Peptide-oligonucleotide Conjugates Research Articles

ChemInform Abstract: Fragment Coupling Approaches to the Synthesis of Peptide—Oligonucleotide Conjugates.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

A New and Efficient Method for Synthesis of 5′-Conjugates of Oligonucleotides through Amide-Bond Formation on Solid Phase

An efficient method for synthesis of oligonucleotide 5′-conjugates through amide-bond formation on solid phase is described. Protected oligonucleotides containing a 5′-carboxylic acid function were obtained by use of a novel non-nucleosidic phosphoramidite building block, where the carboxylic acid moiety was protected by a 2-chlorotrityl group. The protecting group is stable to the phosphoramidite coupling conditions used in solid-phase oligonucleotide assembly, but is easily deprotected by mild acidic treatment. The protecting group may be removed also by ammonolysis. 5′-Carboxylate-modified oligonucleotides were efficiently conjugated on solid support under normal peptide-coupling conditions to various amines or to the N-termini of small peptides to yield products of high purity. The method is well-suited in principle for the synthesis of peptide-oligonucleotide conjugates containing an amide linkage between the 5′-end of an oligonucleotide and the N-terminus of a peptide.

Synthesis of peptide-oligonucleotide conjugates with single and multiple peptides attached to 2'-aldehydes through thiazolidine, oxime, and hydrazine linkages.

2'-Deoxyoligonucleotides and 2'-O-methyloligoribonucleotides carrying one or more 2'-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy, or hydrazide group to give peptide-oligonucleotide conjugates incorporating single or multiple peptides in good yield. The facile conjugation method allows specific coupling in aqueous solution of unprotected oligonucleotides containing aldehyde groups to unprotected N-terminally modified peptides and other small molecules. A 12-mer 2'-O-methyloligoribonucleotide complementary to the HIV-1 TAR RNA stem-loop and containing two conjugated copies of an 8-mer model laminin peptide was hardly affected in TAR RNA binding and showed a similar level of inhibition of HIV-1 Tat-dependent in vitro transcription compared to the unconjugated 2'-O-methyloligoribonucleotide. Advantages of this conjugation method include (1) the ability to attach more than one peptide or other small molecule to oligonucleotide at defined nucleoside residue locations; (2) a conjugation route that does not affect significantly oligonucleotide binding to RNA structures; and (3) three alternative, facile, and mild conjugation reaction types that do not require use of a large excess of peptide reagent.

Evaluating the Specificity of Antisense Oligonucleotide Conjugates: A DNA ARRAY ANALYSIS

Antisense oligonucleotides are potentially powerful tools for selective control of cellular and viral gene expression. Crucial to successful application of this approach is the specificity of the oligonucleotide for the chosen RNA target. Here we apply DNA array technology to examine the specificity of antisense oligonucleotide treatments. The molecules used in these studies consisted of phosphorothioate oligomers linked to the Antennapedia (Ant) delivery peptide. The antisense oligonucleotide component was complementary to a site flanking the AUG of the MDR1 message, which codes for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Using a DNA array of 2059 genes, we analyzed cellular responses to molecules comprised of Ant peptide-oligonucleotide conjugates, as well as to the Ant peptide alone. Besides the expected reduction in MDR1 message level, 37 other genes (approximately 2% of those tested) showed changes of comparable magnitude. The validity of the array results was confirmed for selected genes using Northern blots to assess messenger RNA levels. These results suggest that studies using antisense oligonucleotide technology to modulate gene expression need to be interpreted with caution.

Synthesis of antisense oligonucleotide-peptide conjugate targeting to GLUT-1 in HepG-2 and MCF-7 Cells.

A simple procedure for the preparation of oligonucleotide-peptide conjugate was developed. p-Hydroxy-benzoic acid was used as a linker for the connection of the fragments of peptide and oligonucleotide. It was found that such formed linkage was stable under the conditions of conjugate synthesis. The designed conjugate targeting to GLUT-1 showed up to 50% inhibition of cell proliferation in HepG-2 and MCF-7 cells. Comparing to the results from the expressed antisense RNA in cancer cells, it was proposed that the conjugate of signal peptide mimic and antisense oligonucleotide could improve the permeability of antisense oligonucleotide through cell membrane.

Synthesis of oligonucleotide–peptide conjugates using hydrazone chemical ligation

An oligonucleotide was functionalized on the solid-phase by a tartaramide moiety, which could be converted efficiently in solution into a glyoxylyl group following a mild periodic oxidation. The glyoxylyl-oligonucleotide was found to be very stable upon storage and was successfully engaged in hydrazone ligation with an α-hydrazino acetyl peptide.

Conjugates of antisense oligonucleotides with the Tat and antennapedia cell-penetrating peptides: effects on cellular uptake, binding to target sequences, and biologic actions.

The attainment of effective intracellular delivery remains an important issue for pharmacologic applications of antisense oligonucleotides. Here, we describe the synthesis, binding properties, and biologic properties of peptide-oligonucleotide conjugates comprised of the Tat and Ant cell-penetrating peptides with 2'-O-methyl phosphorothioate oligonucleotides. The biologic assay used in this study measures the ability of the antisense molecule to correct splicing of an aberrant intron inserted into the Luciferase gene; thus, this assay clearly demonstrates the delivery of functional antisense molecules to the splicing machinery within the nucleus. The binding affinities of the conjugates to their target sequences were measured by surface plasmon resonance (BIAcor) techniques. The peptide-oligonucleotide conjugates progressively entered cells over a period of hours and were detected in cytoplasmic vesicles and in the nucleus. Peptide-oligonucleotide conjugates targeted to the aberrant splice site, but not mismatched controls, caused an increase in Luciferase activity in a dose-responsive manner. The kinetics of Luciferase appearance were consistent with the course of the uptake process for the conjugates. The effects of peptide conjugation on the hybridization characteristics of the oligonucleotides were also examined using surface plasmon resonance. The peptide-oligonucleotide conjugates displayed binding affinities and selectivities similar to those of unconjugated oligonucleotides. Conjugation with cell-penetrating peptides enhances oligonucleotide delivery to the nucleus without interfering with the base-pairing function of antisense oligonucleotides.

Sequence-specific RNA cleavage by oligonucleotide-peptide conjugates

This paper considers the site-directed cleavage of the in vitro transcript of human tRNALys3 by the conjugate CNH2Gly(ArgLeu)3ArgLeuN—p-DAG—5"CCCTGGACCCTCAGAT3" (conjugate pep-1A) of the oligodeoxyribonucleotide 1A (CCCTGGACCCTCAGAT) with a peptide [LeuArg]4Gly attached at the 5" terminus of the oligonucleotide through diethylene glycol as a linker. Under physiological conditions, the conjugate pep-1A cleaved the C56-A57 phosphodiester bond of the RNA target with high efficacy. In addition, the conjugate cleaved several other bonds in the D loop, at the basis of the amino-acceptor stem, and in the anticodon hairpin due, probably, to the formation of imperfect complexes of the conjugate with partially complementary sequences in the RNA structure.

Artificial ribonucleases: oligonucleotide-peptide conjugates that cleave RNA at the GpX and PypA phosphodiester bonds.

Artificial ribonucleases: oligonucleotide-peptide conjugates that cleave RNA at the GpX and PypA phosphodiester bonds.

Towards a general method for the stepwise solid-phase synthesis of peptide–oligonucleotide conjugates

Three peptide–oligonucleotide phosphorothioate hybrids were synthesized using a new approach for stepwise solid-phase synthesis of conjugates. This method utilizes commercially available N α-Fmoc amino acids for peptide synthesis and a new solid support. Three specific modifications of the solid-phase were made after the peptide and before the oligonucleotide assembly.

Synthesis of peptide-oligonucleotide conjugates: application to basic peptides.

Efficient methods of synthesis of peptide-oligonucleotide conjugates are badly needed for studies of uptake into cells in culture. We describe improvements to the procedures involved in "native ligation" conjugation in solution to extend the method to basic peptides. Further, we describe progress in development of a total solid-phase synthesis approach that makes use of a L-homoserine linker as the key reagent for growing of oligonucleotide and peptide chains on a single solid support.

Highly efficient synthesis of peptide-oligonucleotide conjugates: chemoselective oxime and thiazolidine formation.

A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-containing peptide with an aldehyde-containing oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatised by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aqueous conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with molecular biology tools, this methodology opens up new prospects for post-functionalisation of high-molecular-weight DNA structures.

Synthesis of Oligonucleotide—Peptide Conjugates and Nucleopeptides

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis of peptide-oligonucleotide conjugates for chromium coordination.

The synthesis of the first peptide-oligonucleotide conjugate designed to coordinate chromium(III) is reported. The overall goal of this work is to synthesize di-deoxynucleotides tethered with chromium(III)-coordinating appendages to model chromium-DNA-protein cross-links, which are a type of DNA lesion that may be involved in chromium-induced cancers. The conjugate dGp(NHCH(2)CH(2)S-Ac-Gly-Ser-Gly-OH)G was made by coupling the peptide, ClAc-Gly-Ser-Gly-OH, and dinucleotide, dGp(NHCH(2)CH(2)SH)G, through a thioether moiety. The conjugate was characterized by HPLC and mass spectrometry. Previously reported methods for small-scale solid-phase synthesis of peptides and dinucleotide were unsuitable; therefore, gram-scale solution-phase methods were developed. We also report the gram-scale syntheses of two other serine-containing peptides, ClAc-betaAla-Ser-Gly-OH and ClAc-Ser-Gly-OH, and three histidine-containing peptides, ClAc-Gly-His-Gly-OH, ClAc-betaAla-His-Gly-OH, and ClAc-His-Gly-OH. The synthesis and characterization of chromium-containing peptide-oligonucleotide conjugates will ultimately help us to understand chromium-DNA interactions at a molecular level, which is necessary before we can determine how chromium causes cancer.

ChemInform Abstract: Peptide Conjugates of Oligonucleotides as Enhanced Antisense Agents

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

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.

Preparation and evaluation of tumor-targeting peptide-oligonucleotide conjugates.

Enormous progress has been made in the development of antisense oligodeoxynucleotides (ODNs) as therapeutic agents inhibiting gene expression. Unfortunately, the therapeutical application of ODNs is still held back because of the low cellular uptake and the lack of specific transport into particular cells. In this paper, we report a drug-targeting system using somatostatin receptors (SSTRs) which are overexpressed in various tumors. Phosphorothioate ODNs were covalently linked to Tyr(3)-octreotate, an analogue of somatostatin. The peptide was assembled by solid-phase synthesis, oxidized to form the cyclic disulfide, and subsequently derivatized with a N-terminal maleimido functionality. 5'-Thiol derivatized phosphorothioate-ODNs directed against the protooncogene bcl-2 were conjugated to this maleimido-modified peptide. Binding studies revealed that the conjugates retain specific binding with nanomolar affinities to SSTRs (IC(50)-values between 1.83 and 2.52 nM). Furthermore, melting studies with complementary DNA revealed that the terminal conjugation of the ODNs did not significantly affect their hybridization affinity.

ChemInform Abstract: A Novel Synthesis of Oligonucleotide-Peptide Conjugates with a Base-Labile Phosphate Linker Between the Two Components According to the Allyl-Protected Phosphoramidite Strategy.

Abstract An efficient synthesis of base-labile nucleotide–peptide conjugates has been accomplished, in which the two components are directly linked between the terminal hydroxyl of a nucleotide and the hydroxyl of a serine or threonine residue of a peptide by a phosphodiester bond. This synthesis utilizes the phosphoramidite method with allyl for the phosphate linkages and the C-terminal of the peptide and allyloxycarbonyl for the nucleoside bases and the N-terminal of the peptide. In this synthesis, the removal of the allylic protecting groups and the detachment of the products was achieved under non-basic or mild basic conditions to bring about no conspicuous decomposition of the labile phosphate linker, and thus the target conjugates were obtained at a high purity and in high yields.

New Phosphoramidite Reagents for the Synthesis of Oligonucleotides Containing a Cysteine Residue Useful in Peptide Conjugation

The preparation is described of four 2-cyanoethyl-N,N-diisopropyl phosphoramidites of N-α-Fmoc-S-protected cysteine hydroxyalkyl amides. The phosphoramidites were used in solid-phase synthesis of 5′-cysteinyl oligonucleotides, useful intermediates in the preparation of peptide-oligonucleotide conjugates through reaction with a maleimide peptide or with a peptide thioester via “native ligation”.

Efficient conjugation of peptides to oligonucleotides by "native ligation".

A new strategy has been developed for conjugation of peptides to oligonucleotides. The method is based on the "native ligation" of an N-terminal thioester-functionalized peptide to a 5'-cysteinyl oligonucleotide. Two new reagents were synthesized for use in solid-phase peptide and oligonucleotide synthesis, respectively. Pentafluorophenyl S-benzylthiosuccinate was used in the final coupling step in standard Fmoc-based solid-phase peptide assembly. Deprotection with trifluoracetic acid generated in solution peptides substituted with an N-terminal S-benzylthiosuccinyl moiety. O-trans-4-(N-alpha-Fmoc-S-tert-butylsulfenyl-L-cysteinyl)aminoc yclohe xyl O-2-cyanoethyl-N,N-diisopropylphosphoramidite was used in the final coupling step in standard phosphoramidite solid-phase oligonucleotide assembly. Deprotection with aqueous ammonia solution generated in solution 5'-S-tert-butylsulfenyl-L-cysteinyl functionalized oligonucleotides. Functionalized peptides and oligonucleotides were used without purification in native ligation conjugation reactions in aqueous/organic solution using tris-(2-carboxyethyl)phosphine to remove the tert-butylsulfenyl group in situ and thiophenol as a conjugation enhancer. A range of peptide-oligonucleotide conjugates were prepared by this route and purified by reversed-phase HPLC.