@Bruno

Amino acids containing alkynes or azides are available from numerous chemical vendors. Your peptide synthesis core facility or preferred peptide manufacturer can provide peptides with Click Groups at either the N- or C-terminus, or on a sidechain.

Here are examples of complex peptides assembled using relatively simple bioorthogonal chemistry, including copper-catalyzed and copper-free Click:

•  Open-access article from California Institute of Technology: "Iterative In Situ Click Chemistry Creates Antibody-like Protein-Capture Agents" 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3716464/

• The Plückthun Group at the University of Zurich is adept at using bioorthogonal chemistry to modify recombinant proteins and artificial peptides. Many of their publications are open-access on their website. For procedural suggestions, it may be prudent to contact them directly. Here some impressive examples:

"Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click and Thiol Chemistries"

http://www.bioc.uzh.ch/plueckthun/pdf/APpub0331.pdf

"Orthogonal Assembly of a Designed Ankyrin Repeat Protein–Cytotoxin Conjugate with a Clickable Serum Albumin Module for Half-Life Extension"

http://www.bioc.uzh.ch/plueckthun/pdf/APpub0355.pdf

"Novel Prodrug-Like Fusion Toxin with Protease-Sensitive Bioorthogonal PEGylation for Tumor Targeting"

http://www.bioc.uzh.ch/plueckthun/pdf/APpub0370.pdf

Plückthun Group's publications listed by year.

http://www.bioc.uzh.ch/plueckthun/index.php?pid=3-1-0

• General troubleshooting guide for copper-catalyzed Click bioconjugation prepared by some of the original inventors of Click Chemistry: "Copper-Catalyzed Azide–Alkyne Click Chemistry for Bioconjugation"

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404492/

• The Bertozzi Group at Stanford University is also adept at synthesizing complicated molecules with bioorthogonal chemistry. Here are their publications:

http://web.stanford.edu/group/bertozzilab/publications.htm

Hope this information is helpful.

Dear Saikat and Lael,

Thank you so much for your input. I gave a quick look into the articles you suggested, and there is indeed a wealth of possibilities that may suit me well. I will read about them carefully. This is extremely helpful, yes!  

Once again, thank you so much!

Bruno

Using isothiocyanates you can avoid the laborious and poorly effective removal of copper ion residuals. Isothiocyanates can react even in aqueous conditions using some base only, enough selective for amines (particularly for primary amines, but they can react with thiols, too). You can find many mono/diisothiocyanates in fine chemical catalogs or some chemist friend can easily prepare you tailor made versions (R. Wong, S. J. Dolman, J. Org. Chem., 2007, 72, 3969-3971). Their reaction is usually as fast as the so-called azide/alkyne reaction - which is not always really click - but without the collateral impurities.

 Thank you Laszlo! The copper is indeed something I would like to avoid if possible. I will definitely look into iso and diisothiocyanates with more detail. 

Best regards,

Bruno

Hi Bruno,

I am just seeing this. Do you require cysteines in your peptides? Those will be problematic with pretty much everything, including the copper in simple azide-alkyne chemistry and the reactive triple bonds used in copper-free azide-alkyne chemistry. You can look at our paper in Bioorg. Med. Chem. Lett. that just came out (DOI: 10.1016/j.bmcl.2016.01.079) for the copper-free click chemistry that I used with peptides. I believe you can buy some of those bicyclononynes that I used (following a personal communication from C. Bertozzi herself - props to her for not pushing her own molecules on me) if for a hefty price tag. Oxime ligation (you can generate aldehyde-groups for that from N-terminal serines) plus azide-alkyne is probably your best bet. Maybe you can use protected versions of cysteine for conjugation and only deprotect them after conjugation? Feel free to contact me directly with your chemistry questions also...

Hi Kai, Many thanks for your reply and suggestions. First thing tomorrow I will check your paper. Cysteines would be desirable but perhaps not absolutely necessary.