Synthesis by Fragment Ligation

Synthesis by Fragment Ligation

The delivery of medium or high molecular weight compounds (including large proteins) and drugs can be delivered to cells by binding to specific peptide carriers, such as osmotic proteins, tate peptides. Similarly, PNA-peptide conjugates may also be used as carriers to deliver drugs to eukaryotic cells or to bacteria. Creative Peptides is committed to advancing the extensive research of PNA and supporting customers' PNA development and application.

About Fragment Ligation

Fragment ligation means synthesizing and purifying individual PNA and peptide sequences, and then chemically selectively ligating in an aqueous environment under conditions compatible with the function of nucleobases and amino acid side chains. Depending on the type of connection between the peptide and the PNA fragment, different methods can be applied.

The Methods of Linkage of Fragments

Connect the fragments through disulfide bond

  • A procedure: both peptides and PNA fragments need to be equipped with a thiol moiety, which is usually obtained by incorporating a cysteine residue at the C-terminal or N-terminal position.
  • A more fruitful procedure: the nucleophilicity of the thiol group of one of the fragments and activation of the thiol of the other. An often applied method to activate a thiol group towards nucleophilic substitution is its attachment to a thiopyridyl moiety. Nucleophilic substitution of this moiety by the incoming free thiol of the other fragment results in formation of the desired product.

Connect the fragments through thioether linkages

  • The thiol-containing fragment can react with the double bond of the maleimide functional group of another fragment in a Michael-type reaction to obtain a covalently linked conjugate.
(a) Conjugation by oxime formation. (b) Chemical ligation of a peptide thioester and cys-PNA.

Fig.1 (a) Conjugation by oxime formation.(b)
Chemical ligation of a peptide thioester and
cys-PNA.(Koning M.; et al.2003)

Connect the fragments through chemoselective oxime

  • A ketone functionality of one fragment was reacted with an amino-oxy group of the other.

Connect the fragments through native chemical ligation

  • A reversible transthioesterification between two unprotected fragments, one containing a C-terminal thioester and the other an N-terminal cysteine.

All compounds were characterized by mass spectrometry, the masses of PNA, peptide, and PNA-peptide conjugates were analyzed. We used PNA extinction coefficient for the concentration determination of the PNA-peptide conjugates. The conjugates were stored at –20 °C.

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  • Koning M.; et al.Synthetic developments towards PNA–peptide conjugates. Current Opinion in Chemical Biology,2003, 7(6):734-740.
  • Awasthi S.K.; Nielsen P.E. Synthesis of PNA-Peptide Conjugates. Methods in Molecular Biology, 2002, 208(3):43.

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