The use of linkers to join two chemical moieties requires their chemical stability towards various conditions as well as an established breaking point which allows for cleavage of the linker. Dde [N-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl] has been introduced as protecting group with cleavage conditions orthogonal to Fmoc/^tBu protocols, thus being stable towards denaturing washing as well as acidic and basic conditions. Advantageously, the cleavage of Dde using a (buffered) aqueous solution of hydrazine or hydroxylamine can be followed spectrophotometrically due to the formation of a chromophoric product.

Dde-based linkers can selectively and temporarily be attached to:

• Appropriately modified biomolecules for binding to streptavidin (with terminal biotin) (A), or conjugation to any solid supports, e.g. via Click reaction (B).
• Solubilizing tags, e.g. hexa-lysine (“helping-hand linkers”, (C)), oligo-arginine, PEGs (D) or other hydrophilic groups improving the solubility of hydrophobic peptides or other compounds when being attached to either the N-terminus or any lysine side chain within a peptide sequence thus facilitating the handling and purification of insoluble and aggregation-prone peptides.
• Dyes and any other conjugate for monitoring, diagnostics, targeting or other purposes.

➔ Please click here to see various examples for Dde-based linkers on our Website.

Dde-based linkers can be utilized for various applications: Attachment of a cleavable biotin tag to proteins for catch-and-release affinity purification over Streptavidin beads (A), reversible labeling or conjugation to other biomolecules, or reversible immobilization on solid supports via Click chemistry (B), temporary attachment of solubilizing tags like oligo-lysine (C) or PEGs (D).

Dde-based linkers with Dde as one of the terminal groups can be incorporated on-resin during SPPS in nearly quantitative steps after orthogonal deprotection of the N-terminus or any other sidechain amino function in the peptide sequence. Linkers with Dde as the connective element may be incorporated by means of their respective functional group(s). Dde can be selectively cleaved by using a (buffered) aqueous solution of hydrazine or hydroxylamine thus releasing the peptide after purification, streptavidin attachment, NCL-based assembly or any other reaction step. The cleavage can be monitored spectroscopically as the resulting pyrazole shows a strong absorption at 290 nm.

➔ Interested in further product details? Please get in contact for a Microsoft Teams product presentation!

➔ Related topics: “Peptide Easy Clean” for chromatography-free peptide purification – our Product of the Month in May 2020

➔ Please also see the Linkerology section on our webpage

References:

• Full Orthogonality between Dde and Fmoc: The Direct Synthesis of PNA-Peptide Conjugates; J. J. Díaz-Mochón, L. Bialy, and M. Bradley; Org. Lett. 2004; 7: 1127-1129. https://doi.org/10.1021/ol049905y
• Synthesis of hydrophobic insulin-based peptides using a helping hand strategy; M. M. Disotuar, M. E. Petersen, J. M. Nogueira, M. S. Kay and D. H. Chou; Org Biomol Chem 2019; 17: 1703-1708.     https://doi.org/10.1039/c8ob01212a  
• Chemical synthesis of Shiga toxin subunit B using a next-generation traceless "helping hand" solubilizing tag; J. M. Fulcher, M. E. Petersen, R. J. Giesler, Z. S. Cruz, D. M. Eckert, J. N. Francis, E. M. Kawamoto, M. T. Jacobsen and M. S. Kay; Org Biomol Chem 2019; 17: 10237-10244. https://doi.org/10.1039/c9ob02012h
• Mapping the Binding Site of BMS-708163 on gamma-Secretase with Cleavable Photoprobes; N. Gertsik, C. W. Am Ende, K. F. Geoghegan, C. Nguyen, P. Mukherjee, S. Mente, U. Seneviratne, D. S. Johnson and Y. M. Li; Cell Chem Biol 2017; 24: 3-8. https://doi.org/10.1016/j.chembiol.2016.12.006
• A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis; M. T. Jacobsen, M. E. Petersen, X. Ye, M. Galibert, G. H. Lorimer, V. Aucagne and M. S. Kay; J Am Chem Soc 2016; 138: 11775-82. https://doi.org/10.1021/jacs.6b05719
• Compounds and methods for purifying peptides produced by solid phase peptide synthesis; Aucagne V., Delmas A.; CNRS; U.S. Patent No. 9,073,969, 2015.
• Cleavable trifunctional biotin reagents for protein labelling, capture and release; Y. Yang and S. H. Verhelst; Chem Commun (Camb) 2013; 49: 5366-8. https://doi.org/10.1039/c3cc42076k
• Investigation on the stability of the Dde protecting group used in peptide synthesis: migration to an unprotected lysine; K. Augustyns, W. Kraas and G. Jung; J Pept Res 1998; 51: 127-33. https://doi.org/10.1111/j.1399-3011.1998.tb00630.x