Total Stepwise Solid-Phase Peptide-Oligonucleotide Conjugate Synthesis on Macroporous Polystyrene

Abstract

An efficient total stepwise solid-phase synthesis of oligonucleotide-peptide conjugates on a macroporous polystyrene is described. Extending our homoserine linker approach, we prepared a range of fluorescein-labelled conjugates containing one of two different peptides together with oligonucleotides containing 2′-deoxynucleoside or 2′-O-methylribonucleoside phosphodiesters, or gapmers containing 2′-deoxyphosphorothioate sequences flanked by 2′-O-methyl wings.     

Targeting the DNA cleavage activity of copper phenanthroline and clip-phen to A.T tracts via linkage to a poly-N-methylpyrrole

To target DNA A.T tracts, a three-ring polyamide containing an N-methylpyrrole amino acid has been linked, on solid support, to carboxylic derivatives of phenanthroline and dimers of phenanthroline: 2-Clip-Phen, 3-Clip-Phen, or 2-Clip-Phen containing a long tether. After metalation by CuCl(2), the DNA cleavage activities of the different conjugates were compared on a restriction fragment. Cleavage patterns showed that the polyamide moiety of conjugates directs the cleavage activity in the vicinity of A.T tracts but the precise cleavage selectivity of these conjugates was dependent on the type of phenanthroline residue linked to the poly-N-methylpyrrole entity.     

Enhanced cellular uptake with a cobaltacarborane-porphyrin-HIV-1 Tat 48-60 conjugate

A series of four porphyrin-cobaltacarborane conjugates have been synthesized, containing three or four cobaltabisdicarbollide anions linked by O(CH(2)CH(2)O)(2) groups to the porphyrin macrocycle and one of them containing a HIV-1 Tat 48-60 peptide sequence linked via a low molecular weight poly(ethylene glycol) (PEG) spacer. The cellular uptake, cytotoxicity, and preferential sites of intracellular localization of the conjugates were evaluated in human HEp2 cells. All conjugates are nontoxic in the dark at the concentrations studied. Upon exposure to low light dose (1 J cm(-)(2)) only the porphyrin-cobaltacarborane-HIV-1 Tat 48-60 conjugate showed 30% inhibition of cell proliferation at a concentration of 10 microM. The cellular uptake was dependent on the number of carborane cages and was significantly enhanced by the presence of the cell penetrating peptide sequence HIV-1 Tat 48-60. All conjugates preferentially localized in the cell lysosomes.     

Synthesis of metallointercalator-DNA conjugates on a solid support

Metallointercalator-DNA conjugates were prepared by amide bond formation between active esters on the nonintercalating ligands of transition metal complexes and primary amines presented at the 5' or the 3' termini of oligonucleotides attached to solid supports. The conjugates were liberated from the support by aminolysis and purified by HPLC on C18 or C4 stationary phases, which separates the two diastereomeric forms of the conjugates containing either the Lambda or the Delta enantiomer of the octahedral metal complex. The coupling reaction proceeds with approximately 75% conversion of the amino-terminated oligonucleotide into the conjugate; the isolated yield is approximately 200 nmol for syntheses initiated on DNA-synthesis columns with a loading of 2 micromol. The conjugates were characterized by ultraviolet-visible and circular dichorism absorption spectroscopy, electrospray ionization mass spectrometry, enzymatic digestion, and polyacrylamide gel electrophoresis (PAGE). Oligonucleotides bearing [Rh(phi)(2)(bpy')](3+) (phi = 9, 10-phenanthrene quinone diimine; bpy' = 4-butyric acid-4'-methyl bipyridyl) form 1:1 duplexes with the complementary strand, and the electrophoretic mobility under nondenaturating PAGE of duplexes containing Delta-Rh is notably different from duplexes containing Lambda-Rh. High-resolution PAGE of DNA photocleavage reactions initiated by irradiation of the tethered Rh complexes reveal intercalation of the complex only near the tethered end of the duplex. Analogous DNA-binding properties were observed with [Rh(phi)(2)(bpy')](3+) tethered to the 3' terminus. By combining the 3' and 5' modification strategies, a mixed-metal DNA conjugate containing both [Os(phen)(bpy')(Me(2)-dppz)](2+) (Me(2)-dppz = 7, 8-dimethyldipyridophenazine) on the 3' terminus and [Rh(phi)(2)(bpy')](3+) on the 5' terminus was prepared and isolated. Taken together, these strategies for preparing metallointercalator-DNA conjugates offer a useful approach to generate chemical assemblies to probe long-range DNA-mediated charge transfer where the redox initiator is confined to and intercalated in a well-defined binding site.     

Synthesis of glycocluster-tumor antigenic peptide conjugates for dendritic cell targeting

The use of dendritic cells (DC) for the development of therapeutic cancer vaccines is attractive because of their unique ability to present tumor epitopes via the MHC class I pathway to induce cytotoxic CD8+ T lymphocyte responses. C-Type membrane lectins, DC-SIGN and the mannose receptor (MR), present on the DC surface, recognize oligosaccharides containing mannose and/or fucose and mediate sugar-specific endocytosis of synthetic oligolysine-based glycoclusters. We therefore asked whether a glycotargeting approach could be used to induce uptake and presentation of tumor antigens by DC. To this end, we designed and synthesized glycocluster conjugates containing a CD8+ epitope of the Melan-A/Mart-1 melanoma antigen. These glycocluster-Melan-A conjugates were obtained by coupling glycosynthons: oligosaccharyl-pyroglutamyl-beta-alanine derivatives containing either disaccharides, a dimannoside (Manalpha-6Man) or lactoside, or a Lewis oligosaccharide, to Melan-A 16-40 peptide comprising the 26-35 HLA-A2 restricted T cell epitope, extended with an oligolysine stretch at the C-terminal end. We showed by confocal microscopy and flow cytometry that fluorescent-labeled Melan-A glycoclusters containing either dimannoside or Lewis oligosaccharide were taken up by DC and concentrated in acidic vesicles; conversely lactoside glycopeptides were not at all taken up. Furthermore, using surface plasmon resonance, we showed that dimannoside and Lewis-Melan-A conjugates bind MR and DC-SIGN with high affinity. DC loaded with these conjugates, but not with the lactose-Melan-A conjugate, led to an efficient presentation of the Melan-A epitope eliciting a CD8+ T-lymphocyte response. These data suggest that synthetically designed glycocluster-tumor antigen conjugates may induce antigen cross-presentation by DC and represent a promising tool for the development of tumor vaccines.     

Synthesis and splice-redirecting activity of branched, arginine-rich peptide dendrimer conjugates of peptide nucleic acid oligonucleotides

Arginine-rich cell-penetrating peptides have found excellent utility in cell and in vivo models for enhancement of delivery of attached charge-neutral PNA or PMO oligonucleotides. We report the synthesis of dendrimeric peptides containing 2- or 4-branched arms each having one or more R-Ahx-R motifs and their disulfide conjugation to a PNA705 splice-redirecting oligonucleotide. Conjugates were assayed in a HeLa pLuc705 cell assay for luciferase up-regulation and splicing redirection. Whereas 8-Arg branched peptide-PNA conjugates showed poor activity compared to a linear (R-Ahx-R)(4)-PNA conjugate, 2-branched and some 4-branched 12 and 16 Arg peptide-PNA conjugates showed activity similar to that of the corresponding linear peptide-PNA conjugates. Many of the 12- and 16-Arg conjugates retained significant activity in the presence of serum. Evidence showed that biological activity in HeLa pLuc705 cells of the PNA conjugates of branched and linear (R-Ahx-R) peptides is associated with an energy-dependent uptake pathway, predominantly clathrin-dependent, but also with some caveolae dependence.     

Ribonuclease activity of the peptides with alternating arginine and leucine residues conjugated to tetrathymidilate

RNA cleaving conjugates have been prepared by attachment of oligodeoxyribonucleotide TTTT to peptides containing arginine, leucine, proline and serine residues. The highest activity was displayed by the conjugates containing peptides with alternating arginine and leucine residues (LR)4G-amide. Ribonuclease activity of the conjugates pep-T4 decreases in the order T4-(LR)4G > T4-(LR)2G > T4-(LLRR)2G > T4-(LR)2PRLRG > S2R3-Hmda-T4 > or = R5 double dagger (LR)3. According to CD spectra, the free peptide (LR)4G-amide in water solution at neutral pH and physiological ionic strength has no pronounced secondary structure whereas conjugated to oligonucleotide it acquires a folding similar to alpha-helix.     

Pentafluorophenyl ester-functionalized phosphorylcholine polymers: preparation of linear, two-arm, and grafted polymer-protein conjugates

Novel pentafluorophenyl (PFP)-ester-functionalized phosphorylcholine (PC) polymers of different architectures were prepared and conjugated to lysozyme as a model protein. Linear and two-arm poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC) structures containing PFP functionality at the chain-end were prepared by atom transfer radical polymerization (ATRP) from novel initiators. Additional conjugates were prepared from phosphorylcholine-substituted cyclooctene (PC-COE) polymers containing PFP-ester bearing comonomers. The polymer-protein conjugates were characterized by HPLC, FPLC, and DLS and were seen to retain most (~80% or greater) of their native enzymatic activity. Pharmacokinetic profiles of the polymer-protein conjugates were studied in mice and found to increase the circulation half-life compared with lysozyme alone.     

Porphyrin-retinamides: synthesis and cellular studies

A series of four porphyrin-retinamides containing either all-trans- or 13-cis-retinoid acid residues, directly linked to the para-phenyl position of meso-tetraphenylporphyrin or via a low-molecular-weight PEG spacer, have been synthesized. The biological properties of these conjugates were evaluated in a model cell line, human HEp2, and in neuroblastoma SK-N-DZ cells, which exhibit moderate expression of retinoic acid receptors and retinoic acid-induced differentiation. The directly linked porphyrin-retinamides were taken up by a greater extent (20-50% more) in SK-N-DZ than in HEp2 cells. However, the PEG-containing conjugates accumulated maximally within both cell lines and approximately by the same amount, probably due to their increased amphiphilicity. Among all conjugates, the porphyrin-PEG-13-cis-retinamide accumulated the most in both cell lines (about 5 times more than the non-pegylated conjugates). None of the porphyrin-retinamide conjugates were toxic toward HEp2 cells at concentrations up to 100 microM, and only the hydrophobic non-pegylated conjugates were moderately toxic to SK-N-DZ cells [IC50 (dark) = 56-92 microM, and IC50 (at 1 J/cm2) = 6-8 microM]. All conjugates preferentially localized within cellular vesicles that correlated well to the lysosomes and, in addition, the PEG-containing porphyrin-retinamides were also found in the ER.     

Oligonucleotide conjugates of Eu(III) tetraazamacrocycles with pendent alcohol and amide groups promote sequence-specific RNA cleavage

Eu(III) complexes of two neutral bifunctional tetraaaza macrocyclic ligands {1-[1-carboxamido-3-(4-nitrophenyl)propyl]-4,7,10-tris(2-hydroxyethyl)-1,4,7,10-tetraazacyclododecane and 2-(4-nitrobenzyl)-1,4,7,10-tetrakis(2-hydroxyethyl)-1,4,7,10-tetraazacyclododecane} are prepared. Eu(III) complexes of the isothiocyanate derivatives of these macrocycles are treated with oligonucleotides containing 2′-O-propylamine linkers to form conjugates. Hydrolytic cleavage of an oligoribonucleotide is promoted by Eu(III) macrocyclic oligonucleotide conjugates containing complementary (antisense) sequences. Cleavage is not observed in the presence of Eu(III) conjugates containing scrambled sequences nor by free complex. Despite the fact that one of the free macrocyclic complexes is more reactive than the other, the extent of cleavage observed is similar for conjugates containing either Eu(III) macrocyclic complex.     

Poly(ethylene glycol) cross-linked hemoglobin with antioxidant enzymes protects pancreatic islets from hypoxic and free radical stress and extends islet functionality

The objective of this study was to investigate the efficiency of multifunctional poly(ethylene glycol)-based hemoglobin conjugates crosslinked with antioxidant enzymes for their ability to protect an oxygen carrier (hemoglobin) and insulin secreting islets from the combination of hypoxic and free radical stress under simulated transplantation conditions. In this study, RINm5F cells and isolated pancreatic islets were challenged with oxidants (H(2)O(2) or xanthine and xanthine oxidase) and incubated with conjugates (hemoglobin-hemoglobin or superoxide dismutase-catalase-hemoglobin) in normoxia (21% oxygen) or hypoxia (6% or 1% oxygen). Hemoglobin protection, intracellular free radical activity and cell viability in RINm5F cells measured by methemoglobin, dichlorofluorescein-diacetate, and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, respectively, showed that cells were better protected by conjugates containing antioxidant enzymes. Insulin secretion from islets and qualitative confocal evaluation of viability showed beta cells were protected by conjugates containing antioxidant enzymes when exposed to induced stress. Our study suggested that antioxidant enzymes play a significant role in hemoglobin protection and thus extended cell protection.     

Separation of Albumin–Ribonuclease Conjugates

A method for separation of albumin–ribonuclease (RNase) conjugates has been proposed based on the use of macroporous silicates. It was established that about 76% of ligand-free human serum albumin (LFHSA) formed complexes with enzymes. It was shown that most of the conjugates of albumin and pancreatic RNase contained up to 2 mol enzyme per 1 mol LFHSA. The conjugates of albumin and bacterial RNase isolated from cells of the strain Bacillus intermedius 7P displayed higher specific activities, containing, on average, 2.3 mol RNase per 1 mol LFHSA (for the conjugates with molecular weights below 92 kDa) or 3.3 mol RNase per 1 mol protein carrier (for the conjugates with higher molecular weight).     

Using small-angle neutron scattering to study the solution conformation of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates

Our past research developed two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates that became the first synthetic polymer-anticancer conjugates to be evaluated clinically. The first, FCE28068, contained Dox bound to the polymeric carrier via a tetrapeptidic linker (glycine-phenylalanine-leucine-glycine (GFLG)) (Mw approximately 30,000 g/mol; approximately 8 wt % drug), and the second, FCE28069, contained additionally galactosamine (Gal) (Mw approximately 30,000 g/mol; approximately 7.5 wt % Dox) again bound by a GFLG linker. Galactosamine was included to promote hepatocyte/hepatoma targeting via the asialoglycoprotein receptor. Both conjugates showed antitumor activity and were clinically less toxic than free Dox (2-5 fold). However, despite their similar chemical characteristics, the conjugates displayed a significantly different maximum-tolerated dose (MTD) in patients. The aim of this study, therefore, was to use small-angle neutron scattering (SANS) to explore the solution behavior of a small library of HPMA polymer conjugates including FCE28068, FCE28069, and their pharmaceutical formulations, plus as reference compounds HPMA copolymer-GFLG conjugates containing aminopropanol (Ap) or galactosamine (Gal) alone (i.e., without Dox). The SANS data obtained showed that HPMA copolymer-GFLG-Ap conjugates (containing 5 and 10 mol % side chains) showed evidence of polymer aggregation, however, no indication of aggregation was observed for FCE28068 and FCE28069 over the concentration range studied (2.5-50 mg/mL). Clear differences in the scattering behavior for the two conjugates were observed at equivalent concentration. Data were best fitted by a model for polydisperse Gaussian coils, and the HPMA copolymer-Dox conjugate with Gal (FCE28069) exhibited a larger radius of gyration (Rg) (by approximately 2.5 nm) compared to FCE28068. In conclusion, we have shown that SANS will be a valuable tool to elucidate conformation-performance relationships for polymer-drug conjugates.     

Preparation, properties and antileukemic activity of arabinosylcytosine polysaccharide conjugates

1. Conjugates of 1-beta-D-arabinofuranosylcytosine (araC) with polysaccharides containing carboxyl groups, such as polygalacturonic acid (PGA) and carboxymethylated yeast beta-D-glucan (CMG) were prepared. 2. Activation of the polysaccharidic carboxyl group by isobutylchloroformiate and formation of a peptide bond via 4-NH2 group of araC was used for a coupling reaction. 3. Elementary analysis, u.v. and i.r. spectra confirmed the structures of the conjugates. 4. The conjugates were most stable against the hydrolysis under the mild acid conditions. 5. It was also shown that under the physiological conditions trypsin catalyze the conjugate hydrolysis and the catalytic effect is higher than that of chymotrypsine. 6. It is suggested that trypsin or trypsin-like proteases could participate in the hydrolysis of the conjugates in vivo. PGA-araC and CMG-araC showed 1.5- or 2.5-times higher antileukemic activity than both free araC or polysaccharides.     

Synthesis and spectroelectrochemistry of N-cobaltacarborane porphyrin conjugates

N-Substituted porphyrins are well-known for the distortion they exhibit of the porphyrin plane through the sp(3) hybridization of one of the pyrrolenic units. They have served as model compounds in investigations of many biochemical processes. In this paper, we developed an efficient route to N-substituted porphyrins, and report the synthesis of a series of new N-substituted cobaltacarborane-porphyrins containing one or two cobaltabisdicarbollide anions linked by (CH(2)CH(2)O)(2) chains to either the core porphyrin nitrogens or to a meso-aminophenyl group. These conjugates show different degrees of distortion of the porphyrin macrocycle, which affect their spectroscopic and electrochemical properties. In particular, the core N-substituted conjugates show significant fluorescence quenching in comparison with the noncore substituted macrocycles. The X-ray structures of two targeted core N-cobaltacarborane porphyrin conjugates are presented. The electrochemical and spectroelectrochemical properties of these porphyrin conjugates were investigated; while the peripheral N-substituted cobaltacarboranylporphyrins undergo three reversible reductions and three reversible oxidations (two attributed to the porphyrin and one to the Co(III) cluster), the core N-substituted porphyrins exhibit complicated electrochemical behavior with coupled chemical reactions.     

Synthesis and properties of cell-targeted Zn(II)-phthalocyanine-peptide conjugates

Two Zn-Pc-peptide conjugates bearing either a short linker or a long PEG-linker between the macrocycle and a bifunctional peptide containing the nucleoplasmin and HIV-1 Tat 48-60 sequences have been synthesized in order to increase the Pc cell-targeting ability and to evaluate the effect of the linker. The presence of the peptide chain increased the water solubility of the Pc macrocycle and, consequently, its fluorescence in aqueous solutions. The highest fluorescence quantum yields were observed at low pH (5.0) for both conjugates and were always higher for the conjugate bearing the short linker. Both conjugates were found to have low dark cytotoxicity toward human HEp2 cells (IC50 > 77 microM) but were highly phototoxic (IC50 < 2 microM at 1 J cm-2). The conjugate bearing the long PEG-linker accumulated the most within cells (26 times more than the unconjugated Zn-Pc), followed by the short linker conjugate (17 times more than the unconjugated Zn-Pc). Both conjugates were found to localized preferentially within the cell lysosomes.     

Inhibition of plasminogen activator inhibitor release in endothelial cell cultures by antisense oligodeoxyribonucleotides with a 5'-end lipophilic modification

A series of conjugates containing residues of lipophilic alcohols covalently bound to 5' end of oligodeoxyribonucleotides targeted against human plasminogen activator inhibitor (PAI-1) mRNA was synthesized via the oxathiaphospholane approach. The highest anti-PAI-1 activity in EA.hy 926 endothelial cell cultures was found for conjugates containing menthyl or heptadecanyl groups linked with an oligonucleotide complementary to a segment of human PAI-1 mRNA. The phosphodiester antisense oligonucleotides, which otherwise exhibit only limited anti-PAI-1 activity, were found to be more active than phosphorothioate oligonucleotides when conjugated to lipophilic alcohol residues. For menthyl conjugates an evidence of antisense mechanism of inhibition was found.     

Targeting DNA mismatches with rhodium intercalators functionalized with a cell-penetrating peptide

Cell-penetrating peptides are widely used to deliver cargo molecules into cells. Here we describe the synthesis, characterization, DNA binding, and cellular uptake studies of a series of metal-peptide conjugates containing oligoarginine as a cell-penetrating peptide. d-Octaarginine units are appended onto a rhodium intercalator containing the sterically expansive chrysenequinone diimine (chrysi) ligand to form Rh(chrysi)(phen)(bpy)(3+)-tethered oligoarginine conjugates, where the peptide is attached to the ancillary bpy ligand; some conjugates also include a fluorescein or thiazole orange tag. These complexes bind and with photoactivation selectively cleave DNA neighboring single-base mismatches. The presence of the oligoarginines is found to increase the nonspecific binding affinity of the complexes for both matched and mismatched DNA, but for these conjugates, photocleavage remains selective for the mismatched site, as assayed using both gel electrophoresis and mass spectrometry experiments. Significantly, the rhodium complex does not interfere with the delivery properties of the cell-penetrating peptide. Confocal microscopy experiments show rapid nuclear localization of the metal-peptide conjugates containing the tethered fluorescein. Mass spectrometry experiments confirm the association of the rhodium with the HeLa cells. These results provide a strategy for targeting mismatch-selective metal complexes inside cell nuclei.     

Quantitative measurements of the specificity and kinetics of conjugate formation between cloned cytotoxic T lymphocytes and splenic target cells by dual parameter flow cytometry

The formation of conjugates between cloned anti-H-2Kb and Dd cytotoxic T lymphocytes (CTL) and splenic target cells has been studied by dual parameter flow cytometry. By varying effector-target combinations and by blocking with anti-MHC class I monoclonal antibodies, we found that the specificity of conjugate formation, in general, paralleled that expected from cytotoxicity studies; however, a significant number of "nonspecific" conjugates was always observed. As expected from previous studies, conjugate formation did not occur below 10 degrees C and was inhibited by cytochalasin B, EDTA, and anti-Lyt-2 antibodies. Conjugate formation followed first-order kinetics. The rate of formation of conjugates increased with temperature from 24 degrees to 37 degrees C; at 37 degrees C, the half-time was 1.4 min. After a 6-min lag period, lysis of target cells could be detected at 37 degrees C but not at 30 degrees C or below. Because target cell lysis proceeded during a period of time when the number of conjugates remained constant, considerable effector cell recycling must have occurred. Comparisons of the fluorescence emissions from conjugated effector or target cells with those from unconjugated cells demonstrated that nearly all conjugates contained one effector cell and one target cell, independent of the ratio of the two cell types in the original mix. Once formed, anti-H-2Kb conjugates were stable when diluted into medium alone, but rapidly disaggregated in medium containing either anti-Lyt-2 or anti-Kb monoclonal antibodies, both of which blocked conjugate formation. This finding suggests that conjugates are normally stabilized by intercellular bonds that are constantly breaking and reforming at the cell:cell interface, and that the antibodies disrupt the conjugates by preventing the reformation of broken bonds.     

Antioxidant properties of conjugates of polyethylene glycols containing terpenophenolic fragments

A series of water-soluble conjugates has been synthesized from polyethylene glycols of various lengths and 4-bromomethyl-2,6-diisobornylphenol. The membrane protective and antioxidant activities of the synthesized products were evaluated on the model of the H₂O₂-induced hemolysis of erythrocytes. It was shown that the studied conjugates have a significant antioxidant activity, and a significant membrane protective effect was shown for the conjugates containing 0.2% and 0.8% of the 2,6-diisobornyl-4-methylenephenolic fragments.