Joel P. Schneider, Yixin Xie, and Tania L. Lopez-Silva at the National Cancer Institute, report a new positively charged azidoamino acid for strain-promoted azide–alkyne cycloaddition, SPAAC, applications that overcomes possible solubility limitations of commonly used azidolysine, especially in systems with numerous ligation sites. The residue is easily synthesized, is compatible with Fmoc-based solid-phase peptide synthesis employing a range of coupling conditions, and offers efficient second-order rate constants in SPAAC ligations employing DBCO (0.34 M–1 s–1) and BCN (0.28 M–1 s–1).
Strain-promoted azide−alkyne cycloaddition has become a widely used tool for selective modifications of biomolecules in vitro and in vivo for numerous chemical biology and materials applications. This reaction is advantageous because of its chemoselectivity, reaction rates, and compatibility with aqueous physiological environments. However, the modification of peptides, proteins, and other macromolecules with azide or strained alkyne groups for SPAAC reactions can affect the macromolecule’s physical, structural, and functional properties, particularly when residues in the native primary sequence are altered or when multiple modifications are required.
For example, incorporating ligation handles can dramatically increase the macromolecular hydrophobicity, which affects the solubility and aggregation propensity. In some cases, organic solvents are needed to aid solubility, but at the risk of influencing the macromolecular structure.
A case in point is azidolysine N3K, which is commonly used to introduce an azide into peptides and proteins by solid-phase peptide synthesis, SPPS, often replacing a Lys residue in the native primary sequence. However, this replacement eliminates the Lys side-chain amine group that provides positive charge, which in turn facilitates solubility. Furthermore, side-chain positive charge may also be required for molecular interactions relevant to structure and function. In our own work, we experienced a problematic decrease in the solubility of peptides when N3K was incorporated at the expense of Lys.
One strategy to improve on commonly used SPAAC-enabling amino acids is to design unnatural residues that not only contain the necessary click chemistry handles but also functional groups that mimic those found in naturally
occurring side chains. In this work, the Schneider Group designed the positively charged azido-containing amino acid 1 to address the issue of decreased macromolecule solubility that often accompanies replacment of Lys with N3K.
Hydrophilic Azide-Containing Amino Acid to Enhance the Solubility of Peptides for SPAAC Reactions
Yixin Xie, Tania L. Lopez-Silva, and Joel P. Schneider
Org. Lett. 2022, 24, 40, 7378–7382
Tania L. Lopez-Silva, Joel P. Schneider, and Yixin Xie