Solid Phase Fragment Condensation Research Articles

Solid-phase fragment condensation for synthesis of peptides from the immunodominant sequence of β1-adrenoreceptor

The P26 peptide corresponding to the 197–222 sequence of the second extracellular loop of the β1-adrenoreceptor (β1-AR) was synthesized by solid-phase fragment condensation on the Wang polymer. Pentapeptide fragments were prepared on the 2-chlorotrityl resin. The racemization degree of the C-terminal alanine residue of the pentapeptide was experimentally evaluated for the synthetic H-Glu-Ser-Asp-Glu-Ala-Arg-OH hexapeptide β1-АR-(202–207) which was prepared by the 5 + 1 fragment condensation with the use of various condensing agents. A content of the diastereoisomeric peptide in the products of the fragment condensation was determined by HPLC on a reversed phase. The D-alanine-containing hexapeptide was specially synthesized and used for a comparison. The minimum racemization degree of the C-terminal alanine residue was observed if complex F was applied to the synthesis of the hexapeptide.

Convergent Solid-Phase Synthesis of Macromolecular MUC1 Models Truly Mimicking Serum Glycoprotein Biomarkers of Interstitial Lung Diseases.

Synthetic macromolecular MUC1 glycopeptides have been used to unravel molecular mechanisms in antibody recognition of disease-specific epitopes. We have established a novel synthetic strategy for MUC1 tandem repeats having complex O-glycosylation states at each repeating unit based on convergent solid-phase fragment condensation under microwave irradiation. We have accomplished the synthesis of 77 amino acid MUC1 glycopeptides (MW = 12 759) having three major antigenic O-glycoforms [Tn, core 1 (T), and core 2 structures] at 10 designated positions out of 19 potential O-glycosylation sites. We demonstrate that the macromolecular MUC1 glycopeptide displaying the essential glycopeptidic neoepitope Pro-Asp-Thr(sialyl-T)-Arg-Pro-Ala-Pro at two different tandem repeats is an excellent serum MUC1 model showing ideal stoichiometric binding with anti-KL6/MUC1 antibody in the sandwich ELISA to quantify human serum KL6/MUC1 levels as a critical biomarker of interstitial lung diseases.

Bidirectional solid phase synthesis of a model oligoglycine bolaamphiphile and purification by rapid self-assembly

We utilised a simple bidirectional (N→C and C→N) solid phase synthesis strategy entailing conventional solid phase peptide synthesis and fragment condensation with a water-soluble carbodiimide to synthesise a model anionic glycylglycine bolaamphiphile containing a suberic acid linker moiety, namely N,N'-suberoyldiglycylglycine. The synthetic suberoyldiglycylglycine was purified using its inherent ability to rapidly self-assemble in an aqueous acidic solution (0.1% trifluoroacetic acid). Monitoring of the rapid assembly process corroborated our visual observation and confirmed packing-directed self-assembly rather than non-specific aggregation or precipitation. The progress of suberoyldiglycylglycine self-assembly was observed to be via the formation of oligomers in the solution, which then self-assembled to form layered β-sheet type macrostructures. Within 24 h, nanotubes grew from these macrostructures and eventually combined to formed microtubes, which we isolated after 5-7 days.

Synthesis of phosphorothioate oligonucleotide–peptide conjugates by solid phase fragment condensation

Phosphorothioate oligonucleotide–peptide conjugates were synthesized by solid phase fragment condensation (SPFC). Arginine rich peptides could be successfully conjugated in 2.8–13.4% isolated yields. All the products were fully characterized by reversed phase HPLC and MALDI–TOF–MS to give satisfactory results.

Suppression of bcr/abl chimeric gene by conjugate DNA enzymes in human cells

Conjugate DNAzymes with polyamines and peptides were successfully prepared by solid phase fragment condensation (SPFC) and showed up to 4.2 times higher catalytic efficiency (kcat/Km). Intracellular localization of DNAzymes could be controlled by conjugated with naturally occurring signal peptides which are responsible for nuclear cytoplasmic transport of proteins. Suppression of bcr/abl chimeric gene on Philadelphia chromosome by conjugate DNA enzymes were largely enhanced in human leukemia cells.

Highly sensitive inhibition of hTERT mRNA expression and telomerase activity by DNA-signal-peptide conjugates

In the present study, we investigated the antisense properties of conjugate oligonucleotides (ODNs) inhibiting human telomerase activity. Conjugate oligonucleotides assembled with signal peptides, artificially designed peptides, amines and sugars were synthesized by solid phase fragment condensation (SPFC) in sufficient yields. Conjugate ODNs showed a high resistance to nuclease degradation and sufficient binding affinity to target RNA, comparatively rapid and sufficient intracellular delivery and specific localization controlled by signal peptides (nuclear localization signals, NLS; nuclear export signals, NES). ODN-NLS conjugates demonstrated high antisense inhibitory effects against human telomerase activity into the nucleus (e. g, phosphorothioate conjugate inhibited the telomerase activity over 95%), whereas ODN-NES conjugates inhibited target mRNA expression into the cytoplasm.

Development of HIV fusion inhibitors

In the past 25 years, the worldwide AIDS epidemic has grown such that roughly 38 million people were estimated to be living with the disease worldwide at the end of 2003. The introduction of antiretroviral-based therapies, beginning in 1987, has enabled many to live with HIV as a chronic, rather than terminal, disease. However, the emergence and spread of drug-resistant strains highlights the continued need for new therapies with novel modes of action. In 2003, the FDA and EMEA approved enfuvirtide (Fuzeon), a 36 amino acid peptide derived from the natural gp41 HR2 sequence, as the first HIV fusion inhibitor. T-1249, a 39 amino acid fusion inhibitor, is active against viruses that develop resistance to enfuvirtide. The development of FIs and the processes to manufacture enfuvirtide and T-1249 on an unprecedented scale for peptide therapeutics are presented. Synthetic routes based on a combination of solid phase peptide synthesis and solution phase fragment condensation as well as the analytical controls necessary to insure a robust process are discussed.

Synthesis of DNA conjugates by solid-phase fragment condensation via aldehyde–nucleophile coupling

Oligodeoxyribonucleotides were synthesized that contain a novel nucleoside, 2′- O-(2,3-dihydroxypropyl)cytidine. Its 2′-diol group was blocked by an allyloxycarbonyl protecting group. Selective deprotection of diol group(s) of the support-immobilized blocked oligodeoxyribonucleotide by Pd(0) followed by periodate oxidation resulted in generation of the 2′-aldehyde group(s) on solid-phase. The modified oligonucleotides were used to prepare a number of conjugates with acridine, biotin and N-modified laminin peptides by oxime, hydrazone and hydrazine formation. The method may be applicable to the synthesis of oligonucleotide–peptide conjugates.

Controlled intracellular localization and enhanced antisense effect of oligonucleotides by chemical conjugation

Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by solid phase fragment condensation. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed peptides with amphipathic character. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugate-RNA hybrids could activate RNase H as effectively as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into the cell nucleus, (f) the conjugates with NES could be localized in the cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells much more strongly than phosphorothioate oligonucleotides.

Efficient cleavage of RNA, enhanced cellular uptake, and controlled intracellular localization of conjugate DNAzymes

Conjugate DNAzymes with polyamines and peptides were successfully prepared by solid phase fragment condensation (SPFC) and showed up to 4.2 times higher catalytic efficiency ( k cat/ K m) and enhanced tolerance against DNase 1digestion. To be pointed out, intracellular localization of DNAzymes could be controlled by conjugated with naturally occurring signal peptides responsible for nuclear cytoplasmic transport of proteins.

Novel Synthesis of 2′-O-Modified Oligonucleotides by Solid Phase Fragment Condensation

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Synthesis of DNA Conjugates by Solid Phase Fragment Condensation

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DNA Conjugates as Novel Functional Oligonucleotides

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Loops on loops: generation of complex scaffolded peptides presenting multiple cyclic fragments.

Scaffolded peptides presenting two different cyclic peptide fragments through a cyclic peptidomimetic scaffold in a site-selective fashion, were generated by stepwise solid phase synthesis and fragment condensation in parallel, demonstrating that either strategy is adequate to generate complex scaffolded peptides.

Synthesis of DNA Conjugates by Solid Phase Fragment Condensation

Development of a novel method for the synthesis of DNA conjugates is described. Oligonucleotides were successfully conjugated with a variety of functional molecules on a solid phase (Solid Phase Fragment Condensation) using an amino, a hydroxyl, a thiol, and a carboxyl group. DNA-peptide conjugate was obtained as a pure from by a single RPHPLC purification approximately in 20% yield. Moreover, it was demonstrated that the present method was effective for the preparation of conjugate molecules, DNA-sugar, DNA-polyamine, DNA-lipid and so on. The study to create new intelligent DNAs by accumulation various biofunctions on the molecule by SPFC is now in progress in our laboratory.

Novel Synthesis of 2′-O Modified Oligonucleotides by Solid Phase Fragment Condensation

Novel Synthesis of 2′-O Modified Oligonucleotides by Solid Phase Fragment Condensation

DNA Conjugates as Novel Functional Oligonucleotides

Oligodeoxynucleotides with RNA cleavage activity 1) were conjugated with amines and peptides by solid phase fragment condensation (SPFC). It was found that 29 mer DNA enzyme conjugated with spermine at its 5′-end showed higher affinity to the target RNA sequence and 40 times higher activity of cleavage than native DNA enzyme. It is also to be noted that conjugate DNA enzymes showed increased resistance against nuclease digestion

Control of intracellular delivery and inhibition of genetic expression by DNA-peptide conjugates.

Various types of DNA-peptide conjugates were synthesized by solid phase fragment condensation (SPFC). DNA-LNS (nuclear localizing signal) peptide conjugate was proved to be delivered and localized into cellular nucleus and exhibited higher antisense inhibitory effect against telomerase than antisense phosphorothioate DNA. In contrast, DNAzymes conjugated with NES (nuclear export signal) peptide was shown to be taken up and localized in cytoplasm. Inhibitory effect of the conjugate DNAzyme against BCR-ABL tyrosine kinase was evaluated to be more significant than the native DNAzyme.

Conjugate DNAzymes.

Conjugate DNAzymes were synthesized by solid phase fragment condensation and their biological properties were characterized. They have increased affinity to target RNA, enhanced stability against DNase 1 digestion and comparable or higher RNA cleaving activity compared with native and also phosphorothioate DNA zymes. It was also demonstrated that conjugate DNAzymes could inhibit BCR-ABL tyrosine kinase in cellular lysis of human leukemia cell line. Consequently DNAzymes can be expected to act effectively in cellular system and also in vivo system.

Antisense effects of DNA-peptide conjugates.

Antisense conjugate DNA covalently bound to functional amines, sugars or peptides such as nuclear localization signals (NLS), nuclear export signals (NES) and artificial amphiphilic alpha-helical and beta-sheet peptides were synthesized by solid phase fragment condensation (SPFC). Inhibitory effects on human telomerase of synthesized antisense conjugate DNAs were evaluated by TRAP assay. Antisense conjugate DNAs showed several times higher inhibitory effects on telomerase activity compared to native antisense DNA in cell lysis solutions. Antisense conjugate DNAs showed comparable or higher as same as inhibitory activity compared with antisense phosphorothioate DNA (S-DNA). Interestingly, antisense conjugate DNA showed much higher inhibition against telomerase activity with native antisense DNA or antisense phosphorothioate DNA in cellular system. We observed enhanced cellular uptake and positively controlled intracellular localization of conjugate DNAs by use of confocal fluorescent microscope.