Peptide Structure Stabilization by Click Chemistry

b) Peptide Synthesis with Azido and Alkyne Amino Acids

As Boc and Fmoc protected derivatives of both azido and alkyne amino acids are available, they can be introduced into peptide sequences through standard SPPS protocols, for example. In an α-helical secondary structure amino acids at positions i and i+4 are above each other. When they carry alkyne and azido function respectively, these groups are close to each other and can undergo cyclo-addition forming the corresponding 1,2,3-triazol moiety and stabilizing the secondary structure in the α-helical conformation. It has been demonstrated with nonapeptides [1,2] that whenever the 1,2,3-triazol bridge carries 5 or 6 methylene groups, the peptide shows a nice regular and ordered secondary structure. However, if the ring size is smaller (m+n= 4) or larger (m+n=7) the secondary structure is rather disordered.

Stabilization of the secondary structure also can be achieved by side chain lactam formation, for example with Lys and Asp at the appropriate positions. Azido and alkyne amino acids have, however, from synthetic and cost points of view some advantages. Using Lys and Asp orthogonal protecting groups have to be selected, which can result in a more complex process and more expensive building blocks. Azido and alkyne functions react highly specific to the 1,2,3-triazol ring. Educts and products are stable and inert at normal peptide synthesis conditions. No additional protection and related deprotection steps are required and the 1,2,3-triazol shows proteolytic stability, which is of high importance, whenever built into biological or pharmacological products.

Protocol for click reaction in peptide synthesis: Successful protocols have been published applying to 3 µmol peptide in 4 ml tBuOH/H2O (1:2) with excess of ascorbic acid (40 µmol) and CuSO4*5H2O (40 µmol) generating Cu(I) in situ. Stirring at room temperature over night is followed by appropriate chromatographic work up. [3]

References:

• [1] CuI-Catalyzed Azide–Alkyne Intramolecular i-to-(i+4) Side-Chain-to-Side-Chain Cyclization Promotes the Formation of Helix-Like Secondary Structures; Mario Scrima, Alexandra Le Chevalier-Isaad, Paolo Rovero, Anna Maria Papini, Michael Chorev, and Anna Maria D’Ursi; Eur. J. Org. Chem. 2010; 446–457; DOI: 10.1002/ejoc.200901157.
• [2] Synthesis and Conformational Analysis of a Cyclic Peptide Obtained via i to i+4 Intramolecular Side-Chain to Side-Chain Azide-Alkyne 1,3-Dipolar Cycloaddition; Sonia Cantel, Alexandra Le Chevalier Isaad, Mario Scrima, Jay J. Levy, Richard D. DiMarchi, Paolo Rovero, Jose A. Halperin, Anna Maria D’Ursi, Anna Maria Papini, and Michael Chorev; J. Org. Chem. 2008; 73: 5663–5674; DOI: 10.1021/jo800142s.
• [3] Side chain-to-side chain cyclization by click reaction; Alexandra Le Chevalier Isaad, Anna Maria Papini, Michael Chorev Paolo Rovero; J. Pept. Sci. 2009; 15: 451–454; DOI 10.1002/psc.1141
• [4] “Click” cyclized gallium-68 labeled peptides for molecular imaging and therapy: Synthesis and preliminary in vitro and in vivo evaluation in a melanoma model system; Molly E. Martin, M. Sue O’Dorisio, Whitney M. Leverich, Kyle C. Kloepping, and Michael K. Schultz; in: A Pathway to Personalized Diagnosis and Treatment Series: Recent Results in Cancer Research, 2012; 194: Richard P. Baum; Frank Rösch (Editors) 2012 ISBN 978-3-642-27993-5.
• [5] Improved synthesis and biological evaluation of chelator-modified -MSH analogs prepared by copper-free click chemistry; Nicholas J. Baumhover, Molly E. Martin, Sharavathi G. Parameswarappa, Kyle C. Kloepping, M. Sue O’Dorisio c, F. Christopher Pigge, Michael K. Schultz; Bioorg. Med. Chem. Lett. 2011; 21(19): 5757-61; DOI:10.1016/j.bmcl.2011.08.017.