Selective Lysine Labeling with Squaric Acid

Covalent labeling of surface-accessible amino acid side chain residues is a versatile tool for protein analysis, e.g. to reveal interactions, study their structure, and develop improved therapeutics. However, available tools often suffer from low selectivity as there are various reactive moieties competing with each other, such as amino groups of Lysines, guanidine functions of Arginines, imidazole rings of Histidines, alcohol functions of Serines and Threonines, thiols of Cysteines, as well as electron rich rings of Tyrosines and Tryptophans.

Lysines are often reported as key interacting amino acids in binding sites. Besides, they are more prevalent in the proteome compared to other nucleophilic amino acids like Cysteine, thus offering a larger range of utility but at the same time making selectivity much harder to achieve.

Squaric esters display a suitable compound class to overcome this limitation as they react chemoselectively with side chain amino functions of surface-accessible Lysines of proteins under mild conditions. Especially monosubstituted squaric acid esters can be used as reagents to couple to amino-functionalized compounds.

Mono amidated squaric acid esters react with extremely high selectivity with epsilon amino functions of surface accessible lysines within proteins. Other nucleophilic groups within biopolymers, such as guanidino functions of arginines, imidazole rings of histidines, alcohol moieties for serine and threonine, as well as electron rich aromatic rings from tyrosine or tryptophane will not be derivatized.

Compared to N-hydroxysuccinimidyl esters, which are also reported for protein labeling, the reaction of squarates with amines is up to four orders of magnitude slower. The use of squaric acid esters, hence, provides an accurate method to derivatize Lysines, only, and reduce side reactions with other amino acid side chains. Within our portfolio, we offer various squaric acid ethyl ester PEGylating reagents – just scroll down to see the related products.

→  For more information about PEGylation, download our brochure!

→  You are looking for a specific squaric acid ethyl ester derivative? Inquire for a custom synthesis!

References:

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Be squared: expanding the horizon of squaric acid-mediated conjugations; F. R. Wurm, H.-A. Klok; Chem. Soc. Rev. 2013; 42: 8220-8236. https://doi.org/10.1039/C3CS60153F

Squaric acid analogues in medicinal chemistry; J. Chasak, V. Slachtova, M. Urban, L. Brulikova; Eur. J. Med. Chem. 2021; 209: 112872. https://doi.org/10.1016/j.ejmech.2020.112872

Anticancer Agents, 15. Squaric Acid Diethyl Ester: A New Coupling Reagent for the Formation of Drug Biopolymer Conjugates. Synthesis of Squaric Acid Ester Amides and Diamides; L. F. Tietze, M. Arlt, M. Beller, K. H. Glüsenkamp, E. Jähde and M. F. Rajewsky; Chemische Berichte 2006; 124: 1215-1221. https://doi.org/10.1002/cber.19911240539

Squaric Esters Applications as Novel Lysine Electrophiles in Molecular Probe Design; J. S. Ho; PhD Thesis; Massachusetts Institute of Technology; 2020.