Pictet-spengler ligation for protein chemical modification

Abstract

Aldehyde- and ketone-functionalized proteins are promising new substrates for the development of chemically modified biotherapeutics and protein-based materials. Their reactive carbonyl groups are typically conjugated with a-effect nucleophiles, such as substituted hydrazines and alkoxyamines, to generate hydrazones and oximes, respectively. However, the resulting C=N linkages are susceptible to hydrolysis under physiologically relevant conditions, which limits their utility in biological systems. Here we introduce a Pictet-Spengler ligation that is based on the classic Pictet-Spengler reaction of aldehydes and tryptamine nucleophiles. The ligation exploits the bioorthogonal reaction of aldehydes and alkoxyamines to form an intermediate oxyiminium ion; this intermediate undergoes intramolecular C-C bond formation with an indole nucleophile to form an oxacarboline product that is hydrolytically stable. The reaction was utilized for site-specific chemical modification of glyoxal- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin. In conjunction with techniques for site-specific introduction of aldehydes into proteins, the Pictet-Spengler ligation offers a new means to generate stable bioconjugates for medical and materials applications.

Description

[0001] Cross References to Related Applications [0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application 61 / 727,501, filed November 16, 2012, which is hereby incorporated by reference in its entirety. [0003] Statement Concerning Federal Government Rights in Inventions Resulting from Sponsored Research or Development [0004] This invention was made with US Government support awarded by the National Institutes of Health under grant number GM59907. The US Government has certain rights in this invention. Background of the invention [0005] introduction [0006] Reaction methodology for protein modification has been an active area of ​​research for decades. Early strategies focused on global modification of the original amino acid, providing access to a variety of modified products (Glazer AN (1970), "Specific Chemical Modification of Proteins", Annu. Rev. Biochem. 39(1): 101- 130). However, various applications require site-...

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Problems solved by technology

Some such reactions have been reported, but they are limited by slow reaction kinetics (Sasaki T, Kodama K, Suzuki H, Fukuzawa S, & Tachibana K (2008), "N-terminal labeling of proteins by the Pictet-Spengler reaction , "Bioorg.Med.Chem.Lett.18(16):4550-4553) or require organic co-solvent constraints (Alam J, Keller TH, & Loh T-P (2010), "Functionalization of Peptides and Proteins by Mukaiyama Aldol Reaction, "J.Am.Chem.Soc.132(28):9546-9548; Alam J, Keller TH, & Loh T-P (2011), "Indium mediated allylation in peptide and protein functionalization", Chem.Commun.47(32): 9066-9068)

A generally accepted C–C bond-forming transformation capable of achieving oxime bioconjugation with versatility and operational simplicity has not yet been reported.

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