Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Feb 14;34(3):e24.
doi: 10.1093/nar/gnj020.

Diels-Alder cycloadditions in water for the straightforward preparation of peptide-oligonucleotide conjugates

Vicente Marchán  1 , Samuel Ortega, Daniel Pulido, Enrique Pedroso, Anna Grandas
Affiliations

Diels-Alder cycloadditions in water for the straightforward preparation of peptide-oligonucleotide conjugates

Vicente Marchán et al. Nucleic Acids Res. .

Abstract

The Diels-Alder reaction between diene-modified oligonucleotides and maleimide-derivatized peptides afforded peptide-oligonucleotide conjugates with high purity and yield. Synthesis of the reagents was easily accomplished by on-column derivatization of the corresponding peptides and oligonucleotides. The cycloaddition reaction was carried out in mild conditions, in aqueous solution at 37 degrees C. The speed of the reaction was found to vary depending on the size of the reagents, but it can be completed in 8-10 h by reacting the diene-oligonucleotide with a small excess of maleimide-peptide.

PubMed Disclaimer

Figures

<b>Figure 1</b>
Figure 1
General scheme for the preparation of peptide–oligonucleotide conjugates by Diels-Alder reaction in water.
<b>Figure 2</b>
Figure 2
Reversed-phase HPLC traces of the conjugation reactions (d, diene-oligonucleotide; cj, conjugate). (A) maleimide-dipeptide-NH2 + diene-TT 1:1 after 4 h (the N-terminal amino acid of the dipeptide is indicated on top of each profile); (B) maleimide-GTSKLNYL-NH2 + diene-CATGGCT 2:1 after 2.5 h; (C) maleimide-GTSKLNYL-NH2 + diene-GATCTAAAAGACTTT, 2:1 after 6 h (a) and 5:1 after 1 h (b); (D) maleimide-KETAAAKFERQHMDSSTSAA-OH + diene-TTTTTTTT 1:1 after 20 h, first (a), second (b) and third (c) assay (see Materials and Methods); (E) maleimide-KETAAAKFERQHMDSSTSAA-OH + diene-TTTTTTTTTTTTTTT 1:1 after 20 h (a), and 8 h later after having added the amount of peptide equivalent to that of unreacted oligonucleotide (b).
<b>Figure 3</b>
Figure 3
Preparation of the maleimide-peptides: (a) assembly of the peptide chain using Fmoc-amino acids on Rink amide (top) or 2-chlorotrityl chloride (bottom) resins; (b) coupling of 3-maleimidepropanoic acid in the presence of DCC (or DIPC) and HOBt; (c) final deprotection with a 90–95% TFA solution.
<b>Figure 4</b>
Figure 4
Solution synthesis of diene-TT: (a) (PhOCH2CO)2O; (b) pTsOH; (c) tetrazole; (d) tBuOOH; (e) conc. aq. NH3.
<b>Figure 5</b>
Figure 5
Solid-phase synthesis of diene-oligonucleotides: (a) oligonucleotide assembly using standard 3′-phosphoramidite nucleoside derivatives; (b) tetrazole-mediated coupling of CH2=CH-CH=CH-CH2-CH2-O-P(OCNE)NiPr2, followed by capping and oxidation (aq. I2); (c) final deprotection with conc. aq. ammonia.
See this image and copyright information in PMC

References

    1. Venkatesan N., Kim S.J., Kim B.H. Novel phosphoramidite building blocks in synthesis and applications toward modified oligonucleotides. Curr. Med. Chem. 2003;10:1973–1991. - PubMed
    1. Kurreck J. Antisense technologies. Improvements through novel chemical modifications. Eur. J. Biochem. 2003;270:1628–1644. - PubMed
    1. Leumann C.J. DNA Analogues: from supramolecular principles to biological properties. Bioorg. Med. Chem. 2002;10:841–854. - PubMed
    1. Bashkin J.K., Gard J.K., Modak A.S. Synthesis and characterization of nucleoside peptides: toward chemical ribonucleases. 1. J. Org. Chem. 1990;55:5125–5132.
    1. Hovinen J., Guzaev A., Azhayeva E., Azhayev A., Lönnberg H. Imidazole tethered oligodeoxyribonucleotides: synthesis and RNA cleaving activity. J. Org. Chem. 1995;60:2205–2209.

Publication types

MeSH terms