TDGR mutants were also shown to be very defective in the transport of lactose. However, certain mutants (i.e., Asn-322) exhibited moderate lactose transport activity. Finally, it was observed that all of the mutant strains were unable to facilitate the uphill accumulation of beta-methylthiogalactopyranoside. The locations of the amino acid substitutions are discussed with regard to their possible role in sugar recognition.     

Dendrimers as carrier protein mimetics for IgE antibody recognition. Synthesis and characterization of densely penicilloylated dendrimers

The synthesis of benzylpenicilloyl-containing dendrimers has been achieved by a convenient procedure involving quantitative functionalization of the terminal amino groups of the three Starbust PAMAM generations used (G(n); n = 0, 1, 2). All these densely penicilloylated dendrimers (G(n)P) exhibit similar, simple NMR spectroscopic data suggesting highly symmetric structures and a monodisperse nature, and the results obtained from MALDI-TOF-MS demonstrate their exact chemical composition. The use of PAMAM dendrimers has allowed us to synthesize, for the first time, carrier benzylpenicilloyl conjugates (G(n)P) of precisely defined chemical structure. The attempts to synthesize G(2)P show that forced experimental conditions are not always useful for the functionalization of the dendrimer, especially in introducing bulky groups. The initial results with sera from patients with different RAST levels were positive and thus suggestive that inhibition occurs, so recognition exists; we can therefore conclude that the hapten-carrier (dendrimer) conjugates studied mimic recognition with natural hapten-carrier (protein) conjugates.     

ATP-insulin conjugates and their use for immunofactor analysis

A method resulting in ATP-insulin conjugates by covalent binding of ATP modified at C(6) amino group of the adenine residue with insulin was developed. The modified ATP was bound to insulin by means of metha-p-toluene sulfonate-N-cyclohezyl Nf [2-morpholinyl(4)ethyl]-carbodiimide. The ATP analogs and ATP-insulin conjugates possess the coenzyme activity in a reaction of luciferin oxidation by luciferase from the fireflies Luciola mingrelica. the catalytic properties of soluble and immobilize on CNBR-activated. Sepharose enzymes in reactions with native ATR, its modified derivatives and ATP--insulin conjugates were compared. The bioluminescence reaction involving ATP--insulin conjugate is inhibited by antibodies against insulin. This effect can form a basis for insulin detection in solution, which is based on competitive binding of free and antibody-labelled ATP--insulin conjugates.     

Humoral and cellular responses to native antigen following oral and parenteral immunization with lipid-conjugated bovine serum albumin

Humoral and cellular immune responses of rabbits to bovine serum albumin (BSA) were measured following oral and parenteral immunization with either BSA or one of two dodecanoic acid conjugates of BSA. The first consisted of a mixture of lightly and heavily conjugated BSA-molecules (L-BSA-mix), while the second (L-BSA) was a homogeneous preparation of heavily conjugated BSA with more than 95% of the 60 available amino groups covalently bound to dodecanoic acid. Animals ingesting L-BSA-mix had a similar humoral immune response but enhanced cellular reactivity to BSA in comparison to animals ingesting the native antigen. No systemic immunologic responses to BSA were detected following ingestion of L-BSA in spite of the demonstration of circulating BSA antigenic groups. This lack of a detectable immune response after oral administration was not due to masking of antigenic sites by the lipid residues since both humoral and cellular immune responses to BSA were obtained in animals injected with L-BSA. Ingestion of L-BSA did not induce tolerance since a subsequent injection of BSA elicited a normal primary immune response. The differences in immunogenicity between BSA, L-BSA and L-BSA-mix following oral administration may be related to different modes of antigen recognition by the gut-associated lymphoid tissues.     

Vinyl sulfone bifunctional derivatives of DOTA allow sulfhydryl- or amino-directed coupling to antibodies. Conjugates retain immunoreactivity and have similar biodistributions.

We have synthesized a bifunctional vinyl sulfone-cysteineamido derivative of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) that can be conjugated to the sulfhydryls of mildly reduced recombinant antibody (chimeric anti-CEA antibody cT84.66) at pH 7 or to the amino groups of lysine residues at pH 9. The conjugation is sulfhydryl specific at pH 7 (case 1), and amino specific at pH 9 (case 2) as long as the antibody has no free sulhydryl groups. At a molar ratio of 50 BCA (bifunctional chelating agent) to mAb, the number of chelates conjugated is 0.8 for case 1, and 4.6 for case 2. The resulting conjugates can be radiolabeled with (111)In to high specific activity (5 mCi/mg) with high efficiency (>95%) at 43 degrees C in 60 min. The radiolabeled conjugates retained >95% immunoreactivity and are stable in serum containing 1mM DTPA over 3 d. When the radiolabeled conjugates were injected into nude mice bearing LS174T human colon tumor xenografts, over 40% ID/g accumulated in tumors during the period 24-72h. Tumor-to-blood ratios were 4.5, 3.5, and 2.5 for the sulfhydryl coupled conjugate at 24, 48, and 72 h, respectively, and 2.7, 2.5, and 2.3 for the amino-coupled conjugate at the same time points. For other organs the biodistributions were nearly identical whether the conjugates were attached via sulfhydryl or amino groups. These novel BCAs are easy to synthesize, offer versatile conjugation options, and give equivalent biodistributions that result in high tumor uptake and good tumor-to-blood ratios.     

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.     

Mutants of the Lactose Carrier of Escherichia coli Which Show Altered Sugar Recognition Plus a Severe Defect in Sugar Accumulation

Lactose and melibiose are actively accumulated by the wild-type Escherichia coli lactose carrier, which is an integral membrane protein energized by the proton motive force. Mutants of the E. coli lactose carrier were isolated by their ability to grow on minimal plates with succinate plus IPTG in the presence of the toxic lactose analog β-thio-o-nitrophenylgalactoside (TONPG). TONPG-resistant mutants were streaked on melibiose MacConkey indicator plates, and red clones were picked. These melibiose positive mutants were then streaked on lactose MacConkey plates, and white clones were picked. Transport assays indicated that the mutants had altered sugar recognition and a defect in sugar accumulation. The mutants had a poor apparent K _m for both lactose and melibiose in transport. One mutant had almost no ability to take up lactose, but melibiose downhill transport was 58% (V _max ) of normal. All of the mutants accumulated methyl-α-d-galactopyranoside (TMG) to only 8% or less of normal, and two failed to accumulate. Immunoblot analysis of the mutant lactose carrier proteins indicated that loss of sugar transport activity was not due to loss of expression in the membrane. Nucleotide sequencing of the lacY gene from the mutants revealed changes in the following amino acids of the lactose carrier: M23I, W151L, G257D, A295D and G377V. Two of the mutants (G257D and G377V) are novel in that they represent the first amino acids in periplasmic loops to be implicated with changes in sugar recognition. We conclude that the amino acids M23, W151, G257, A295 and G377 of the E. coli lactose carrier play either a direct or an indirect role in sugar recognition and accumulation. Received: 12 October 1999/Revised: 21 December 1999     

A universal approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A universal approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with amino group in aglycone moiety can be conjugated with PBC carboxy derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a condensing agent. Both N-and O-glycosides differing in the conformation mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides isolated from natural sources can be transformed into N-glycosides with a functional group in aglycone. The approach was tested by conjugation of the carboxy derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand of lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

The phospholipid requirement for activity of the lactose carrier of Escherichia coli

The transport activity of the lactose carrier of Escherichia coli has been reconstituted in proteoliposomes composed of different phospholipids. The maximal activity was observed with the natural E. coli lipid as well as mixtures containing phosphatidylethanolamine or phosphatidylserine. Phosphatidylcholine or mixtures of phosphatidylcholine with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activity. The lactose carrier reconstituted with amino phospholipids of increasing degrees of methylation (dioleoylphosphatidylethanolamine, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine, and dioleoylphosphatidylcholine) revealed a progressive decrease in both counterflow and proton motive force-driven lactose uptake activities. Trinitrophenylation of phosphatidylethanolamine in the E. coli proteoliposomes resulted in a marked reduction in lactose carrier activity. Partial restitution of transport activity was obtained by detergent extraction of the carrier from these inactive proteoliposomes and reconstitution of the carrier into proteoliposomes containing normal E. coli lipid. These results suggest that the amino group of the amino phospholipids (e.g. phosphatidylethanolamine and phosphatidylserine) is required for the full function of the lactose carrier from E. coli.