SYNTHESIS OF PEPTIDE-OLIGONUCLEOTIDE PHOSPHOROTHIOATE CONJUGATES BY CONVERGENT OR STEPWISE SOLID-PHASE STRATEGIES

A convergent strategy was employed to link eight 10–27-mer peptides to oligonucleotide phosphorothioates, resulting in twenty-six various conjugates. A stepwise synthesis strategy for the preparation of peptide-oligonucleotide phosphorothioate conjugates, employing Fmoc peptide chemistry, was developed and applied to the synthesis of four conjugates. Three of these conjugates contained either a 10 or 16-mer peptide, incorporating either 2 or 3 arginine residues, respectively.     

Synthesis of peptide-oligonucleotide phosphorothioate conjugates by convergent or stepwise solid-phase strategies

A convergent strategy was employed to link eight 10-27-mer peptides to oligonucleotide phosphorothioates, resulting in twenty-six various conjugates. A stepwise synthesis strategy for the preparation of peptide-oligonucleotide phosphorothioate conjugates, employing Fmoc peptide chemistry, was developed and applied to the synthesis of four conjugates. Three of these conjugates contained either a 10 or 16-mer peptide, incorporating either 2 or 3 arginine residues, respectively.     

Syntheses and DNA binding of new cationic porphyrin-tetrapeptide conjugates

Recently cationic porphyrin-peptide conjugates were synthesized to enhance the cellular uptake of porphyrins or deliver the peptide moiety to the close vicinity of nucleic acids. DNA binding of such compounds was not systematically studied yet.We synthesized two new porphyrin-tetrapeptide conjugates which can be considered as a typical monomer unit corresponding to the branches of porphyrin-polymeric branched chain polypeptide conjugates. Tetra-peptides were linked to the tri-cationic meso-tri(4-N-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin and bi-cationic meso-5,10-bis(4-N-methylpyridyl)-15,20-di-(4-carboxyphenyl)porphyrin. DNA binding of porphyrin derivatives, and their peptide conjugates was investigated with comprehensive spectroscopic methods. Titration of porphyrin conjugates with DNA showed changes in Soret bands with bathocromic shifts and hypochromicities. Decomposition of absorption spectra suggested the formation of two populations of bound porphyrins.Evidence provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and induced CD signals reveals that peptide conjugates of di- and tricationic porphyrins bind to DNA by two distinct binding modes which can be identified as intercalation and external binding. Tri-cationic structure and elimination of negative charges in the peptide conjugates are preferable for the binding. Our findings provide essential information for the design of DNA-targeted porphyrin-peptide conjugates.     

How changes in the sequence of the peptide CLPFFD-NH2 can modify the conjugation and stability of gold nanoparticles and their affinity for beta-amyloid fibrils

In a previous work, we studied the interaction of beta-amyloid fibrils (Abeta) with gold nanoparticles (AuNP) conjugated with the peptide CLPFFD-NH2. Here, we studied the effect of changing the residue sequence of the peptide CLPFFD-NH2 on the efficiency of conjugation to AuNP, the stability of the conjugates, and the affinity of the conjugates to the Abeta fibrils. We conjugated the AuNP with CLPFFD-NH 2 isomeric peptides (CDLPFF-NH2 and CLPDFF-NH2) and characterized the resulting conjugates with different techniques including UV-Vis, TEM, EELS, XPS, analysis of amino acids, agarose gel electrophoresis, and CD. In addition, we determined the proportion of AuNP bonded to the Abeta fibrils by ICP-MS. AuNP-CLPFFD-NH2 was the most stable of the conjugates and presented more affinity for Abeta fibrils with respect to the other conjugates and bare AuNP. These findings help to better understand the way peptide sequences affect conjugation and stability of AuNP and their interaction with Abeta fibrils. The peptide sequence, the steric effects, and the charge and disposition of hydrophilic and hydrophobic residues are crucial parameters when considering the design of AuNP peptide conjugates for biomedical applications.     

Conjugate formation in urea: coupling of insoluble peptides to alkaline phosphatase for ELISA and in situ detection of antibody-forming cells

We report here a new method to produce synthetic peptide/alkaline phosphatase (AP) conjugates in the presence of urea. The method allows the use of peptides that are not soluble to a sufficient degree in aqueous buffers. The presence of 8 M urea during the construction of the synthetic peptide/AP conjugates does not influence enzyme activity nor the affinity of the anti-peptide antibodies for the conjugated peptide. We demonstrate that these synthetic peptide/AP conjugates can be used for detection of specific antipeptide antibody-forming cells (AFC) in vivo. This method for constructing enzyme conjugates with insoluble proteins or peptides suggest not only new possibilities for detection of specific AFC in vivo but also for applications in receptor-ligand studies, ELISA (enzyme-linked immunosorbent assay), and spot ELISA for detection of antibody-secreting cells in vitro.     

G-specific RNA-cleaving conjugates of short peptides and oligodeoxyribonucleotides

Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.     

Cellular uptake and biological activity of peptide nucleic acids conjugated with peptides with and without cell‐penetrating ability

A 12‐mer peptide nucleic acid (PNA) directed against the nociceptin/orphanin FQ receptor mRNA was disulfide bridged with various peptides without and with cell‐penetrating features. The cellular uptake and the antisense activity of these conjugates were assessed in parallel. Quantitation of the internalized PNA was performed by using an approach based on capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF). This approach enabled a selective assessment of the PNA moiety liberated from the conjugate in the reducing intracellular environment, thus avoiding bias of the results by surface adsorption. The biological activity of the conjugates was studied by an assay based on the downregulation of the nociceptin/orphanin FQ receptor in neonatal rat cardiomyocytes (CM). Comparable cellular uptake was found for all conjugates and for the naked PNA, irrespective of the cell‐penetrating properties of the peptide components. All conjugates exhibited a comparable biological activity in the 100 nM range. The naked PNA also exhibited extensive antisense activity, which, however, proved about five times lower than that of the conjugates. The found results suggest cellular uptake and the bioactivity of PNA‐peptide conjugates to be not primarily related to the cell‐penetrating ability of their peptide components. Likewise from these results it can be inferred that the superior bioactivity of the PNA‐peptide conjugates in comparison with that of naked PNA rely on as yet unknown factors rather than on higher membrane permeability. Several hints point to the resistance against cellular export and the aggregation propensity combined with the endocytosis rate to be candidates for such factors. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

G-specific RNA-cleaving Conjugates of Short Peptides and Oligodeoxyribonucleotides

Abstract

Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.     

Can nuclear localization signals enhance nuclear localization of plasmid DNA?

Nonviral vectors are safer and more cost-effective than viral vectors but are significantly less efficient, and thus, increasing the efficiency of nonviral vectors remains an important objective. One way to overcome this problem is by stimulating the nuclear localization of exogenous genes. Nuclear localization signals (NLSs) are known to be involved in the active transport of exogenous proteins and probes into the nucleus. However, stimulation of nuclear localization of plasmid DNA has yet to be confirmed completely. In the present study, we prepared plasmid DNA-NLS peptide conjugates and adjusted spacer length and number introduced in an attempt to increase transfection efficiency. In comparison to conjugates with unmodified plasmid DNA and short spacers, we found that NLS-plasmid DNA conjugates with covalent bonding by diazo coupling through PEG chain (MW 3400) stimulated complexation with the nuclear transport proteins importin alpha and importin beta. Evaluation of transfection showed higher expression efficiency with plasmid DNA-NLS peptide conjugates than with unmodified plasmids. However, evaluation of intracellular trafficking after microinjection into the cytoplasm showed plasmid DNA-NLS peptide conjugates only within the cytoplasm; there was no NLS-plasmid stimulation of nuclear localization. Our findings suggest that stimulation of plasmid nuclear localization cannot be achieved merely by changing spacer length or chemically modifying plasmid DNA-NLS peptide conjugates. An additional mechanism must be involved.     

EFFICIENT SYNTHESIS OF OLIGONUCLEOTIDE-PEPTIDE CONJUGATES ON LARGE SCALE

The conjugation of oligonucleotide phosphorothioates with antennapedia peptide was studied in detail to allow efficient preparation of the conjugates on up to 15 μmol scale. Under optimized conditions, the use of oligonucleotides and the peptide in an equimolecular ratio gave the desired conjugates in more than 60% isolated yield.     

Synthesis, solution conformation, and antibody recognition of oligotuftsin-based conjugates containing a beta-amyloid(4-10) plaque-specific epitope

One possible therapeutic approach to treat or prevent Alzheimer's disease (AD) is immunotherapy. On the basis of the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Abeta(4-10) epitope alone or flanked by alpha- or beta-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the beta-amyloid (1-17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Abeta(4-10) B-cell epitope with the beta-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Abeta with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.     

Synthesis of tolerogenic monomethoxypolyethylene glycol and polyvinyl alcohol conjugates of peptides

Recent studies from this laboratory showed that tolerogenic peptide conjugates are very effective reagents for obtaining epitope-specific immunosuppression of antibody responses to immunopathogenic sites on multideterminant complex protein antigens. This paper describes the procedure for synthesis of well-defined conjugates of peptides to monomethoxypoly-ethylene glycol (mPEG) or to polyvinyl alcohol (PVA). The first step involves succinylation of the hydroxyl groups on the polymers by reaction with succinic anhydride. The polymer is then coupled via the carboxyl of the succinyl group to the -NH₂ of the completed peptide on the synthetic resin, while maintaining intact all the side-chain protecting groups on the peptide. The mPEG or PVA-peptide conjugates are cleaved from the resin and purified by standard procedures. This method results in the preparation of conjugates in which one molecule of tolerogenic polymer is coupled to the N-terminal of an otherwise unaltered peptide molecule.     

Structure-activity relationships of carboxymethylpullulan-peptide-doxorubicin conjugates--systematic modification of peptide spacers

A series of carboxymethylpullulan (CMPul)-doxorubicin (DXR) conjugates bound by peptide spacers of different compositions and lengths were prepared and evaluated for their in vivo antitumor effects. Systematic study of the peptide spacers indicated that CMPul-DXR conjugates bound via appropriate dipeptide spacers were more potent than DXR.     

Identification of CD21-binding peptides with phage display and investigation of binding properties of HPMA copolymer-peptide conjugates

Cancer targeting with peptides has become promising with the emergence of combinatorial peptide techniques such as phage display. Using phage display under stringent screening conditions, we selected five distinct peptides that specifically recognized the CD21 receptor, a cell surface marker of malignant B cell lymphoma. Two highly hydrophobic sequences were excluded (RLAYWCFSGLFLLVC and PVAAVSFVPYLVKTY). The binding affinity toward CD21 of the other three selected peptides (RMWPSSTVNLSAGRR, PNLDFSPTCSFRFGC, and GRVPSMFGGHFFFSR) was analyzed with fluorescence quenching. Their dissociation constants were determined to be within the micromolar range. On the basis of the results of phage ELISA, competitive phage ELISA, and fluorescence quenching, the binding sites of the three selected peptides were found to reside within the first four short consensus repeats of CD21 (SCR1-4). The peptide RMWPSSTVNLSAGRR (P1) was bound to the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, a potential drug carrier for chemotherapeutic agents, and the surface binding properties of HPMA copolymer-P1 conjugates were investigated. Specific interactions were observed between HPMA copolymer-P1 conjugates and surface-bound receptor. Binding of HPMA copolymer-P1 conjugates was directly related to the amount of surface (MaxiSorp plate) bound receptor, and the binding of the conjugates could be inhibited by the application of a 3-4 orders-of-magnitude excess of free peptide over the peptide concentration in conjugates. The enhanced binding of polymer-bound peptide was ascribed to multivalent interactions between the HPMA copolymer-P1 conjugate and the surface-bound CD21 receptor.     

Synthesis of peptide conjugates with vitamins for induction of antigen‐specific immunotolerance

In this report, we designed conjugates of an antigen peptide with the immunosuppressive vitamins all‐trans retinoic acid (ATRA) and vitamin D3 for efficient induction of antigen‐specific immunotolerance. We established a synthetic scheme for the preparation of the peptide‐vitamin conjugates, which the chemically unstable vitamins tolerated. Among the obtained conjugates, the ATRA conjugate successfully suppressed inflammatory effects in macrophages and dendritic cells and induced antigen presentation in dendritic cells. This synthetic method of conjugate is conceivably applicable to other antigen peptides for induction of antigen‐specific immunotolerance.     

Enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption

Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and other antisense agents. Cellular uptake of most CPP conjugates occurs mainly through endocytotic pathways, and most CPP conjugate is retained in the endosomal compartments of the cell. Several methods to induce endosome disruption have been shown to improve the bioavailability of CPP conjugates to the cytosol and/or nucleus by facilitating escape from the endosomal compartments. Here we describe protocols for the delivery of CPP-PNA conjugates to adherent cultured cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment to potentiate the antisense effects of CPP-PNA conjugates through increased release of CPP conjugates into the cytoplasm. This protocol, consisting of CPP-mediated delivery assisted by an endosome-disruption agent, allows the delivery of the CPP-PNA conjugates to the nucleus and/or cytosol of cultured cells. The endosome-disruption treatment improves the nuclear antisense effects of CPP-PNA conjugates by up to two orders of magnitude using 24-hour delivery.     

Conjugates of catecholamines. III. Synthesis and characterization of monodisperse oligopeptides conjugates related to isoproterenol

A series of monodisperse oligopeptide conjugates related to the catecholamine, isoproterenol, has been synthesized. The peptide carrier molecules used were synthesized by stepwise and fragment condensation techniques and ranged in size from a single, blocked amino acid derivative to isomeric pentapeptides. The amino acid compositions and sequences of the carriers were chosen so as to provide specific information concerning the effects of molecular weight, hydrophilic/hydrophobic balance, charge, etc., on the biological activity of the final conjugates. The common point of attachment for the drug in all carriers was a p-aminophenylalanine residue. The peptide-catecholamine conjugates were prepared via the attachment of carboxyl-containing catecholamine congeners, to the peptide carriers by techniques described previously. The conjugates were purified rigorously by chromatographic techniques and characterized by high-field n.m.r. spectroscopy.     

Immobilized ligands of high stability: Hydroxyalkyl methacrylate gel conjugates with oligo- and polypeptides

The long-time stability of conjugates prepared from epoxy derivatives of hydroxyalkyl methacrylate gels and peptides or proteins has been investigated at various pH values. These conjugates were found to be extremely stable. The observed slow release of nitrogen is due mainly to a splitting of the peptide bond adjacent to the covalent bond anchoring the peptide to the matrix. This peptide bond is partially labilized by microenvironmental influences of the matrix. Since in all experiments only a fraction of the immobilized ligand became detached, it is suggested that there is a subpopulation of fixed ligands which are susceptible to a matrix-induced bond splitting.     

Studies Towards the Synthesis of Peptide-Oligonucleotide Conjugates

Abstract

We describe a peptide fragment, solid-phase coupling strategy for synthesis of peptide-oligonucleotide conjugates. Model conjugates contained a hydrophobic tetrapeptide, a hydrophobic influenza virus fusion nonapeptide, or a basic octapeptide of the HIV-1 Tat protein coupled to either dT₁₂ or a 16-mer anti-Tat oligodeoxyribonucleotide. Conjugation yields were improved by removal of internucleotide 2-cyanoethyl groups prior to peptide coupling and by use of a C12 spacer between peptide and oligonucleotide.     

Design and biological activity of β-sheet breaker peptide conjugates

The sequence LPFFD (iAβ₅) prevents amyloid-β peptide (Aβ) fibrillogenesis and neurotoxicity, hallmarks of Alzheimer’s disease (AD), as previously demonstrated. In this study iAβ₅ was covalently linked to poly(ethylene glycol) (PEG) and the activity of conjugates was assessed and compared to the activity of the peptide alone by in vitro studies. The conjugates were characterized by MALDI-TOF. Competition binding assays established that conjugates retained the ability to bind Aβ with similar strength as iAβ₅. Transmission electron microscopy analysis showed that iAβ₅ conjugates inhibited amyloid fibril formation, which is in agreement with binding properties observed for the conjugates towards Aβ. The conjugates were also able to prevent amyloid-induced cell death, as evaluated by activation of caspase 3. These results demonstrated that the biological activity of iAβ₅ is not affected by the pegylation process.